Survival, physiological response and recovery of enteric bacteria exposed to a polar marine
environment
by James Joseph Smith
A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in
Microbiology
Montana State University
© Copyright by James Joseph Smith (1995)
Abstract:
This report describes an assessment of the survival, physiological response and recovery of common
human fecal indicator and enteropathogenic bacteria with long-term polar marine exposure. The
maintenance and expression of antibiotic-resistance (R) and conjugative (F) plasmids were also
examined. Direct (non-culture-based) methods for assessing bacterial viability were employed to
determine fractions of viable-but-nonculturable (VBNC) bacteria. A systematic study of the
mechanisms behind one of these techniques (5-cyano-2,3-ditolyl tetrazolium chloride [CTC] reduction)
was also investigated in a model prokaryote Polar marine exposure experiments were performed using
in situ diffusion chambers filled with cultures of 2 indicator, (Escherichia coli, Enterococcus faecalis),
and 2 pathogenic bacteria (Salmonella typhimurium, Yersinia enterocolitica). These were placed in situ
at Winter Quarters Bay, McMurdo Station, Antarctica for periods of 54-56 days (-1.8°C, 34.5 ppt
salinity). E. coli cells harboring R-(pUC19), as well as F-(pFamp), plasmids were exposed for 54 and
21 days, respectively. Exposure significantly extended the survival of all enteric bacteria examined
when compared to survival studies in more temperate marine environments. Little cell lysis or
decreases in total cell numbers were noted. A progressive increase in amounts of VBNC and injured
cells with exposure was noted in E. coli, S. typhimurium, and Y. enterocolitica. These organisms also
became markedly thermosensitive after 48 days exposure, and would no longer form colonies on
plating media at 37°C. E. coli, S. typhimurium, and Y. enterocolitica respiratory activity was limited by
nutrient availability rather than temperature in the environment. R-, and F- plasmids were maintained
and expressed for up to 54 days.
Experiments demonstrated that both CTC and INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5 -phenyl
tetrazolium chloride) were reduced in response to bacterial respiratory activity. CTC appeared to be
reduced by the primary aerobic dehydrogenases (NADH, succinate), while INT was reduced by these
as well as ubiquinone and possibly cytochromes b555,556. Both CTC and INT were reduced under all
anaerobic conditions examined. However, CTC appears to form a nonfluorescent formazan in the
presence of high concentrations of inorganic phosphate.
SURVIVAL, PHYSIOLOGICAL RESPONSE AND RECOVERY OF ENTERIC
BACTERIA EXPOSED TO A POLAR MARINE ENVIRONMENT
by
Jam es Jo sep h Sm ith
A th esis su b m itte d in p a rtia l fu lfillm en t
o f th e re q u ire m e n ts fo r th e d eg ree
of
D octor o f P hilosophy
in
M icro b io lo g y
MONTANA STATE UNIVERSITY, BOZEMAN
Bozem an, M o n tan a
May 1995
© COPYRIGHT
by
Jam es Jo sep h Sm ith
1995
All R ights R eserved
JXIt
Svvx Lr!3
ii
APPROVAL
o f a thesis su b m itted by
Jam es Joseph Sm ith
This thesis h as b e en re a d by each m e m b e r of th e th esis com m ittee a n d
h a s b e e n fo u n d s a tis fa c to ry re g a rd in g c o n te n t, E nglish u sag e, fo rm a t,
c itatio n s, b ib lio g rap h ic style, a n d con sisten cy , a n d is re a d y fo r subm ission to
th e College of G raduate Studies.
Ce -
>n, G rad u ate Com m ittee
Date
A pproved fo r th e M ajor D e p artm en t
H ead, Mmor D ep artm en t
Date
A pproved fo r the College o f G rad u ate Studies
7/V ^
D ate
G ra d u a te D ean
SURVIVAL, PHYSIOLOGICAL RESPONSE AND RECOVERY OF ENTERIC BACTERIA
EXPOSED TO A POLAR MARINE ENVIRONMENT
Jam es Jo se p h Sm ith
Advisor: G ordon A. M cFeters
M ontana S tate U niversity
1994
A b stra c t
This re p o rt d escrib es a n assessm en t o f th e survival, physiological
re sp o n se a n d re c o v ery o f com m on h u m a n fecal in d ic a to r a n d
e n te ro p a th o g e n ic b a c te ria w ith lo n g -term p o la r m a rin e ex p o su re. The
m a in te n a n c e a n d ex p re ssio n o f a n tib io tic -re sistan c e (R) a n d co n ju g ativ e (F)
plasm ids w ere also exam ined. D irect (n o n -cu ltu re-b ased ) m e th o d s fo r
assessing b a c te ria l v iab ility w ere em p lo y ed to d e te rm in e frac tio n s of viableb u t-n o n c u ltu ra b le (VBNC) b acteria. A system atic stu d y o f th e m echanism s
b e h in d o n e o f th e se tech n iq u es (5-cyano-2,3-ditolyl te tra z o liu m ch lo rid e [CTC]
re d u c tio n ) was also in v estig ated in a m o d el p ro k a ry o te
Polar m a rin e ex p o su re e x p erim en ts w ere p e rfo rm e d u sin g in situ
diffusion c h am b ers filled w ith c u ltu res o f 2 in d ic a to r, (Escherichia coli,
Enterococcus faecahs), a n d 2 p ath o g en ic b a c te ria (Salm onella typhim urium ,
Yersinia enterocolitica). T hese w ere p la c e d in situ a t W inter Q u arters Bay,
M cM urdo Station, A ntarctica fo r p erio d s o f 54-56 d ay s (-1.8°C, 34.5 p p t
salinity). E. coh cells h a rb o rin g R-(pUC19), as well as F-(pFam p), plasm ids
w ere exposed fo r 54 a n d 21 days, respectively. Exposure significantly ex ten d ed
th e survival o f all e n te ric b a c te ria e x am in ed w h en c o m p a re d to survival
stu d ies in m o re te m p e ra te m a rin e e n v iro n m e n ts. Little cell lysis o r d ecreases
in to ta l cell n u m b e rs w ere n o te d . A p ro g ressiv e in crease in a m o u n ts of VBNC
a n d in ju re d cells w ith exposure was n o te d in E. coli, S. typhim urium , a n d Y.
enterocolitica. T hese organism s also becam e m a rk ed ly th erm o sen sitiv e a fte r
48 days exposure, a n d w ould n o lo n g e r fo rm colonies o n p latin g m e d ia a t 37°C.
E. coli, S. typhim urium , a n d Y. enterocolitica re s p ira to ry a ctiv ity was lim ited
b y n u trie n t a v ailab ility r a th e r th a n te m p e ra tu re in th e e n v iro n m e n t. R-, a n d
F- plasm ids w ere m a in ta in e d a n d expressed fo r u p to 54 days.
E xperim ents d e m o n stra te d th a t b o th CTC a n d INT (2-(4-iodophenyl)-3(4 -n itro p h en y l)-5 -p h e n y l te tra z o liu m ch lo rid e) w ere re d u c e d in re sp o n se to
b a cte ria l re s p ira to ry activity. CTC a p p e a re d to b e re d u c e d b y th e p rim ary
aerobic d eh y d ro g e n ases (NADH, succinate), w hile INT was re d u c e d b y th ese as
well as u b iq u in o n e a n d possibly cytoch ro m es 6555,556- Both CTC a n d INT w ere
re d u c e d u n d e r all an aero b ic conditions exam ined. However, CTC a p p ea rs to
fo rm a n o n flu o re sc e n t fo rm az a n in th e p re se n c e of h ig h c o n c e n tra tio n s of
in o rg a n ic p h o s p h a te .
iii
STATEMENT OF PERMISSION TO USE
In p re s e n tin g th is th e sis in p a rtia l fu lfillm e n t o f th e re q u ire m e n ts fo r
a d o c to ra l d e g re e a t M o n ta n a S ta te U n iv e rsity , B ozem an, I a g re e th a t th e
lib ra ry sh all m ake i t a v a ila b le to b o rro w e rs u n d e r th e ru le s o f th e L ib rary , I
f u r th e r a g re e th a t c o p y in g o f th is th e sis is allo w ab le o n ly fo r sc h o la rly
p u rp o se s, c o n siste n t w ith “fa ir u s e ” as p re s c rib e d in th e U.S. C o p y rig h t Law.
R eq u ests fo r e x te n siv e co p y in g o r r e p r o d u c tio n o f th is th e s is s h o u ld b e
r e f e r r e d to U n iv e rsity M icrofilm s In te rn a tio n a l, 3 00 N o rth Zeeb R oad, A n n
A rb o r, M ichigan 4 8 1 0 6 , to w h o m I h a v e g ra n te d “th e e x clu siv e r ig h t to
re p ro d u c e a n d d is trib u te m y d is se rta tio n fo r sale in a n d fro m m icro film o r
electronic fo rm at, along w ith th e rig h t to re p ro d u c e a n d d istrib u te m y a b s tra c t
in a n y fo rm a t in w hole o r in p a rt.”
Signature.
Date,
/
iv
This w ork is d e d ic a te d to m y la te little -b ro th e r, A ndrew W illiam Smith.
ACKNOWLEDGEMENTS
I w o u ld firs t like to ex p ress m y d e e p a p p re c ia tio n to m y m e n to r, D r.
G ordon A. McFeters, w ho k e p t m y som etim es b lu rry v isio n fo c u se d o n m y goal,
a n d alw ays h a d sage w o rd s o f e n c o u ra g e m e n t.
Second* I w o u ld like to give
sincere th an k s to Dr. Gill G. G eesey, fo r his e n co u rag e m e n t o f m y re s e a rc h a n d
life’s goals, a n d fo r serv in g o n m y g ra d u a te co m m ittee.
I w o u ld also like to
th a n k all m y com m ittee m em bers, Dr. Clifford Bond, Dr. W a rre n Jones, Dr. Jo an
H enson, a n d Dr. D onald Schiem ann.
D uring m y w ork in A ntarctica, I w o u ld like to sin c e re ly th a n k m y “seaice p a r tn e r ”, Jam es H ow ington, as w ell as D r. Jim B arry, D r. J o h n Priscu, Rich
B artlett, S hona H. Hastie, Barb Kelley, th e crew s of S-025/S-004, a n d th e staff o f
A n ta rc tic S u p p o rt A ssociates fo r t h e n a ss is ta n c e in w ays to o n u m e ro u s to
m e n tio n .
I w o u ld a lso lik e to th a n k th e B rem ers, G eeseys, G illis’, a n d Jam ie
S om m ers (a n d fa m ily ) fo r th e ir f r ie n d s h ip a n d m a n y g o o d tim e s s p e n t
to g e th e r.
My fellow g ra d u a te s tu d e n ts a n d th e s ta ff a t th e M icro b io lo g y
D e p artm en t h a v e alw ays b e e n a source o f e n co u rag e m e n t a n d assistance.
My re s e a rc h a ctiv itie s h a v e b e e n s p o n s o re d b y th e N a tio n a l S cience
F o u n d a tio n Office o f P o la r P ro g ram s, a n d N a tio n al A e ro n a u tic s a n d Space
A d m in istratio n . T h e ir fin a n c ia l s u p p o rt is g ra te fu lly acknow ledged.
Finally, b u t m o st im p o rtan tly , I sh o u ld like to th a n k w ith d e e p love a n d
g ra titu d e th e s u p p o rt o f m y p a re n ts a n d fam ily .
T h e ir e n c o u ra g e m e n t, a n d
love th ro u g h o u t m y e d u ca tio n a n d life h a v e m a d e it all possible.
vi
TABLE OF CONTENTS
P age
LIST OF TABLES......................................................................................................................... ix
LIST OF FIGURES........................................................................................................................ x
ABSTRACT...........................
CHAPTER I . GENERAL INTRODUCTION....................................................
xiv
I
ro in
In d ic a to rs ...................................................................................................
I
Survival o f Enteric B acteria in th e M arine E n v iro n m e n t................
Starvation, Cold Shock, a n d th e “V iable-but-N onculturable” State
Effects of C ulture Physiology o n Survival a n d C u ltu ra b ih ty ..........
E n u m eratio n a n d A ctivity M easurem ents o f B acteria in A queous
E n v iro n m e n ts ................
9
Plasm id M aintenance a n d E x p re ssio n .............................................................;. 10
Enteric B acteria in Polar M arine E n v iro n m e n ts ........................................... 11
R esearch Goals a n d O b je ctiv es.............................................................................. 12
V ahdation o f T etrazo h u m R eduction M e th o d s ..................................... 12
Exposure o f Enteric B acteria to a Polar M arine E n v iro n m e n t.... 13
R e fe re n c e s...................................................................................
13
CHAPTER 2. MECHANISMS OF INT (2-(4-IODOPHENYL)-3(4-NITROPHENYL)-5-PHENYL TETRAZOLIUM CHLORIDE), AND
CTC (5-CYANO-2,3-DITOLYL TETRAZOLIUM CHLORIDE) REDUCTION IN
ESCHERICHIA COU K -1 2 ........................................................................................................21
In tr o d u c tio n ................................................................................................................ 21
M aterials a n d M e th o d s ........................................................................................... 27
27
D eterm ination o f INT a n d CTC Chem ical P ro p e rtie s ...............
G row th C o n d itio n s ......................
27
S ubstrates, Inhibitors, a n d U n c o u p le rs .................................................28
In v e rte d M em brane V e sic le s.................................................................... 30
Oxygen C o n su m p tio n ..................................................................*...............32
C alcu la tio n s..................................................................................................... 32
Results a n d D iscu ssio n ...................................................................................
33
Chem ical P roperties o f CTC/INT, a n d T heir F o rm a z a n s ................... 33
R esp iratio n /T etrazo liu m R eduction R a tio s ...........................................35
Effects o f In h ib ito rs o n T etrazo h u m R e d u c tio n ................................. 37
D irect C o u n ts .......................................................................................
43
CTC a n d INT R eduction U n d er A naerobic C onditions........................ 45
R e fe re n c e s.................................................................................................................... 50
v ii
TABLE OF CONTENTS (Continued)
Page
CHAPTER 3. EFFECTS OF SUBSTRATES AND PHOSPHATE ON INT (2-(4IODOPHENYL) - 3 - (4-NITROPHENYL) -5 -PHENYL TETRAZOLIUM
CHLORIDE, AND CTC (5-CYANO-2,3-DITOLYL TETRAZOLIUM CHLORIDE)
REDUCTION IN ESCHERICHIA COLI..................................................................................
55
In tr o d u c tio n ................................................................................................................ 55
M aterials a n d M e th o d s .........................................................................................
57
C h em ica ls.....................................................................................
G row th C o n d itio n s ........................................................................................57
Incubation o f Cells w ith CTC a n d IN T ..................................................... 57
Exam ination o f Cells for CTC a n d INT R ed u c tio n ................................58
M easurem ent o f Form azan P ro d u c tio n .................................................. 59
M easurem ent o f Oxygen C o n su m p tio n .................................................. 59
R e su lts.................................................................................
Effect o f S ubstrates o n Form azan P ro d u c tio n ......................................60
Effect o f S ubstrates on D irect C o u n ts ..................................................... 60
Effect o f P hosphate o n CTC a n d INT R e d u c tio n ................................. 63
Effect o f P h osphate on Oxygen C o n su m p tio n ...................................... 63
D iscu ssio n .....................................................................................................................66
Effect o f S ubstrates o n CTC a n d INT R e d u c tio n ..........................
66
Effect o f P hosphate o n CTC a n d INT R e d u c tio n ................... j.......... .. 68
R e fe re n c e s...................................................... *....................................................... . 71
57
CHAPTER 4. SURVIVAL, PHYSIOLOGICAL RESPONSE, AND RECOVERY OF
ENTERIC BACTERIA EXPOSED TO A POLAR MARINE ENVIRONMENT......................... 74
In tr o d u c tio n ............................................................................
M aterials a n d M e th o d s ............................................................................................. 77
O rganism s, G row th C onditions, a n d E x p o su re......................................77
Plate Counts a n d I n ju r y .......:...............................................
DVCs.................................................................................................................. 79
CTC R e d u c tio n ................................................................................................ 79
D iffusion R a te s ..........................................................
Chem icals, M edia, a n d E q u ip m e n t........................................................... 81
R e s u lts ................................................................................................. ,....................... 81
R ecoverability a n d I n j u r y .......................................................................... 81
DVCs, a n d CTC R e d u c tio n ............................................................................90
D iffusion R a te s .............................................................
D iscu ssio n ........... ......................................................................................................... 94
R eferen c es................................................................................................................... 101
78
v iii
TABLE OF CONTENTS (Continued)
P age
CHAPTER 5. PLASMID MAINTENANCE AND EXPRESSION IN
ESCHERICHIA COLL EXPOSED TO A POLAR MARINE ENVIRONMENT........................ 106
In tro d u c tio n .............................................................................................................. 106
M aterials a n d M ethods..............................................................................
108
Organism s, G row th C onditions, a n d E x p o su re ................................. 108
Plasm id E x p re ssio n ............................................
109
Plasm id M a in te n a n c e ................................................................................. 109
D irect V iable C ounts a n d S u b leth al I n ju r y .........................................109
R e su lts..........................................................................................................................H O
Plasm id M aintenance a n d E x p re ssio n ..................................................H O
R ecoverabihty a n d S ubleth al I n j u r y ................................................. H O
DVCs, a n d CTC R ed u c tio n ..........................................................................114
D iscu ssio n ........................................................................................................
114
R e fe re n c e s...................................................
117
CHAPTER 6. SUMMARY AND DISCUSSION..................................................................... 119
G eneral Results a n d C o n c lu sio n s........................................................................ 119
Survival a n d Physiological R esponse o f
Enteric B acteria to Polar M arine E x p o su re ......................................... 119
T etrazolium R eduction M ethod V a h d a tio n ......................................... 123
D iscu ssio n ................................................................................................................... 126
Enteric B acteria a n d Polar M arine E x p o s u re ......................................126
T etrazohum R eduction a n d V ia b ih ty ........................
128
U nresolved Is s u e s .........................................................................................129
CTC/INT R ed u c tio n ......................................................................... 129
Enteric B acteria a n d Cold M arine E x p o su re ............................130
C oncluding R e m a rk s ..........................:..................................................................... 131
R e fe re n c e s..........................
132
ix
LIST OF TABLES
Table
1. S ubstrates, in h ib ito rs, a n d sites o f in h ib itio n fo r
in v e rte d m e m b ra n e vesicle, te tra z o liu m re d u c tio n
assays...............................................................................................................
Page
31
2. Physical a n d chem ical p ro p e rtie s o f CTC a n d INT
re d u c tio n . M olar e x tin c tio n coefficients,
solubilities, a n d CTC re d o x p o te n tia l w ere
d e term in e d in this stu d y .......................................................................................... 33
3. Ratios o f re sp ira tio n a n d ETS activity fo r w hole cell
a n d in v e rte d m e m b ra n e vesicle te tra zo liu m
re d u c tio n assays...........................................................
36
4 Effect o f p h o sp h a te c o n c e n tra tio n o n re s p ira tio n
ra te s in Escherichia coli ........................................................................................66
5. C o n cen tratio n s o f in o rg a n ic p h o s p h a te in n a tu ra l
w aters as well as com m only u se d b u ffers a n d
grow th m ed ia...........................
69
LIST OF FIGURES
F ig u re
Page
I . R eduction reactio n o f (A) CTC to CTF, a n d (B) INT to INF
show ing in te rm e d ia te species b etw een te tra zo liu m
a n d f o r m a z a n ............................................................................................................. 22
2. Schem atic of th e E. coli a ero b ic re s p ira to ry c h a in
show ing th e sites o f a c tio n o f th e v a rio u s
in h ib ito rs u se d in th is stu d y . M id-point re d o x
p o te n tia ls fo r th e v a rio u s c y to ch ro m es, iro n su lfu r c e n te rs (Fe-S), a n d d e h y d ro g e n a se s a re
given. Sites o f re d u c tio n o f CTC a n d INT fro m this
stu d y a re encircled. D ashed lines in d ic a te sites o f
in h ib itio n of e le c tro n tra n s p o rt p ro cesses. See
m a te ria ls a n d m e th o d s fo r chem ical ab b re v iatio n s.
C om piledfrom (4 ,1 8 ,1 9 ,2 3 , 25, 27, 29, 3 0 ,3 2 ,4 8 ,5 0 , 51).
26
3. Effects o f vario u s in h ib ito rs o n CTC (sh a d e d b ars),
a n d INT (solid bars) re d u c tio n in E. coli in v e rte d
m e m b ra n e vesicles. D ata a re ex p ressed as p e rc e n t
d ev iatio n s in fo rm az a n p ro d u c tio n fro m su b strate s
(NADH o r succinate) alo n e. Sites o f in h ib itio n a re
listed in T able 2. P-values a re < 0.05 ex cep t w here
n o te d . E rror b a rs re p re s e n t s ta n d a rd d ev iatio n s
(n = 2). C alculations a n d a b b re v iatio n s a re as
d escrib ed in m aterials a n d m e th o d s...... *.............................................................39
4. Effects o f in h ib ito rs o n aerobic CTC (s h a d e d b ars), a n d
INT (solid bars) re d u c tio n in w hole cells o f E. coli.
F orm azan p ro d u c tio n values a re e x p re ssed as
p e rc e n t d e v iatio n s fro m c o n tro l w ith o u t
in h ib ito rs (A). P-values a re s 0.05 ex cep t w here
n o te d . P ercen t CTC (+) cells b y epiflu o rescen ce
m icroscopy (B). H orizontal lin e in d ic a te s th e
p e rc e n t o f to ta l AODC cells e n u m e ra te d b y R2A
p la te counts. E rror b a rs re p re s e n t s ta n d a rd
d eviations (n = 2). See m aterials a n d m e th o d s fo r
calculations a n d ab b re v iatio n s..............................................................................40
xi
LIST OF FIGURES (C o n tin u ed )
F ig u r e
Page
5. Effects o f v ario u s su b strate s a n d e le c tro n acceptors
o n CTC (sh ad ed bars) a n d INT (solid bars) re d u c tio n
b y w hole E. coli cells u n d e r a n a e ro b ic conditions.
F orm azan p ro d u c tio n values a re e x p ressed as
p e rc e n t d ev iatio n s fro m c o n tro l w ith o u t
su b strate (s) (A). P v alu es a re <0.05 ex cep t w here
n o te d . P ercen t CTC (+) cells b y ep iflu o rescen ce
m icroscopy (B). H orizontal lin e in d ic a te s th e
p e rc e n t o f to tal AODC cells e n u m e ra te d b y R2A p la te
counts. E rror b a rs re p re s e n t s ta n d a rd d ev iatio n s
(n = 2). See m aterials a n d m eth o d s fo r calculations
a n d ab b rev iatio n s...................................................................................................... 46
6. Effects o f d ifferen t su b strate s o n CTC (sh a d e d bars),
a n d INT (solid bars) re d u c tio n to e th a n o l
ex tractab le fo rm azan (A), a n d p e rc e n t CTC/INT (+)
cells b y ep iflu o rescen ce m icro sco p y (B). Plate
co u n ts w ere o n R2A ag ar. E rro r b a rs re p re s e n t
s ta n d a rd deviations (n = 2 )..................................................................................... 61
7. Effects o f p h o sp h ate o n CTC (+) (A), a n d INT (+) (B)
cell n u m b ers, a n d to ta l fo rm az a n p ro d u c tio n .
Symbols: P ercent CTC (+) o r INT (+) cells (solid
circles), to ta l fo rm az a n p ro d u c e d (o p e n circles),
to ta l CTF d ep o sit co n tain in g cells (flu o rescen t +
n o n flu o resc en t) (solid trian g le s). E rro r b a rs
re p re se n t s ta n d a rd deviations (n = 2 ).................................................................64
8. Plate (n = 5), CTC (+), DVC (+), a n d to ta l d ire c t counts
fo r E coli exposed to a p o la r m a rin e e n v iro n m e n t
fo r 54 days. E rror b a rs re p re se n t s ta n d a rd d ev iatio n s................................ 82
9. Plate (n = 5), CTC (+), DVC (+), a n d to ta l d ire c t counts
fo r S. typ h im u iiu m exposed to a p o la r m a rin e
e n v iro n m e n t fo r 54 days. E rro r b a rs re p re s e n t
s ta n d a rd d eviations.........................................................
83
x ii
LIST OF FIGURES (C o n tin u ed )
F ig u re
Page
10. Plate (n = 5), CTC (+), DVC (+), a n d to tal d ire c t counts
fo r Y. enterocolitica exposed to a p o la r m arin e
e n v iro n m e n t fo r 54 days. E rro r b a rs re p re s e n t
s ta n d a rd d eviations...................................................................................................85
11. Plate (n = 5), CTC (+), a n d to tal d ire c t counts fo r
S. typ h im u riu m exposed to a p o la r m a rin e
e n v iro n m e n t fo r 56 days. E rro r b a rs re p re s e n t
s ta n d a rd deviations...................................................................................................86
12. P ercen t su b le th a l in ju ry su sta in e d b y v a rio u s en teric
b a c te ria w ith ex p o su re to a p o la r m a rin e
en v iro n m e n t fo r 54 to 56 d ay s..............................................................................87
13. Selective (TLYD; b lack b a rs) a n d n o n selectiv e
(TLY; stip led bars) p la te c o u n t re c o v eries a t
d iffe re n t te m p e ra tu re s fo r (A) E. coli, (B)
S. typhim urium , a n d (C) Y. enterocolitica exposed
to a p o la r m a rin e e n v iro n m e n t fo r 54 d ay s. E rror
b ars re p re se n t s ta n d a rd deviations (n = 5 )....................................................... 88
14. P ercent CTC (+) E. coli (A), S. typ h im u riu m (B), a n d
Y. enterocolitica (C) cells w ith a n d w ith o u t n u trie n t
a d d itio n a t -L S 0C (u n sh a d e d b a rs), 8°C (stip p led
b a rs), 20°C (diagonal strip e d b a rs), a n d 37°C
(black b a rs) a fte r ex p o su re to th e p o la r m a rin e
e n v iro n m e n t fo r 48 days. E rro r b a rs re p re s e n t
s ta n d a rd d eviations.............................1.................................................................... 92
15. Survival, p lasm id expression, su b stra te
resp o n siv en ess (DVC) a n d re s p ira to ry activ ity
(CTC [+]) o f R coU TX-432 (pUC 119) exposed to a
p o la r m a rin e e n v iro n m e n t fo r 54 d a y s (d). Solid
circles w ith h a tc h e d lin es in d icates TLY p la te
co u n ts o f w ild-type E. coli TX-432 fo r com parison.
Plate co u n ts (n = 5) w ere o n TLY (nonselective),
TLYD (selective), a n d TLYA (selective fo r pUC19
expression) ag ars. E rro r b a rs re p re s e n t s ta n d a rd
d eviations............................................................. .'.................................................. . I l l
x iii
LIST OF FIGURES (C o n tin u ed )
F ig u re
Page
16. Survival, p lasm id expression, s u b stra te resp o n siv en ess
(DVC) a n d re s p ira to ry activity (CTC [+]) o f w ild-type
E. coli TX-432 exposed to a p o la r m arin e
e n v iro n m e n t fo r 54 days (d). Plate co u n ts (n = 5)
w ere o n TLY (nonselective) a n d TLYD (selective)
agar. E rror b ars re p re se n t s ta n d a rd d ev iatio n s........................................ ,. 112
17. R ecoverability a n d conjugative F-plasm id ex p ressio n
in E. coli K-12 (pFam p) exposed to a p o la r m a rin e
e n v iro n m e n t fo r 21 d ay s (d). E rro r b a rs re p re se n t
s ta n d a rd deviations (n = 5 ).................................................................................. 113
I
CHAPTER I
GENERAL INTRODUCTION
In d ic a to rs
In th e la st few d ecad es th e use o f m a rin e outfalls fo r th e disposal of
sa n ita ry w astes h a s in c re a se d greatly, as h a v e th e u rb a n p o p u la tio n s
in h a b itin g co astal a re a s (12, 89). T he su rv iv al o f h u m a n e n te ric p ath o g en s
a n d in d ic a to r organism s in m a rin e e n v iro n m e n ts h a s su b se q u e n tly assu m ed
in creasin g p u b lic h e a lth significance w ith re s p e c t to th e p re se n c e a n d
e n u m e ra tio n o f th e se ty p es o f m icro o rg an ism s in com m ercial fish a n d
shellfish, m a rin e m am m als, a n d re c re a tio n a l w aters (14, 69). T ech n iq u es fo r
th e e n u m e ra tio n o f in d ic a to r organism s (i.e. E. coli, coliform s) a n d specific
p a th o g en s fro m m a rin e e n v iro n m e n ts h a v e b e e n d ev elo p ed a n d acc e p ted as
s ta n d a rd m eth o d s ( I ) . However, th e se p ro c e d u res, such as th e m u ltip le-tu b e
fe rm e n ta tio n (MTF o r MPN), a n d m e m b ra n e filtra tio n (ME), as w ell as in d ic a to r
organism s, w ere la rg e ly d e v elo p e d fo r th e ex am in atio n o f re la tiv e ly w arm
(>10°C), fresh w ate r system s (m ost com m o n so u rces o f b a th in g a n d d rin k in g
w ater) (14, 69). It h a s b e e n recognized th a t th e b eh av io r o f in d ic a to rs a n d
p a th o g e n s in th e m a rin e en v iro n m en t d iffers g re a tly fro m th a t o f fresh w ate r
(9, 43, 53). In a d d itio n , th e survival o f th e p rim a ry in d ic a to r o f fecal pollution,
E. coli , in seaw ater h a s b e e n show n to b e in v e rsely re la te d to te m p e ra tu re .
2
w ith co ld er te m p e ra tu re s e x ten d in g su rv iv al tim e by a fa c to r o f 2 fo r each
IO0C decrease. In fact, n u m e ro u s stu d ies h a v e d e term in e d th a t w ater
te m p e ra tu re is th e m o st im p o rta n t fa c to r in p re d ic tin g fecal coliform survival
in m a rin e en v iro n m en ts (2, 5, 26, 38, 84). This is o f p a rtic u la r re lev an ce since
sewage is o fte n d isc h a rg e d u n tre a te d in to lo w -tem p eratu re m a rin e
en v iro n m en ts a n d o n ly ca. 10% o f m a rin e e n v iro n m e n ts a re > 5°C (58).
C u rre n t " d ie -o ff stu d ies o f h u m a n e n te ric a n d g en etically e n g in e e re d
m icroorganism s in th e e n v iro n m e n t h a v e d e te rm in e d th a t fo r m an y , if n o t
m ost b acteria, th e re a p p e a r to b e w h a t a re te rm e d “in ju re d ” a n d /o r "viablebut-nonC ulturable" (VBNC) states (67, 77). R ecent stu d ies d escrib in g th e
p e rsiste n c e o f h u m a n e n te ric b a c te ria in aq u atic e n v iro n m e n ts h a v e
d e m o n stra te d th a t m a n y o f th e se o rg an ism s e n te r a n a lte re d physiological
state te rm e d v ia b le-b u t-n o n cu ltu rab le (VBNC) (11, 24, 44). VBNC is com m only
d e fin e d as th e in a b ility to fo rm colonies o n a given solid m e d iu m w hile
rem aining active b y d ire c t v iab le-co u n t (DVC) m eth o d s (67, 77). Stressors
w ith in a q u atic e n v iro n m e n ts also in d u c e su b le th a l p h ysiological a n d
s tru c tu ra l changes, te rm e d in ju ry , in e n te ric b a c te ria (51). In th is in ju re d
state, b a c te ria a re u n a b le to re p ro d u c e u n d e r co n d itio n s w h ich allow fo r
grow th o f u n in ju re d cells, in clu d in g re s tric tiv e te m p e ra tu re s a n d th e
p re sen c e o f selective a g en ts (79). As a n exam ple, a b a c te riu m show n to b e
v iable b y DVC m eth o d s b u t w hich will n o t fo rm a colony o n nonselective
m ed iu m is te rm e d VBNC. A b a c te riu m w hich w ill fo rm a co lo n y o n
nonselective m edium , b u t n o t o n this sam e m ed iu m co n tain in g a selective
a g en t to w hich it is n o rm a lly re s ista n t is te rm e d in ju red . It sh o u ld b e
em phasized th a t b o th in ju re d a n d VBNC resp o n ses a re com m only o b serv ed in
b a c te ria exposed to e n v iro n m e n ta l stress. Since th e m a jo rity o f e n u m e ra tio n
3
te ch n iq u es fo r e n te ric b a c te ria re q u ire c u ltu rin g , re su lts m a y serio u sly
u n d e re stim a te n u m b e rs o f v iable b a c te ria in th e e n v iro n m en t. This is
especially tru e in e n v iro n m e n ts w h ere stre sso rs such as cold a n d starv atio n
m a y p ro m o te e n try in to th e VBNC state. M arine studies using la b o ra to ry
m icrocosm s o r in situ d iffu sio n c h am b ers h a v e show n th a t cold shock,
starv atio n , a n d possibly osm otic shock a re in d u c ers o f this state (2, 18, 38, 63,
66). M ost o f these ex perim ents h a v e u se d la b o ra to ry , b a tch -cu ltu re
m icrocosm s w hich a re su b ject to "b o ttle effects" ( I , 17). This co n fin em en t
te rm in a te s exchange o f n u trie n ts a n d m e ta b o lite s w ith th e a m b ie n t w ater,
w hich is im p o rta n t fo r a n e v alu a tio n o f in situ survival a n d physiological
processes in n a tu ra l aq u atic e n v iro n m e n ts. A stu d y b y M cFeters a n d T erzieva
fo u n d sig n ifican t d ifferen c es in E. coli a n d Yersinia enterocolitica survival
a n d su b le th a l in ju ry levels betw een ex p erim en ts using b a c te ria l suspensions
in d iffusion ch am b e rs in la b o ra to ry c o n ta in e rs, in situ in a m eso tro p h ic
stream , a n d in sam ple bo ttles (50). T he u se o f diffusion ch am b ers d ep lo y ed in
situ larg ely alleviates th e se p ro b le m s (49). E xperim ents using in situ diffusion
c h am b ers fo r th e stu d y o f e n te ric b a c te ria l su rv iv al asso ciated w ith sewage in
th e m a rin e e n v iro n m e n t a re few (2, 39), a n d n o n e u se d te ch n iq u es to
d e term in e th e ex ten t o f b a c te ria in th e VBNC state.
Survival o f E nteric B acteria in th e M arine E n v iro n m en t
M arine w aters p re s e n t u n fa v o ra b le co n d itio n s fo r th o se co p io tro p h ic
b a c te ria fo u n d in ra w sew age, w hich co m m o n ly p e rsist in te m p e ra te
fre sh w a te r e n v iro n m e n ts.
Salinity, p re s su re , U V -irradiation, c o m p etitio n
w ith, a n d p re d a tio n b y th e a u to c h th o n o u s flo ra a n d fau n a, te m p e ra tu re
4
flu ctu atio n s, a n d (in m a n y cases) o lig o tro p h ic co n d itio n s all e x e rt effects o n
th e survival a n d physiology o f sewage b a c te ria (4, 9, 10, 23, 39, 77). Reviews of
th e lite ra tu re h av e show n th a t th e p rim a ry effecto rs o f p ath o g en ic a n d
in d ic a to r b a c te ria l su rvival a n d activ ity in seaw ater a re th e re la tiv e ly
o ligotrophic n a tu re o f m o st m a rin e e n v iro n m e n ts, as well as te m p e ra tu re (14,
53, 60). G razing by larv ae a n d h e te ro tro p h ic zo o p lan k to n m a y also red u ce
n u m b e rs o f e n teric b a c te ria fro m th e e n v iro n m e n t. Some a u th o rs feel th a t
p ro tis ta n grazing m a y b e th e p rim a ry fa c to r in flu en cin g e n te ric b a c te ria l
survival in a q u eo u s en v iro n m e n ts (23). H ow ever, it a p p e a rs th a t te m p e ra tu re
p lay s a n im p o rta n t ro le in re g u la tin g ra te s o f b a c te ria l rem o v al th ro u g h
grazing, w ith low er ra te s a t low er te m p e ra tu re s, h i a n e stu a rin e stu d y of th e
effect o f eu k ary o tic grazing p re s su re a n d te m p e ra tu re o n R coli survival in
d iffusion ch am b ers, A n d e rso n e ta L (2) fo u n d th a t grazing ex erted little o r n o
d ecline in cell n u m b e rs below 3°C o v er 6 d ay s. However, a t 13 a n d 24°C
grazing d id e x ert a n effect. In a d d itio n , sev e ra l studies in th e A ntarctic
m a rin e e n v iro n m e n t h a v e fo u n d th a t g razin g ra te s a re sig n ifican tly re d u c e d
co m p ared to m o re te m p e rate regions (7 1 ,7 3 ) .
S tudies o n th e effects o f n u trie n t lev els o n e n te ric b a c te ria l survival
a re conflicting. L essard e t a k (39) o b serv ed n o c o rrelatio n betw een diel
cycles, o r d issolved organic c a rb o n a n d E. coli su rv iv al o v e r a ra n g e of
te m p e ra tu re s (0-25°C) in a salt m arsh . T e m p e ra tu re was fo u n d to b e th e m o st
significant fa c to r in th a t stu d y . In a d d itio n , C h am b erlain a n d M itchell (10)
d e sc rib e d littie effect o f n u trie n ts on e n te ric b a c te ria l su rv iv al below ca. 12 °C.
However, studies b y C arlucci a n d P ram er (9), as well as M unro e t al. (61) fo u n d
th a t in c re a se d levels o f n u trie n ts p ro lo n g e d e n te ric b a c te ria l su rv iv al in
seaw ater a t ca. 24-28°C using p la te co u n ts a s a m easu re o f v iability. Also,
5
W eibe e t a l. re c e n tly show ed th a t in c re ase d su b stra te c o n ce n tra tio n s a re
re q u ire d to m a in ta in grow th ra te s of E coli a t m inim al (10°C), c o m p ared to
h ig h e r grow th te m p e ratu res (86). Thus, it a p p e a rs th a t w hile in c re ase d
n u trie n t levels m a y allow fo r th e in c re a se d p e rsiste n c e o f e n te ric b a c te ria in
te m p e rate en v iro n m en ts, th is effect m ay be re d u c ed , o r n o n e x iste n t a t
te m p e ra tu re s below ca. 10°C.
S tarvation, Cold Shock. A daptation, a n d th e "V iable-but-N onculturable" State
C onsiderable re se a rc h h as b e e n d o n e o n th e sta rv a tio n o f b o th
a u to c h th o n o u s b a c te ria fro m aq u atic e n v iro n m e n ts a n d o rg an ism s o f pu b lic
h e a lth significance (57). H ow ever, it a p p e a rs th a t th e starv atio n -su rv iv al
re sp o n se of e n teric b a c te ria is n o t id e n tic al to th a t o f m a n y au to ch th o n o u s
m a rin e b a c te ria (16, 35, 77). A q uestio n arises as to w h e th e r som e sewage
b a cte ria , su ch as Vibrio cholerae a n d Salm onella spp. p e rsist in d e fin ite ly in
th e m a rin e e n v iro n m e n t (85). Reviews b y Stevenson (81), M orita (59, 60),
K jelleberg e t al. (35), a n d M atin e t al. (44) in d ic a te th a t th e p e rsiste n c e of
m a rin e a u to c h th o n o u s m icro rg an ism s in sea w ater is p rim a rily a fu n c tio n o f
th e ir a b ility to a d a p t to th e e n v iro n m e n t, a n d /o r th ro u g h th e a b ility to fo rm
"d orm ant" cells. Several term s h av e b e e n p ro p o se d fo r this sta te including
"m icrocysts", "som nicells", "d o rm an t", a n d "v iab le-b u t-n o n cu ltu rab le" (VBNC).
T he la tte r te rm re fe rs to th e in ab ility o f cells to d iv id e a n d fo rm recognizable
colonies o r tu rb id ity in p la tin g a n d MPN assay s u sin g c o n v en tio n al selective
a n d /o r no n selectiv e m edia. T he o th e r te rm s re fe r larg ely to a spore-like state
o f n o n sp o re fo rm in g b a c te ria , larg ely as a re s u lt o f sta rv a tio n a n d /o r cold
shock. In this state b a c te ria a re seen to re d u c e th e ir size c o n sid erab ly
6
("dw arfing"), a n d sw itch fro m specific, low -affinity m e m b ra n e u p ta k e system s
to non-specific, re la tiv e ly high-affinity u p ta k e system s (35, 59, 60). O th er
physiological changes in c lu d e in c re a se d in tra c e llu la r levels o f ppG pp a n d
in d u c tio n o f th e strin g e n t re sp o n se (35, 6 4 ), in c re a se d a d h e s io n to surfaces
(33), agg reg atio n (40), su b leth al in ju ry (79), n o n -c u ltu ra b ility (67), as well as
in c re a se d re sista n c e to g astric pH, cell w all sy n th esis in h ib ito rs, a n d
c h ao tro p h ic agents (21, 45). It is also a p p a re n t th a t sy n th esis o f specific
starv atio n a n d cold-shock p ro te in s tak es p la ce as a n aly z e d b y 2-D gel
electrophoresis (3 5 ,4 5 ) . A lterations in m e m b ra n e fa tty acid s h a v e b e en n o te d
in Vibrio vulnificus (41), a n d Vibrio cbolerae (25, 28) in re sp o n se to n u trie n t
d e p riv a tio n in m a rin e m icrocosm s. It h as long b e en re co g n ized th a t
Escherichia coh m a rk e d ly in c re ase s th e p ro p o rtio n o f u n s a tu ra te d fa tty acids
in th e cell m em b ra n e in re sp o n se to low te m p e ra tu re s (< ca. 10-20°C) (13, 30,
65). This is a n a p p a re n t a d a p ta tio n to allow cells to re ta in a flu id ( “n o n o rd e re d ” lip id phase) m e m b ra n e a t low er te m p e ra tu re s (30, 31). Loss of
m e m b ra n e flu id ity le a d s to segregation o f p ro te in /lip id in th e m e m b ra n e a n d
ap p ears to le a d to a loss o f c ertain enzym atic fu n ctio n s (83).
It h a s b e e n h y p o th e siz e d th a t v irtu a lly all b a cte ria , in clu d in g
co p iotrophs, can po ssib ly u se b o th K- a n d r strateg ies d u rin g p e rio d s o f th e ir
life cycle (3). T h e r-stra te g ists re ly o n h ig h re p ro d u c tiv e ra te s fo r survival
w hile K -strategists d e p e n d o n a d a p ta tio n to th e carry in g cap a c ity o f th e
e n v iro n m e n t. This w o u ld h e lp ex p lain th e p e rsiste n c e o f e n te ric ,
allo ch th o n o u s, c o p io tro p h ic b a c te ria w h en ex p o sed to o lig o tro p h ic co n d itio n s.
It is em p h asized th a t u n d e r co n d itio n s o f m a rin e starv atio n a n d /o r cold shock,
su bpo p u latio n s of e n teric b a c te ria a re seen to becom e n o n c u ltu ra b le (11, 76,
78). This p h e n o m e n o n h a s b e e n o b serv ed in : E. coli (57), Klebsiella aerogenes,
7
Vibrio choleras (6 ,1 1 , 78, 81, 88),V ibrio vu ln ificu s (63, 66), Shigella sonnei,
S h ig e lla fle x n e ri (11), C am phylobacter jejtm i, Cam pylobacter p ylo ri (72),
Salmonella enteriditis (78), Legionella p n eum ophila ,A ero m o n a s spp.,
Aerom onas salm onicida (56), A lterom onas esperjiana ,E nterococcus spp.,
Micrococcus spp., a n d N itrobacter spp. (77). E n v iro n m en tal stressors,
p a rtic u la rly cold-shock, h a v e also b e en fo u n d to in d u c e su b le th a l
physiological a n d s tru c tu ra l changes, te rm e d in ju ry , in e n te ric b a c te ria (S I,
79). This is d e fin e d as th e in a b ility o f a n o rg an ism to re p ro d u c e u n d e r
c o n d itio n s w hich allow fo r g row th o f u n in ju re d cells (re stric tiv e
te m p e ra tu re s, selective ag en ts, etc.). T he p u b lic h e a lth im p licatio n s a re
obvious as e n u m e ra tio n o f th e p ath o g en ic a n d in d ic a to r b a c te ria fisted above
a re b a se d larg ely u p o n c u ltu re m ethod s, a n d VBNC b a c te ria h a v e b e e n show n
to re ta in th e ir p ath o g en icity a n d p lasm id -en co d ed a n tib io tic re sista n c e (7, 79).
T he u sefu ln ess o f th e cofiform a n d fecal co lifo rm indices fo r p u b lic h e a lth
w ater q u a lity m o n ito rin g m a y th u s b e serio u sly co m p ro m ised especially w ith
re sp e c t to co ld er m a rin e e n v iro n m en ts ( I , 69, 76, 84).
Effects o f C ulture Physiology o n Survival a n d C u ltu rab ility
A q u e stio n re m a in s as to th e effects o f in ju ry (i.e. su b le th a l stress) o n
fo rm atio n o f th e VBNC state in th e m a rin e e n v iro n m en t. G a u th ie r e t al. (20),
O liver e t a l . (66), M eynell (52), a n d G au th ier e t a l. (19) resp ectiv ely , h a v e
show n th a t survival a n d th e fo rm atio n o f VBNC cells a re fu n c tio n s o f grow th
p h ase, p rio r starv atio n , p rio r ex p o su re to cold, a n d in c re a se d salinity.
U nstarved, n o n -salin ity o r cold exposed, log-phase cells seem to b e m o st
susceptible to cell d e a th w ith o u t fo rm atio n o f VBNC cells (46). S tatio n ary
8
p h ase, p re -sta rv ed , -cold shocked, a n d -saline exposed cells h a v e a n in creased
ab ility to fo rm VBNC cells. R ecent w ork b y O hver e t al. h a s show n th a t th e re
m ay be a n antag o n istic effect o f cold shock a n d starv atio n o n th e fo rm atio n o f
VBNC cells, w ith tim e o f p re sta rv a tio n as well as cu ltu re O.D. d ire c tly re la te d to
fo rm atio n o f VBNC Vibrio vu ln ificu s in 5°C seaw ater (66). In ad d itio n , Nilsson
e t al. h a v e d e m o n stra te d th a t fo rm atio n o f th e VBNC state is in d e p e n d e n t of
n u trie n t levels fo r th is o rg an ism (63). A rise in te m p e ra tu re to 25°C was also
d e m o n stra te d to in c re a se c u ltu ra b ility to in itia l levels.
Specific p ro te in s a re sy n th esized in re sp o n se to cold sh o ck a n d
starv atio n . However, a q u e stio n re m a in s as to w h e th e r a n d to w h a t deg ree
th e se p ro te in s a n d th e ir fu n ctio n s o v erlap in th e in d u c tio n o f th e VBNC state
(22, 34, 44). Indiv id u al p ro te in functio n s a re difficult to assess, b u t
tra n sc rip tio n a l, a n d m etabolic re g u la to rs h a v e b e e n im p licated . Studies b y
M atin (44, 46) d escrib e th e c u rre n t know ledge o f th e genetic b a sis o f
sta rv a tio n responses. M cCann e t aL (48) h a v e re c en tly d isco v e re d th e KatF
(rpoS) gene in E. coli is a m a jo r sw itch co n tro llin g ex p ressio n o f som e 32
starv atio n -m e d iate d p e x genes. In d u ctio n o f p e x genes is in d e p e n d e n t of
cyclic AMP, a n d is cro ss-p ro tectiv e fo r h e a t, osm otic a n d o x id ativ e stresses.
KatF h as also b e e n id e n tifie d as a p u ta tiv e sigm a facto r (o5) c o n tro llin g
ex pression o f stress-in d u ced genes (42). IKatF hom ologues h a v e also b e e n
fo u n d in Salm onella typ h im urium , Klebsiella pneum oniae, Shigella flexneri
a n d Pseudom onas aeruginosa. T his c o o rd in a te co n tro l o f m u ltip le u n lin k e d
genes a n d o p ero n s b y a com m on re g u la to ry e lem en t a p p e a rs to b e a fo rm o f
“global c o n tro l system ” re sp o n d in g to e n v iro n m e n ta l stress (35, 64). Sets of
c o o rd in a tely re g u la te d genes in d u c e d u n d e r a given e n v iro n m e n ta l stim ulus
a re te rm e d stim ulons (64). However, w hile resp o n ses to h e a t sh o ck a n d
9
oxidative stress h a v e b e e n s tu d ie d extensively, th e physiologic a n d genetic
re g u la tio n o f b a cte ria l a d a p ta tio n to su rv iv al u n d e r cold m a rin e co n d itio n s
re m a in s la rg e ly u n e x p lo re d .
E n u m eratio n a n d A ctivity M easu rem en ts o f B acteria in A queous E nvironm pnrs
V arious m eth o d s h a v e b e e n em p lo y ed w ith re sp ec t to th e e n u m eratio n
o f e n v iro n m e n ta lly stre sse d b a c te ria . T he m o st w idely u se d tech n iq u es fo r
e n u m e ra tio n o f total, active, o r v iable cells in a p o p u la tio n are; acrid in e
o ran g e d ire c t counts (AODC, to ta l cells), sp read -, o r d ro p -p latin g (p late count,
re c o v era b le o r c u ltu ra b le cells), d ire c t v iab le co u n ts b y th e m e th o d o f K ogure
e t a l . (36, 80), p -io d o n itro te tra zo liu m ch lo rid e (INT) o r 5-cyano-2,3-ditolyl
tetrazo liu m ch lo rid e (CTC) re d u c tio n (32, 74, 90) (active cellu lar re sp ira tio n ),
a n d p latin g o n n o n -selectiv e a n d selective m e d ia (injury) (79). A som ew hat
typical assay o f a p o p u la tio n o f cells w ould involve to tal AODC (DAPI can also b e
used) com b in ed w ith INT re d u c tio n to d e te rm in e to ta l c o u n t a n d th e frac tio n o f
actively re sp irin g cells (82). CTC re d u c tio n in p a rtic u la r is b eg in n in g to b e
w idely u s e d as a n in d ic a to r o f actively re sp irin g , a n d h e n ce v ia b le cells.
However, th e use o f CTC a n d INT fo r a ssessm en t o f b a cterial activ ity is
h a m p e re d b y a lack o f k n o w led g e re g a rd in g th e specific c e llu la r p ro cesses
th e y m easu re. This in fo rm a tio n is esse n tial fo r a ccu rate in te rp re ta tio n of
physiological a n d v ia b ility d a ta o b ta in e d u sin g th e se in d ic a to rs. A system atic
stu d y o f re d u c tio n o f th e se in d ic a to rs in p ro k a ry o te s is n e ed e d .
A com parison o f DVC a n d ra d io la b e le d su b stra te u p ta k e
(m icro au to rad io g rap h y ) m e th o d s using VBNC Salmonella enteriditis a n d E. coll
in m a rin e m icrocosm s show ed th a t ca. 90% o f cells resp o n siv e to th e DVC
10
m e th o d w ere m etab o lically active, a n d n o sig n ifican t d ifferen ce was o b serv ed
b etw een th ese m e th o d s (76). R adioisotopic m easu rem en ts discu ssed b y H obbie
(27) h a v e b e e n u sed to d e te rm in e m o re p recisely th e u p tak e kinetics an d
m etab o lism o f b a cte ria l p o p u latio n s. Several a u th o rs h av e also u sed ATP assays
as well as tritia te d th y m id in e a n d /o r 14C -Substrate u p tak e to m easu re
p o p u la tio n activities, DNA synthesis a n d e n d o g en o u s m etab o lism (33, 60, 62).
T he use of tritia te d u rid in e /le u c in e to m e a su re R N A /protein syn th esis ra te s
c an give a n estim atio n o f b io sy n th esis in a n exposed b a c te ria l p o p u latio n ,
w hile u p ta k e o f 14C-Iabeled a -m eth y l-g lu co se allow s fo r e stim atio n o f
m e m b ra n e tra n s p o rt of exogenously p ro v id e d c arb o h y d ra te . E lucidation o f
w h e th e r e n te ric b a c te ria ex p o sed to e x trem e lo w -tem p eratu re m a rin e
e n v iro n m e n ts c o n tin u e to sy n th esize m acro m o lecu les a n d re ta in th e ab ility to
tra n s p o rt exogenously su p p lie d n u trie n ts m ay h e lp to d e fin e th e m etabolic
s ta te o f th e s e organism s w hile try in g to survive.
P lasm id M ain ten an ce a n d Expression
f!
P ro lo n g ed su rv iv al o f p la sm id -h a rb o rin g e n te ric b a c te ria in sewage
re le a s e d in to lo w -te m p e ra tu re m a rin e e n v iro n m e n ts p re s e n ts th e p o ssib ility
o f h o riz o n ta l tra n s fe r v iru le n c e a n d /o r a n tib io tic re sista n c e (R) genes to
a u to ch th o n o u s b a c te ria (89). V irulen ce-asso ciated genes in E coli, Y.
enterocolitica, S. typhum urium , a n d S. flexneri a re also u n d e r te m p e ra tu re
reg u latio n , a n d a re m o st com m only re p re ss e d a t low te m p e ra tu re s (47).
N a tu ra l g en etic e x ch an g e th ro u g h tra n s fo rm a tio n , c o n ju g atio n , a n d
tra n sd u c tio n h a s b e e n d e sc rib e d in m a rin e e n v iro n m en ts, as w ell as tra n s fe r
o f R plasm ids fro m e n teric to m a rin e b a c te ria a n d fish p a th o g en s (24, 37, 70,
11
89). Since cold-shock as well as sta rv a tio n h a v e b e en show n to in d u ce
su b leth al in ju ry a n d th e VBNC resp o n se, th e p o ssib ility of u n d e re stim a tio n o f
p lasm id h a rb o rin g e n teric b a c te ria l n u m b e rs also exists. Plasm ids h a v e b een
d e m o n strated to be stably m a in ta in e d a n d ex p ressed in VBNC £ coli, as well as
£ coli exposed to well, lake, a n d m a rin e w aters a t te m p e ratu res o f 15-25°C (7,
8). W h eth er e n teric b a c te ria will m a in ta in a n d express R-, a n d F-plasm ids,
w hich code fo r congugative fu n c tio n s a n d c e rta in b a c te rio p h a g e re c ep to rs, in
situ u n d e r lo w -tem p eratu re stress a n d m a rin e ex p o su re is un k n o w n , ha
ad d itio n , it re m a in s to b e see n w h e th e r p lasm id -co n tain in g a n d w ild-type
e n te ric b a c te ria h a v e sim ilar su rv iv al re sp o n se s u p o n e n v iro n m e n ta l
ex posure.
E nteric B acteria in P o lar M arin e F nvironm ents
R ecent in te re s t in th e biological a n d chem ical p o llu tio n p ro b lem s
asso ciated w ith th e U.S. A ntarctic bases h a s p ro v id e d th e im p etu s fo r re se a rc h
in to th e fa te o f p o llu ta n ts in th e m a rin e en v iro n m e n t, a n d in m a rin e anim al
life (39). N ow here h a s th is d ra w n as m u c h focus as th e la rg e st A ntarctic b ase,
M cM urdo statio n (29, 54). U n tre a ted ra w sewage m ixed w ith ~ 4 0 p p t salinity, =
37°C desalin izatio n b rin e is d isch a rg e d (ca. 20-40,000 g a l/d a y ) a fte r
co m m in u ta tio n (g rin d in g w ith a n im p e lle r b lad e) th ro u g h a h e a te d ,
su b m erg ed o u tfall lo c ated ~ 15 m below th e w a te r surface. This o utfall is
lo cated alm ost d irectly seaw ard fro m th e statio n . A m bient seaw ater conditions
in th e g e n eral a re a a re a n e a rly c o n sta n t -1.8°C (seaw ater is p o ise d a t its
freezing p o in t), 34.5 p p t salinity, w ith a n a v erag e c u rre n t (m e a n tid a l speed)
o f 180 m /d a y , a n d a m e an n e t sp eed o f 26 m / d (close to th e low er lim it of
12
detection). Effluent conditions have b e e n d escrib ed as 33-38°C, 30-36.5 p p t
salinity, a n d 0.0060-0.0081 w ? /s e c (72). E nteric b a c te ria in th e e fflu e n t are
ch allen g ed b y v e ry cold te m p e ra tu re s, osm otic shock, a n d g ra d u a lly
in creasin g oligotrophic co n d itio n s as th e sew age p lu m e d isp erses. In a d d itio n ,
solar ra d ia tio n is highly a tte n u a te d b y 2-3 m o f fa st ice (a n d snow) cover fo r 10
m o n th s of th e year, a n d is effectively a b se n t fo r u p to 4 m o n th s d u rin g p o lar
w inters. Y early in p u ts of o rg an ic n u trie n ts th ro u g h p rim a ry a n d sec o n d a ry
p ro d u c tiv ity a re som e o f th e h ig h e st re p o rte d fo r m a rin e su rface w aters d u e to
u n d e r-ic e p h y to p la n k to n a n d d ia to m bloom s, b u t a re h ig h ly seasonal (68). T he
bloom s re g u la rly la st fo r ca. I m onth, a n d h a v e b e e n fo u n d to p ro d u c e am ino
acid a n d sugar c o n cen tratio n s in th e w a te r co lu m n o f u p to 4 9 0 a n d 25 ><M,
re sp ec tiv e ly (87).
T hus, th e A ntarctic m a rin e e n v iro n m e n t re p re se n ts a u n iq u e setting in
w hich to stu d y th e survival p a tte rn s a n d physiological co n seq u en ces o f
su b leth al stress in en teric sew age b a c te ria ex p o sed to cold, osm otic stress, a n d a
“fe a st a n d fa m in e ” existence.
R esearch Goals a n d O bjectives
V alidation o f T etrazolium R eduction M ethods
1) E lucidate th e site(s) a n d m echanism (s) b y w hich 5-cyano-2,3-ditolyl
te tra zo liu m ch lo rid e (CTC) a n d (2 -(4 -io d o p h e n y l)-3-(4 -n itro p h e n y l)-5p h e n y l te tra zo liu m c h lo rid e (INT) a re re d u c e d to in tra c e llu la r fo rm azan
crystals in a m odel p ro k ary o te.
2) D eterm ine w h e th er a n d to w h a t e x te n t CTC a n d INT a re biologically re d u c e d
u n d e r a n aero b ic co n d itio n s w ith d iffe re n t su b strates in a m o d e l p ro k ary o te.
13
3) D efine th e su b strates w hich give op tim al CTC a n d INT re d u c tio n u n d e r
aero b ic co n ditions w ith d iffe re n t su b stra te s in a m o d el p ro k a ry o te .
4) See how d iffe re n t su b strates effect CTC (+)/IN T (+) cell n u m b e rs in re la tio n
to p la te counts.
Exposure o f E nteric B acteria to a Polar M arine E n v iro n m en t
1) D eterm ine th e survival kinetics, in ju ry , a n d loss o f c u ltu ra b ility o f
representative e n te ric organism s (in d ic a to rs a n d p a th o g en s) w h en ex p o sed
to cold (-1.8°C) seaw ater.
2) D eterm in e w h e th e r in d ic a to rs a n d p a th o g e n s s h a re sim ilar su rv iv al
resp o n ses, a n d w hich in d ic a to rs a re o p tim a l fo r this e n v iro n m e n t.
3) Use th e above d a ta to d e te rm in e if ex p o su re to th is ty p e o f en v iro n m e n t
e n h an c e s s u rv iv a l/p e rsiste n c e o f e n te ric o rg an ism s.
4) E lucidate th e specific cellu lar resp o n ses o f b a c te ria , in te rm s o f su b strate
u p ta k e a n d m acro m o lecu lar sy nthesis to co ld m a rin e e n v iro n m e n ta l
exposure, focusing o n co ld shock a n d sta rv a tio n resp o n ses.
5) D eterm ine w h e th e r n o n c u ltu ra b ility o f v ia b le e n te ric b a c te ria fo u n d in
sewage is p ro m o te d u n d e r cold m a rin e cond itio n s, a n d , if so, th e d eg ree to
w hich th ese b a c te ria p e rsist ov er tim e a n d c o u ld je o p ard ize p u b lic h ealth .
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20
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JFor a b ro a d review o f th e p rin c ip a l co n cep ts I w ould re fe r re a d e rs to
references 23, 3 0 ,4 2 , a n d 49.
21
CHAPTER 2
MECHANISMS OF IN T ( 2 - ( 4-IODOPHENYL)-3 - ( 4-NITROPHENYL)-5PHENYL TETRAZOLIUM CHLORIDE), AND CTC (5 -CYANO-2,3 -DITOLYL
TETRAZOLIUM CHLORIDE) REDUCTION IN ESCHERICHIA COLI K-12
In tro d u c tio n
T etrazolium salts a re o fte n u se d a s in d ic a to rs o f m icro b ial re s p ira to ry
activity, as well as viability, p a rtic u la rly in physiological a n d ecological
studies. T hey a re also u tiliz e d as d iffe re n tia l ag en ts in b acterio lo g ical m ed ia
(TTC [2,3,5-triphenyl-2H -tetrazolium chloride] in Tergitol-7, a n d mE) (2). INT
(2 -(4 -io d o p h e n y l) -3 - (4 -n itro p h e n y l) -5-p h e n y l te tra z o h u m c h lo rid e ), a n d
m o re re c e n tly CTC (5-cyano-2,3-ditolyl te tra zo liu m ch lo rid e), h a v e b e en used
to in d ic a te th e frac tio n o f re sp irin g b a c te ria in p o p u latio n s. T his is d o n e
th ro u g h re d u c tio n o f th e soluble te tra z o h u m salts to th e ir co rre sp o n d in g
c o lo re d (INT), o r flu o resc en t (CTC), in so lu b le, in tra c e llu la r fo rm a z a n cry stals
w hich c an b e view ed using epifluorescen ce m icroscopy (Fig. I) (14, 15, 33, 45,
52). INT re d u c tio n h a s also b e e n u s e d to q u a n tify re s p ira to ry p o te n tia l in
m ixed p o p u la tio n s b y organic ex tractio n , a n d colorom etric d e te rm in a tio n o f
fo rm azan p ro d u c tio n (6, 17, 26). T he INT te c h n iq u e p ro d u c e s d a rk re d ,
in tra c e llu la r IN T -form azan (INF) cry stals w h ich c an b e d ifficu lt to visualize,
p a rtic u la rly ag ain st a n o p aq u e b ack g ro u n d (12, 44, 45, 46). U n fo rtu n ately , th is
is o fte n th e ty p e o f b a ck g ro u n d e n c o u n te re d in stu d ies o f m icro b ial ad h esio n ,
biofilm s, a n d sedim ents, a s w ell as b a c te ria l e n u m e ra tio n s o n m e m b ra n e filters
22
STABLE RADICAL CATION
\
//
H - N ----- N •
INF
INT-formazan
F ig u re 1(A ). R eduction re a ctio n o f INT to INF, show ing interm ediate, species
betw een te trazo liu m a n d form azan.
23
F ig u re 1(B). R eduction re a ctio n o f CTC to CTF, showing in te rm e d ia te species
betw een tetrazo liu m a n d form azan.
24
using epiflu o rescen ce m icroscopy. INF cry stals also a p p e a r to b e soluble in
im m ersio n oil (45, 46). However, th e re c e n tly d ev elo p ed cy an o -su b stitu ted ,
2,3-diaryl te tra zo liu m salt CTC form s a flu o rescen t fo rm azan (CTF) w hich is
re la tiv e ly e a sy to visualize a g a in st a n o p a q u e b ack g ro u n d b y ep iflu o rescen ce
m icroscopy (33). In ad d itio n , CTF is in so lu b le in im m ersion oil, a n d re ta in s its
flu o rescen ce u p o n sto ra g e (41). D irect co u n tin g o f activ ely re sp irin g b a c te ria
is sim plified considerably, a n d CTF p ro d u c tio n can b e m o n ito re d by
flu o rescen ce-activ ated cell sorting (FACS) an aly sis (14, 33, 38, 52).
W ith th e in creasin g use o f te tra zo liu m salts fo r stu d ies o f p ro k ary o tic
p h y sio lo g y a n d ecology, q u e stio n s a rise co n ce rn in g th e specific cellu lar
process(es) th e ir re d u c tio n m easures. A lthough sites o f re d u c tio n fo r INT a n d
CTC in eu k ary o tic e le c tro n tra n s p o rt system s h a v e b e e n fa irly w ell d efin ed ,
th ose in p ro k a ry o tic cells re m a in largely u n re so lv ed (25, 26, 27, 37). In
eu karyotic m ito ch o n d ria, INT h a s b e e n fo u n d to b e re d u c e d b y th e
su ccinaterubiq uinone o x id o red u ctase (SDH) com plex, o r cy to ch ro m e
(21,26,
27). Stellm ach a n d S everin fo u n d th a t CTC is re d u c e d p rim arily b y m em b ran eb o u n d NAD( P)H -dehydrogenase, a n d p o ssib ly su p ero x id e a n io n (NADH-D) in
Ascites tu m o r cells (43). K aprelyants a n d Kell fo u n d th a t CTC was re d u c e d
d ire c tly b y re s p ira to ry c h a in d e h y d ro g e n ases in M icrococcus lu teu s (15).
A lthough n o t d e m o n stra te d experim en tally , SDH h a s b e e n su g g ested as th e
p rim a ry site o f CTC a n d INT re d u c tio n in p ro k a ry o te s (14, 26, 33). It is well
k now n th a t d iffe re n t te tra z o liu m salts h a v e d iffe re n t re d u c tio n p o te n tia ls, a n d
th e red o x p o te n tia l o f CTC is u n re p o rte d (16, 25, 37), Since th e u se o f d ifferen t
te tra zo liu m salts a n d assay co n d itio n s fo r th e d e te rm in a tio n o f re s p ira to ry
p o te n tia l a n d activ ity m a y y ie ld d iffe re n t fin d in g s, resu lts w o u ld b e easier to
25
in te rp re t if th e ir red o x p o te n tia ls a n d site(s) o f re d u c tio n in th e p ro k a ry o tic
e le c tro n tra n s p o rt c h a in w ere know n.
In o rd e r to d e te rm in e th e m echanism s o f INT and. CTC re d u c tio n in a
p ro k a ry o tic re s p ira to ry c h a in we ex am in ed th e effects o f v a rio u s in h ib ito rs o f
e lec tro n tra n s p o rt a n d u n c o u p lers o f o x idative p h o sp h o ry la tio n w ith well
d e sc rib e d sites of actio n . In g en eral, blockage o f th e e le c tro n tra n s p o rt c h ain
a t a p o in t b efo re th a t a t w hich th e tetrazo liu m salt is re d u c e d will re s u lt in
d e crea se d re d u c tio n to fo rm azan , w hile blockage a fte r th e site o f re d u c tio n
will increase, o r p ro d u c e n o change in tetrazo liu m re d u c tio n (25, 27, 38, 43).
In o rd e r to d e te rm in e th e e x te n t o f coupling betw een te tra zo liu m re d u c tio n
a n d re sp ira tio n , fo rm az a n fo rm atio n was c o rre la te d w ith oxygen co n su m p tio n
m easu rem en ts. T hese e x p erim en ts w ere c a rrie d o u t in w hole cells a n d
in v e rte d m e m b ra n e vesicles o f Escherichia coli K-12, a p ro k a ry o te w ith
re la tiv e ly w ell-d escrib ed a ero b ic a n d a n a e ro b ic e le c tro n tra n s p o rt c h ain s (4,
1 8 ,1 9 , 20, 2 3 ,3 0 , 50, 51) (Fig. 2). To determ in e w hether, a n d to w h at ex ten t CTC
a n d INT a re re d u c e d u n d e r an ae ro b ic co n d itio n s, w hole cells w ere assay ed
u n d e r co n d itio n s d e sig n e d to stim u late sev eral d iffe re n t a n ae ro b ic re s p ira to ry
ch ains, as w ell as glucose fe rm e n ta tio n . In o rd e r to f u r th e r c h a ra c te riz e th e
chem ical p ro p e rtie s o f INT a n d CTC, th e ir m id -p o in t re d o x p o te n tia ls ( E v 2),
solubilities, a n d m o la r e x tin c tio n coefficients (e) in 95% e th an o l o f CTC a n d ESTT
w ere also d e term in e d .
26
|NAOH~>NAD+ (-320 mV) |
|SUCCINATE~>FUMARATE (+30 ~^V)]
MALONATE
S u c c in a te D e h y d ro g e n a s e C om plex
Cytochrome Zr556 (+36 mV)
FeS-2 (-220 mV)
FAD (+30 to -60 mV)
FeS-I (-20 mV)
FeS-3 (+100 mV)
NADH D e h y d ro g e n a s e
FeS-2 (-240 mV)
FeS-I (-220 mV)
FAD (+30 to -60 mV)
ROTENONE-
T ra n slo c a tlo n
U biquinone (+70 mV)
M enaquinone (-74 mV)
HQNO
High Oxygen
Low O xygen
Cytochrome O55
(+129 t o +46 mV)
Cytochrome b.
(+140 mV)
ANTIMYCIN A
CYTOCHROME
COMPLEX
CYTOCHROME d
’ ' com plex
O
Cytochrome b;62
Cytochrome o
(+165 to +260 mV)
Cytochrome O558 (+180 mV)
Cytochrome Zr595 (+160 mV)
Cytochrome d
(+260 to 280 mV)
KCN .
AZIDE
KCN .
AZIDE
I e -+ 0 ---------------- > O2-
1C"+ (I,-----------------> O2-
2e + 0 , ----------------> 0 |
2e +
2H*+ 2e + 1/2 O2— >H20
2H*+ 2e + 1/2 Q2— >H20
(+820 mV)
----------------- > 0^"
(+820 mV)
N O T E : E1/2 = -90 mV (INT)1 -200 mV (CTC)
F ig u re 2. Schem atic o f th e E. coli a ero b ic re s p ira to ry c h a in show ing th e sites
of action of th e v ario u s in h ib ito rs u sed in th is study. M id-point red o x
p o te n tia ls fo r th e v ario u s c y to ch ro m es, iro n -su lfu r c e n te rs (Fe-S), a n d
d ehy d ro g en ases a re given. Sites o f re d u c tio n o f CTC a n d INT fro m th is stu d y
a re encircled. D ashed Unes in d icate sites o f in ib itio n o f e le c tro n tra n s p o rt
processes. See m aterials a n d m eth o d s fo r chem ical a b b rev iatio n s. Com piled
from (4 ,1 8 ,1 9 , 23 ,2 5 , 2 7 ,2 9 , 30, 3 2 ,4 8 , 5 0 ,5 1 ).
27
M a te ria ls a n d
M e th o d s
D eterm in atio n o f INT a n d CTC C hem ical P ro p erties
All chem icals a n d su b strates u sed w ere re a g e n t g rad e o r b e tte r. CTC was
o b ta in e d fro m Polysciences, Inc. (W arrin g to n , Pa.). INT a n d INF, as well as all
su b strate s, flu o resc en t stains, a n d in h ib ito rs w ere p u rc h a se d fro m Sigma
Chem ical Co. (St Louis, Mo.).
D upU cate re d o x titra tio n s w ere c a rrie d o u t in a n a n ae ro b ic te n t u n d e r
a n N2/OO2/H2 atm o sp h ere using sodiu m d ith io n ite as U tran t a n d a n O rion
9 6 7 SBN co m b in atio n re d o x electrode. AU d ire c tly m e a su re d re d o x p o ten tials
w ere c o rre c te d b y re fe re n c e to th e n o rm a l h y d ro g e n e lec tro d e (NHE). INT was
titra te d a s a co ntrol, as its re d o x p o te n tia l h a s b e e n re p o rte d p re v io u sly (E1/2 =
-90 mV) (16, 25, 37). TetrazoU um salts w ere dissolved a t 0.1 mM in 5 0 m l, 0.1 M
p h o sp h a te b u ffer, pH 7.0 as d e sc rib e d b y K arm ark er e t a l . (16). T h e ascorbic
acid (0.1 M)-pH (pH ra n g e 2.5-11.5) m e th o d o f Seidler was also u se d to
d e te rm in e th e re la tiv e re d u d b iU tie s o f INT a n d CTC (36). M olar extinction
coefficients w ere d e te rm in e d b y dissolving INT-(INF), a n d CTC-form azan (CTF)
in 95% e th an o l. T hese values w ere su b se q u e n tly u s e d to calcu late fo rm azan
p ro d u c tio n fro m ab so rb an ce d a ta. CTF was o b ta in e d b y re d u c tio n o f 50 ml, 0.1
mM CTC in double-distiU ed w a ter (dd-H2Q) usin g sodium d ith io n ite. T he
p re c ip ita te was w ashed 3 tim es w ith dd-H2G a n d lyophitized.
G row th C onditions
Escherichia coli K-12 w as grow n aerobicaU y w ith v ig o ro u s a e ra tio n a t
25°C to late-log p h a se in glycerol (8 g /l)-m in e ra l m ed iu m su p p le m e n te d w ith I
28
jM selenic acid a n d sodium m o ly b d ate (9). E. coli was also grow n
an aero b ically , a n d all su b se q u e n t m a n ip u la tio n s p e rfo rm e d in a N2/OO2/H2
a tm o sp h e re fo r all a n ae ro b ic ex p erim en ts. T he sam e grow th m ed iu m as above
was used, except glycerol w as re p la c e d b y glucose. Cells w ere w ashed 3 tim es
a n d resu sp e n d ed in 0.85% NaCl (pH 6.5) a t ca. lO^O/ml. Subsam ples fo r aerobic
te tra zo liu m salt ex p erim en ts w ere rem o v ed a fte r vigorous a e ra tio n fo r 10 m in
a t 25°C. Plate counts w ere p e rfo rm e d b y sp o t-p latin g subsam ples (exposed fo r
th e sam e p erio d , in th e sam e b u ffe r as resp ectiv e assays) o n to R2A m ed iu m a n d
in c u b a tio n fo r 48 h a t 25°C. Plates fo r a n ae ro b ic assays w ere in c u b a te d in th e
sam e N2/GO2/H2 a tm o sp h ere as above.
S u b strates. In h ib ito rs, a n d U ncouplers
In h ib ito rs w ere a d d e d as e ith e r 0.1 m l IOX solutions, o r 10 m icro liter
IOOX solutions to th e following fin a l co n cen tratio n s: KCN (2 m m ), sodium azide
(50 mM), ro te n o n e (ROT, I mM ), m alo n ate (MAT, 100 mM),
th e n o ly ltriflu ro a ce to n e (TTF, I mM ), c a rb o n y l cy an id e mch lo ro p h e n y lh y d ra zo n e (CCCP, 10 ^M ), a n tim y d n A (20 /<M), a n d 2-heptyl-4hydroxyquinoline-N -oxide (HQNO, 0.2 mM). R otenone, TTF, CCCP, HQNO, a n d .
an tim y cin A w ere m a d e a s fresh ly p re p a re d 100X solutions in a b so lu te eth an o l
a n d 10 jA a d d e d to assays. Final e th a n o l c o n ce n tra tio n d id n o t exceed 1%.
Superoxide dism utase (SOD) w as u s e d a t a fin a l c o n ce n tra tio n o f 175 u n its/m l.
C oncentrations of su b strate s fo r a n ae ro b ic in c u b atio n s w ere 10 mM each of
glucose, fo rm ate, glycerol, a n d fu m a ra te . C om binations w e re as show n in
Figures 3, a n d 4. R2A b ro th w ith o u t p h o sp h a te (R2A-P) was u s e d a t 0.1stre n g th , fin al c o n cen tratio n . N itrate w as p re s e n t as 40 mM KNO3. Assays
w ere p e rfo rm ed in 10 mM p o tassiu m p h o s p h a te b u ffer co n tain in g 0.85% NaCl,
29
5 m M MgCl2, a t pH 6.5. T he pH o f all w hole cell assay solutions was ad ju sted to
6.5 p rio r to cell a n d tetrazo lim n salt a d d itio n . Cells w ere u se d a t a final
c o n c e n tra tio n o f ca. IO^ cells/m l. Cells fo r n u trie n t assays w ere allow ed to
re a c t fo r I h p rio r to tetrazo liu m salt a d d itio n . Freshly p re p a re d CTC a n d INT
solutions w ere a d d e d a t a final c o n c e n tra tio n o f I mM. In cu b atio n s w ere
p e rfo rm e d in d u p lic a te a t 2 5 °C w ith v ig o ro u s a e ra tio n fo r 2 h in th e d a rk
(an aero b ic in cu b atio n s w ere 4 h ). R eactions w ere sto p p ed b y a d d itio n of 3.7%
fo rm a ld e h y d e (final c o n ce n tra tio n ) a n d im m ersio n in a n ice b a th . Sam ples
fo r R/ETS ra tio m easu rem en ts w ere sto p p ed b y a d d itio n o f 5% trich lo ro acetic
a cid (TCA) fin al c o n cen tratio n . F o rm ald eh y d e k illed (15 m in exposure)
co ntrols w ere p e rfo rm e d fo r all assays. Subsam ples (91 /d) fo r CTC-reduction
assays w e re d ilu te d in filter-sterilized d istille d w ater, filte re d th ro u g h 0.22 ^ m
p re -b la c k en e d N uclepore p o ly c arb o n ate m e m b ra n es (no. 110656), a n d
c o u n te rsta in e d w ith 10 /vg/ml DAPI (35). Subsam ples fo r INT assays w ere
d ilu te d as above a n d a ir-d rie d as heat-fixed sm ears using 20 ^ l aliquots. These
w ere c o u n te rsta in e d w ith 100 m g/1 a c rid in e o ra n g e (AO) in 2 mM T iis b u ffe r
(pH 7.0) fo r 3 m in. a n d view ed u n d e r b o th ep iflu o rescen ce a n d b rig h t field
m icroscopy. Sam ples fo r ep iflu o rescen ce d ire c t c o u n ts w ere view ed using a
Leitz O rtholux II m icroscope e q u ip p ed w ith Leitz filter blocks B2, N2.1, a n d H3
to visualize DAPI, CTC, a n d INT/AO, respectively. INT (+) cells c o n tain ed re d
IN T -form azan d eposits w h e n view ed u n d e r b rig h t-fie ld m icroscopy, w hile CTC
(+) cells c o n ta in e d flu o rescen t-o ran g e CTC-form azan d eposits. A t le a st 600 cells
in a 10 fields w ere c o u n ted a t 1,250 X fo r e ac h filter. E rror w as calcu lated as
d e sc rib e d p re v io u sly (40).
S am ples fo r sp ec tro p h o to m etric d e te rm in a tio n o f fo rm az a n (0.9 ml)
w ere c en trifu g e d in a h ig h -sp eed m icro cen trifu g e (5,000 X g, 10 m in) a n d
30
su p e rn a ta n ts d iscard ed . Pellets w ere th e n re su sp e n d ed in 95% EtOH a n d
d is ru p te d b y so n ieatio n fo r 15 s using a so n ica to r m icro tip (F isher Scientific
Sonic D ism em brator 50) a t 30% pow er, a n d sto re d a t 4°C o v ern ig h t.
Suspensions w ere th e n cen trifu g ed (9,000 X g, 10 m in) a n d a b so rb an c e of
su p e rn a ta n ts d e te rm in e d a t 450 run fo r CTC, a n d 485 n m fo r INT using a n HP
8452A sp ectro p h o to m eter. Cell pellets re ta in e d n o visible fo rm az a n a fte r th e
ab o v e e x tra ctio n p ro c e d u re .
In v e rte d M em b ran e Vesicles
T h ree lite r c u ltu res o f E. coli K-12 grow n aero b ically as d e sc rib e d ab o v e
w ere h a rv e ste d in la te log p h a se a n d in v e rte d m e m b ra n e vesicles, as well as
vesicles d e p le te d in Mg2+-d e p e n d e n t ATPase, w ere p re p a re d b y th e m eth o d o f
B urstein e t al. (9). D epleted inverted-vesicles lack Mg2+ -d e p e n d en t ATPase
activity, b u t re ta in m e m b ra n e -b o u n d d e h y d ro g e n a se a n d e le c tro n tra n s p o rt
activities (9, 34). Study o f in h ib itio n o f th e se m e m b ran e p ro cesses is
fa c ilita te d th ro u g h th e u se o f in v e r te d m e m b ra n e vesicles w hich avoids
p ro b lem s re la te d to in h ib ito r p e rm e ab ility th ro u g h in ta c t cell m e m b ra n es (11,
29). D uplicate assays w ere p e rfo rm e d in a fin a l volum e o f 1.0 m l, 10 mM
p h o sp h a te b u ffe r (pH 6.5), co n tain in g 5 m M m ag n esiu m a ce ta te. In h ib ito r
c o n ce n tra tio n s u s e d w ere th e sam e as th o se fo r in ta c t cells ab o v e, a n d sites o f
action a re show n in Figure 2 a n d Table I . Vesicle p ro te in (0.25 mg) was ad d ed ,
a n d allow ed to re a c t w ith a g en ts fo r 30 m in b efo re te trazo liu m salt ad d itio n .
Freshly p re p a re d INT a n d CTC w ere a d d e d to a fin al c o n cen tratio n o f I mM.
S u b strates w ere th e n a d d e d to fin al co n ce n tra tio n s o f 10 mM succinate, o r 0.6
mM NADH w hich was g e n e ra te d using 50 ^g alcohol d e h y d ro g e n ase, 1%
ab so lu te ethanol, a n d 0.6 mM NAD+ (29).
31
T a b le I . S ubstrates, in h ib ito rs, a n d sites o f in h ib itio n fo r in v e rte d m e m b ra n e
vesicle, te tra zo liu m re d u c tio n assays.
S u b s tra te
In h ib ito r
NADH
R o te n o n e
NADH d e h y d ro g e n ase
S u c c in a te
M alonate
S u ccin ate d e h y d ro g e n a se
S ite
o f In h ib itio n
(c o m p e tetiv e in h ib itio n )
S u c c in ate
TIF
Q uinone b in d in g su b u n it o f
su ccin ate d e h y d ro g e n a s e
NADH
HQND
U b iq u in o n e -c y to c h ro m e b
NADH
A n tim y cin A
C ytochrom e b -cy to ch ro ih e o /d
NADH
KCN
C ytochrom e o /d com plexes
NADH
S uperoxide dism utase
S u p ero x id e a n io n s
NADH
D epleted m em b ran es
M g + -d ep en d en t ATPase
NADH
CCCP
T ra n sm e m b ra n e p ro to n
g r a d ie n t
NADH
aADP
Stim ulation o f ATPase
aADP is co n sid e re d a stim u la to r r a th e r th a n a n in h ib ito r.
T h ere w as n o d e tec ta b le fo rm az a n p ro d u c tio n b y th e se su b stra te s in th e
ab sen ce o f vesicles. O xygen c o n su m p tio n ra te s re m a in e d c o n sta n t th ro u g h o u t
th e I h in c u b atio n s w ith b o th succinate a n d NADH. R eactions w ere p e rfo rm e d
a t 25°C fo r I h r in th e d ark , a n d w ere sto p p ed b y th e a d d itio n o f 5% TCA (final
c o n c e n tra tio n ). T C A -treated c o n tro ls w ere p e rfo rm e d u sin g su ccin ate a n d
NADH as re d u c tan ts. T h ere was n o detectab le fo rm azan p ro d u c e d b y TCAtre a te d vesicles, o r vesicles in th e ab sen ce o f su b strate . M em b ran es w ere
p elleted a t 175,000 X g fo r I h, resu sp en d ed , a n d form azans d issolved in 95%
32
e th a n o l as d e sc rib e d fo r in ta c t cells above. D ebris was p e lle te d b y re p e a tin g
c en trifu g atio n , a n d th e s u p e rn a ta n ts u s e d fo r a b so rb an c e m e asu re m e n ts.
O xygen C onsum ption
Oxygen co n su m p tio n w as m e a su re d using a G ilson 5 \6 o x y g rap h w ith a
C lark-type e lec tro d e (29). All ex p erim en ts w ere p e rfo rm ed in a 2 ml- w aterja ck eted cell a t 25°C. O xygen sa tu ra tio n w as m e a su re d using d o u b le-d istilled
w ater a n d co rre cted fo r a ltitu d e a n d p re ssu re (2). AU assays w ere c a rrie d o u t
in th e sam e solutions u s e d fo r te tra z o h u m re d u c tio n assays. P ro tein was
d e te rm in e d by th e micro-BCA m e th o d (39). TCA -(m em branes), a n d
fo rm aldehyde-(w hole ceUs) tre a te d c o n tro ls show ed n o d e te c ta b le oxygen
co n su m p tio n o r fo rm az a n fo rm atio n .
C alculations
In o rd e r to c o rre la te oxygen c o n su m p tio n w ith fo rm az a n p ro d u c tio n it
was n ecessary to c o n v e rt v alu es o b ta in e d to th e n u m b e r o f e le c tro n e q u iv alen ts
(eq) re q u ire d fo r re d u c tio n . The values 2 e q /m o l fo rm azan , a n d 4 e q /m o l 0 2
w ere u se d (17, 26, 37). T he d eg ree o f coupling betw een oxygen co n su m p tio n
a n d te tra z o h u m re d u c tio n (nm ol 0 2 -e q u iv a le n ts /n m o l fo rm a z a n -e q u iv a le n ts)
was ex p ressed as th e re s p ira tio n /e le c tro n tra n s p o rt system (R/ETS) ra tio (26).
To d e te rm in e th e re la tiv e effects o f v a rio u s ag en ts o n te tra zo liu m re d u c tio n , a h
w hole ceU- o r v esicle-fo rm azan v alues w ere c o m p ared to n o -in h ib ito r o r n o ­
su b stra te co n tro ls. P e rc e n t d ev iatio n s fro m c o n tro ls w ere c alc u la te d usin g th e
eq uation: [mol form azan^inhibitor or substrate)~niol form azan^control)/m ol
form azan(control)] X 100 = % d ev iatio n . S tatistical significance o f d ifferen ces
33
betw een tre a tm e n ts a n d c o n tro ls w as d e te rm in e d using a o n e -ta ile d stu d e n t’s tte st assum ing u n e q u a l v a ria n c e (a = 0.05) (24).
R e s u lts a n d
D is c u s s io n
Chem ical P roperties of CTC/INT. a n d T h e ir Fnrm azans
T he physical a n d chem ical p ro p e rtie s o f CTC a n d INT, a n d th e ir
c o rresp o n d in g form azans d e te rm in e d in th is stu d y a re listed in T able 2.
T a b le 2. Physical a n d chem ical p ro p e rtie s o f CTC a n d INT re d u c tio n . M olar
extin ctio n coefficients, solubilities, a n d CTC re d o x p o te n tia l w ere
d e te rm in e d in th is study.
C hem ical
T etrazo h u m Salt
T etrazo h u m F orm azan
P ro p e rty
CIC
INT
E1/2 (mV)a
-200 (-50 ?)
-90
S o lu b ility ^
ca. 50 mM
ca. 5 mM
ca. 2 0 0 ^M
ca. 2 5 0 ^M
Color
Colorless to
Colorless to
R ed -o ra n g e
P u r p le - re d
lig h t yellow
lig h t y ello w
^m ax
NDc
ND
4 50 n m
485 n m
«(90% EtOH)
ND
ND
1.624X 104
6.781 XIO3
1/m olocm
1/m olocm
Ex: 380 n m
NF
F lu o resc en c e
NFd
NF
CIF
—
INF
—
Em: 602 n m
a M id-point re d u c tio n p o te n tia l (pH 7.0, 250C) w ith re fe re n ce to th e n o rm al
h y d ro g e n electrode. Possible CTF p ro d u c tio n a t ca. -50 mV (see text),
k T etrazo h u m salt solubilities a t 25°C w ere d e te rm in e d in d istilled w ater,
fo rm azan solubilities w ere in 95% e th a n o l (form azans a re w a te r in so lu b le).
CTC solubility v a rie d w ith so lv en t (i.e. seaw ater, d istilled w ater, etc.)
c ND, N ot determ ined.
^N F, n o t flu o rescen t.
34
D uring re d u c tio n CTC u n d e rw e n t a d is tin c t th re e -p h a se tra n sitio n , from
soluble a n d d a rk orange, to a b rillia n t o ra n g e colloidal su sp en sio n (ca. -50
mV), a n d finally to a d a rk o ra n g e -re d fo rm az a n p recip itate. D uring th e
colloidal p h ase, re d o x p o te n tia l re a d in g s to o k ca. 10 m in u tes to in crease a n d
stabilize a fte r d ith io n ite a d d itio n . No su ch effects w ere n o te d w ith INT w hich
u n d e rw e n t a tw o-phase tra n sitio n , fro m d a rk r e d a n d soluble, to th e in so lu b le
p u rp le -re d form azan. In th e p re se n c e o f asco rb ic a cid as a re d u c in g agent,
CTC was re d u c ed a t pH ;> 4, w hile INT was re d u c e d a t pH & 5, indicating CTC was
m o re easily re d u c e d th a n INT (37). F orm azan fo rm atio n w as g re a te st betw een
pH 5.5-8.5 fo r ENT, a n d pH 5-10 fo r CTC.
T etrazolium salts a re c o n v e rte d to fo rm azan s b y all re d u c in g system s
possessing a c tu a l re d o x p o te n tia ls m o re n e g ativ e th a n th e
te tra zo liu m /fo rm az a n system (37). T h erefo re, use o f CTC a n d INT in red u cin g
e n v iro n m e n ts (sed im en ts, m e d ia c o n ta in in g re d u c in g a g en ts [thioglycollate,
ascorbate, etc.]) w h ere in situ re d o x p o te n tia ls a re low er th a n th e E1 / 2 values
o f th e te tra zo liu m salts will le a d to non -b io lo g ical te tra zo liu m re d u c tio n (33,
37). This co u ld ex p lain o b serv atio n s o f INF o n non-living d e trita l m aterial in
b e n th ic suspensions, a n d CTC re d u c tio n b y som e grow th m e d ia (8, 31). In
a d d itio n , b a c te ria l exopolym eric m a te ria l (alg in ate fro m Pseudomonas
aeruginosa) a n d a g a r h a v e b e e n re p o rte d to re d u c e CTC a n d INT, respectively
in th e absence o f v iable cells ( 1 ,5 ) . This m a y b e d u e to rion-biological
re d u c tio n b y io n iz e d carboxyl-, o r p y ru v y l -g ro u p s in th e se p o ly sacch arid es,
p a rtic u la rly u n d e r acidic co n d itio n s.
W hile d ifferen t te tra z o liu m salts c a n b e co m p ared o n th e b asis o f m id ­
p o in t re d u c tio n p o te n tia ls (E1/2) a n d re d u c ib ilitie s o b ta in e d u n d e r id en tical
assay conditions, a ctu al re d u c tio n p o te n tia ls will v a ry d e p e n d e n t o n te st
35
co n d itio n s (pH, co n cen tratio n ) (16, 37). In gen eral, th e less n eg ativ e a
te tra z o h u m salt re d o x p o te n tia l th e m o re easily it is re d u c e d (36, 37). However,
resu lts in d ic a te d th a t CTC was slightly m o re re d u cib le th a n INT. This m ay be
d u e to th e fo rm atio n o f a colored, colloidal in te rm e d ia te d u rin g CTC re d u ctio n
a t ca. -50 mV. T etrazolium salts u n d e rg o tetrazo lin y l-rad ical cation
in te rm e d ia te stages d u rin g re d u c tio n , a n d i t a p p e a rs th a t th is fo rm is m ore
stable a n d p ro d u ces a diffuse, p o o rly IocaUzed fo rm azan a t re d o x potentials
h ig h e r th a n ca. -200 mV d u rin g CTC re d u c tio n (Fig. I) (27, 37). This w ould
e x p lain th e e v en ly -d isp e rse d , w e ak ly -flu o re sc e n t in tra c e llu la r d ep o sits
o b se rv e d in w hole ceU CTC re d u c tio n assay s in th e p re sen c e o f c e rta in
in h ib ito rs (an tim y cin A, KCN, CCCP) (see below ). Sim ilar re su lts h a v e b e en
o b serv ed using m e n d o la b lu e (E1/2 = -H O mV) as a n artificial e le c tro n d o n o r in
Listeria m o n o cyto g en es a n d ErUch ascites tu m o u r ceUs (8, 28, 42). W e h av e
also o b se rv e d th is p h e n o m e n o n in th e p re se n c e o f p h o sp h a te o v e r ca. 10 mM
in R coli (41, see foUowing c h a p te r). E th an o l-ex tractab le CTF in th e absence o f
CTC (+) ceUs m ay also b e d u e to th is p h en o m en o n .
R esn iratio n /T etrazo U u m R eduction Ratios
R/ETS ra tio s in d ic a te th e m oles o f re s p ira to ry e le c tro n flow d e term in e d
b y oxygen co nsum ption (R) p e r m ole o f tetrazoU um salt re d u c e d (ETS). Thus,
R/ETS ra tio s re fle c t th e d e g re e o f coupling b etw ee n th e se tw o p ro cesses (see
m aterials a n d m ethods) (26). A lthough R/ETS ra tio s v a rie d w ith tim e of
in c u b atio n , in g en eral, te tra z o h u m re d u c tio n re fle c te d a fa irly c o n sta n t
fra c tio n o f re s p ira to ry activ ity o v e r I h in c u b a tio n (p a rtic u la rly a fte r 20
m ih). Using in v e rte d m e m b ra n e vesicles, R/ETS valu es w ith NADH as th e
su b strate w ere ca. 45-60 fo r CTC, a n d 8-14 fo r INT o v er I h in c u b atio n (Table 3).
36
T a b le 3. Ratios o f re sp ira tio n a n d ETS activ ity fo r w hole cell a n d in v e rte d
m e m b ra n e vesicle te tra zo liu m re d u c tio n assays.
R e s p ir a tio n Z E T S a
T im e
W h o le C ells
In v e rte d
E ndogenous
NADH
SUCCINATE
(Oxidant)
(O xidant)
(Oxidant)
CDC
M em b ran e
V e sic le s
INT
CDC
INT
CDC
INT
2.4 + 0.24
47.7 ± 3.2
42.2 ± 3.5
11.8 ± 3 .4
59.2 ± 4.1
10.5 ± 1.0
5.2 ±0.79
74.3 ± 4.5
46.2 ± 2.5
13.7 ±2.1
57.0± 3.7
8.7 ±0.77
5.7 ± 0.54
76.5 ± 5.1
47.8 ± 2.2
13.1 ± 1.3
38.2 ± 2 .4
4:9 ±1.2
7.8 ±0.97
115± 7.1
58.0± 3.2
7.8 ± 1.1
34.7 ± 3.3
2.9 ±0.97
8.9 ± 0.76
91.3 ±4.3
57.6 ± 2.8
9.1 ± 1.9
39.2 ± 3.1
3.6 ± 1.4
(m in )
5
10
20
40
60
aRespirationZETS = m ol O2 e q /m o l fo rm azan eq.
Using succinate as th e su b stra te RZETS ra tio s fo r CTC a n d DNT w ere ca. 35-60,
a n d 4-10, respectively. T hus, th e d e g re e o f coupling was ro u g h ly sim ilar fo r
in d iv id u a l te tra zo liu m salts w ith e ith e r su ccin ate, o r NADH a s th e p rim a ry
e lec tro n d o n o r. These re su lts also in d ic a te d DNT re d u c tio n was m o re tig h tly
c o u p led th a n CTC to re s p ira to ry e le c tro n flow in in v e rte d m e m b ra n e vesicles
b y a fa c to r o f ca. 5-10 (T able 3). In c o n tra st, RZETS ra tio s fo r w hole cells
in d ic a te d CTC re d u c tio n w as m o re tig h tly c o u p led to re s p ira to ry e le c tro n
tra n s p o rt th a n INT b y a facto r of ca. 10 (T able 3). RZETS ratio s o b ta in e d in
th e se system s w ere also sig n ifican tly h ig h e r th a n th o se r e p o rte d p rev io u sly
37
using b a cte ria l cell ho m o g en ates (17, 26). T hese resu lts suggest CTC a n d /o r INT
re d u c tio n m a y be lim ite d b y p e n e tra tio n th ro u g h th e in ta c t cell envelope to
th e site(s) o f re d u c tio n in w hole cells. A lternatively, CTC re d u c tio n m ay b e
m o re closely lin k ed th a n INT to d e h y d ro g e n ases active d u rin g endogenous
re sp ira tio n , o r m ay be in h ib ite d b y re la tiv e ly low A/<h + in in v e rte d vesicles
c o m p ared to w hole cells (see below ). O ne a d d itio n a l p o ssib ility is d ifferen tial
NADH-D a n d SDH activities d u e to changes in m em b ra n e lipids d u rin g vesicle
fo rm atio n (1 0 ). S hghtly low er R/ETS ra tio s a fte r 20 m in . in c u b a tio n using
succinate as c o m p ared w ith NADH as th e re s p ira to ry su b stra te in d ic a ted
re d u c tio n o f CTC a n d DStT was m o re closely co u p led to SDH th a n NADH-D activity
in vesicles, h i o th e r w ords, n o rm a liz a tio n o f fo rm azan p ro d u c tio n values to
re s p ira to ry e lec tro n flow as m e a s u re d b y oxygen co n su m p tio n in d ic a te d th a t
SDH activity re d u c e d m o re INT a n d CTC to fo rm azan th a n NADH-D.
Effects o f In h ib ito rs o n T etrazo h u m R ed u ctio n
T he m ethods o f m id-, a n d te rm in a l-c h a in blockage w ere u sed to
d e te rm in e sites o f CTC a n d INT re d u c tio n . In general, blockage o f electro n
tra n s p o rt below (i.e. closer to th e te rm in a l oxidase) th e site o f tetra zo h u m
re d u c tio n sh o u ld e ith e r in crease, o r leav e u n c h a n g e d th e a m o u n t o f fo rm azan
p ro d u c e d co m p ared to th e co n tro l (no in h ib ito r) (25, 27). Blockage p rio r to th e
site o f re d u c tio n (i.e. clo ser to th e p rim a ry d eh y d ro g en ase) sh o u ld decrease
fo rm azan p ro d u c tio n (Fig. 2). Effectors o f ATP synthesis (ADP, ATPase d ep leted
m em b ran es), a n d a p ro to n c o n d u cto r (CCCP) w ere also u tilized to d eterm in e
th e ir effects o n te tra z o h u m re d u c tio n . In h ib itio n o f re s p ira to ry e lec tro n
tra n s p o rt processes using in v e rte d m e m b ra n e vesicles in d ic a te d site(s) o f CTC
a n d INT re d u c tio n u n d e r d e fin e d d e h y d ro g e n a se (SDH o r NADH-D) activities,
38
w hile experim ents using w hole cells in d ic a te d site(s) o f re d u c tio n d u rin g
endogenous re sp ira tio n (Figs. 2, 3, 4A, a n d Table I) (29).
The p ro d u c tio n o f b o th INF a n d CTF in in v e rte d m e m b ra n e vesicles
using succinate o r NADH as th e re d u c ta n t in d ic a ted b o th CTC a n d INT re d u c tio n
was m e d ia ted th ro u g h m em b ra n e-b o u n d SDH-, a n d NADH-D activities.
R otenone tre a tm e n t stro n g ly in c re a se d CTF, b u t d e c re a se d INF p ro d u c tio n in
w hole cells (a n d vesicles), indicatin g b o th CTC a n d INT w ere re d u c e d b y NADHD in b o th vesicles a n d w hole cells (Figs. 3 a n d 4A). However, a d ecrease in CTF
fo rm atio n in vesicles using ro te n o n e suggests CTC was re d u c e d p rio r to th e
FeS-2 c e n te r o f NADH-D in th is system (Figs. 2 a n d 4A) (29, S I). M alonate
in h ib itio n (n e a rly 100% fo r CTC), a n d TTF stim u latio n o f CTF a n d INF
p ro d u c tio n in vesicles in d ic a te d b o th CTC a n d INT w ere also re d u c e d b y SDH,
p rio r to th e ubiquin o n e-b in d in g su b u n it (Figs. 2 a n d 3) (19, 32, 48). The
n e a rly 7-fold in c re ase in INF fo rm atio n w ith TTF in d ic a te d INT re d u c tio n was
tig h tly co u p led to SDH activity, c o n siste n t w ith R/ETS ra tio re su lts (see below)
(Fig. 3). Using w hole Cells, SDH in h ib itio n b y m alo n ate o r TTF h a d n o
significant effect o n CTC-, w hile in c re asin g IN T -reduction 10-fold (Fig. 4A).
Thus, w hile CTC a n d INT w ere re d u c e d b y b o th SDH a n d NADH-D in vesicles, CTC
re d u c tio n d id n o t a p p e a r to b e m e d ia te d p rim arily b y SDH in w hole cells.
In creased INF fo rm atio n in m a lo n a te -tre a te d w hole cells m a y b e d u e to
stim u latio n o f o th e r d e h y d ro g e n ases activ e d u rin g e n d o g en o u s re sp ira tio n
w ith E1/2 values h ig h e r th a n th a t o f ESJT, su ch as fo rm ate d e h y d ro g e n a se (E1/2
= -105 mV) (30). In h ib itio n a t u b iq u in o n e (HQNO) a n d cy to ch ro m e b
(a n tim y cin A) in c re a se d CTC re d u c tio n in vesicles, w hile n o sig n ifican t effect
was o b serv ed using w hole cells (Figs. 3 a n d 4A). This in d ic a ted th a t CTC was
re d u c e d p rio r to u b iq u in o n e in th e re s p ira to ry c h ain as h a s b e e n re p o rte d fo r
39
Micrococcus luteus (Fig. 2, Table 2) (14, 29). In b o th vesicles a n d w hole cells
INF fo rm atio n was d ecreased by HdNO, b u t stro n g ly in creased b y in h ib itio n a t
p o in ts fu rth e r along the tra n s p o rt c h ain (an tim y cin A, cy an id e, azide),
0
u
c
0
700
600
500 400 /
200 /
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C
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I 50
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£f
<ti
CL
50
0
-50
-50
-100
-
-I 50
-
I 00
Percent Deviation from Control
in d icatin g INT re d u c tio n by u b iq u in o n e a n d possibly cy to ch ro m e b 555 555
-I 50
F ig u re 3. Effects o f v arious in h ib ito rs o n CTC (sh ad ed b ars), a n d INT (solid
b a rs) re d u c tio n in E. coli in v e rte d m e m b ra n e vesicles. D ata a re ex p ressed as
p e rc e n t d eviations in form azan p ro d u c tio n fro m su b strates (NADH o r
succinate) alone. Sites of in h ib itio n a re listed in T able 2. P valu es a re 5 0.05
except w here n o te d . E rror bars re p re se n t s ta n d a rd d ev iatio n s (n = 2).
C alculations a n d ab b rev iatio n s a re as d e sc rib e d in m aterials a n d m ethods.
40
O
L_
C
O
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£
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2000
I 5 0 0 F1000
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IOOz
2000
I 500
1000
z/5 0 0
100
80
60
80
60
40
40
20
20
0
0
-20
-40
-
20
-
40
-60
-
60
-80
-
80
-I 0 0
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F ig u r e 4 (A ). Effects o f in h ib ito rs o n aerobic CTC (sh a d ed b ars), a n d INT
(solid b ars) re d u c tio n in w hole cells o f £ coli. Form azan p ro d u c tio n values a re
ex p ressed as a p e rcen tag e d ev iatio n s fro m c o n tro l w ith o u t in h ib ito rs (A). Pv alu es a re s 0.05 except w h ere n o te d . P ercen t CTC (+) cells b y epifluorescence
m icroscopy (B). H orizontal line in dicates th e p e rc e n t of to ta l AODC cells
e n u m e ra te d b y R2A p la te counts. E rror b a rs re p re se n t s ta n d a rd d ev iatio n s (n =
2). See m aterials a n d m e th o d s fo r calcu latio n s a n d a b b rev iatio n s.
41
F ig u r e 4 (B ). Effects o f in h ib ito rs o n aero b ic CTC (sh a d ed b a rs), a n d INT
(solid bars) re d u c tio n in w hole cells o f £_ coli. Form azan p ro d u c tio n values a re
e x p ressed as a p e rc en ta g e d ev iatio n s fro m c o n tro l w ith o u t in h ib ito rs (A). Pvalues a re s 0.05 except w here no ted . P ercen t CTC (+) cells b y epifluorescence
m icroscopy (B). H orizontal lin e in d icates th e p e rc e n t o f to ta l AODC cells
e n u m e ra te d b y R2A p late counts. E rror b a rs re p re se n t s ta n d a rd d ev iatio n s (n =
2). See m aterials a n d m eth o d s fo r calcu latio n s a n d ab b rev iatio n s.
42
(Figs. 2, 3, a n d 4A) (29, 30). In co n trast, KCN a n d azide in h ib itio n o f
cy to ch ro m es o a n d d stro n g ly d e c re a se d CTF fo rm atio n in w hole cells,
suggesting CTC re d u c tio n b y th e te rm in a l oxidases (Fig. 4A) (4, 30). Sim ilar
resu lts h a v e b e e n re p o rte d w ith a z id e -tre a te d Pseudomonas dim inuta (33).
C onsidering th e E1/2 o f CTC (-200 mV), it a p p e a re d u n lik ely th a t th e term in al
oxidases co u ld d irectly re d u c e CTC, a s th e ir m id -p o in t p o ten tials a re ca. +70 to
+280 mV (Fig. 2 a n d Table 2). SDH a n d NAD-H a re in d irectly in h ib ite d b y
blockage o f te rm in a l oxidases (27, 31). In a d d itio n , cy an id e is know n to block
FeS-centers in several enzym es d ire c tly (22, 35). However, SDH a n d NADH-D
in h ib itio n b y KCN a re n o t co n sisten t w ith in c re a se d re d u c tio n o f b o th CTC a n d
INT in K CN-treated vesicles, a n d in h ib itio n b y azid e (w hich d o es n o t effect FeScen ters) in w hole cells (Figs. 3 a n d 4A). In h ib itio n o f th e te rm in a l oxidases
also in h ib its th e m a jo r site o f p ro to n c o n su m p tio n (in th e fo rm ation o f H zO by
te rm in a l oxidases) a n d tra n s m e m b ra n e electro ch em ical g ra d ie n t (Apg+)
g e n e ra tio n in E coli cells (Fig. 2). This g rad ien t, fo rm ed across th e
m e m b ra n e, c a n b e significant (-80 to -1 8 0 mV, n eg ativ e in sid e) a n d
c o n trib u te s to th e o v erall re d o x e n v iro n m e n t in w hich te tra z o liu m re d u c tio n
takes place (4, 23, 30, 37). CTC re d u c tio n is expected to b e m o re sensitive to a n
in crease in red o x p o te n tia l th a n INT d u e to CTCs low er E1/2, a n d th u s m ay b e
selectively in h ib ite d to som e degree. T h e o b se rv e d d e crea se d CTF-, a n d
in c re a se d IN F-form ation b y collapsing Apg+ in w hole cells w ith CCCP su p p o rt
th is h y p o th esis (Fig. 4A). A lthough CTC re d u c tio n in c re ase d in CCCP-treated
in v e rte d vesicles, p ro to n co n su m p tio n is o n th e o u tsid e su rface o f th e
m e m b ra n e in b u ffe re d so lu tio n , in h ib itin g n o rm a l (Le. w h o le cell, o r n o n in v e rte d vesicle) A^g+ fo rm atio n , b u t still allow ing som e d e g re e o f u n co u p lin g
(Fig. 3) (9). However, i t sh o u ld b e n o te d th a t in trac ellu lar CTC re d u c tio n b y
43
superoxide a n io n in w hole cells c a n n o t b e ru le d out. D ism utation o f superoxide
a n io n s in vesicles using SOD h a d n o effect o n CTC re d u c tio n , w hile slightly
d ecreasing INF fo rm atio n (Figs. 2 a n d 3, T able 2). INT re d u c tio n m ay th u s be
m e d ia ted to som e ex ten t b y p ro d u c tio n o f superoxide an io n s as h y p o th esized by
Seidler (37). U ncouphng (CCCP), d ep letio n o f ATPse, a n d ADP a h in creased CTF
a n d INF fo rm atio n in vesicles (Fig. 13). Since th ese co n d itio n s a re all know n to
in crease re s p ira to ry e le c tro n tra n s p o rt activity, it a p p ea rs CTC a n d INT
re d u c tio n w as lin k e d to e le c tro n tra n s p o rt p ro cesses w hich w ere, in tu rn ,
lin ked to ATPase activity (9, 29, 34).
D irect C ounts
In g eneral, d ire c t co u n ts o f CTC a n d INT (+) aero b ic cells re fle c ted
fo rm azan p ro d u c tio n tre n d s co m p ared to c o n tro ls (Figs. 4A a n d B). However,
cell CTC (+) co u n ts w ere d e c re a se d significantly using HQNO a n d an tim y cin A,
w hile CTF p ro d u c tio n w as u n a ffe cted (Fig. 4A a n d B). This suggests th a t factors
o th e r th a n th e to ta l a m o u n t o f CTF p ro d u c e d affect fo rm atio n o f flu o rescen t
CTF crystals. Size a n d n u m b e r o f flu o resc en t CTF crystals p e r cell was h ig h ly
v a ria b le d e p e n d in g o n th e p a rtic u la r in h ib ito r u sed . S everal in h ib ito rs,
n o ta b ly HQNO, antim y cin A, cyanide, azide, a n d CCCP re su lte d in v e ry fain tly
flu o resc en t CTF w hich w as e v en ly d is trib u te d th ro u g h o u t cells. F o rm atio n o f
visible INF crystals a p p e a re d less sensitive to th e effects o f in h ib ito rs th a n CTF
(T able 3). H ow ever, v isu alizatio n o f INF cry stals w as d ifficu lt c o m p ared to
flu o rescen t CTF, p a rtic u la rly w h en d ep o sits w ere sm all. Using c o n tro l cehs,
p late counts o n R2A in d ic a te d 52.6 ± 1.4% o f to ta l cells (AODC) w ere ab le to fo rm
colonies o n this m ed iu m w hile INT a n d CTC (+) cell n u m b ers w ere 97.0 ± 2.2,
a n d 89.9 ± 0.9% o f to ta l cell n u m b e rs, resp ectiv ely (Fig. 4B). INT (+) cell
44
n u m b e rs w ere c o n siste n tly h ig h e r th a n p la te co u n ts, ev en in th e p resen ce Of
in h ib ito rs (Fig. 4B). TTF, ro te n o n e , a n d azid e w ere th e m o st effective agents
fo r increasing INT (+) cell co u n ts (Fig. 4B). CTC (+) cell n u m b e rs w ere g reatest
using TTF, a n d in th e n o in h ib ito r co n tro l (Fig. 4B). AU CTC (+) ceU n u m b ers
w ere significantly below p la te counts, e x ce p t fo r th e co n tro l, m alo n ate, a n d
TTF sam ples (Fig. 4B). This m ay h av e b e en d u e to th e use o f I mM CTC, as
m axim al fo rm azan p ro d u c tio n h as b e e n fo u n d a t 2-6 mM in sev eral b a cte ria (8,
33). The use o f cyanide o r azid e to in crease tetrazo liu m re d u c tio n (as h as b een
suggested fo r INT) wiU in c re ase INF fo rm a tio n significantly (24, 26, 38, 45, 54)
(Fig. 4B). However, th is te ch n iq u e m a y n o t alw ays b e a p p ro p ria te fo r CTC
re d u c tio n assays in in ta c t b a c te ria l ceUs (42, 43).
T he re d u c tio n of INT a n d CTC a p p e a re d closely lin k ed to b a cterial
re s p ira to ry activity. H ow ever, sig n ifican t in traceU u lar re d u c tio n o f b o th
tetrazoU um salts w as o b se rv e d in fo rm a ld e h y d e -tre a te d ceUs if sufficient tim e
(5-15 m in) h a d n o t p assed b efo re CTC o r INT a d d itio n (d a ta n o t shown). This
effect was m u c h m o re p ro n o u n c e d fo r BSfT th a n CTC, re q u irin g ca. 15 min
c o n tact w ith fo rm ald e h y d e to com pletely elim in ate INT-, a n d 5 m in . to
elim in ate C TC -reducdon (CTF p ro d u c e d w as d iffuse a n d p o o rly fluorescent).
Sim ilar resu lts w ere n o te d using INT in a re c e n t stu d y o f tetrazoU um red u c tio n
in C am pylobacter jeju n i b y B oucher e t a h (7). T his m ay b e re la te d to “no th in g
d e h y d ro g en ase" activ ity n o te d in sev eral h isto ch em ical stu d ies a ttrib u te d to
re sid u a l re d u c tio n p o te n tia l in fixed cells (3, 21, 37). Since INT w o u ld co n tin u e
to b e re d u c e d a t h ig h e r re d o x p o te n tia ls th a n CTC as th e p o te n tia l dissipates
(i.e. >100 mV, <90 mV), th is m a y explain m o re p e rsiste n t INT re d u c tio n after
fixation. No d e te c ta b le tetrazoU um re d u c tio n was o b serv ed in th e p resen ce of
n u trie n ts o r fo rm alin w ith o u t ceUs. T h erefo re, to p re v e n t re s id u a l fo rm azan
45
fo rm atio n i t is im p o rta n t to fix cells w ith fo rm ald e h y d e fo r a t le a st 10-15 m in
p rio r to tetrazo liu m salt a d d itio n . T his differs fro m resu lts using R2A as a
n u trie n t source re p o rte d b y Bovill e ta l., a lth o u g h th e c o n c e n tra tio n o f R2A in
th e p re s e n t stu d y was 10-fold low er (8).
R eduction o f te tra zo liu m salts b y in v e rte d m e m b ra n e vesicles suggests
th e ir sp o n tan o u s re d u c tio n b y “n o n v ia b le ” cell m em b ran es. However, it
sh o u ld be em phasized th a t vesicles a re p ro d u c e d u n d e r re d u c in g conditions
(d ith io tre ito l) in o rd e r to m a in ta in th e re d u c e d state o f e le c tro n -tra n sp o rt
co m ponents (9). Indeed, a re d u c in g e n v iro n m e n t is im p o rta n t to o b ta in
vesicles w ith active d e h y d ro g e n ases (3, 11, 29). In th e e n v iro n m e n t (u n d e r
n o n -red u cin g conditions) ETS com p o n en ts w o u ld b e ex p ected to oxidize,
d e g ra d e, a n d becom e n o n fu n ctio n al. However, re d u c tio n o f CTC o r INT b y
m e m b ra n e -b o u n d ETS c o m p o n en ts (in th e ab se n c e o f v iab le cells) in red u cin g
e n v iro n m e n ts such as sed im en ts c a n n o t b e ru le d out.
CTC a n d INT R eduction U nder A naerobic C onditions
T etrazolium re d u c tio n in w hole cells w as a ssay ed u n d e r a n aero b ic
co n d itio n s w hich in d u c e 4 d iffe re n t e le c tro n tra n s p o rt pathw ays: Glucose
fe rm e n ta tio n , fo rm ate d e h y d ro g e n ase-fu m a ra te re d u c ta se (> +30 mV), glycerol
d eh y d ro g en ase-fu m arate re d u c tase , a n d n itra te re d u c tase (+20 to +220 mV)
u n d e r endogenous re s p ira tio n (Figs. 5A a n d B) (20). R2A-P w as a d d e d a s a
g en eral so u rce o f su b strates. G lucose fe rm e n ta tio n a n d R2A-P m e d iu m (w hich
co n tain s glucose a n d p y ru v a te ) stim u la te d re d u c tio n o f b o th te tra zo liu m salts
o v er 100%, indicating th a t th e v ario u s d eh y d ro g en ases p o stu la te d to b e active
d u rin g glucose fe rm e n ta tio n re d u c e CTC, a n d p a rtic u la rly INT. In d u c tio n of
th e fo rm ate d e h y d ro g e n ase-fu m a ra te re d u c ta s e p ath w ay in c re a se d CTC
Percent Deviation from Control
46
F ig u r e 5 (A ). Effects of various su b strate s a n d electro n accep to rs on CTC
(sh a d e d bars) a n d INT (solid b ars) re d u c tio n b y w hole E. coli cells u n d e r
an aero b ic conditions. Form azan p ro d u c tio n valu es a re ex p ressed as a
p e rc e n ta g e d ev iatio n s fro m c o n tro l w ith o u t su b strate (s) (A). P-values are
^0.05 except w here no ted . P ercent CTC (+) cells b y epiflu o rescen ce m icroscopy
(B). H orizontal line in d icates th e p e rc e n t of to ta l AODC cells e n u m e ra te d by
R2A plate counts. E rror b ars re p re se n t s ta n d a rd deviations (n = 2). See
m aterials a n d m ethods fo r calculations a n d a b b rev iatio n s.
47
Plate Count (R2A)
100
90
90
0)
80
80
Q)
70
70
+
60
60
U
50
50
40
40
0)
u
U
0)
30
30
CL
20
U
S-V
H
U
Percent INT (+) Cells
100
I 0
0
Uu Li.
CD Lu
F ig u r e 5(B) . Effects o f vario u s su b strates a n d electro n accep to rs o n CTC
(sh a d e d bars) a n d ENT (solid bars) re d u c tio n b y w hole E. coli cells u n d e r
a n aero b ic conditions. Form azan p ro d u c tio n valu es a re ex p ressed as a
p e rc en ta g e d ev iatio n s fro m c o n tro l w ith o u t su b strate (s) (A). P v a lu es a re
s0.05 except w here n o te d . P ercent CTC (+) cells b y ep iflu o rescen ce m icroscopy
(B). H orizontal line in d icates th e p e rc e n t o f to ta l AODC cells e n u m e ra te d by
R2A plate counts. E rror b ars re p re se n t s ta n d a rd deviations (n = 2). See
m aterials a n d m eth o d s fo r calculations a n d a b b rev iatio n s.
48
re d u c tio n to th e g reatest deg ree (Fig. 5A a n d B). CTC is m o st likely re d u c e d b y
fo rm ate d eh y d ro g en ase (-145 to -360 mV) in th is system (20). Stim ulation of
CTC re d u c tio n b y fo rm ate is sim ilar to resu lts fo u n d w ith aerobic
C am pylobacter je ju n i cells (7). G lycerol-fum arate a n d n itr a te h a d little, o r n o
effect o n CTF fo rm atio n , b u t CTC (+) cell n u m b e rs w ith g ly cero l-fu m arate
in c re a se d 5-10% over co n tro l values (Figs. 4A a n d B). In c o n tra st, INF
fo rm atio n , a n d INT (+) cell n u m b e rs d e crea se d w ith fu m a ra te o r n itra te as th e
te rm in a l e lec tro n acc e p to r (Fig. 5A). N um bers o f CTC (+) a n d INT (+) cells w ere
ab o v e ca. 70% u n d e r all an aero b ic in c u b atio n co n d itio n s te ste d (except INT in
th e p re se n c e o f fo rm ate-fu m arate) (Fig. SB). G lucose a n d fo rm ate -fu m arate
p ro d u c e d th e la rg e st n u m b e rs o f CTC (+) Cells, w hile INT (+) n u m b e rs w ere
g re a te st in th e c o n tro l a n d glucose sam ples (Fig. SB). W ith th e ex ception o f th e
control, CTC (+) n u m b e rs w ere eq u al to, o r g re a te r th a n p la te co u n ts o n R2A in
all cases. Thus, s u b stra te a d d itio n a p p e a re d n ecessary fo r CTC (+) counts to
exceed p la te counts w h en using I mM CTC. INT (+) cells n u m b e rs w ere 5-20%
g re a te r th a n p la te c o u n ts in th e c o n tro l, glucose, a n d n itra te sam p les (Fig. SB).
INT a n d CTC w ere re d u c e d to significant d eg rees u n d e r all a n ae ro b ic conditions
e m p lo y e d in th is stu d y , w ith glucose p a rtic u la rly stim u latin g fo rm az a n
p ro d u c tio n .
In sum m ary, stu d ie s using b o th in v e rte d m e m b ra n e v esicles a n d w hole
cells in d ic a ted CTC a n d INT w ere re d u c e d b y b o th SDH a n d NADH-D (Fig. 2).
However, th e specific sites in v o lv ed i n th e re d u c tio n o f CTC a n d INT w ere
som ew hat d ifferen t since CTC a p p e a re d to b e re d u c e d b y d eh y d ro g en ases o th e r
th a n SDH in w hole cells. In a d d itio n , INT c o u ld b e re d u c e d b y u b iq u in o n e,
p o ssib ly cy to ch ro m e 6555,556, su perox id e a n io n , a n d p e rh a p s o th e r p rim a ry
d e h y d ro g e n ases w hich d o n a te electro n s to u b iq u in o n e (Fig. 2). T hese resu lts
49
a re co n sisten t w ith re d u c tio n sites in fe rre d fro m INT a n d CTC m id -p o in t red o x
p o ten tials, as well as being sim ilar to sites d e te rm in e d fo r eu k ary o tic
m ito c h o n d ria l e le c tro n tra n s p o rt chains, a n d M icrococcus lu teu s (Fig. 2, Table
2) (14, 26, 27, 30, 37). Both CTC a n d INT w ere re d u c ed u n d e r m o st anaerobic
co n d itio n s tested , p a rtic u la rly glucose fe rm e n ta tio n (Fig. 5A). C oupling o f
te tra zo liu m re d u c tio n to re s p ira tio n (as m e a su re d b y oxygen co nsum ption)
d iffered b etw een w hole cells (CTC > INT) a n d in v e rte d m e m b ra n e vesicles (INT >
CTC), b u t in g en eral ra n g e d betw een 10 a n d 4 0 p airs o f re s p ira to ry electrons
fo r ev ery tetrazo liu m m olecule re d u c ed . CTC re d u c tio n a p p e a re d m o re
sensitive to effectors o f in tra c e llu la r re d o x p o te n tia l th a n INT in w hole cells,
a n d a p p e a re d to p ro d u c e a weakly-, o r n o n -flu o rescen t fo rm azan a t red o x
p o te n tia ls h ig h e r th a n ca. -200 mV. Also, u se o f in h ib ito rs o f te rm in a l oxidases
(he. cyanide, azide), a n d u n c o u p lers m ay n o t b e u seful to m axim ize w hole cell
p ro k ary o tic CTC re d u c tio n assays.
In fo rm atio n o b ta in e d using CTC a n d /o r INT to assess re s p ira to ry activity
in p ro k ary o tic cells will v a ry d e p en d in g o n th e o rg an ism a n d assay
conditions. For in stan ce, it a p p e a rs som e activ e b a c te ria d o n o t re d u c e INT, o r
CTC u n d e r c e rta in conditions (40, 47). This co u ld be re la te d to low tetrazolium
sa lt su b sta n tiv ity (a sso c ia tio n w ith cell co m p o n en ts, p e rm e ab ility ), u n iq u e
re s p ira to ry p athw ays, o r low cellu lar A^h + (p articu larly using CTC) in som e
b a c te ria (37). To d a te CTC re d u c tio n h a s b e e n re p o rte d in R coli, Salm onella
typ h im u riu m , Yersinia enterocolitica, Enterococcus faecalis (40), K lebsiella
p n eu m o n ia e (52), Paracoccus d en itrificans (41), Micrococcus luteus (14),
Pseudomonas fluorescens (13), Pseudomonas p u tid a (3 3 ), C am pylobacter jeju n i
(I), Listeria m onocytogenes (8), Listeria innocua (41), S higella fle x n e r i, a n d
Vibrio cholerae (non-01) (4 9 ). H ow ever, co n sid erin g th e re la tiv e ly v a ria b le
50
n a tu re o f p ro k a ry o tic re s p ira to ry ch ain s co m p ared to eu k ary o tes, d ifferen ces
in te tra zo liu m re d u c tio n betw een organ ism s a re to b e expected. T herefore,
selection o f a n a p p ro p ria te te tra zo liu m salt fo r a n ap p licatio n , a n d
in te rp re ta tio n o f d a ta sh o u ld take in to a cc o u n t th e h isto ch em ical p ro p e rtie s o f
th e se in d ic a to rs o f re s p ira tio n in p ro k a ry o tic system s.
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47. Tuovila, B. J., a n d P. A. LaRock. 1985. Effect o f species d ifferen ce a n d grow th
ra te in th e u se o f INT as a n in d ic a to r o f b a c te ria l re sp ira tio n . J. M icrobiol.
M eth. 4:185-188.
48. W ebb, J. L. 1966. M alonate, p . 1-235. I n J. L. W ebb (ed.), Enzym e a n d
m etabolic in h ib ito rs, voL 2. A cadem ic Press, New York.
54
49. W om m ack, E. 1994. (COMB, U niversity o f M aryland, B altim ore). Personal
c o m m u n ic atio n .
50. Yagi, T. 1991. Bacterial NADH-quinone oxidoreductases. J. Bioener. Biomemb.
23:211-223.
51. Yagi, T. 1993. T he b a cte ria l e n erg y -tra n sd u c in g NADH-quinone
oxidoreductases. Biochim. Biophys. Acta. 1141:1-17.
52. Yu, F. P., a n d G. A. M cFeters. 1994. R apid in situ assessm en t o f physiological
activities in b a c te ria l biofilm s usin g flu o re sc e n t p ro b e s. J. M icrobiol.
M eth. 20:1-10.
53. Yu, F. P., a n d G. A. M cFeters. 1994. Physiological resp o n ses o f b a c te ria in
biofilm s to disinfection. Appl. Environ. M icrobiol. 60:2462-2466. .
54. Zim m erm an, R., R. Itu rriag a, a n d J. Becker-Black. 1978. S im ultaneous
d e te rm in a tio n o f th e to ta l n u m b e r of aq u atic b a c te ria a n d th e n u m b e r
th e re o f in v o lv ed in re sp ira tio n . A ppl. Environ. M icrobiol. 36:926-935.
55
CHAPTER 3
EFFECTS OF SUBSTRATES AND PHOSPHATE ON INT (2-(4-IODOPHENYL)3 - ( 4-NITROPHENYL)- 5 -PHENYL TETRAZOLIUM CHLORIDE), AND CTC
(5-CYANO-2,3-DITOLYL TETRAZOLIUM CHLORIDE) REDUCTION IN
ESCHERICHIA COLI
In tro d u c tio n
T h e te tra z o liu m salts INT (2 -(4 -io d o p h en y l)-3 -(4 -n itro p h en y l)-5 p h e n y l te tra zo liu m ch lo rid e) a n d CTC (5-cyano-2,3,ditolyl te tra z o liu m ch lo rid e)
h a v e o fte n b e e n u se d as in d ic a to rs o f b a c te ria l re s p ira to ry activ ity a n d
viability (3 ,4 , 5, 7 ,1 0 ,1 1 ,1 3 , 20, 21, 2 3 ,2 6 ,2 9 , 30, 35, 36). Both salts a re red u ced
to th e ir in so lu b le r e d (INF), o r flu o resc en t-o ran g e (CTF) fo rm azan s b y
c o m p o n en ts o f th e p ro k a ry o tic re s p ira to ry d r a in (16, see c h a p te r 2).
F orm azan cry stals can su b se q u e n tly b e view ed u n d e r ep iflu o rescen ce
m icroscopy, o r e x tra c te d in a n o rg an ic so lv en t (com m only e th an o l) a n d
L
q u a n tifie d sp ectrophotom etrically. CTC a p p e a rs to b e re d u c e d b y th e p rim ary
d e h y d ro g e n ases (succinate, NAD(P)H, a n d p o ssib ly o th ers) in Escherichia coii,
w hile INT m a y also b e re d u c e d b y u b iq u in o n e , a n d p o ssib ly cytochrom es
£>555,556 (see c h a p te r 2). T hese sites o f re d u c tio n a re so m ew h at sim ilar to
those suggested fo r eukaryotic cells ( 1 5 ,1 6 ,1 7 , 24, 27, 28). T he re d o x state of
cell m e m b ra n es also a p p e a rs to p la y a ro le in CTC re d u c tio n to flu o rescen t
fo rm azan cry stals (see c h a p te r 2). T he a m o u n t o f e le c tro n flow th ro u g h th e
re s p ira to ry c h a in is d ic ta te d b y th e a v ailab ility a n d q u a lity o f oxidizable
56
su b strate s, w h e th e r exogenous o r en d o g en o u s. Indeed, it a p p e a rs th a t several
o rganism s in c re a se te tra z o liu m re d u c tio n in re sp o n se to in c re a se d n u trie n t
c o n cen tratio n s (3, 5, 20, 21,23, 29). C onsidering th e in c re ase d use of INT a n d
CTC as in d ic a to rs in studies o f p ro k a ry o tic cell physiology a n d viability, a n
u n d e rs ta n d in g o f w hich su b stra te s p ro m o te optim al te tra zo liu m re d u c tio n is
n eeded.
S tudies w ere u n d e rta k e n to exam ine th e effects o f su b stra te a d d itio n o n
CTC a n d INT re d u c tio n in aerobic Escherichia coli, a p ro k a ry o te w ith re lativ ely
well d e sc rib e d d e h y d ro g e n ase a n d re s p ira to ry c h ain co m p o n en ts (2, 6, 18, 34,
Fig. 2). Substrates, fo r th e p rim a ry aero b ic E. coli d eh y d ro g en ases (sin­
gly cerol-3 -p h o sp h a te, succinate, fo rm ate, la c ta te , fo rm ate, a n d D -am ino acid),
as well as glucose w ere a d d e d a t eq u im o lar co n cen tratio n s to cu ltu res in saline,
a n d gross fo rm az a n p ro d u c tio n , as well as INT a n d CTC (+) cell n u m b e rs
co m p ared . This la b o ra to ry h a s also n o te d a significant in h ib ito ry effect o f
in o rg an ic p h o sp h a te o n CTC re d u c tio n to visible, flu o rescen t CTF d ep o sits in
som e b a c te ria l stra in s (Pyle e t al. su b m itte d ). This in h ib itio n resu lts in
sig n ific a n t u n d e re stim a tio n s o f a ctiv ely re s p irin g cell n u m b e rs w h e n c e rta in
b uffers a n d /o r m e d ia a re u sed fo r CTC re d u c tio n assays. In o rd e r to investigate
th is p h e n o m e n o n w e c o rre la te d fo rm a z a n p ro d u c tio n , o x y g en consu m p tio n ,
a n d CTC/INT (+) cell n u m b e rs w ith p h o sp h a te co n cen tratio n s com m only u sed
in biological b u ffe rs a n d g ro w th m edia.
57
M a te ria ls a n d M e th o d s
C hem icals
5-cyano-2,3-ditolyl te tra zo liu m c h lo rid e (CTC) was p u rc h a se d fro m
Polysciences, Inc., W arrin g to n , Pa., U.S.A. 4 ,,6 -d iam id in o -2 -p h en y lin d o le
(DAPI), a c rid in e o ran g e (AO), d iso d iu m su ccin ate, glucose, lactic acid, glycerol,
a n d potassiu m p h o sp h a te w ere all p u rc h a se d fro m Sigm a C hem ical Co., St.
Louis, Mo. U.SA. R2A m ed iu m a n d casam ino a d d s w ere o b ta in e d fro m Difco
L aboratories, D etroit, MI, U.SA.
G row th C onditions
Escherichia coli K-12 w as grow n aero b ically w ith v ig o ro u s a e ra tio n a t
25°C to e a rly -sta tio n a ry p h a se in glycerol (8 g /l)-m in e ra l m e d iu m
su p p lem en ted w ith I ^m o l I" I selenic acid a n d sodium m o ly b d ate (6). G row th
o n glycerol will s u p p o rt p ro d u c tio n o f all th e p rim a ry E. coli d e h y d ro g e n ases
(18). Cells w ere w ashed 3 tim es a n d re su sp e n d ed a t IO10 c ells/m l in 0.85% NaCl
(pH 6.5). Plate co u n ts w ere p e rfo rm ed b y spot-plating su b sam p les (exposed fo r
th e sam e p e rio d , in th e sam e b u ffer a s resp ectiv e assays) o n to R2A m ed iu m a n d
in c u b atio n fo r 48 h a t 25°C.
In cu b atio n o f Cells w ith CTC a n d INT
O ne-tenth m l o f re su sp e n d e d cells fro m above w ere a d d e d to 0.8 m l o f
saline c o n tain in g 10 mM o f e ith e r glucose, glycerol, su ccin ate, fo rm ate , la ctate .
58
o r 0.2% casam ino acids (final c o n cen tratio n s). Cells w ere also re su sp e n d e d in
saline containing 10, 25, 50, 75 a n d 100 mM p o tassiu m p h o sp h a te buffer, pH 6.5.
Suspensions w ere sh ak e n vigorously a t 250 re v /m in fo r 30 m in , th e n fresh ly
p re p a re d CTC o r INT was a d d e d to a fin al co n cen tratio n o f I mM . This
c o n c e n tra tio n was ch o sen to m axim ize d ifferen ces in te tra zo liu m re d u c tio n
betw een substrates, r a th e r th a n to optim ize CTC (+), o r INT (+) cell n u m b e rs (5,
21). Incubations w ere p e rfo rm e d in d u p lic a te a t 25°C w ith vigorous a e ra tio n
fo r 2 h in th e dark. This tim e o f in c u b a tio n w as fo u n d to give o p tim al CTC a n d
INT (+) cell n u m b ers. R eactions w ere sto p p ed b y a d d itio n o f 3.7% fo rm ald eh y d e
(final c o n ce n tra tio n ) a n d im m ersio n in a n ice b a th . F o rm ald eh y d e k illed (15
m in exposure) co ntrols w ere p e rfo rm e d fo r all assays.
Fxam ination o f Cells fo r CTC a n d INT R eduction
Subsam ples fo r C TC -reduction assays w ere d ilu te d in filter-sterilized
saline, filte re d th ro u g h 0.22 ^ m p re -b la c k e n e d N uclepore p o ly c a rb o n a te
m em branes (no. 110656), a n d c o u n te rsta in e d w ith 10 /*g/ml DAPI fo r 10 m in.
Subsam ples fo r INT assays w ere d ilu te d a s a b o v e in d istilled w a ter a n d aird rie d , heat-fixed sm ears p re p a re d using 20 jA aliquots. T hese w ere
c o u n tersta in e d w ith 0.01% a crid in e o ra n g e (AO) in 2 mM T ris b u ffe r (pH 7.0)
fo r 3 m in a n d view ed u n d e r b o th ep iflu o rescen ce a n d b rig h t fie ld m icroscopy.
A sim ilar b rig h t fie ld te c h n iq u e w as u s e d to visualize to ta l (i.e. flu o rescen t a n d
non-fluorescent) CTF dep o sits. However, DAPI was u sed as th e fluorochrom e.
Sam ples fo r epifluorescence d ire c t c o u n ts w ere view ed usin g a Leitz O rtholux
II m icroscope eq u ip p ed w ith Leitz filter blocks B2, N2.1, a n d H3 to visualize
DAPI, CTC, a n d DSTT/AO, respectively. INT (+) cells c o n tain ed r e d IN T-form azan
deposits w hen view ed u n d e r b rig h t-field m icroscopy, w hile CTC (+) cells
59
c o n tain ed flu o rescen t-o ran g e CTC-form azan deposits. At le a st 600 cells in a: 10
fields w ere c o u n te d a t 1,250 X fo r each filter. E rro r was calcu lated a s d escrib ed
b y Jones (9).
M easurem ent o f F orm azan P ro d u ctio n
Sam ples fo r sp ectro p h d to m etric d e te rm in a tio n o f fo rm azan (0.9 ml)
w ere c e n trifu g e d in a h ig h -sp e ed m icro cen trifu g e (5,000 X g, 10 m in).
S u p ern atan ts w ere th e n d iscard ed , a n d p ellets re su sp e n d ed in 95% EtOH an d
d is ru p te d b y so n icatio n a t h ig h p ow er fo r 15 s using a so n ica to r m icro tip
(Fisher Scientific Sonic d ism e m b ra to r 50) a t 30% pow er a n d s to re d a t 4°C
o v ernight. Suspensions w e re th e n c e n trifu g e d (9,000 X g, 10 m in ) a n d
ab sorbance o f su p e rn a ta n ts d e te rm in e d a t 4 5 0 n m fo r CTC, a n d 485 n m fo r INT
using a n HP 8452A sp ectro p h o to m ete r. Cell p ellets re ta in e d n o visible
fo rm azan a fte r th e a b o v e e x tra c tio n p ro c e d u re . M olar ex tin ctio n coefficients
w ere d e te rm in e d in 95% e th a n o l b y dissolving INF a n d CTF a t know n
co n cen tratio n s. T hese v alu es w ere su b se q u e n tly u se d to c o n v e rt optical
d en sity m e asu re m e n ts to m oles o f fo rm azan .
M easurem ent o f O xygen C onsum ption
O xygen c o n su m p tio n w as m e a su re d usin g a Gilson 5 \6 o x y g rap h w ith a
C lark-type electro d e. AU ex p erim en ts w ere p e rfo rm e d in a 2 m l- w aterja ck eted ceU a t 25°C. O xygen sa tu ra tio n w as m e asu re d usin g double-distiU ed
w ater a n d co rre cted fo r a ltitu d e a n d p re s su re (I ) . AU assays w ere c a rrie d o u t
in saline (pH 6.5) alone, o r w ith a d d e d p h o sp h a te as d escrib ed above.
RepUcation w as a ch ie v e d b y re a e ra tin g th e sam ple 3 tim es w ith a 5-m l sy rin g e
60
a n d 22 gauge need le. The re su sp e n d e d cells fro m F o rm ald eh y d e (3.7%) tre a te d
c o n tro ls show ed n o d e tec ta b le oxygen co n su m p tio n .
R e s u lts
Effect o f S ubstrates o n F orm azan P ro d u ctio n
In all cases CTF p ro d u c tio n w as c o n sid e ra b ly g re a te r th a n INF b y a
facto r o f 30-150, d e p en d in g o n th e su b stra te (Fig. GA). In o rd e r of
effectiveness in p rom oting CTF fo rm atio n su b stra te s w ere: la c ta te > fo rm ate >
glucose > succinate ~ glycerol > casam ino acids > control. In o rd e r of
p ro d u c tio n o f INF su b strates w ere: fo rm ate »
casam ino acids > la ctate >
su ccin ate = co n tro l > glycerol = glucose.
Effect o f S ubstrates o n D irect C ounts
All su b strates te sted re su lte d in a t le a st 85% CTC (+) cells, w ith form ate,
lactic acid, a n d casam ino a d d s yieldin g 100% o f cells show ing flu o rescen t CTF
deposits (Fig. 6B). All CTC (+) counts w ere 30-50% g re a te r th a n p la te counts
w ith th e m o st effective su b stra te s in th e o rd e r la ctate = fo rm ate = casam ino
acids > succinate ~ glycerol > glucose > co n tro l. CTF cry stal size a n d in ten sity o f
flu o rescen ce v a rie d c o n sid e ra b ly b e tw ee n su b stra te s. In g e n eral, succinate,
lactate, a n d casam ino a d d s p ro d u c e d th e larg est, m o st b rig h tly flu o rescen t
crystals, w ith m o st cells c o n tain in g o n e d ep o sit. G lucose, fo rm ate, glycerol,
a n d endogenous re sp ira tio n re s u lte d in som ew hat sm aller CTF crystals. These
cells o ften c o n ta in e d 1-2 d e p o sits p e r cell, som e o f w hich w ere p o o rly
nmol INF Produced
61
F ig u r e 6 (A ). Effects o f d ifferen t su b strate s o n CTC (sh a d ed b a rs), a n d INT
(solid bars) re d u c tio n to e th a n o l ex tra cta b le fo rm azan (A), a n d p e rc e n t
CTC/INT (+) cells by epifluorescence m icroscopy (B). Plate c o u n ts w ere o n R2A
ag ar. E rror b ars re p re se n t s ta n d a rd d ev iatio n s (n = 2).
62
Plate Count
I
I 00
90
(ft
0)
80
U
70
+
60
U
100
I 90
V
80
(ft
70
U
60
£
CD
H-
50
50
z
C
40
40
I
£f
30
30
&
20
20
10
I 0
U
0)
0)
o
CL
CL
0
0
SUBSTRATES
F ig u r e 6 (B ). Effects o f d ifferen t su b strates o n CTC (sh ad ed b ars), a n d INT
(solid b ars) re d u c tio n to e th a n o l ex tra cta b le fo rm azan (A), a n d p e rc e n t
CTC/INT (+) cells by epifluorescence m icro sco p y (B). Plate c o u n ts w ere o n R2A
ag ar. E rror b a rs re p re se n t s ta n d a rd d ev iatio n s (n = 2).
63
flu o resc en t (Fig. 6B). This effect was n o t as n o ticeab le fo r INF d ep o sits, except
th a t c ry stal size was n o tic e ab ly la rg e r in sam ples w ith fo rm a te a n d casam ino
acids. G lycerol re su lte d in th e le ast INT (+) cells, 10% below p la te counts (Fig.
6B). INT (+) counts w ith o th e r su b strate s w ere all 25-50% g re a te r th a n p late
counts w ith th e m ost effective su b strate s in th e o rd e r casam ino acids >
su ccin ate = lactate = fo rm ate = co n tro l > glucose > glycerol. G lucose re su lte d in
INT (+) counts 15% below th a t o f th e co n tro l, w hich w ere a p p ro x im ately 95%.
Effects o f P hosphate o n CTC a n d INT R eduction
P hosphate in creased CTC a n d INT re d u c tio n a t 10 mM c o m p ared to
c o n tro ls co ntaining n o p h o sp h a te (Figs. 7A a n d B). However, i t d e c re a se d b o th
INT a n d CTC re d u c tio n above 10 mM. INT a n d CTC (+) cell counts also in creased
5 a n d 10% w ith 10 mM p h o sp h a te c o m p ared to saline alo n e (Fig. 7A a n d B). CTC
(+) cell n u m b e rs d e c re a se d w ith in creasin g p h o sp h a te c o n c e n tra tio n ,
declining sh arp ly ab o v e 50 mM to less th a n 5% a t 100 mM (Fig. 7B). In
co n trast, INT (+) cells re m a in e d betw een 90 a n d 95% u p to 75 mM p h o sp h ate,
w ith c o u n ts d ro p p in g to 85% a t 100 mM (Fig. 7A). B right fie ld m icroscopic
exam in atio n o f CTF d eposits show ed th a t, w h ile flu o rescen t p lu s
n o n flu o re sc e n t CTF (+) cell n u m b e rs d e c re a se d 20% w ith in c re asin g
p h o sp h a te , th is effect was m u c h less p ro n o u n c e d th a n th e re d u c tio n in CTC (+)
(i.e. flu o resc en t CTF cry stals) cell n u m b e rs (Fig 7B).
Effect o f P h osphate o n O xygen C onsum ption
In creasin g p h o s p h a te c o n c e n tra tio n d e c re a se d ra te s o f re s p ira tio n as
m e a su re d b y oxygen con su m p tio n , a lth o u g h th is was n o t see n w ith m id-log
p h ase cells (Table 4). This effect was m ost p ro n o u n ced above 50 mM p h o sp h ate
w ith O2 c o n su m p tio n d ecreasing a p p ro x im ately 40%.
-80
0.8
-
O.e.r
-10
0
10 20 30 4 0 50 6 0 70 80 9 0 100
Phosphate (mM)
F ig u re 7 (A ). Effects o f p h o sp h ate o n CTC (+) (A), a n d INT (+) (B) cell
n u m b ers, a n d total fom aazan productio n .. Symbols: P ercent CTC (+) o r INT (+)
cells (solid circles), to tal form azan p ro d u c e d (o p en circles), to ta l CTF d eposit
co n tain in g cells (flu o re sc en t + n o n flu o resc en t) (solid trian g les). E rro r b a rs
re p re se n t s ta n d a rd d eviations (n = 2).
65
O
10 20 30 4 0 50 60 70 80 9 0 100
Phosphate (mM)
F ig u r e 7 (B ). Effects o f p h o sp h a te o n CTC (+) (A), a n d INT (+) (B) cell
num bers, a n d total fo rm azan p ro d u ctio n . Symbols: Percent CTC (+) o r INT (+)
cells (solid circles), to tal fo rm azan p ro d u c e d (o p en circles), to ta l CTF d eposit
co n tain in g cells (flu o re sc en t + n o n flu o resc en t) (solid trian g le s). E rror b ars
re p re se n t s ta n d a rd d ev iatio n s (n = 2).
66
T a b le 4 . Effect o f p h o sp h a te c o n c e n tra tio n o n re s p ira tio n
ra te s in Escherichia coli.
P h o sp h a te (mM)
a R esp iratio n ra te (nm ol O /m l m in '2 )
k S ta tio n a ry p h a se
Log p h a se
0
11.23 ±0.92
10.38 ±1.71
10
8.47 ± 0.8 4
8.79 ±0.11
25
9.87 ±0.83
11.41 ±1.35
50
9.02 ± 0.59
13.11±0.13
75
5.63 ±0.83
12.93 ± 0.60
100
4.99 ±0.23
4.99 ±0.23
a E rrors re p re s e n t s ta n d a rd d ev iatio n s (n > 3).
k Cells u sed in tetrazo liu m re d u c tio n assays.
D is c u s s io n
Effect of S ubstrates o n CTC a n d INT R eduction
T he p ro d u c tio n o f 30-150 tim es less INF co m p ared to CTF o n a m o la r b asis
in d ic a te d re d u c tio n o f INT to fo rm azan was significantly less tig h tly co u p led to
re s p ira to ry activity th a n CTC in w hole K coli cells. This w as tru e re g a rd le ss o f
su b strate a d d itio n o r ty p e (Fig. 6A). H ow ever, ro u g h ly e q u iv alen t EStT a n d CTC
(+) cell n u m b e rs w ere n o te d , e v e n th o u g h m u c h less INF th a n CTF was
p ro d u c e d (Fig. 6B).
A d d itio n o f n u trie n ts to en d o g en o u sly resp irin g E. coli cells in c re a se d
red u ctio n o f CTC to CTF, a n d CTC (+) cell n u m b e rs in all cases (Fig. 6B). Thus it
67
a p p ea rs th a t pro v isio n o f exogenous su b strate s e n h an c e d CTC re d u c tio n
re la tiv e to en d o g en o u s re sp ira tio n . W hile a d d itio n o f n u trie n ts also g en erally
in c re ase d INT re d u c tio n to INF, a n d INT (+) cell n u m b ers, glucose a n d glycerol
b o th d e c re a se d th ese valu es (Fig. 6B)). T hese su b strates a re b o th glycolytic
p ath w ay in te rm e d ia tes, a n d glucose is k now n to re p re ss th e activ ity o f th e
tricarboxylic a cid (TCA) cycle (18). O ne o f th e suggested sites o f re d u c tio n of
INT is succinate d eh y d ro g en ase (SDH) in th e TCA cycle (13, 15, 17, 29). Thus,
in h ib itio n o f TCA activity b y glucose m ay le a d to d ecrea se d INT re d u ctio n .
G lycerol is a cte d u p o n b y sn -g ly c ero l-3 -p h o sp h ate d e h y d ro g e n a se , a n d th is
su b stra te a p p e a rs to re p re ss re d u c tio n o f INT c o m p ared to en d o g en o u s
re sp ira tio n (18). Succinate, th e su b stra te o f th e enzym e suggested to m e d ia te
b o th INT a n d CTC re d u c tio n , h a d n o effect o n INT re d u c tio n , w hile increasing
CTC re d u c tio n n e a r values fo r glucose a n d glycerol (15, 21). Form ate p ro d u c e d
th e g re a test g en eral stim u latio n of fo rm az a n p ro d u c tio n b y INT a n d CTC, a n d is
a cte d u p o n b y fo rm ate d e h y d ro g e n ase in E coli (Fig. GA) (18). In c re a se d
te tra zo liu m re d u c tio n c o u ld b e m e d ia te d th ro u g h th e activity o f th is enzym e
d irectly, o r b y its a b ility to tra n slo c a te p ro to n s effectively (4 H + /fo rm ate
oxidized), g e n eratin g a lo w er in tra c e llu la r re d o x p o te n tia l (18). Lactate
in creased re d u c tio n o f b o th CTC a n d INT, th e fo rm e r to th e h ig h e st v alu e o f all
su b strate s te ste d (Fig. GA). la c ta te is o x id ized b y lactate d e h y d ro g e n ase (LDH)
in E coli, prod u cin g b o th NADH a n d p y ru v a te . CTC m ay b e re d u c e d d irectly b y
LDH activity, o r th ro u g h in c re a se d NADH d e h y d ro g e n ase (NADH-D), o r TCA
cycle activities (18). C asam ino acids stim u lated b o th CTC a n d INT re d u c tio n a n d
in c re ase d CTC a n d INT (+) cells n u m b e rs to 100% (Fig. GA). A m ino acids a re
acted u p o n b y D -am ino a cid d eh y d ro g en ase in aero b ic E coli cells (18). In
general, it a p p e a re d th e m o st effective su b stra te s fo r in creasin g CTC a n d INT
68
re d u c tio n a n d CTC/INT (+) cell n u m b e rs w ere fo rm ate, casam ino acids, a n d
lactate. F orm ate h a s b e e n p re v io u sly fo u n d to p ro m o te tetrazo liu m re d u c tio n
in Cam pylobacter je ju n i (4).
Effect of P hosphate o n CTC a n d INT R eduction
Inorganic p h o sp h a te h a d a re p re ssiv e effect o n CTC a n d INT re d u c tio n
above 10 mM. This effect h a s also b e en n o te d in Klebsiella pneum oniae, a n d
Salm onella typ h im u riu m (19). T hese re su lts w ere co n siste n t w ith decreases in
re sp ira tio n ra te s as m e a s u re d b y oxygen co n su m p tio n , p a rtic u la rly above 50
mM p h o sp h ate (Table 4). H ow ever, a d d itio n o f 10 mM p h o sp h a te in creased INT
a n d CTC (+) cell n u m b e rs as w ell as CTF a n d INF fo rm atio n in E. coli, w hile
re sp ira tio n ra te s d e crea se d slightly (Fig. 7, T able 4). Little effect was n o te d o n
INT (+) cell n u m b e rs u p to 7 5 mM p h o sp h ate . Levels o f flu o rescen t CTC (+)
cells p ara lleled gross CTF p ro d u c tio n tre n d s o v er 0-100 mM p h o sp h ate.
However, levels o f n o n flu o re sc e n t p lu s flu o re sc e n t [CTF (+)] cells o n ly
d ecreased fro m 100 to 80%, w hile flu o resc en t CTC (+) cells n u m b e rs d ecreased
to < 5% w ith in creasin g p h o s p h a te c o n c e n tra tio n s (Fig. 7B). This suggests th a t,
w hile CTF fo rm atio n a n d ra te s o f re s p ira tio n w ere d e c re a se d b y in creasin g
p h o sp h a te , fo rm atio n o f flu o r e s c e n t CTF cry stals was in h ib ite d to a g re a te r
d egree. P hosphate th e re fo re a p p e a rs to in h ib it CTC re d u c tio n to flu o rescen t
fo rm azan e ith e r d ire c tly , o r in d ire c tly b y lo w erin g ra te s o f re s p ira to ry
activity. P rod uction o f a w eakly flu o rescen t, o r m o re d iffuse CTF h as b e en
o b serv ed using m e ld o la b lu e a s a n artificia l e lec tro n d o n o r in b o th
p ro k ary o tic a n d eu k aryotic cells (5, 28). This resp o n se was also seen in
p re v io u s w ork (see p re v io u s c h a p te r) u sin g sev e ra l in h ib ito rs o f e le c tro n
tra n s p o rt a n d oxidative p h o sp h o ry la tio n . Pyle e ta l. (19) h a v e also show n th a t
69
CTC re d u c tio n to flu o rescen t fo rm azan is in h ib ite d to som e d eg ree above pH 6.5
in E. coli, K. pneum oniae, a n d S. typhim urium , a n d th a t th e effect is b o th
p h o sp h a te a n d pH d e p en d e n t. Since th e CTC red u c tio n tech n iq u e is d e p e n d e n t
u p o n th e p ro d u c tio n o f visible, in tra c e llu la r flu o resc en t-fo rm a z an crystals, it
is suggested th a t p h o sp h a te c o n c e n tra tio n b e k e p t a t o r below 10 mM d u ring
CTC assays w ith som e b a cte ria . Buffers a n d grow th m e d ia fo r bacteriological
use com m only c o n tain p h o sp h a te a t ap p ro x im ately 1-150 mM (Table 5).
P h o sp h a te c o n c e n tra tio n s in u n p o llu te d fre s h w aters g e n e ra lly ra n g e fro m
approxim ately 10 nM to 2 /<M, w hile th o se in seaw ater ra n g e betw een 1-5 nM
(8, 33). However, it sh o u ld b e n o te d th a t seaw ater pH is com m only S.2-8.4, a n d
fresh w ate r pH can b e significantly alk alin e, w hich m ay also h a v e a n effect o n
CTC a n d INT reduction.
T a b le 5 . C o n ce n tra tio n s o f in o rg a n ic p h o s p h a te in n a tu ra l
w aters as well as com m only u sed b u ffers a n d
grow th m edia.
M edium
P h o sp h ate (m m ol T 1)
M9
64
M63
100
MOPS
132
R2A
1.7
TSB
14.4
P h o sp h a te B uffered W ater
0.62
P h o sp h a te B uffered Saline
1 0 - ISO m M
S e a w ate r
10 nM - 2 ^M
F resh w ater (u n p o llu te d )
1-5 nM
,1
70
A lthough INT (+) cell n u m b e rs p a ra lle le d th e co n ce n tra tio n s o f INF
p ro d u c e d , in creasin g p h o sp h a te d id n o t re d u c e INT (+) n u m b e rs to th e sam e
d eg ree as w ith CTC (Figs. 7A a n d B). At 100 mM p h o sp h a te INT (+) cell n u m b ers
w ere o n ly d e crea se d 10% fro m m axim um values. This in d ic a te d th a t fo rm atio n
o f visible INF crystals a n d fo rm az a n re d u c tio n w ere less sensitive to p h o sp h a te
c o n ce n tra tio n , d e c re a se d re s p ira tio n ra te , a n d re d u c e d fo rm a z a n p ro d u c tio n
th a n CTC. In a d d itio n , it a p p e a re d th a t INT fo rm e d visible in tra c e llu la r crystals
a t low er fo rm azan co n cen tratio n s th a n CTC. As n o te d above, th is m a y b e d u e to
th e n e e d fo r p ro d u c tio n o f flu o re s c e n t CTF crystals, w hich a p p a re n tly was
!;
in h ib ite d b y p h o s p h a te a n d sev e ra l o th e r a g en ts k now n to affect in tra c e llu la r
!
iIl
I
re d o x processes (see p rev io u s c h a p te r).
D ue to th e v a ria b le n a tu re o f p ro k a ry o tic re s p ira to ry d e h y d ro g e n ase
activities, a n d e le c tro n tra n s p o rt chain s in v ario u s b a cte ria , d ifferen ces in CTC
a n d INT re d u c tio n a re to b e expected. This is p a rtic u la rly tru e w ith d ifferen t
organism s, a n d physiological co n ditio n s (31).. It sh o u ld b e em p h asized th a t
tetrazo liu m -fo rm azan system s are, in th e m o st basic sense, re d o x in d icato rs
w ith in so lu b le re d u c tio n p ro d u c ts. W hile it a p p e a rs th a t re d u c tio n of
in d iv id u a l te tra zo liu m salts is m e d ia te d p rim a rily b y specific re d u c ta n ts (i.e.
d eh y d ro g en ases a n d ETS com ponents), th e y c an also b e re d u c e d b y electro n
d o n o rs such as asco rb ate, d ith io n ite, th io su lfate, a n d som e u n d e fin e d m ed iu m
co m p o n en ts (4, 5, see p rev io u s c h a p te r). C onditions w hich stro n g ly affect
in tra c e llu la r a n d tra n sm e m b ra n e re d o x p o te n tia l w ould also b e expected to
effect tetrazo liu m re d u c tio n . In a d d itio n , fo r m axim al CTC (+) cell n u m b ers,
;,
su b strate a d d itio n a p p e a re d to p ro m o te CTC re d u c tio n to flu o rescen t CTF
c o m p ared to end o g en o u s re sp ira tio n . Use o f selected su b strates, a n d avoidance
j
o f h ig h in o rg an ic p h o sp h a te co n ce n tra tio n s (> 50 mM) m a y th u s h e lp to
-I
71
m axim ize re d u c tio n o f INT a n d p a rtic u la rly CTC to visible, in tra c e llu la r
fo rm azan deposits.
R eferen c es
1. A m erican Public H ealth A ssociation (1992) S ta n d a rd m e th o d s fo r th e
exam in atio n of w ater a n d w astew ater. 1 9 th ed. W ashington, D.C. A m erican
Public H ealth A ssociation.
2. A nraku, Y., a n d G ennis, R. B. (1987) T he aero b ic re s p ira to ry c h ain o f
Escherichia coli. T rends Biochem. Sci. 12, 262-266.
3. Blenkinsopp, S. A. a n d Lock, M. A. (1990) T he m e asu re m e n t o f electro n
tra n s p o rt activity in riv e r biofilm s. W ater Res. 24:441-445.
4. B oucher, S. N., Slater, E. R., C ham berlain, A. H. L. a n d A dam s, M. R. (1994)
P roduction a n d viability o f coccoid form s of C am pylobacter jejuni. J. Appl.
Bacteriol. 77:303-307.
5. Bovill, R. A., Shallcross, J. A. a n d M ackey, B. M. (1994) C om parison o f th e
flu o re sc e n t re d o x d y e 5 -cy an o -2,3 -d ito ly ltetrazo liu m c h lo rid e w ith p io d o n itro tetraz o liu m v io let to d e te c t m etab o lic activ ity in h e a t-stre sse d
Listeria m o nocytogenes cells. J. AppL B acteriol. 77:353-358.
6. B urstein, C., T iankova, L. a n d A. Kepes, A. (1979) R esp irato ry co n tro l in
Escherichia coli K 12. Eur. J. Biochem. 94:387-392.
7. H arvey, R. W. a n d Young, L. Y. (1980) E n u m eratio n o f p a rtic le -b o u n d a n d
u n a tta c h e d re sp irin g b a c te ria in th e sa lt m a rsh e n v iro n m e n t. AppL
Environ. M icrobiol. 40:156-160.
8. H olland, H. D. (1984) T he ch em istry o f th e earliest a tm o sp h ere a n d oceans,
p p. 89-127. I n T he chem ical evo lu tio n o f th e a th m o sp h e re a n d oceans ed.
H olland H. D. P rinceton, N. J., P rin ceto n U niversity Press.
9. Jones, J. G. (1979) C ounts using m e m b ra n e filters, p p . 19-31. I n A guide to
m e th o d s fo r estim ating m icro b ial n u m b e rs a n d biom ass in fre s h w ater
Jones, J. G (ed.). Scientific p u b lic a tio n n u m b e r 39. C um bria, England,
F resh w ater Biological A ssociation.
10. Jorgensen, F., N ybroe, O. a n d K nochel, S. (1994) Effects o f sta rv a tio n a n d
osm otic stress o n v iab ility a n d h e a t re sista n c e o f Pseudom onas Huorescens
AH9. J. Appl. BacterioL 7 7 :340-347.
o
72
11. K aprelyants, A. S. a n d Kell, D. B. (1993) T he use o f 5-cyano-2,3-ditolyl
tetxazolium ch lo rid e a n d flow c y to m e try fo r th e v isu alisatio n o f
re s p ira to ry activity in in d iv id u a l cells o f Micrococcus luteus. J. M icrobiol.
M eth. 17:115-122.
12. K aprelyants, A. S. a n d Kell, D. B. (1993) D orm ancy in statio n ary -p h ase
c u ltu re s o f Micrococcus luteus: flow cy to m etric an aly sis o f starv atio n a n d
resu scitatio n . AppL Environ. M icrobiol. 59:3187-3196.
13. K enner, R. A. a n d A hm ed, S. I.. (1975) M easurem ents o f e lec tro n tra n s p o rt
activities in m a rin e p h y to p la n k to n . M ar. Biol. 33, 119-127.
14. Lippold, H. J. (1982) Q uantitative succinic d eh y d ro g en ase h isto ch em istry . A
com parison o f d ifferen t tetrazo lim n salts. H istochem . 76:381-405.
15. N achlas, M. M., M argulies, S. I. a n d Seligm an, A. M. (1960) Sites of electro n
tra n s fe r to tetrazo liu m salts in th e succinoxidase system . J. Biol. Chem.
235:2739-2743.
16. Packard, T. T. (1985) M easurem ent o f e le c tro n tra n s p o rt activ ity o f
m icro p lan k to n . In A dvances in aq u atic m icrobiology ed . Jan n asch , H. W.
a n d P. J. W illiams, P. J. Vol. 3, pp. 207-262. London, A cadem ic Press Inc.
17. Pearse, A. G. E. (1972) Principles o f ox id o red u ctase h isto ch em istry , pp. 880920 I n H istochem istry, th e o re tic al a n d ap p lied . Pearse, A. G. E. (ed.) Vol. 2.
Baltim ore, U.S.A., W illiams a n d W ilkins Co.
18. Poole, R. K., a n d Ingledew , W. J. (1987) Pathw ays o f elec tro n s to oxygen, p p .
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biology. N eid h ard t, F. C., Ingraham , J. L., Low, K. B., M agasanik, B.,
S chaechter, M. a n d U m barger5 H. E. (eds.), Vol. 2. W ashington, D.C., U.S.A.,
A m erican Society fo r M icrobiology.
19. Pyle, B. H., Broadaw ay, S. C., a n d M cFeters, G. A. (1995) Factors affecting th e
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filtra tio n . A ppl. E nviron. M icrobiol, (su b m itte d fo r p u b lic a tio n ).
20. Q uinn, J. P. (1984) T he m odificatio n a n d ev alu atio n o f som e cytochem ical
te ch n iq u es fo r th e e n u m e ra tio n o f m etab o lically activ e h e te ro tro p h ic
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21. Rodriguez, G. G., Phipps, D., Ishiguro, K. a n d Ridgway, H. F. (1992) Use o f a
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32. TuovUa, B. J., a n d P. A. LaRock. (1985) Effect o f species d ifferen ce a n d
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74
CHAPTER 4
SURVIVAL, PHYSIOLOGICAL RESPONSE, AND RECOVERY OF ENTERIC
BACTERIA EXPOSED TO A POLAR MARINE ENVIRONMENT
In tro d u c tio n
In th e la st few d ecad es th e p e rsiste n c e o f h u m a n e n te ric p a th o g en s a n d
in d ic a to r o rg an ism s h a s a ssu m e d in c re asin g e n v iro n m e n ta l a n d p u b lic h e a lth
significance, p a rtic u la rly in en v iro n m e n ts c o n sid e re d p ris tin e (11, 20, 33, 49).
Sewage is o fte n d isc h a rg e d u n tre a te d in to lo w -te m p e ra tu re m a rin e
e n v iro n m e n ts. A pproxim ately 90% o f m a rin e e n v iro n m e n ts a re 5°C o r less,
w ith p o la r reg io n s co n stitu tin g ro u g h ly 14% o f th e e a r th ’s su rface (34). In
p o la r e n v iro n m en ts, re le a se o f u n tr e a te d sew age is p rim a rily d ic ta te d b y th e
logistical difficulties a n d expenses a sso c iate d w ith tre a tm e n t in isolated,
relativ ely sm all com m unities (6, 19, 20). P o lar regions (> 6 6 3 3 ' latitu d e) also
re p re s e n t th e lo w -te m p e ra tu re ex tre m es fo r co astal m a rin e e n v iro n m e n ts,
w ith seaw ater fre q u e n tly p o ised at, o r n e a r, ,its freezing p o in t (ca. -1.8°C).
P a rticu la rly in a re a s w h e re fa s t ice form s, w a te r co lu m n te m p e ra tu re is stable,
w ith solar ra d ia tio n h ig h ly a tte n u a te d a n d seaso n al (38). In ad d itio n ,
co m m u n ity p rim a ry a n d s e c o n d a ry p ro d u c tio n a re re la tiv e ly la rg e a n d h ig h ly
seasonal (3, 17, 48). Survival o f e n teric b a c te ria in th e se e n v iro n m e n ts is
g e n e ra lly e n h a n c e d b y re d u c e d grazin g ra te s o f h e te ro tro p h ic n a n o fla g ellate s
75
a n d larvae, in a d d itio n to th e effects o f low er seaw ater te m p e ra tu re s (2, 39, 40,
42).
F actors affecting th e su rv iv al o f allo c h th o n o u s b a c te ria in m a rin e
e n v iro n m e n ts in c lu d e p re d a tio n , osm otic stress, so lar-ra d ia tio n , n u trie n t
av ailability, b a cte rio p h a g e , algae, a u to c h th o n o u s m icro b ial toxins, h y d ro sta tic
p ressu re, grow th phase, a n d te m p e ra tu re (8, 11, 14, 25). Lower te m p e ratu res
h a v e b e e n show n to e x te n d survival o f e n teric b acteria, a n d it h a s b e e n
fre q u e n tly re p o rte d th a t te m p e ra tu re is th e m o st im p o rta n t fa c to r in
p re d ic tin g fecal coliform su rv iv al in m a rin e e n v iro n m e n ts (2, 5, 12, 28, 51).
T echniques fo r th e e n u m e ra tio n o f fecal in d ic a to r o rg an ism s a n d
specific p a th o g e n s fro m m a rin e e n v iro n m e n ts a re w idely a c c e p te d (I ).
R ecent stu d ie s d escrib in g th e p e rsiste n c e o f h u m a n e n teric b a c te ria in aq u atic
e n v iro n m e n ts h a v e d e m o n stra te d th a t m a n y o f th e se o rg an ism s e n te r a n
a lte re d physio logical,state te rm e d v iab le b u t n o n c u ltu ra b le (VBNC) (11, 24, 44).
This is com m only d e fin e d a s th e in a b ility to fo rm colonies o n a given solid
m e d iu m w hile re m a in in g active b y d ire c t v iab le-co u n t (DVC) m eth o d s.
S tressors w ith in a q u atic e n v iro n m e n ts also in d u c e su b le th a l p h y sio lo g ical a n d
s tru c tu ra l ch an g es, te rm e d in ju ry , in e n te ric b a cte ria . In th is in ju re d state,
b a c te ria a re u n a b le to re p ro d u c e u n d e r c o n d itio n s w hich allo w fo r grow th o f
u n in ju re d cells, in clu d in g re stric tiv e te m p e ra tu re s a n d th e p re se n c e o f
selective a g en ts (30). As a n exam ple, a VBNC b a cte riu m m a y a p p e a r v iab le b y
DVC m e th o d s y e t n o t fo rm colonies o n a n o n selectiv e grow th m e d iu m w hich
n o rm a lly su p p o rts its grow th. If th is b a c te riu m can fo rm co lo n ies o n
n onselective m e d iu m b u t n o t o n m e d iu m w h ich co n tain s selective ag en ts to
w hich th e o rg a n ism is n o rm a lly re sista n t, i t is te rm e d in ju re d . Since th e v a st
m a jo rity o f e n u m e ra tio n te ch n iq u es fo r fecal in d ic a to r a n d p a th o g e n ic
76
b a c te ria re q u ire cu ltu rin g using selective m ed ia, th e a cc u ra te d e te c tio n o f
v iable o rg an ism s in th e e n v iro n m e n t m a y b e serio u sly co m p ro m ised .
Previous m a rin e studies h a v e d e m o n stra te d th a t cold shock, starv atio n , a n d
possibly osm otic shock re s u lt in su b leth al in ju ry a n d th e VBNC re sp o n se (25,
29, 35, 44). M ost o f th e se experim ents h a v e u sed la b o ra to ry m icrocosm s, w hich
a re su b ject to “b o ttle effects”, in clu d in g th e te rm in a tio n o f so lu te exchange
w ith th e e n v iro n m e n t (13, 32). In a d d itio n , th e re la tio n sh ip b etw een
e n v iro n m e n ta l stress, su b le th a l in ju ry , a n d th e VBNC sta te re m a in s larg ely
u n re so lv e d .
C onsidering th a t th e m in im u m te m p e ra tu re a t w hich g row th o f
E scherichia coli h a s b e e n re p o rte d is ca. 7.5°C, th e q u estio n w ith v e ry cold
m a rin e e n v iro n m e n ts is n o t w h e th e r m o st sew age b a c te ria c a n activ ely grow
in situ b u t w h e th e r th e y c a n a d a p t a n d p e rsist th ro u g h th e fo rm a tio n o f
d o rm a n t, o r m etab o lically q u iescen t, cells w hich can re s p o n d to su b se q u e n t
in creases in te m p e ra tu re a n d /o r su b stra te c o n ce n tra tio n s (16, 24, 47, 52). It is
also of in te re s t w h e th er, a n d to w h a t d eg ree, th e se cells d isp lay in ju re d o r
VBNC responses.
In o rd e r to d e te rm in e th e lo n g -term physiological re sp o n se s a n d
re c o v era b ility of e n te ric b a c te ria ex p o sed to ex trem ely -lo w -tem p eratu re
m a rin e e n v iro n m e n ts w e a sse ssed su rv iv al, su b le th a l in ju ry , re c o v era b ility ,
a n d th e VBNC state o f e n te ric b a c te ria u n d e r p o la r m a rin e co n d itio n s.
E xperim ents w ere p e rfo rm e d b y using d iffu sio n c h am b e r e x p o su re o f two
in d ic a to r (E. coh a n d Enterococcus faecalis) a n d two p ath o g en ic (Salm onella
typ h im u riu m a n d Y ersinia enterocolitica) b a c te ria l species in situ f o r p e rio d s
o f 54 to 56 days a t M cM urdo Station, A ntarctica. The E. coh s tra in u s e d was
en terotoxigenic, re p re se n tin g b o th a n in d ic a to r a n d a p a th o g e n . R ecoverable
Tl
a n d in ju re d cells w ere e n u m e ra te d by p la te counts o n selective a n d
n o n selectiv e m edia. V iable cells n u m b e rs a n d su b stra te resp o n siv en ess w ere
d e te rm in e d b y use o f th e DVC m e th o d (27). In addition, tetrazo liu m red u c tio n
w ith 5 -cyano-2,3 -ditolyl te tra z o liu m c h lo rid e (CTC), w hich in d ic a tes active
re sp ira tio n , was u s e d to id e n tify re sp irin g cells (43).
M a te r ia ls a n d M e th o d s
O rganism s. G row th C onditions, a n d Exposure
By using 0.1 m l o f o v e rn ig h t s ta r te r c u ltu re inocula, 100-m l c u ltu res o f
en tero to x ig e n ic E. coli TX-432 (078:H12), S. typhim urium SL320, Y.
enterocolitica 0:8, a n d E faecalis w ere grow n in try p to n e-lac to se-y e a st
e x tra ct (TLY) in 250-m l E rlenm eyer flasks a t 37°C, w ith shaking a t 150 rp m .
C ultures w ere grow n to e a rly sta tio n a ry p h a se (20 h) as m o n ito re d b y A ^Q .
Cells w ere p elleted a t 3,000 X g fo r 10 m in a n d w ashed twice a t ca. 20°C in filtersterilized (I - a n d 0.22-><m-pore-size filters), a g ed sea w ater (FSASW) collected
w eekly fro m W inter Q u arters Bay, A ntarctica. Cells w ere th e n re su sp e n d e d a t a
c o n ce n tra tio n o f ca. IO9 c e lls /m l (1010 CFU/ml fo r E. faecalis) a n d in jected
slowly in to du p licate 30-m l diffusion c h am b ers (31). Tim e zero sam ples w ere
taken, a n d ch am b ers w ere th e n sim ultan eo u sly su sp en d ed a t a d e p th o f I m in
a constant-flow , 1240-liter circulating a q u a riu m (D [dilution ra te ] = 3.92 I r 1) a t
M cM urdo Station, A n tarctica (166° 4 0 ’ E, 77° 5 1 ’ S) d u rin g th e a u stra l sum m er
1992 (O ctober th ro u g h D ecem ber) fo r p e rio d s o f u p to 64 days. T he a q u ariu m
w as fe d w ith seaw ater d ire c tly fro m W in ter Q u arters Bay, a n d th e ta n k
co n tain ed n o m etal fittings. T e m p e ra tu re a n d salin ity w ere -1.8°C a n d 34.5 p p t,
78
respectively, a n d re m a in e d c o n sta n t th ro u g h o u t th e ex p erim en t. Subsam ples
fo r all assays (total, 0.5 ml) w ere rem o v ed usin g a sterile I- o r 5-m l syringe,
k e p t a t th e in situ te m p e ratu re, a n d tra n s p o rte d to th e la b o ra to ry im m ediately.
Cells w ere d ilu te d in -1.8°C FSASW, allow ed to re a c h ca. 25°C o v e r a p e rio d o f I
h, a n d u sed fo r all assays d escrib ed below.
Plate C ounts a n d In ju ry
D ilutions o f all c u ltu res w ere m e m b ra n e filte re d o r spot- o r spreadp la te d o nto various m edia. E. coli, S. typhim urium , a n d Y. enterocolitica w ere
p la te d on TLY a g ar w ith 0.1% d eoxych o late (TLYD; selective) a n d o n TLY ag ar
w ith o u t d eoxycholate (nonselective). E. faecalis was p la te d o n b ra in h e a rt
in fu sio n a g a r (BHI agar; nonselective), a n d mE a g a r (selective). S ta n d a rd
in c u b atio n was a t 25°C, except fo r R faecalis, w hich was in c u b a te d a t 3TC. All
colonies w ere c o u n ted a t 2 4 ,7 2 , a n d 144 h , o r u n til CPU’s n o lo n g e r increased.
A typical o r slow ly grow ing colonies w h ich fo rm e d w ere p erio d ic ally pick ed
fro m p la te s a fte r v ario u s p e rio d s o f ex p o su re a n d stre ak e d o n to e ith e r Tergitol7 a g ar (R coli), salm onella-shigella (SS) a g a r (S. typhim urium ), Yersiniaselective a g a r (Y. enterocolitica), o r mE a g a r {E. faecalis) to ch eck fo r
c o n tam in atio n . A typical colonies o f E. coli a n d S. typ h im u riu m w ere also
in o c u la ted o n trip le su g ar iro n (TSI) a g ar. No co n tam in atio n w as in d ic a ted in
a n y o f th e c h am b ers b y th is m e th o d w ith in cu b atio n s a t 37°C.
E coli, S.
typ h im u riu m , a n d Y. enterocolitica cells ex p o sed fo r 5 4 d ay s w ere in cu b ated a t
-1.8, 8, 20, a n d 37°C fo r u p to 15 days to d e term in e optim al p latin g recovery
te m p e ra tu re s. S ublethal in ju ry w as c alc u la te d a s (CFUn 0 n s e le c tiv e C FU selective/C FU nonselective) X 100 as d escrib ed previously (45). Tgo a n d T99
d ecay ra te s (tim e re q u ire d fo r I - a n d 2 -o rd er-o f-m ag n itu d e re d u c tio n s in
79
re c o v e ra b le cell n u m b e rs, resp ectiv ely ) a n d d iffu sio n c h a m b e r volum e
tu rn o v e r tim es w ere calcu lated as d e sc rib e d p rev io u sly a n d a re in c lu d e d h e re
fo r com parative pu rp o ses (28, 51).
DVCs
A liquots of E. coli, S. typhhxmrixxm, a n d Y. enterocolitica (0.1 ml) w ere
d ilu te d in to 0.9 m l FSASW a t -1.8°C a n d assay ed fo r su b stra te resp o n siv en ess b y
th e m e th o d o f Kogure e t al. (27), a s m o d ified b y Singh e t al. (46). Final
c o n ce n tra tio n s of com ponents w ere as follows: 0.025% y e a st ex tract, 0.3%
Casam ino Acids, a n d 0.002% naladixic acid. A fter a 2 -h p e rio d o f acclim ation a t
25°C, in c u b atio n was c o n tin u e d a t 37°C fo r 8 h a t 100 rp m . In cu b atio n s w ere
sto p p ed b y a d d itio n o f 0.1 m l o f 37% fo rm ald eh y d e. Cells w ere th e n filtered a n d
stain ed w ith 0.01% acrid in e o ran g e (AO) in 2 mM Tris b u ffer (pH 7.0) fo r 3 m in.
E longated (DVC-positive) [DVC(+)] as well a s to ta l cells (AODC) w ere e n u m e ra te d
b y ep iflu o rescen ce m icroscopy.
CTC R eduction
A liquots o f all organism s (0.1 m l) w ere assay ed fo r e le c tro n tra n s p o rt
(resp iratio n ) b y th e m e th o d o f R odriguez e t al. (43) w ith a fin a l co n ce n tra tio n
o f 5 mM CTC in FSASW. CTC in cu b atio n s w ere a t 37°C a n d 100 r p m fo r 4 h a fte r a
2-h p e rio d o f acclim ation a t 25°C. E. coli, S. typhhnurium , a n d Y. enterocolitica
cells exposed fo r 48 days w ere in c u b a te d a s ab o v e a t -1.8, 8, 20, a n d 37°C w ith
a n d w ith o u t in itial a d d itio n o f 0.1 X TLY (fin al co n cen tratio n , ca. 3.3 g of
organic c a rb o n p e r lite r) to d e te rm in e w h e th e r te m p e ra tu re o r n u trie n t
availab ility lim ited in situ cellu lar re s p ira tio n . In cu b atio n s w e re te rm in a te d
w ith fo rm ald e h y d e a s d e sc rib e d above. B acterial suspensions w ere filte re d a n d
80
c o tm te rsta in e d w ith 4 % 6-diam idino-2 -p h en y lin d o le (DAPI) (0.1 ^ g /m l) fo r 10
m in, a n d cells co n tain in g flu o re sc e n t C T C dbrm azan d ep o sits (CTC-positive
cells) [CTC(+)], as well as to tal cells, w ere e n u m e ra te d b y epifluorescence
m icro sco p y .
All epiflu o rescen ce c o u n ts w ere p e rfo rm e d u sin g N uclepore 0.22-pm pore-size, p re b la c k e n e d p o ly c a rb o n a te filtra tio n m e m b ra n es (no. 110656) a n d
a Zeiss s ta n d a rd 16 m icroscope e q u ip p e d w ith a 100-W U.V. m e rc u ry lam p (Optiquip 1500). Zeiss filter set no. 48-77-09 (KP 490, FT 510, a n d LP 520) fo r AO was
u se d fo r DVC counts, v M le no. 48-77-01 (BP 3 6 5 /1 1 , FT 395, a n d LP 397) was u sed
fo r CTC a n d DAPI counts. D uring CTC co u n ts, to tal cells w ere first c o u n ted w ith
th e DAPI filte r set a n d th e n CTC(+) cells w ere e n u m e ra te d b y sw itching to th e
DVC filter set.
F orm aldehyde (3.7% )-killed CTC a n d DVC assay controls w ere
u s e d fo r all organism s. C ontrol e x p erim en ts also in d ic a te d n o o b serv ab le
in creases in to ta l cell n u m b e rs w ith 8-h (DVC) a n d 4-h (CTC) in cu b atio n s as
d e sc rib e d above. A m inim um o f 800 cells in a t le ast 8 fields w ere c o u n ted fo r
each sam ple, a n d cell size was n o te d b y using a c alib ra te d o c u la r grid . E rror
w as calc u la te d as d e sc rib e d p rev io u sly (22).
D iffusion R ates
In o rd e r to d e te rm in e d iffu sio n ra te s o f m etab o lites acro ss ch am b e r
m e m b ra n es in situ, rh o d a m in e d iffusio n acro ss th e c h a m b e r m e m b ra n es was
assay ed using a sterile c o n tro l ch am b er. Also, in o rd e r to d e te rm in e decreases
in d iffusion ra te s d u e to m e m b ra n e fouling, d iffu sio n c h am b e rs in w hich E.
coli, S. typhim urium , a n d E. faecalis h a d b e e n exposed fo r 6 4 d ay s w ere
em ptied, b u t n o t flushed, a n d 30 m l o f I mM rh o d a m in e 6G in FSASW (m olecular
w eight, 479.0) w as in jected . C ham bers w e re p la c e d in situ, a n d diffu sio n was
81
m o n ito re d b y m easu rin g A546 o f subsam ples. R eadings w ere c o rre c te d fo r
d ilu tio n d u e to sam pling a n d p lo tte d ag ain st tu n e in situ. A Beckm an DU 7400
sp e c tro p h o to m e te r was u se d fo r all a b so rb a n c e m easu rem en ts.
C hem icals. M edia, a n d E quipm ent
R hodam ine 6G, a-lactose, T ris-base, Tris-HCL, DAPI, AO, 37%
fo rm ald eh y d e, naladbtic acid, sodium h y d ro x id e, a n d d eo xycholate w ere
p u rc h a se d fro m Sigm a C hem ical Co. (St. Louis, M o.). Yeast ex tract, casam ino
acids, agar, try p tic soy b ro th w ith o u t d ex tro se, BHI b ro th , a n d mE a g a r w ere
p u rc h a se d fro m Difco L aboratories (D etroit, M ich.). CTC was o b ta in e d fro m
Polysciences, Inc. (W arrin g to n , Pa.). All cell m a n ip u la tio n s fo r lowte m p e ra tu re in c u b atio n s (8 a n d -1.8°C) w ere p e rfo rm e d in a re frig e ra te d
e n v iro n m e n ta l ro o m .
R e su lts
R eco v e ra b ih ty a n d In ju ry
U pon ex p o su re to th e p o la r m a rin e e n v iro n m e n t, all b a c te ria ex am in ed
d e m o n stra te d d eclining reco v erab ility , d e te rm in e d b y co lo n y fo rm atio n , in
a d d itio n to in creases in su b leth al in ju ry a s assessed b y th e TLY/TLYD CPU ra tio
(Figs. 8-12). T he frac tio n o f slowly grow ing colonies th a t re q u ire d >24 h fo r
colony fo rm atio n also g e n e ra lly in c re a se d w ith tim e o f e x p o su re fo r all
organism s o n all m ed ia u s e d (d a ta n o t show n).
E. coli re c o v era b ility o n TLYD d e c re a se d ra p id ly th ro u g h o u t th e 5 4 -d ay
exposure, w ith a T99 o f ap p ro x im ately 336 h (Fig. 8). R ecoverabiU ty o n TLY
82
in d icated a Tgg of ap p ro x im ately 480 h. Sublethal in ju ry ra p id ly in creased to
96% a fte r 4 d ay s of exposure a n d th e n re m a in ed betw een 92 a n d 98.5%
th ro u g h o u t th e re m a in d e r o f th e 54-day ex posure (Fig. 12).
O TLY
• TLYD
e AODC
♦ DVC (+)
V CTC (+)
5
10 1 5 2 0 2 5 3 0 3 5 4 0 4 5 50 55
Time (d)
F ig u re 8. Plate (n = 5), CTC (+), DVC (+), a n d total d irect co u n ts fo r E. coli
exposed to a p o la r m arin e en v iro n m e n t fo r 54 days. E rro r b a rs re p re se n t
sta n d a rd deviations.
S. typhim wrim n reco v erab ility o n TLYD a n d TLY d e c re a se d b y I log u n it
a fte r 4 a n d 11 d ay s exposure, respectively. This was follow ed b y continuing
decreases in re co v erab ility o n b o th m ed ia (Fig. 9). Tgg v a lu es fo r TLYD an d
83
TLY reco v eries w ere 408 a n d 528 h, respectively. Sublethal in ju ry rap id ly
in creased to a m axim um of 87% a t 4 days, a fte r w hich a g en eral decreasing
tre n d was observed. A fter 11 days, in ju ry a p p ea re d low er fo r th e S.
ty p h im u riu m p o p u la tio n th a n for th e o th e r te st organism s th ro u g h o u t th e 54d a y exposure (Fig. 12). A fter 54 days o f exposure, plate counts o n both TLY and
TLYD w ere approxim ately 5 X IO3 CFU/ml.
E
"X
Cfi
0)
U
O
CD
O
□
•
o
♦
V
0
5
TLY 1
TLYD
AODC
DVC (+)
CTC (+)
10 1 5 20 25 3 0 35 4 0 4 5 5 0 55
Time (d)
F ig u re 9. Plate (n = 5), CTC (+), DVC (+), a n d total d irect co u n ts fo r S.
ty p h im u riu m exposed to a p o la r m arin e en v iro n m e n t fo r 54 days. E rror b ars
re p re se n t s ta n d a rd dev iatio n s.
84
It was also o b serv ed th a t S. typ h im u riu m colonies ch an g ed fro m convex a n d
ro u n d to fla t a n d sp read in g a fte r 7 day s o f exposure. These colonies show ed
re a ctio n s typical o f S. typ h im u riu m w h en grow n o n TSI a n d SS ag ar.
Y. enteroeohtica p la te co u n ts o n TLY show ed little d e crea se in th e first
4 days, w hile counts o n TLYD d ecrea se d b y ap p ro x im ately I o rd e r o f m ag n itu d e
(Fig. 10). A fter 4 days, co u n ts o n b o th TLY a n d TLYD c o n tin u e d to d ecrease.
A fter 15 days o f exposure p la te counts o n TLYD a n d TLY d ecrea se d steadily,
w ith T99 values o f 360 a n d 504 h, respectively.
As w ith E. coli a n d 5. typhim urium , Y enteroeohtica in ju ry re a c h e d its
h ig h est in itial v alu e o f 80% a t 4 d ay s a n d th e n in creased to 99% b ecau se o f a
sh a rp 2-log-unit d ecrea se in TLYD cou n ts b y 15 days, w hile TLY counts
re m a in e d stab le (Fig. 12). A fter 54 d a y s o f exposure, p late co u n ts o n TLYD a n d
TLY w ere IO3 a n d IO4 CFU/ml, resp ectiv ely .
E. faecahs re c o v era b ility o n BHI a g a r d ecrea se d I log o v e r th e firs t 27
d ays of exposure a n d d e crea se d som ew hat m o re rap id ly to a to ta l d ecrease o f 3
o rd e rs o f m a g n itu d e a fte r 56 d a y s (Fig. 11). In co n trast, mE a g a r p la te counts
d ecreased 0 .4 o rd e r o f m a g n itu d e u n til 4 days, a fte r w hich CPU’s o n this
m edium ra p id ly d e crea se d to <0.2 CFU/ml b y 27 days. In ju ry in creased
g rad u ally to 99% a fte r I w eek o f expo su re a n d re m a in e d effectively 100% a fte r
ap proxim ately 27 d ay s (Fig. 12). T99 valu es av erag ed 192 a n d 8 40 h o n mE a g a r
a n d BHI a g ar, resp ectiv ely .
A fter 54 d a y s o f e n v iro n m e n ta l ex p o su re, R coli, S. typhim urium , a n d Y.
enteroeohtica cells failed to fo rm colonies o n TLY o r TLYD a t 37°C (Fig. 13).
O ptim al colony-form ing te m p e ra tu re s o n TLY a n d TLYD w ere -1.8° a n d 8°C (E.
coli), 20 a n d 20°C (S. typhim urium ), a n d 8 a n d -1.8°C (Y. enteroeohtica),
respectively. S. typ h im u riu m w ould n o t fo rm colonies a t 8 a n d -1.8°C o n TLYD
85
(Fig. I SB), w hile Y. enterocolitica show ed g re a te r colony-form ing a b ility on
this m edium a t these te m p e ra tu re s (Fig. ISC)
F o
o ~ 1—o
□
•
o
♦
TLY
TLYD
AODC
DVC (+)
V CTC (+)
5
10 1 5 20 25 30 35 4 0 4 5 50 55
Time (d)
F ig u re 10. Plate (n = 5), CTC (+), DVC (+), a n d total d irect co u n ts fo r Y.
enterocolitica exposed to a p o la r m a rin e e n v iro n m e n t fo r 54 days. E rror b ars
re p re se n t s ta n d a rd d ev iatio n s.
86
□ BHI
• mE
o AODC
V CTC (+)
0
5
10 15 2 0 25 3 0 3 5 4 0 45 5 0 5 5 6 0
Ti me (d)
F ig u re 11. Plate (n = 5), CTC (+), a n d to tal d irect counts fo r E. faecalis exposed
to a p o la r m arin e e n v iro n m e n t fo r 56 d ay s. E rror b ars re p re s e n t sta n d a rd
d ev iatio n s.
87
O
5
♦
E. col i
•
S. t y p h i m u r i um
■
Y. e n t e r o c o l i t i c a
a
E. f a e c a l i s
10 1 5 20 25 30 35 40 45 50 55 60
Time (d)
F ig u re 1 2 . P ercent su b leth al in ju ry su sta in e d b y v a rio u s e n te ric b acteria
w ith exposure to a p o la r m arin e e n v iro n m e n t fo r 54 to 56 days.
88
- 1 .8
8.0 2 0 .0 37.0
Temperature (0 C)
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Temperature (0 C)
F ig u re 13. Selective (TLYD; black bars) a n d n o n selectiv e (TLY; stipled bars)
p la te c o u n t re co v eries a t d iffe re n t te m p e ra tu re s fo r (A) E. coli, (B) S.
typ h im u riu m , a n d (C) Y. enterocolitica ex p o sed to a p o la r m a rin e en v iro n m e n t
fo r 54 days. E rror b ars re p re se n t s ta n d a rd d ev iatio n s (n = 5).
89
T e m p e r a t u r e ( 0 C)
F ig u re 13 ( c o n tin u e d ) . Selective (TLYD; black bars) a n d n o n selectiv e
(TLY; stipled bars) plate c o u n t recoveries a t d ifferen t te m p e ra tu re s fo r (A) E.
coli, (B) S. typhim iiriiim , a n d (C) Y. enterocolitica exposed to a p o la r m arin e
e n v iro n m e n t fo r 54 days. E rror b ars re p re s e n t s ta n d a rd d ev iatio n s (n = 5).
G row th a t 8°C re q u ire d a n av erag e o f 6 d a y s fo r th e m a jo rity o f colony
form ation, a n d a n a d d itio n a l 2-3 days fo r visible colony fo rm atio n by slowgrow ers. S ubculturing o f ra n d o m ly pick ed colonies o n to m e d ia u sed to check
fo r co n tam in atio n (see M aterials a n d M ethods) in d ic a ted th a t th e ob serv ed
grow th was n o t d u e to c o n tam in atio n o f c h am b e rs w ith a u to c h th o n o u s m a rin e
b a cte ria . In c o n tro l e x p erim en ts using fre sh ly grow n cells, n o n e o f th e
organism s te ste d fo rm ed colonies a t -1.8°C a n d o n ly Y. enterocolitica h a d th e
ability to form colonies a t 8°C. Also, d ifferen ces betw een TLY a n d TLYD
recoveries a t 20 a n d 37°C fo r all b a c te ria te ste d w ere n o te d in th is experim ent.
By co n trast, rein o cu latio n of 100 m l of TLY p rew arm ed to 37°C w ith 0.5 m l 64-
90
day-exposed E. coli, S. typhim urium , a n d E. faecalis p ro d u c e d grow th curves
v e ry sim ilar to th o se o b ta in e d w ith fre sh o v e rn ig h t in o c u la (i.e., sim ilar
grow th ra te s a n d cell d ensities), except th a t E. coli and E. faecalis show ed
p ro lo n g e d lag phases (d a ta n o t show n).
DVCs. a n d CTC R eduction
E. coli CTC (+) cell n u m b e rs w ere co n sisten tly low er th a n DVC positive E.
coli n u m b e rs b y 0.2 to 1.5 o rd e rs o f m ag n itu d e following e n v iro n m e n ta l
e x p o su re (Fig. 8). DVC (+) cell n u m b e rs g ra d u a lly d eclin ed o v e r th e first 15
d ays o f exposure th e n stabilized a t ca. 10% o f in itial values. A fter 25 days of
exposure, th e se n u m b e rs stead ily in c re a se d to with in I o rd e r o f m ag n itu d e o f
inoculum values b y 54 days. This sam e g en eral tre n d was o b serv ed in CTC (+)
counts, w hich stabilized a t ca. 1% b y 25 d ay s th e n ro se stead ily to 90% o f in itial
values b y 54 days.
CTC (+) a n d DVC (+) cell n u m b e rs c o rre la te d well fo r th e S. typhim urium
a n d Y. enterocolitica cu ltu res th ro u g h o u t th e 5 4 d ay s o f ex p o su re (Figs. 9 a n d
10). T hese n u m b e rs d ecrea se d steadily b etw een d ay s 4 a n d 20. CTC (+) a n d DVC
(+) n u m b e rs in each c u ltu re th e n stab ilized a t 1% o f in itia l values. A fter 40
days, CTC (+) S. typ h im u riu m cell n u m b e rs g ra d u a lly in c re a s e d to w ith in 79%
o f to ta l cell n u m b e rs b y 54 days w hile Y. enterocolitica CTC (+) cell n u m b ers
in c re ase d to w ith in 10% o f in itia l v a lu es (Figs. 9 a n d 10). T h e VBNC re sp o n se
w as g re a te s t fo r S. typhim urium , w ith 2- to 4 -o rd er-o f-m ag n itu d e
u n d e re stim atio n s o f DVC (+) a n d CTC (+) n u m b e rs a fte r 11 day s. Y.
enterocolitica and E. coli nonselective p la te co u n ts u n d e re stim a te d DVC (+) a n d
CTC (+) n u m b e rs b y I to 3 o rd e rs o f m a g n itu d e a fte r 2 0 d ay s a n d b y I to 4.5
o rd e rs o f m ag n itu d e a fte r ap p ro x im ately 25 days, respectively.
91
E. faecalis is re sista n t to naladixic a c id a n d th e re fo re c a n n o t b e assay ed
fo r su b stra te resp o n siv en ess b y th e DVC m e th o d . A fter a tra n s ie n t in c re ase in
E. faecalis CTC (+) cell n u m b ers to 100% o f to tal cells a t 4 days, th ese cell
n u m b e rs d e c re a se d ra p id ly (Fig. 11). This w as follow ed b y a s h a rp decline in
th e n u m b e rs of reco v erab le cells o n mE ag ar. A fter 7 days, n o CTC (+) E.
faecalis cells w ere observ ed , a lth o u g h p la te c o u n ts o n BHI a g a r re m a in e d ca.
IO6 CFU/ml (see above).
T otal cell n u m b e rs fo r all b a c te ria te s te d re m a in e d re la tiv e ly co n sta n t
(only AODC values a re p re s e n te d fo r c la rity [Figs. 8-11]). Slight in creases in
to ta l cell n u m b e rs a n d d ecreases in cell size w ere o b serv ed w ith in th e first
w eek o f exposure.
A ddition of 0.1X TLY to cell p o p u latio n s o f E. coli a n d S. typ h im u riu m
exposed fo r 48 d ay s significantly in c re a se d th e p e rcen tag e o f cells displaying
re s p ira to ry activity a t -1.8, 8, 20, a n d 37°C in com p ariso n w ith v a lu es fo r
co ntrols w ith o u t n u trie n t a d d itio n (Fig. 14). £ coli show ed th e g re a te st
rela tiv e in crease, fro m ca. 8% to g re a te r th a n 90% a t 20°C (Fig. 14A). S.
ty p h im u riu m h a d th e g re a te s t p e rc en ta g e s o f re sp irin g cells a t all
te m p e ra tu re s a fte r n u trie n t a d d itio n , w ith v alu es reach in g o v e r 90% fo r m o st
te m p e ra tu re s. S. typ h im u riu m also show ed th e g re a test p e rc e n ta g e o f
re sp irin g cells in th e a b se n c e o f a d d e d n u trie n ts , av erag in g a p p ro x im a te ly
30% re sp irin g cells a t all te m p e ra tu re s ex am in ed (Fig. 14B). Y. enterocolitica
h a d th e le a s t in crease, w ith v e ry slig h t in c re a se s in p e rc en ta g e s o f CTC (+)
cells a t 8 a n d 20°C in th e p resen ce o f a d d e d n u trie n ts. W ith n u trie n t a d d itio n ,
Y. enterocohtica re a c h e d a m axim um o f o n ly ca. 12% CTC (+) cells a t 20°C (Fig.
14C). No in creases in cell n u m b e rs w ere o b serv ed d u rin g 4 a n d 8 h
in c u b a tio n s w ith a n d w ith o u t
92
30 -
NO TLY
NO TLY
F ig u re 14. P ercent CTC (+) E. coli (A), S. typhim urium (B), a n d Y.
enterocolitica (C) cells w ith a n d w itho u t n u trie n t a d d itio n a t -1.8°C (u n sh a d ed
bars), 8°C (stip p led b ars), 20°C (diagonal strip e d b ars), a n d 3T C (black bars)
a fte r exposure to th e p o la r m arin e e n v iro n m e n t fo r 48 days. E rro r b a rs
re p re se n t s ta n d a rd d eviations.
93
C
U
H
U
j
TLY
NO TLY
F ig u re 14 ( c o n tin u e d ) . P ercent CTC (+) E. coli (A), S. typ h im u riu m (B), a n d
Y. enterocolitica (C) cells w ith a n d w ith o u t n u trie n t ad d itio n a t -1.8°C
(u n sh a d e d b a rs), 8°C (stip p led b ars), 20°C (d iag o n al strip e d b ars), a n d 3TC
(black bars) a fte r ex p o su re to th e p o la r m a rin e e n v iro n m e n t fo r 48 days.
E rror b a rs re p re s e n t s ta n d a rd dev iatio n s.
n u trie n t ad d itio n s. This in d ic a te d th a t in creases in CTC (+) cell n u m b ers w ere
n o t d u e to grow th o f new cells.
D iffusion Rates
Rate o f diffusion o f rh o d a m in e 6G fro m th e ch am b er a n d tim e re q u ire d
fo r 90 a n d 99% exchange w ere calculated as d escrib ed previously (51). Times
fo r 90 a n d 99% exchange o f c o n tro l c h a m b e r volum e u n d e r in situ conditions
w ere 31 a n d 63 h, respectively. C ham bers in w hich b a cte ria h a d b e en exposed
fo r 64 days show ed 90 a n d 99% exchange tim es o f 41 a n d 83 h (£. coli), 43 an d 87
94
h (S. typ h im u riu m ), a n d 44 a n d 88 h (R faecalis), resp ectiv ely . D iffusion ra te s
in in o c u la ted ch am b ers d e c re a se d b y 24% ( £ coli), 27% (S. typhim urium ), a n d
'28% (£. faecalis) in co m p ariso n w ith c o n tro l levels a fte r 6 4 d a y s o f exposure.
D is c u s s io n
T otal b a c te ria l c o u n ts re m a in e d rela tiv e ly c o n sta n t a n d n e a r th e level o f
th e o rig in al in o cu lu m fo r a ll org an ism s, in d ic a tin g m in im al cell lysis w ith
en v iro n m e n ta l exposure o f 54 to 56 d ay s. Slight increases in to ta l cell
n u m b e rs a n d d ecreases in cell size w ith in th e first w eek o f e x p o su re w ere
likely d u e to re d u ctiv e division (Figs. 8-11; cell size d a ta n o t show n) (25).
D ecreases in d iffusion ra te s (ca. 30%) w ere ex p ected b e ca u se o f fo u lin g
o f th e diffusion c h a m b e r m e m b ra n e b y m icro b ial cells a n d su sp e n d e d
p a rtic u la tes. C ontrol c h a m b e r d iffu sio n ra te s in th is stu d y w e re slightly
h ig h e r th a n th o se re p o rte d b y V asconcelos a n d Swartz fo r a m a rin e
e n v iro n m e n t (51). From th e d iffusion ra te s a n d to ta l cell c o u n ts o b ta in e d in
th is study, w e conclude th a t m o st m etab o lites diffused o u t o f th e cham bers.
AU organism s, ex cep t E. coli o n TLYD a n d E. faecalis o n mE, rem ain ed
re c o v era b le o n selective a n d n o n sele c d v e m e d ia th ro u g h o u t th e exposures
(Figs. 8-11). R ecoverable ceU c o u n ts o v e r th e 5 4 -day ex p o su re fo r th e 3 gram ­
n e g ativ e o rg an ism s (E. coli, S. typhim urium , Y. enterocolitica), y ield ed
som ew hat sim ilar T90 values (216 to 259 h ; TLYD T90 v alu es w e re alw ays low er
th a n TLY) fo r b o th th e selective a n d n o n selectiv e m edia. T his is low er th a n
th e v a lu e o b ta in e d in a n e a rly p o la r m a rin e m icrocosm stu d y ad d ressin g
re c o v e ra b ility o f E. coli a t O0C (T90 ~ 395 to 5 40 h o n tergitol-7) (18) b u t
95
a p p ro x im ately two to six tim es g re a te r th a n th o se o b ta in e d in m o re re c e n t
m a rin e survival studies in <5°C m a rin e e n v iro n m e n ts (2, 5, 28). A lthough
d ire c t com p ariso n s betw een th e se re su lts a re difficult, th e p rev io u sly o b serv ed
tre n d s o f p ro lo n g e d survival a t low te m p e ra tu re s a n d re d u c e d reco v eries o n
selective m e d ia w ere o b serv ed in th e p re s e n t study.
E. faecalis also re ta in e d h ig h levels o f re c o v era b le cells o n n o n selecd v e
m ed iu m (BHI agar) (T90 = 576 h) a fte r 56 d a y s o f exposure, a lth o u g h this
o rg an ism b ecam e n o n re c o v e ra b le o n mE a g a r a fte r o n ly 14 d ay s (T90 = 96 h)
(Fig. 11). This indicates th a t th e u se o f th is selective m ed iu m lead s to low levels
o f d e te c tio n o f in ju re d cells, signified b y th e d ra m a tic u n d e re stim a tio n of
v iab le cells d e te c te d using BHI ag ar. In p rev io u s studies, Tgos fo r E. faecalis of
269 h a t 4°C w ith TSA a g a r (5) a n d 122 h a t O0C using mE a g a r w ere fo u n d (28).
Collectively, th ese re su lts in d ic a te th a t w hile E. faecalis survives a t least as
well o r b e tte r th a n p a th o g e n s w h e n re c o v e ry is m e a su re d o n n o n sele c d v e
m ed ia, reco v eries o n mE a g a r in c re asin g ly u n d e re stim a te d n u m b e rs o f viable
E. faecalis cells in <10°C m a rin e e n v iro n m e n ts w ith tim e o f exposure.
R ecoverability o f all organism s e x am in ed was c o n siste n tly low er o n
selective th a n o n n o n sele c d v e m e d ia . T h is in d ic a te s th a t in th is en v iro n m en t,
sig nificant p ro p o rtio n s o f th e e n te ric b a c te ria l p o p u la tio n s su sta in e d
su b le th a l in ju ry w hich w as g re a te s t w ith in th e first w eek o f e x p o su re (Fig.
12). O rganism s in o rd e r o f m axim al levels o f in ju ry th ro u g h o u t th e stu d y a re
a s follows; E. faecalis > E. coli > Y. enterocolitica > S. typ h im tu iu m . However,
th e system u sed fo r assessing E. faecalis in ju ry was d iffe re n t fro m th a t u sed
fo r th e gram -n eg ativ e org an ism s. B oth in d ic a to r o rg a n ism s te ste d g en erally
d e m o n stra te d g re a te r in itia l a n d su sta in e d in ju ry th a n th e p a th o g e n s u p o n
ex p o su re to th e p o la r m a rin e e n v iro n m e n t (45). T hese re s u lts m a y explain th e
%
re la tiv e ly p o o r recoveries o f th e se b a c te ria o n h ig h ly selective m e d ia in th e
p re v io u s lo w -te m p e ra tu re m a rin e su rv iv al stu d ies d e sc rib e d above.
In cu b atio n of 54-day-exposed cells o f E coli, S. typhim urium , a n d Y.
en terocolitica a t v ario u s te m p e ra tu re s in d ic a te d th a t p o la r m arin e exposure
sh ifte d o p tim al a n d p erm issiv e grow th te m p e ra tu re s dow nw ards, allowing E.
coli a n d S. typ h im u riu m cells to fo rm colonies slowly a t -1.8 a n d 8°C (Figs. 13A
a n d B). This suggests th a t lo n g -term co ld ex p o su re m ay in d u c e physiological
ch an g es in th e se o rg an ism s w hich p e rm it g ro w th below ca. 7.5°C. T he
o b serv ed loss o f colony-form ing a b ility o f th e se organism s a t 37°C in d icates
th a t ex p o su re also in d u c e d m a rk e d th e rm o sen sitiv ity w ith re sp e c t to colony
fo rm atio n a t n o rm a l in c u b a tio n te m p e ra tu re s. E n v iro n m en tal a d a p ta tio n
th ro u g h ch an g es in m e m b ra n e lip id s a t low te m p e ra tu re s a n d ex p ressio n o f
cold shock p ro te in s is know n to occur in E. coli (16, 21, 2 3 ,4 1 ). These
a d a p ta tio n s m ay p ro d u c e physiological c h a n g e s w hich re s u lt in in c re a se d
th e rm o sen sitiv ity as w ell as allow ing lo n g -te rm su rv iv al a t low te m p e ra tu re s.
M utational ev en ts in c u ltu re su b p o p u latio n s d u rin g ex p o su re c o u ld also h av e
p ro d u c e d th e o b serv ed grow th te m p e ra tu re resp o n ses. C ontrol ex perim ents
w ith fre sh ly grow n cells in d ic a te d th a t n o n e o f th e b a c te ria te s te d (w ith th e
exception o f Y. enterocolitica) c o n ta in e d su b p o p u latio n s cap ab le o f grow th a t
<8°C o v er ca. 15 d a y s o f ex p o su re o n a g a r p lates. These re su lts in d ic a te th a t
su b p o p u latio n s cap ab le o f grow th a t <8°C w ere n o t p re s e n t in c u ltu re s u p o n
in itia l ex p o su re b u t d e v e lo p e d o n ly a fte r lo n g -term , in situ ex p o su re.
A lthough th e tre n d o f in creasin g th e rm o se n sitiv ity a n d lo w ered o p tim al
g row th te m p e ra tu re w as o b se rv e d in all s tra in s te ste d {E. coli, S. typhim urium ,
a n d Y. enterocoUtica), m ech an ism s o f a d a p ta tio n m ay d iffer b etw een
o rganism s a n d w ith in c u ltu re su b p o p u latio n s. A sig n ifican t p ro p o rtio n o f th e
97
e x p o sed p ath o g en ic b a c te ria (en tero to x ig en ic E. coli, S. typhim urium , an d Y.
enterocolitica) becam e n o n re c o v era b le o n n o n selectiv e m ed iu m a t 37°C.
H ow ever, th e se b a c te ria re ta in e d re s p ira to ry activ ity a n d th e cap a b ility fo r
re g ro w th a t th is sam e te m p e ra tu re in liq u id m edium . This in d ic a tes th e
p o ssibility o f re g ro w th a n d p athogenesis in a n a p p ro p ria te h o st. T he
in c re asin g fra c tio n o f slow ly grow ing co lo n ies is c o n siste n t w ith p re v io u s
o b serv atio n s o f d e lay e d reg ro w th o f R coli u p o n reco v ery fro m exposure to
re d u c e d te m p e ra tu re s (21). R elatively h ig h -lev el reco v eries o f Y.
enterocolitica o b serv ed a t low er te m p e ra tu re s in th e co n tro l a n d exposed
p o p u la tio n s a re th o u g h t to b e d u e to th e p sy ch ro tro p h ic n a tu re o f this
o rg an ism (50).
G ood g en eral a g re em e n t betw een DVC (+) a n d CTC (+) v a lu es in d icated
th a t th e se assays w ere sim ilar in th e ir a b ility to assess p h ysiological activity,
a lth o u g h th e y m e a su re d iffe re n t cellu lar processes. T he stab ility o f CTC (+)
a n d DVC (+) cell n u m b e rs (a t ca. 1% o f in oculum ) fo r th e e n te ric bacilli
in d ic a te s th a t th e se o rganism s p e rsiste d in a n activ e a n d su b strate-resp o n siv e
state a t ca. 0.1% o f in o cu lu m n u m b ers fo r a t le ast 54 days. W hen th ese results
a re c o m p a re d w ith n o n selectiv e p la te c o u n t reco v eries, i t is c le a r th a t th e re
w as a progressive sh ift to a VBNC state u p o n p o la r m a rin e ex p o su re in
su sp en sio n s o f E. coli, S. typhim urium , and Yr enterocolitica (Figs. 8-11). This
g e n eral tre n d h a s b e e n p rev io u sly o b se rv e d in m a n y e n te ric b a c te ria o n
ex p o su re to aq u atic e n v iro n m e n ts (4, 26, 4 4 ). T hese o b serv atio n s a re also
c o n sisten t w ith th o se o f Dawe a n d Penrose, w ho fo u n d n o d e crea se in ATP
levels b u t d ecreasing p la te c o u n ts in colifofm suspensions e x p o sed to a n 8- to
12°C m a rin e e n v iro n m e n t (10). The p re s e n t re su lts in d ic a te th a t p la te c o u n t
e n u m e ra tio n s, p a rtic u la rly o n selective m e d ia , m a y sig n ifican tly a n d
98
p ro g ressiv ely u n d e re stim a te n u m b e rs of v ia b le en teric b a c te ria in co ld (ca.
<10°C) m a rin e e n v iro n m en ts. Results o b serv ed w ith E faecalis in d ic a ted th a t
CTC re d u c tio n assays significantly u n d e re stim a te d v iab le cell n u m b e rs o n
n o n selectiv e m e d iu m follow ing e n v iro n m e n ta l ex p o su re (Fig. 11). Thus, CTC
re d u c tio n is a p o o r in d ic a to r o f E faecalis v iab ility in th is e n v iro n m e n t.
A d d itio n of re la tiv e ly h ig h n u trie n t c o n ce n tra tio n s to cells exposed fo r
48 d ay s significantly in c re a se d p e rc en ta g e s o f re sp irin g E coll a n d S.
ty p h im u riu m cells o v er levels o b serv ed fo r c o n tro ls w ith n o n u trie n t a d d itio n
a t all te m p e ra tu re s exam ined (Fig. 14A a n d B). Slight in creases w ere also n o te d
fo r Y. enterocolitica cells (Fig. 14C). This in d ic a tes th a t la rg e n u trie n t in p u ts
in c re ase re s p ira to ry activities o f d o rm a n t (i.e., show ing n o re s p ira to ry
activity b y CTC red u ctio n ) E. coli, a n d S. typhim urium , a t th e low in situ
te m p e ra tu re , revealing th a t th e m a jo rity o f th e se cells m a in ta in e d th e
cap acity fo r u p ta k e a n d m etabolism o f a d d e d n u trie n ts. C oastal p o la r m a rin e
e n v iro n m e n ts a re c h a ra c te riz e d b y seaso n ally h ig h p ro d u c tiv ity a n d som e o f
th e h ig h e st re p o rte d p a rtic u la te a n d d isso lv ed org an ic c a rb o n c o n c e n tra tio n s
in m a rin e surface w aters (38, 54). It w as n o te d th a t in creases in resp irin g
n u m b e rs o f E coli, S. typhim urium , a n d Y. enterocohtica a fte r 25 d ay s o f
exposure c o rre sp o n d e d w ith th e a n n u a l p h y to p la n k to n b lo o m o b se rv e d d u rin g
c o n c u rre n t stu d ies (20, 39). T hus, la rg e n u trie n t in p u ts m a y allow E coli, S.
typ h im u riu m , a n d possibly Y. enterocohtica to p e rsist in a nongrow ing,
m etab o lically active, VBNC s ta te b y p ro v id in g su b stra te s fo r cellu lar
m a in te n a n c e e n e rg y re q u ire m e n ts. In c re a se d levels o f n u trie n ts h a v e b e e n
fo u n d to in c re ase su rv iv al ra te s o f e n te ric b a c te ria in seaw ater (b y usin g p la te
counts as a m easu re o f viability) (8 ,3 5 ) . In ad d itio n , W eibe e t al. recen tly
d e m o n stra te d th a t in c re a se d s u b stra te c o n ce n tra tio n s a re re q u ire d to m a in ta in
99
g row th ra te s o f b o th a u to c h th o n o u s m eso p h ilic m a rin e h e te ro tro p h ic b a c te ria
a n d E. coli a t m inim al g row th te m p e ra tu re s (IO 0C fo r £ coU) (53). However,
several re p o rts h a v e ch allen g ed th a t th is o b serv atio n h o ld s tru e a t
te m p e ra tu re s below ca. 12°C (9, 28). P revious sh o rt-te rm co ld exposure
ex p erim en ts h av e also show n th a t tra n s la tio n in itia tio n , as w ell as b in d in g a n d
tra n s p o rt o f am in o acids, is in h ib ite d in £ coli below 8°C (7, 15). However,
tra n sp o rt, in c o rp o ra tio n , a n d m in eralizatio n o f a d d e d am in o acid s w ere n o t
com pletely in h ib ite d . Cold survival in th e VBNC state w ith re d u c e d ra te s of
re s p ira to ry activ ity h a s also b e e n re p o rte d fo r th e e n teric p a th o g e n Vibrio
v u ln ific u s (37). Low te m p e ra tu re , r a th e r th a n m inim al n u trie n ts , a p p e a rs to
b e a n im p o rta n t d e te rm in in g fa c to r lim itin g re s p ira to ry a ctiv ity a n d
p ro m o tin g th e VBNC sta te in th is o rg an ism (36). In th e p re s e n t study,
re s p ira to ry activ ity o f £ coli a n d S. typ h im u riu m a p p ea rs lim ited b y n u trie n t
a v ailab ility r a th e r th a n b y te m p e ra tu re in th is co ld e n v iro n m e n t (Fig. 4A a n d
B).
In cu b atio n o f 54-day-exposed £ coli, S. typhim urium , arid Y.
e n te rocolitica a t 37°C in liq u id m ediu m (DVC, CTC, a n d re g ro w th experim ent)
re v e a le d la rg e n u m b e rs o f cells c a p a b le o f d isp lay in g o r re g a in in g m etab o lic
activity, w hile n o cells co u ld b e re c o v ere d a s colonies o n th e so lid m e d ia u sed
a t th a t te m p e ra tu re (Fig. 13). Evidently, th e r e w as som e re s p o n se in 54-dayexposed cells w hich p re c lu d e d colony fo rm a tio n o n solid m e d ia , b u t n o t
re sp ira tio n o r grow th in liq u id m ed iu m a t 37°C. This m ay b e d u e to th e
d iffe re n t osm otic e n v iro n m e n t of a liq u id m e d iu m in c o m p ariso n w ith th e
su rface o f a n a g a r p la te .
In sum m ary, e x p o su re o f e n te ric b a c te ria to a v e ry cold, p o la r m a rin e
e n v iro n m e n t in c re a se d th erm o sen sitiv ity , as d e m o n stra te d b y a su b se q u e n t
' 100
re d u c tio n in optim al te m p e ra tu re fo r co lo n y fo rm atio n fro m 37 to <20°C.
R esp ira to ry activ ity in E. coli, S. typhim urium , a n d Y. enterocolitica was
lim ite d b y n u trie n t a v ailab ility r a th e r th a n te m p e ra tu re in th is e n v iro n m e n t.
D ecay ra te s o f E. coli, S. typhim urium , a n d Y. enterocohtica w ere two to fo u r
tim es g re a te r in th e p re s e n t stu d y th a n in p re v io u s in situ m a rin e studies of E.
coli survival a t <10°C. Exposure also re s u lte d in a p ro g ressiv e in crease in th e
n u m b e r o f in ju red , VBNC, a n d inactive cells in th ese p o p u la tio n s w ith time,
re su ltin g in sig nificant u n d e re stim a tio n s o f a c tu a l n u m b e rs o f v iab le
organism s in th e e n v iro n m e n t b y th e p la te c o u n t m eth o d . W hile E. faecalis
d e ca y ra te s w ere th e low est o f all organism s te ste d o n n o n selectiv e m edium ,
th is o rg an ism ra p id ly becam e n o n re c o v e ra b le o n th e selective m ed iu m
com m only u se d fo r its d etectio n . T h erefo re, th is o rg an ism (using mE a g a r fo r
en u m eratio n ) is u n su ita b le as a n in d ic a to r o f th e p re sen c e o f
e n te ro p a th o g e n ic b a c te ria in th is e n v iro n m e n t. W hile th e d e c a y ra te s o f
re c o v era b le cells a p p e a re d sim ilar fo r E. coli, S. typhim urium , a n d Y.
en tero co h tica o n all m e d ia tested , it is su g g ested th a t in c u b a tio n o f sam ples fo r
coliform e n u m eratio n fro m <10°C m a rin e e n v iro n m e n ts b e re d u c e d to <20°C if
p la tin g tech n iq u es a re u sed . M o st-p ro b ab le-n u m b er e n u m e ra tio n s using
liq u id m e d ia m ay also give b e tte r reco v eries th a n a g a r p la te assays. However,
d a ta th u s o b ta in e d sh o u ld b e co n sid e re d m in im u m estim ates o f v iab le o rganism
n u m b e rs in th is ex trem e e n v iro n m e n t b e ca u se o f th e c o n sid e ra b le n u m b e r o f
VBNC a n d in ju re d coliform s expected o n th e b a sis o f th e p re s e n t stu d y .
101
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105
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106
CHAPTER 5
PLASMID MAINTENANCE AND EXPRESSION IN ESCHERICHIA COII
EXPOSED TO A POLAR MARINE ENVIRONMENT
In tro d u c tio n
Sewage d isch a rg e d in to m a rin e e n v iro n m e n ts is e x p ec te d to c o n tain
e n te ric b a c te ria h a rb o rin g a n tib io tic -re sista n c e , as w ell as v iru len c eassociated plasm ids (5, 8). In tro d u ctio n o f la rg e n u m b ers o f p lasm id ­
h a rb o rin g h u m a n e n te ric b a c te ria fro m a p o in t so u rce in c re ase s th e
likelih o o d o f h o riz o n ta l tra n s fe r o f th e se g en es to n a tu ra lly o ccu rrin g
b a cte ria l p o p u latio n s. T h e te rm “genetic p o llu tio n ” h a s b e e n u s e d to d escrib e
th is in tro d u c tio n o f new genetic m a te ria l, o r th e tra n sfe r o f genes in th e
e n v iro n m e n t re su ltin g fro m a n th ro p o g e n ic activities (14). T ra n sfe r of
v iru len ce, co njugative, a n d /o r an tib io tic re sista n c e (R) genes m a y re s u lt in
th e in c re a s e d d isse m in a tio n o f a n tib io tic -re sista n c e a n d /o r v iru le n c e
d e te rm in a n ts to b a c te ria in th e e n v iro n m e n t, w ith u n k n o w n im p acts o n th e
in d ig en o u s m a rin e m a c ro fa u n a (18). This is o f p a rtic u la r c o n c e rn in th e
a n ta rc tic m a rin e e n v iro n m e n t w h ere m a c ro fa u n a m ay h a v e little n a tu ra l
re sista n c e to a n th ro p o g e n ic a lly in tro d u c e d p a th o g en ic m icro o rg an ism s.
Plasm ids a p p e a r to b e m a in ta in e d a n d expressed in b a c te ria exposed to
m arin e, lak e a n d well w aters a t 15-25°C, a n d h a v e even b e e n fo u n d in b a c te ria
from subsurface sedim ents as d eep as 360 m (1-3, 6). Plasm ids also a p p e a r to b e
107
m a in ta in e d in v ia b le -b u t n o n c u ltu ra b le (VBNC) E. coli a fte r m a rin e ex posure
(2). T he a u to ch th o n o u s a n ta rc tic m a rin e b a c te ria o f M cM urdo Sound,
A n ta rc tica h av e p re v io u sly b e e n fo u n d to h a rb o r a n tib io tic-resistan ce
p lasm id s (9). However, th e e x te n t to w hich th e se plasm ids w ere th e re su lt of
h o riz o n ta l tra n s fe r fro m M cM urdo S tatio n sewage b a cte ria is n o t know n.
Several stu d ies h a v e show n th a t n a tu ra l genetic ex ch an g e
(tran sfo rm atio n , con ju g atio n , a n d tra n sd u c tio n ) takes place in m arin e
e n v iro n m e n ts, a n d th a t R plasm id s a re tra n s fe rre d fro m e n te ric (i.e. E. coli) to
m a rin e b a c te ria (V ibrio spp.), as well as fish p ath o g en ic species (Aerom onas
sahnonicida) (I, 10, 14). Low te m p e ratu res (& 4°C), a n d /o r olig o tro p h ic
co n d itio n s a p p e a re d to h a v e little effect o n freq u en cies o f gen e tra n sfe r in
th e se m a rin e studies. T hese co n d itio n s h av e also b een fo u n d to p ro m o te
in d u c tio n o f th e VBNC state in en teric b a c te ria (15). In a d d itio n , low
te m p e ra tu re s e x te n d su rv iv al o f e n te ric b a c te ria in m a rin e e n v iro n m e n ts (11,
16, see c h a p te r 2). T hese studies suggest th a t release of p lasm id -h arb o rin g
e n te ric b a c te ria in to p o la r m a rin e e n v iro n m e n ts (c h a ra c te riz e d b y low
te m p e ra tu re s, low grazing ra te s, as w ell as h ig h ly seasonal p rim a ry a n d
sec o n d a ry p ro d u c tiv ity ) m a y re s u lt in p ro lo n g e d survival, a n d h e n c e g re a te r
o p p o rtu n ity fo r gene tra n s fe r to a u to c h th o n o u s m icro o rg an ism s, o r in fectio n
o f in d ig e n o u s m a c ro fa u n a .
In o rd e r to d e te rm in e w h e th e r p la sm id s a re m a in ta in e d a n d expressed
in en teric b a c te ria exposed to p o la r m a rin e conditions, E. coli h a rb o rin g e ith e r
a n R p la sm id (pUC19) o r conju g ativ e F-plasm id (w hich also co d es fo r an tib io tic
resistance) w ere exposed in situ to a p o la r m a rin e e n v iro n m e n t fo r 21-54 days.
Plasm id expression w as m o n ito re d b y p la tin g o n m ed ia w ith a n d w ith o u t th e
antibiotics w hose re sista n c e was co d ed o n th e respective plasm ids. Plasm id
108
m a in te n an c e was exam ined b y e x tractio n fro m exposed cells a n d visualization
b y m in ip re p a ra tiv e gel elec tro p h o resis. S u b le th a l in ju ry , s u b stra te
responsiveness, a n d re s p ira to ry activ ity w ere also m o n ito re d in exposed E. coli
(pUC19), a n d resu lts c o m p ared w ith th e w ild-type strain .
M a te ria ls a n d M e th o d s
O rganism s. G row th C onditions, a n d Exposure
T he w ild-type (WT) entero to x ig en ic s tra in Escherichia coli TX-432
(078:H12) w asC aC l2-transform ed w ith th e h ig h -c o p y n u m b e r, am picillinresistance plasm id pUC19 to p ro d u c e s tra in E. coli TX-432 (pUC19) (12). This
o rg an ism was grow n to e a rly -sta tio n a ry p h a s e in try p to n e-lac to se-y e a st
e x tra c t (TLY) b ro th su p p lem en te d w ith am p icillin (50 mg/1) (TLYA) a t 37°C.
T he p a re n t s tra in w as grow n in th e sam e m e d iu m w ith o u t am picillin.
Escherichia coli K-12 (pFam p) (Str i A m pr ) h a rb o rin g th e c o n ju g ad v e Fp la sm id (th e F-plasm id also e n co d es am p icillin resistan ce) w as grow n to earlysta tio n a ry p h ase in th e following m edium ; 13 g try p to n e, I g glucose, 8 g NaCl,
0.3 g MgS04° 7 H 2 0 ,0.3 g CaClz 0 H2O, a n d 15 m g each am picillin a n d
strep to m y cin p e r 1,000 m l d eio n ized w a te r (TRY-AS). All organism s (E. coh TX432 [WT], E. coli TX-432 [pUC19], £ coli K-12 [pFam p]) w e re su b se q u e n tly p la ce d
in m e m b ra n e diffusion ch am b ers a n d ex p o sed in situ to th e a n ta rc tic m arin e
e n v iro n m e n t a t M cM urdo Station, A n tarctica as d escrib ed p rev io u sly (16, see
c h a p te r 2). Exposure tim es w ere 54 d ay s fo r £ coh TX-432 (WT) a n d £ coh TX432 (pUC19), a n d 21 days fo r £ coh K-12 [pFam p]. Strain £ coh K-12 (pFam p)
109
was o b ta in e d from Dr. M ark Sobsey (Univ. o f N. C arolina), a n d is com m only
u s e d as a n F-specific b a cte rio p h a g e h o s t (4).
Plasm id Expression
At tim e zero, as well as a fte r v a rio u s tim es of exposure, cells w ere d ilu te d
a n d spot-, o r sp read -p lated o n to th e sam e m ed iu m u sed fo r grow th (see above)
w ith a n d w ith o u t antib io tics (i.e., E. coli TX-432 [pUC19] o n TLY/TLYA, E. coli K12 [pFamp] o n TRY/TRY-AS). All d ilu tio n s w ere p e rfo rm e d in filter-sterilized ,
ag ed seaw ater a t ca. -1°C. Subsam ples o f E. coli TX-432 (WT) w ere p la te d on
TLYA th ro u g h o u t 54 days exposure a n d n o A m pr m u ta n ts w ere n o ted . All assay
in c u b atio n s w ere a t 25°C. T his te m p e ra tu re w as p rev io u sly fo u n d to give
o ptim al re c o v ery o f £ coli TX-432 (WT) (16. see c h ap te r 2).
P lasm id M ain ten en ce
O ne-m l subsam ples o f £ coli TX-432 [pUC19] (ca. 10& cells) w ere
cen trifu g ed a n d pellets sto re d a t -70°C. Plasm id DNA was e x tra c te d b y th e
alk alin e lysis m in ip re p a ra tiv e p ro c e d u re in c lu d in g RNAse (20 p g /m l)
tre a tm e n t (12). Plasm ids w ere d e te c te d b y ag aro se (0.9%) gel electro p h o resis
r u n in Tris-acetate-EDTA b u ffe r (12). Gels co n siste d o f I la n e HindlII-X
stan d a rd , I la n e of pUC19 sta n d a rd , a n d sam p le lanes fro m v ario u s exposure
tim es.
D irect V iable C ounts a n d S u b leth al In ju ry
S u b strate responsiveness (DVC), re s p ira to ry activity (as m e a su re d b y 5cyano-2,3 -ditolyl tetrazo liu m c h lo rid e [CTC] re d u c tio n to flu o rescen t,
in tra c e llu la r fo rm azan ), a n d su b leth al in ju ry (TLY -vs- TLYD [TLY + 0.1%
HO
deoxycholate] platin g recoveries) w ere a ssa y ed as d e sc rib e d p rev io u sly (16, see
c h a p te r 2).
R e s u lts
Plasm id M aintenence a n d Expression
Isolation o f pUC19 fro m R coli TX-432 [pUC19] a n d E coli K-12 [pFamp]
cells a fte r v ario u s p erio d s o f exposure in d ic a te d p lasm id m a in te n an c e
th ro u g h o u t 54 d a y exposure (gels n o t show n). Plate c o u n ts o n m e d ia w ith
(TLYA) a n d w ith o u t (TLY) antibiotics in d ic a te d th a t b o th pUC19 a n d F-plasm ids
w ere ex p ressed th ro u g h o u t exposures in th e v a st m a jo rity o f c u ltu ra b le E coli
TX-432 [pUC19] an d E . coli K-12 [pFamp] cells, resp ectiv ely (Figs. 15 a n d 17). A
sm all fra c tio n (ca. 1-10%) o f E. coli K-12 [pFamp] cells d id n o t express th e Fp lasm id p rio r to d a y 8, a fte r w hich it w as ex p ressed in 100% o f exposed cells
(Fig. 17). This sam e g en eral p a tte rn was o b serv ed w ith E. coli TX-432 [pUC19]
betw een 7 a n d 15 days (Fig. 15).
R eco v erab ility a n d S u b le th a l In iu ry
E coli K-12 [pFamp] show ed g re a te r su rv iv al (tim e fo r 100-fold d ecrease
in re c o v era b le cell n u m b e rs [T99] = 30 d) th a n E coli TX-432 [pUC19] (T99= 15 d)
w h en m e a su re d using no n selectiv e p la tin g m ed ia. H ow ever, su rv iv al o f E coli
TX-432 [pUC19] o n nonselective m ed iu m w as p o o re r th a n th e w ild-type
plasm idless stra in (T99= 20 d) (Figs. 15 a n d 16).
Sublethal injury ([CFUnonselective’CFUselective/CFUnonselectivel X100
= % injury) increased rapidly and progressively in the E. coli TX-432 [pUC19]
Ill
p o p u la tio n , reach in g effectively 100% a fte r 11 days (Fig. 15). R ecoverability
o n selective m edium (TLYD) d ecreased m uch m o re rap id ly in th e pUC19c o n tain in g s tra in (T99 = 3.5 d) com pared to w ild-type (T99 ~ 14 d) containing no
plasm id (Figs 15 a n d 16).
• WT TLY
■ TLY
□ TLYD
a
TLYA
♦ AODC
0 DVC (+)
a
CTC (+)
0
5
10 15 2 0 2 5 3 0 3 5 4 0 45 5 0 5 5
Time (d)
F ig u re 15. Survival, plasm id expression, su b strate resp o n siv en ess (DVC) a n d
re sp ira to ry activity (CTC [+]) o f E. coliTX-432 (pUC119) exposed to a po lar
m arin e e n v iro n m e n t fo r 54 d ay s (d). Solid circles w ith h a tc h e d lines in d icates
TLY p la te c o u n ts o f w ild-type E. coli TX-432 fo r cam parison. Plate counts (n = 5)
w ere on TLY (nonselective), TLYD (selective), a n d TLYA (selective fo r pUC19
expression) agars. E rror b a rs re p re se n t s ta n d a rd dev iatio n s.
112
□ TLY
• TLYD
o AODC
♦ DVC (+)
V CTC (+)
5
10 1 5 2 0 2 5 3 0 35 4 0 4 5 5 0 55
Time (d)
F ig u re 1 6 . Survival, p lasm id expression, su b strate resp o n siv en ess (DVC) a n d
re sp ira to ry activity (CTC [+]) of w ild-type E m tiTX -432 exposed to a polar
m arin e en v iro n m e n t fo r 54 d ay s (d). Plate co u n ts (n = 5) w ere o n TLY
n onselecdve) a n d TLYD (selective) agar. E rro r b ars re p re se n t s ta n d a rd
d ev iatio n s.
CFU/ml
113
o TRYA
1
O
2
4
6
8
,
1
,
1
Jl___ I___L
10 1 2 14 1 6 1 8 2 0 22
Time (d)
F ig u re 1 7. R ecoverability a n d conju g ativ e F-plasm id ex p ressio n in E. coli K12 (pFam p) exposed to a p o la r m arin e en v iro n m e n t fo r 21 d ay s (d ). E rror b a rs
re p re se n t s ta n d a rd d eviations (n = 5).
114
DVCs, a n d CTC R eduction
E. coM TX-432 [pUC19] to tal cell n u m b e rs b y acrid in e o ran g e d ire c t
counts (AODC) re m a in e d re lativ ely c o n sta n t th ro u g h o u t th e 54 d a y exposure. E.
coli TX-432 [pUC19] CTC (+) cell n u m b e rs d ecreased steadily d u rin g th e first
w eek o f exposure, th e n a p p e a re d to stabilize a t ca. 1% o f to ta l cell n u m b ers
(Fig. 15) However, a fte r 27 days exposu re n o CTC (+) cells could b e d etected . In
co n trast, w ild-type CTC (+) cell n u m b e rs re m a in e d w ith in 1% o f to ta l cell
n u m b e rs th ro u g h o u t 54 days ex p o su re (Fig. 16).
E. coli TX-432 [pUC19] su b strate resp o n siv e (DVC [+]) cells decreased
steadily, th e n stabilized a fte r 7 days a t ca. 1% of to tal cell n u m b e rs (Fig. 15).
DVC (+) n u m b ers th e n re m a in e d a t ca. 1% o f to tal cell n u m b ers (AODC)
th ro u g h o u t th e 54 d a y exposure. This sam e g en eral tre n d w as n o te d in th e
w ild-type stra in (Fig. 16).
D isc u ssio n
Isolation of plasm ids, a n d e x p ressio n o f an tib io tic re sista n c e genes
en co d e d b y them , in d ic a te d an tib io tic -re sistan c e a n d co n ju g ativ e plasm ids
w ere m a in ta in e d a n d ex p ressed in E. coli w h en exposed in situ to th e A ntarctic
m a rin e e n v iro n m en t. In ad d itio n , low te m p e ra tu re (-1.8°C) a p p e a re d to
significantly e x te n d surv iv al o f all stra in s w h en co m p ared to te m p e ra te
m arin e en v iro n m en ts (11, 16, see c h a p te r 4 ). However, co m p ariso n betw een
w ild-type E. coli TX-432, a n d p lasm id-co n tain in g E. coli TX-432 (pUC19)
in d ic a te d sig n ifican tly p o o re r su rv iv al (m e a su re d b y colo n y -fo rm in g ability)
o f th e la tte r w ith exposure to th e p o la r m a rin e e n v iro n m e n t (Figs. 15 a n d 16).
115
W hile th e F-plasm id c o n tain in g s tra in sh o w ed th e b e st re c o v era b ility , overall,
th e m e d iu m u s e d fo r its e u m e ra d o n (TRY-AS/TRY), was d iffe re n t fro m th a t
u sed fo r th e R coli TX-432 stra in s (TLYA/TLY), m aking d ire c t com parisons
difficult. T he p lasm id -co n tain in g s tra in o f E. coli TX-432 show ed m u ch g re a ter
p ro g ressiv e su b leth al in ju ry o v e r th e p e rio d o f ex p o su re th a n th e w ild-type
(Figs. 15 a n d 16). It is n o t know n w h e th e r th is was d u e to th e p resen ce a n d
expression o f th e plasm id, o r d u e to th e p re sen c e o f th e cell-w all-synthesis
in h ib ito r am picillin in th e in itia l grow th m e d iu m u se d fo r th e plasm idco n tain in g stra in . G enerally, re sista n c e to a n an tib io tic is n o t a n ab so lu te in a
b acteriu m , e v en if it expresses th e re sista n c e p h e n o ty p e . T h e selective a g en t
u sed (deoxycholate) is a su rfa ctan t, w h ich p re su m ab ly acts a t th e cell
w a ll/m e m b ra n e . T hus, th e g re a te r in ju ry n o te d in th e p lasm id -co n tain in g
stra in could h a v e b e e n a re s u lt o f d a m a g e d cell-w all c o m p o n en ts in this stra in
d u rin g grow th w ith a n tib io tic. H ow ever, if th is is th e case, it suggests th e
p o ssib ility o f sig n ifican t u n d e re stim a tio n o f R -p h en o ty p e e n te ric b a c te ria
fro m th is e n v iro n m e n t usin g s ta n d a rd m e th o d s fo r e n u m e r a tio n w hich u se
su rfa ctan ts in
selective ag en ts in p la tin g m ed ia).
T he sam e g en eral su b stra te re sp o n siv e n e ss p a tte rn (DVC) w as n o te d fo r
b o th w ild-type, a n d pUC IS -co n tain in g E. coli d u rin g ex p o su re (Figs. 15 a n d
16). However, CTC (+) E. coli (pUC19) n u m b e rs d ecreased ra p id ly to n o n d etectab le levels a fte r 27 days exposure (Fig. 15). This is in c o n tra st to th e
w ild-type strain , w hich m a in ta in e d ca. 1% CTC (+) cell n u m b e rs th ro u g h o u t 54
days exposure (Fig. 16). As CTC re d u c tio n is sensitive to b o th in trac ellu lar
re d o x p o te n tia l a n d re s p ira to ry activ ity , i t a p p e a rs th a t th e se cellu lar
p a ra m e te rs d e c re a se d sig n ifican tly in th e p lasm id -co n tain in g stra in co m p ared
to th e w ild-type. As suggested above, i t is n o t know n w h e th e r th is w as a re su lt
116
o f actu a l p la sm id expression, o r p a rtia l c ellu lar d am ag e in th e in itial grow th
m e d iu m c o n tain in g am p icillin .
Plate co unts o n all m e d ia u se d p ro g ressiv ely u n d e re stim a te d DVC (+) cell
n u m b e rs a fte r 13 days ex p o su re, in d icatin g a n in creasin g p ro p o rtio n of VBNC
p la sm id -h a rb o rin g R coli d u rin g exposure (Fig. 15). T he p ro p o rtio n of VBNC R
coli TX-432 (pUC19) in c re ase d fro m 2 to n e a rly 5 o rd ers-o f-m ag n itu d e betw een
13 a n d 54 days exposure respectively. T h ese re su lts suggest th a t n o t only will
c o n v en tio n al p la te c o u n t assays u n d e re stim a te th e d en sity o f v iab le b a cte ria
in p o la r m a rin e e n v iro n m e n ts d u e to su b leth al in ju ry , b u t also d u e to a sh ift in
cell populatio n s to a VBNC state. These cells, w h e th er in ju re d a n d /o r VBNC
a p p e a r to m a in ta in p la sm id DNA in th e a b se n c e o f selective p re ssu re . These
resu lts a re co n siste n t w ith th e p rev io u s re su lts o f Byrd a n d Colwell w hich
show ed pla sm id (pBR322 a n d pUC8) m a in te n an c e in VBNC E. coli in te m p e ra te
m a rin e m icrocosm s (2).
W hile it h a s b e e n reco g n ized in p re v io u s stu d ies th a t p la sm id s are
m a in ta in e d in e n teric b a c te ria ex p o sed to v a rio u s aq u eo u s e n v iro n m en ts, th e
m ech an ism w h e reb y a b a c te riu m w ill m a in ta in seem in gly su p erflu o u s DNA in
th e ab sen ce o f selective p re s su re is u n c le a r. H ow ever, re c e n tly several
a u th o rs h a v e suggested th a t plasm id s c o n stitu te a “selfish” g e n e system w hich
leads to cell d e a th if plasm ids a re n o t m a in ta in e d (13, 17). H ow ever, th e
p lasm id s in th e se Studies c o n ta in e d re strictio n -m o d ificatio n system s, a n d th is
th e o ry is b a se d o n a co m p etitio n betw een th e se gene fu n ctio n s. N onetheless, it
d o es a p p e a r th a t plam id s a r e stab ly m a in ta in e d a n d ex p ressed in R coli u p o n
exposure to a p o la r m a rin e e n v iro n m e n t, seem ingly in th e a b se n c e o f d ire c t
selective (an tib io tic) p re s s u re .
117
Experim ents to show tra n sfe r o f p la m id DNA to th e a u to ch th o n o u s
m a rin e b a c te ria of M cM urdo Sound, A n tarctica w ere n o t p e rfo rm e d in th e
p re s e n t study. However, th e expressio n a n d m a in te n an c e o f antibioticre sista n c e a n d conjugative plasm id s in R coli in th e e n v iro n m e n t raises th e
p o ssib ility o f tra n s fe r of th e se genes to th e a u to c h th o n o u s m a rin e b a cte ria
th ro u g h conjugation, tra n sfo rm a tio n , o r tra n sd u c tio n . In a d d itio n , th e
likelihood o f gene tra n s fe r is in c re a se d d u e to th e lo n g e r p e rsiste n c e o f
e n te ric b a c te ria in a v iable state in th is co ld m a rin e e n v iro n m e n t (16).
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A ntarctic n a tu ra l m icro b ial assem blages. AppL Environ. M icrobiol. 48:515518.
10. Kruse, H., a n d H. S erum . 1994. T ran sfer o f m u ltip le d ru g resistan ce plasm ids
b etw een b a c te ria o f d iv e rse orig in s in n a tu ra l m ic ro e n v iro n m e n ts. AppL
Environ. M icrobiol. 60:4015-4021.
11. Lessard, E. J., a n d J. M. S ieburth. 1983. Survival o f n a tu ra l sewage
p o p u la tio n s o f e n te ric b a c te ria in d iffu sio n a n d b a tc h c h am b e rs in th e
m a rin e en v iro n m e n t. AppL Environ. M icrobiol., 45, 950-959.
12. M aniatis, T., E. F. Fritsch, a n d J. Sam brook. 1982. M olecular cloning: a
la b o ra to ry m a n u al. Cold S pring H arb o r L aboratory, Cold Spring H arbor,
N.Y.
13. N aito, T., K. K usano, a n d I. KobayashL 1995. Selfish b e h a v io r o f restrictio n m odification system s. Science 267:897-899.
14. Paul, J. H., M. E. F iischer, a n d J. M. T h u rm o n d . 1991. G ene tra n s fe r in
m a rin e w a ter colum n a n d sed im en t m icrocosm s b y n a tu ra l p lasm id
tra n sfo rm a tio n . AppL Environ. M icrobiol. 57:1509-1515.
15. Roszak, D. B., a n d R. R. Colwell. 1987. Survival strategies o f b a c te ria in th e
n a tu ra l en v iro n m e n t. M icrobiol. Rev. 51:356-379.
16. Sm ith, J. J., J. P. H ow ington, a n d G. A. M cFeters. 1994. Survival,
physiological resp o n se, a n d re c o v ery o f e n te ric b a c te ria ex p o sed to a p o la r
m a rin e en v iro n m e n t. AppL Environ. M icrobiol. 60:2977-2984.
17. Y arm olinsky, M. B. 1995. P rogram m ed cell d e a th in b a c te ria l po p u latio n s.
Science. 267:836-837.
18. Young, H.-K. 1993. A ntim icrobial re sista n c e sp re a d in aq u atic
en v iro n m en ts. J. A ntim icrob. C hem other., 31:627-635.
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CHAPTER 6
SUMMARY AND DISCUSSION
G e n e r a l R e s u lts a n d
C o n c lu s io n s
S urvival a n d Physiological R esponse o f E nteric B acteria to P o lar M arine
Exposure
M icrobes exist in all b u t th e m o st ex trem e en v iro n m e n ts o n e a rth .
P resum ably, th e y h a v e evolved to survive a n d , u n d e r a p p ro p ria te
co n d itio n s, m u ltip ly in p erm issiv e e n v iro n m e n ts. This is p a rtic u la rly th e
case w ith h u m a n e n te ric b a cte ria , w hich a re m o st com m only d isch arg ed
fro m w astew ater ou tfalls in to aq u eo u s en v iro n m en ts. It is n o w w idely
reco g n ized th a t, th e m a jo rity o f th e tim e, m o st o f m icroorganism s o n th e
p la n e t a re m e re ly su rviving r a th e r th a n activ ely grow ing a n d m u ltip ly in g
in th e e n v iro n m e n t (11, 22, 24, 30). U n fo rtu n ately , th e h isto ry o f
m icrobiology h a s focused o n grow th o f m icro o rg an ism s, w h e th e r fo r
e n u m e ra tio n , la b o ra to ry m an ip u la tio n , o r b io technological p u rp o se s (30).
C onsequently, i t is a n im p o rta n t c o n ce p t in e n v iro n m e n ta l m icrobiology
th a t, u n d e r e n v iro n m e n ta l co n ditions (i.e. o u tsid e th e la b o ra to ry o r host),
co p io tro p h ic b a c te ria a re m o st o fte n s ta rv e d fo r n u trie n ts, a n d n o t actively
in creasin g biom ass o r m ultiplying (11, 22, 30. 34). For th e p u rp o se s o f this
discussion, grow th w ill b e d e fin e d as m u ltip licatio n o f b a c te ria l n u m b e rs,
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r a th e r th a n a n in crease in biom ass, a lth o u g h th e fo rm er o fte n tak es p lace
w ith o u t th e la tte r. T he “n o n -g ro w th ” o r “d o rm a n t” state in b a c te ria h a s
b een th e su b ject o f num ero u s re c e n t studies (11, 26, 30). The g e n eral aims
o f th e se stu d ies w ere th e elu cid atio n o f th e a d ap tiv e m ech an ism s involved
in survival, as well as th e m etabolic processes w hich re m a in activ e a n d
allow th e cell to re sp o n d ra p id ly to a p p ro p ria te e n v iro n m e n ta l c o n d itio n s
fo r grow th. W ork b y M orita, a n d K jelleberg, am ong o th ers, (11, 23, 30)
fo cu sed larg ely o n th e survival of a u to c h th o n u o s m a rin e b a c te ria in th e
e n v iro n m e n t. O th er re c e n t w ork h a s fo cu sed m o re o n th e su rv iv al o f
b a c te ria o f p u blic h e a lth significance su ch a s E. coli, th e Salm onellae,
Vibrio cholera, a n d Vibrio vulnificus in a q u a tic e n v iro n m e n ts (4 ,1 5 ,2 6 ,
31). T he d e g re e to w hich e n u m e ra tio n o f th e se organism s in th e
e n v iro n m e n t is co m p ro m ised b y e n v iro n m e n ta l exposure, p o ssib ly
th ro u g h a d a p tiv e resp o n ses, is o f c o n sid e ra b le c o n ce rn to p u b lic h e a lth
a u th o ritie s. T hese officials re ly o n a c c u ra te e n u m e ra tio n o f in d ic a to r
b a c te ria in th e e n v iro n m e n t in o rd e r to assess pu b lic h e a lth risk . Since
cold te m p e ra tu re s h av e long b e e n reco g n ized to e x ten d th e su rv iv al o f m ost
b a c te ria in a q u eo u s e n v iro n m en ts, it w as o f in te re s t to d e te rm in e th e
p hysiological resp o n ses of h u m a n e n tero p a th o g e n ic a n d fecal in d ic a to r
b a c te ria to n a tu ra lly occurring cold e n v iro n m e n ts (2, 3, 8, 20, 30). It sh o u ld
b e em p h asized th a t m o st aq u atic e n v iro n m e n ts a re c o n sid e ra b ly co ld er
th a n th e h u m a n g u t (37°C), a n d a la rg e m a jo rity of m a rin e e n v iro n m e n ts
(ca > 90%) a re 5°C o r less (23).
C oncerns re la te d to th e p e rsiste n c e o f e n teric b a c te ria a n d th e
im p a c t o f u n tre a te d sew age d isch a rg e in to c o ld m a rin e e n v iro n m e n ts w ere
ra ise d a t M cM urdo Station, A ntarctica in th e la te 1980’s (9, 21). Use of
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d esalin ized seaw ater o b ta in e d n e a r th e M cM urdo S tation sew age o u tfall fo r
d rin k in g w ater, as w ell a s u n k n o w n effects o f p o la r m a rin e e x p o su re o n
e n teric b a c te ria l surv iv al a n d p ersiste n c e p ro v id e d th e im p etu s fo r this
study.
T he m ain goal o f th e p reced in g s tu d y w as to elu cid ate th e
physiological re sp o n se o f h u m a n e n te ro p a th o g e n ic a n d fecal in d ic a to r
b a c te ria to a v e ry cold, n a tu ra l m a rin e e n v iro n m en t. Em phasis was p laced
o n th e u se o f m eth o d s fo r th e d ire c t e n u m e ra tio n of v iable b a cte ria l
n u m b e rs usin g ep iflu o re sce n c e m icro sco p y r a th e r th a n th o se re q u irin g
b acte ria l grow th. T hese in c lu d e d re d u c tio n o f 5-cyano-2,3-d ito ly l
te tra zo liu m c h lo rid e (CTC) to flu o resc en t in tra c e llu la r fo rm az a n cry stals to
in d ic a te re s p ira to ry activ ity (29). N aladixic acid, a n in h ib ito r o f DNA
gyrase a n d cell division, was also u se d w ith a d d e d n u trie n ts (casam ino
a c id s/y e a st extract) to assess v iable a n d su b stra te resp o n siv e cells (DVC)
(12). M ethods re q u irin g g ro w th (i.e., co lo n y fo rm atio n ) h a v e b e e n show n
to significantly u n d e re stim a te n u m b e rs o f v iab le b a c te ria in a q u eo u s
e n v iro n m e n ts, w h e th e r th ro u g h su b le th a l in ju ry , o r th e VBNC re sp o n se
(30, 33). However, p la te counts w ere also p e rfo rm e d in o rd e r to assess
reco v erab ility , in ju ry , p la sm id expression, a n d th e VBNC re s p o n se in
exposed p o p u la tio n s o f e n te ric b a c te r ia .'
W hile p o la r m a rin e e n v iro n m e n ts re p re s e n t th e ex tre m es fo r low
te m p e ra tu re seaw ater (p o ised a t its freezin g p o in t, ca. -1.9°C), th e y
p ro v id e d a u n iq u e setting in w hich to stu d y th e effects o f low te m p e ra tu re
o n b a c te ria l su rvival a n d physiology in situ.
T h ree h u m a n e n te ric p a th o g e n s (e n tero to x ig en ic E. coli, Salm onella
typ h im u riu m , a n d Yersinia enterocolitica), tw o in d icato rs o f fecal
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c o n ta m in a tio n (Enterococcus faecalis, a n d E coli), a n d a p lasm id h arb o rin g
E coli s tra in w ere grow n to e a rly sta tio n a ry p h a se in ric h m e d iu m (TLY), a t
37°C a n d exposed lo n g -term (54-56 d) to th e A ntarctic m a rin e e n v iro n m e n t
(-1.8°C, 34.5 p p t salinity) using in situ d iffu sio n ch am b ers (18) (c h a p te r 4).
W ith re sp e c t to th ese experim ents, th e re su lts a re su m m arized as follows:
1.
AU o f th e b a c te ria u s e d d e m o n stra te d g re a te r p ersisten ce in this
e n v iro n m e n t c o m p ared to p rev io u s stu d ies in m o re te m p e rate m a rin e
reg io n s using p la te c o u n ts to assess v iab ih ty .
2.
All b a c te ria show ed sig n ifican t a m o u n ts o f su b leth al in ju ry ,
in d icatin g th e in a b ility o f som e b a c te ria to fo rm colonies o n selective
v e rsu s n o n selectiv e m ed ia.
3.
In d ic a to rs a n d p la sm id -h a rb o rin g E coli show ed g re a te r su b leth al
in ju ry th a n p a th o g e n s (S. typhim urium , Y. enterocolitica) d u rin g
exposure, a lth o u g h th e E coli stra in u s e d w as also a p a th o g en
(e n te ro to x ig e n ic ).
4.
D irect e n u m e ra tio n o f re sp irin g (CTC [+]) a n d s u b stra te resp o n siv e
(DVC [+]) ceUs in d ic a ted th a t ca. 10% o f E coli, S, typhim urium , a n d Y
enterocolitica p o p u la tio n s re m a in e d activ e th ro u g h o u t 54 d a y s o f exposure.
H ow ever, CTC re d u c tio n significantly u n d e re stim a te d n u m b e rs o f v iable E
faecalis a n d p la sm id -h a rb o rin g E coh a fte r I a n d 3 weeks exposure,
re s p e c tiv e ly .
5.
D irect m e th o d s in d ic a te d th a t e x p o su re p ro g ressiv ely in c re a se d
n u m b e rs o f VBNC E coli, S,. typhim urium , a n d Y. enterocolitica. Plate
co unts o fte n u n d e re stim a te d activ e cells u sin g d ire c t m eth o d s b y 3-5
o rd e r s-o f-m a g n itu d e .
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6.
Plasm ids coding fo r b o th a n tib io tic resistan ce (pUC19) a n d
co n ju g ativ e fu n c tio n s /a n tib io tic re sista n c e (Fam p) w ere sta b ly m a in ta in e d
a n d expressed in exposed E. coli. (c h a p te r 5).
Two significant physiological re sp o n se s w ere n o te d in E. coli, S,
typhim urium , a n d Y. enterocolitica o v e r th e p e rio d o f exposure:
1. Cells becam e m a rk e d ly th erm o sen sitiv e b y failing to fo rm colonies a t
37°C, a n d a p p e a re d to low er perm issive g row th te m p e ratu res to ca. 2 0°C.
2. A ddition o f n u trie n ts (0.1 X TLY) a t v a rio u s te m p e ratu res in d ic a ted
re s p ira to ry activity, as m e a su re d b y CTC re d u c tio n , was lim ited b y n u trie n t
a v ailab ility r a th e r th a n te m p e ra tu re in t h is e n v iro n m e n t.
T etrazo h u m R eduction M ethod V alidation
In th e stu d y o f b a cte ria l survival su m m arized above, CTC w as u sed a s
a n in d ic a to r o f b a c te ria l re s p ira to ry activ ity . T his no v el a n d relativ ely
n ew te tra zo liu m salt form s a flu o r e s c e n t in tra c e llu la r fo rm a z a n cry stal
w hich is m u c h e asie r to view th a n its n o n flu o re sc e n t relativ e, INT (29).
However, in th e co u rse o f lite ra tu re in v e stig atio n s to d e te rm in e th e exact
m echanism s o f re d u c tio n o f th e se c o m p o u n d s in b a c te ria it b ecam e
a p p a re n t th a t th is a re a w as larg ely u n in v e stig a te d (25, 32). H aving u sed
CTC to d e term in e “re s p ira to ry activity” in th e previous studies, a n d
c o n sid erin g i t is bein g u s e d w ith in c re asin g fre q u e n c y a s a n in d ic a to r of
b a c te ria l viability, a q u e stio n a ro se as to e x actly w h a t cellu lar process(es)
CTC red u ctio n indicated.
Using th e w ell-d escrib ed E. coli K-12 aero b ic re s p ira to ry c h ain as a
m odel, th e effects o f specific in h ib ito rs a n d stim u lato rs o f e le c tro n
tra n s p o rt a n d oxidative p h o sp h o ry la tio n o n CTC a n d INT re d u c tio n w ere
124
e x am in ed w ith b o th w hole cells a n d in v e rte d m e m b ra n e vesicles (25, 28).
Results in d ic a te d m echanism s a n d site(s) o f re d u c tio n o f INT a n d CTC u n d e r
b o th en d o g en o u s (w hole cells) a n d d e fin e d (vesicles) d e h y d ro g e n ase
activities. T etrazolium re d u c tio n in re la tio n to oxygen c o n su m p tio n
m e asu re m e n ts in d ic a te d th e d eg ree o f co u p lin g o f aero b ic re sp ira tio n to
CTC/INT red u ctio n . R eduction o f b o th CTC a n d INT w as also exam ined u n d e r
several a n ae ro b ic co n d itio n s d e sig n e d to te st re d u c tio n u n d e r fe rm e n ta tio n
a n d a n ae ro b ic re s p ira tio n (c h a p te r 2). S u b stra te s o f th e p rim a ry aerobic E.
coli re s p ira to ry c h ain w ere also u sed to assess w hich gave o p tim al
fo rm az a n p ro d u c tio n a n d CTC/INT (+) cell values. A erobic a n d an aero b ic
CTC/INT (+) values w ere also c o m p ared to p la te counts o n R2A a g ar
(c h a p ters 2 a n d 3). Finally, th e effect o f in o rg an ic p h o sp h a te o n CTC
re d u c tio n was inv estig ated .
R esults o f th e se experim ents stu d y in g p ro k a ry o tic CTC a n d INT
re d u c tio n a re sum m arized as follows:
1.
CTC a n d INT w ere re d u c e d b y co m p o n en ts o f p ro k a ry o tic re sp ira to ry
chains, w ith CTC re d u c tio n m o re tig htly c o u p led to aero b ic re s p ira to ry
a ctiv ity (oxygen consum ption) th a n INT in w hole cells (c h a p te r 2).
2.
CTC was re d u c e d b y b o th p rim a ry d eh y d ro g en ases [NADH (NADH-D),
a n d succinate (SDH)] activities, w hile INT w as re d u c e d b y th e se , as well as
u b iq u in o n e a n d possib ly cy to ch ro m es 6556,555 (c h a p te r 2).
3.
CTC re d u c tio n to fo rm azan [i.e., CTC (+) cells] was stro n g ly d ecreased
b y in h ib ito rs o f th e te rm in a l oxidases (KCN, azide) a n d tra n sm e m b ra n e
p o te n tia l (CCCP, 2, 4 -d in itro p h en o l). T h is is in c o n tra st to INT re d u c tio n to
form azan, w hich w as stro n g ly stim u la te d b y th e se in h ib ito rs (C hapter 2).
125
4.
CTCs s ta n d a rd m id -p o in t re d u c tio n p o te n tia l was -200 mV. However,
CTC a p p e a re d to fo rm a diffuse, poorly-, o r n o n -flu o rescen t fo rm az a n u n d e r
c e rta in co n ditions w hich a p p e a re d to re la te to h ig h e r (m o re positive) red o x
p o ten tials (> c a . -100 mV) (c h a p ter 2).
5.
INT a n d CTC b o th ex h ib ited re d u c tio n a fte r fixing cells w ith
fo rm alin , p re su m ab ly re la te d to re sid u a l in tra c e llu la r re d u c tio n p o te n tia l.
It is reco m m en d ed th a t cells be killed fo r a t le a s t 15 m in. w ith th is ag en t
p rio r to tetrazo liu m re d u c tio n assays (c h a p te r 2).
6.
Both CTC a n d ENT w ere re d u c e d to significant d eg rees u n d e r
a n ae ro b ic conditions, p a rtic u la rly glucose fe rm e n ta tio n . G lucose, R2A
w ith o u t ph o sp h ate, a n d fo rm ate a p p e a re d to optim ally p ro m o te CTC
re d u c tio n , w hile en d o g en o u s re s p ira tio n p ro d u c e d g re a te st INT re d u ctio n .
N u trien t ad d itio n was necessary fo r CTC (+) counts to exceed p la te counts
u n d e r a n ae ro b ic co n d itio n s, w hile e n d o g e n o u s re s p ira tio n w as re q u ire d
fo r INT (+) cells to exceed p la te counts (c h a p te r 2).
7.
S u b stra te s of th e p rim a ry aero b ic d e h y d ro g e n ases (su ccin ate, am ino
acid, fo rm ate, lactate, glycerol) as well as glucose stro n g ly p ro m o te d CTC
re d u c tio n c o m p ared to en d o g en o u s re sp ira tio n . INT re d u c tio n w as
d ecrea se d b y glucose a n d glycerol as su b stra te s (c h a p te r 3). A erobic
CTC/INT (+) cell n u m b e rs exceeded p la te co u n ts b y 0.5-2 tim es, except w ith
INT + glycerol,(c h a p te r 3).
8.
CTC re d u c tio n to flu o rescen t fo rm az a n w as in h ib ited a b o v e 20 mM
p h o sp h a te , a n d a p p e a re d to p rogressiv ely fo rm m o re n o n flu o re sc e n t
fo rm a z a n w ith in c re asin g p h o s p h a te c o n c e n tra tio n (c h a p te r 3).
9.
CTC a n d INT w ere re d u c e d ab io d cally b y red u cin g ag en ts such as
d ith io n ite, thioglycollate, a n d asco rb ate. In d e e d , a n y re d u c in g system s
126
p re s e n t w ith E1/2 values low er th a n th a t o f INT o r CTC w ould b e expected to
le a d to re d u c tio n of th e tetrazo liu m salts (32). In a d d itio n it a p p e a rs th a t
m icrobial poly sacch arid es such as a g ar a n d alg in ate m ay re d u c e INT a n d
CTC resp ectiv ely u n d e r a p p ro p ria te co n d itio n s (c h a p te r 2).
D is c u s s io n
Enteric B acteria a n d Polar M arine Exposure
S ta n d a rd m e th o d s o f re c o v ery a n d e n u m e ra tio n o f fecal in d ic a to r
b a c te ria fro m m a rin e e n v iro n m e n ts u tilize co lo n y form in g a b ility o r
grow th a t 37°C using selective m e d ia (I ) . H ow ever, th e se m e th o d s largely
ig n o re th e physiological state o f b a c te ria in th e e n v iro n m e n t w ith re g a rd s
to th e in ju re d state, a n d cells w hich a re v ia b le -b u t-n o n c u ltu ra b le . In
a d d itio n , th e th e rm a l stress im posed o n b a c te ria u p o n te m p e ra tu re upshifts
o f ca. 20 to n e a rly IOO0C betw een n a tu ra l e n v iro n m e n t a n d in c u b a to r
sh o u ld b e ex p ected to h a v e som e effect o n su b se q u e n t grow th.
C onventional defin itio n s o f d e a th in p ro k a ry o te s a re also la rg e ly b a se d o n
grow th a s m e a su re d b y cell division (26). A b a c te riu m m a y re s p o n d to
n u trie n ts a n d re sp ire , b u t n o t fo rm colonies o n a p late, p a rtic u la rly in th e
p re se n c e o f selective ag en ts. A b a c te riu m in th is state is o b v io u sly n o t
“d e a d ”, ju s t in ju re d , a n d /o r n o n c u ltu ra b le ,...o r m o re a p p ro p ria te ly
“n o n re c o v e ra b le ”. B acteria in th e se sta te s h a v e b e e n sh o w n to re ta in th e ir
p a th o g en icity , a n d h e n c e re p re s e n t a p o te n tia l fo r sig n ifican t
u n d e re stim a te s o f fecal p o llu tio n (16, 33). It is th e re fo re im p o rta n t to
/
127
reco g n ize th e e n v iro n m e n ta l c o n d itio n s w hich p ro m o te in ju ry a n d
n o n c u ltu ra b ility in e n te ric b a c te ria l in d ic a to rs a n d e n te ro p a th o g e n s.
W hen sewage is re le a se d in to aq u atic e n v iro n m en ts, th e com m on
p e rc e p tio n is th a t d ilu tio n a n d d e a th la rg e ly elim in ate th e asso ciated
e n te ric b a c te ria l flora. H ow ever, in th e p o la r m a rin e e n v iro n m e n t
c o n d itio n s su ch as g e n erally low c u rre n t sp eed , low p re d a tio n ra te s,
extrem ely cold te m p e ratu res, a n d a tte n u a te d so lar ra d ia tio n coinbine to
allow th e e x te n d e d surv iv al a n d p e rsiste n c e o f e n te ric b a c te ria l in d ic a to rs
a n d p a th o g en s (ch ap ters 4 a n d 5).
R esults fro m th is s tu d y in d ic a te th a t su rv iv al o f h u m a n e n te ric
b a c te ria com m only a sso c iate d w ith sew age is sig n ifican tly e x te n d e d in
p o la r v e rsu s te m p e ra te m a rin e e n v iro n m e n ts. P erh ap s m o s t significantly,
th is e n v iro n m e n t in d u c e s large, p ro g ressiv e a m o u n ts o f in ju ry a n d
n o n c u ltu ra b ility in exposed b a c te ria . N um bers o f v iab le b a c te ria
d e te rm in e d b y d ire c t m eth o d s in d ic a te d th a t ca. 10% o f all b a c te ria
re m a in e d v iable th ro u g h o u t 54 d ay s o f exposure. This le d to la rg e
u n d e re stim a tio n s o f v iable cell n u m b e rs using p la te counts c o m p ared to
d irect-v iab le d e tec tio n m eth o d s. In a d d itio n , th e b a c te ria a p p e a re d to
b eco m e m a rk e d ly th erm o sen sitiv e w ith ex p o su re re la tiv e to co lo n y
fo rm atio n . This m a y b e d u e to a n a d a p ta tio n p ro d u cin g g re a te r
u n s a tu ra tio n in m e m b ra n e lip id s in o rd e r to re ta in m e m b ra n e flu id ity in
som e b a c te ria a t low te m p e ra tu re s, how ever, th is a sp e c t re m a in s
u n in v e stig ate d (13). In a d d itio n , re s p ira to ry activ ity was lim ite d b y
n u trie n ts r a th e r th a n te m p e ra tu re . T h ese re su lts in d ic a te d th a t en teric
b a c te ria m a y re m a in v iab le fo r significantly e x te n d e d p e rio d s in p o la r
m a rin e e n v iro n m e n ts. U tilization o f o rg an ic m a tte r fro m a n n u a l
128
p h y to p la n k to n bloom as w ell as p rim a ry sew age efflu en t m ay allow th ese
b a c te ria to fu r th e r p ro lo n g v ia b ility in th e e n v iro n m e n t b y p ro v id in g
n e e d e d c e llu la r m a in te n a n c e en erg y .
T ra n sfe r o f a n tib io tic re sista n c e a n d /o r v iru len ce p la sm id s m a y b e
p ro m o te d in cold m a rin e e n v iro n m e n ts th ro u g h p lasm id m a in te n an c e ,
ex p ressio n , a n d p ro lo n g e d b a c te ria l su rv iv al (13) (c h a p te r 5). Presum ably,
in c re a se d surv iv al also in creases th e lik elih o o d o f h o riz o n ta l tra n s fe r o f
g enetic elem en ts to a u to c h th o n o u s b a c te ria .
T he re le ase o f sew age in to m a rin e en v iro n m e n ts is a n in creasin g
p h e n o m e n o n w orldw ide. In o rd e r to p ro te c t public h e alth , as well as living
m a rin e reso u rces, it is im p o rta n t to reco g n ize th e lim itatio n s o f c u rre n t
m e th o d s fo r d e tec tin g fecal in d ic a to r b a c te ria in th e e n v iro n m e n t a n d
m o d ify tech n iq u es a cc o rd in g ly (27).
T etrazolium R eduction a n d V iability
T he u se o f tetrazo liu m salts fo r assessm en t of th e physiological
activ ity o f b a c te ria a n d th e ir v iab ility m u s t b e view ed w ith so m e caution.
U n d e r d e fin e d co n d itio n s, w ith know n o rg an ism s, th ey re p re s e n t a v e ry
u se fu l te c h n iq u e fo r e n u m e ra tin g a ctiv ely re sp irin g , a n d h e n c e v ia b le
b acteria. H ow ever, th e ir chem ical n a tu re as, sim ple re d o x in d ic a to rs w ith
in so lu b le re d u c tio n p ro d u c ts sh o u ld b e em p h asized (32). C ertain
co n d itio n s, such as h ig h c o n c e n tra tio n s o f p h o sp h a te a n d specific
n u trie n ts a p p e a r to significantly in h ib it CTC a n d /o r INT re d u c tio n . In
add itio n , abiotic re d u c tio n o f CTC a n d INT to fo rm azan in red u cin g
e n v iro n m e n ts, a n d b y re d u c in g a g en ts is to b e expected u n d e r c e rta in
co n ditions This raises sev eral im p o rta n t q u estio n s. Is a b a c te riu m
129
in c ap a b le o f re d u c in g CTC o r INT to visible in tra c e llu la r fo rm az a n
n onviable? For exam ple, E. faecalis a n d E. coli (pUC19) exposed to th e p o la r
m a rin e e n v iro n m e n t d id n o t p ro d u c e CTC (+) cells a fte r ca. 1-2 weeks, even
th o u g h n o nselective p la te c o u n ts in d ic a te d p o p u la tio n s o f v ia b le b a c te ria
as h igh as ca. IO5 CFU/ml. C onversely, is a b a c te riu m w hich m ain tain s
en o u g h in tra c e llu la r re d o x p o te n tia l to re d u c e CTC o r INT alw ays viable?
F orm alin -treated cells re ta in e d th e a b ility to re d u c e CTC a n d INT to visible
in tra c e llu la r fo rm az a n fo r u p to 15 m in . W hat exactly co n stitu tes
“v iab ility ”? This is a n issue th a t is o fte n d e b a te d am ong m icro b ial
physiologists. B acteria in a “d o rm a n t sta te m a y b e carry in g o u t v ery low
levels o f activ ity (b io sy n th esis, re s p ira tio n ), m aking d e te c tio n o f these
processes difficult. T h erefo re, w hich m easu re(s) of activ ity b e s t estim ate
viability? T hese q u estio n s em phasize th e im p o rtan c e o f a th o ro u g h
u n d e rsta n d in g o f th e processes in d ic a te d b y CTC a n d INT re d u c tio n in o rd e r
to a cc u ra te ly in te rp re t re su lts o b ta in e d b y th e ir use.
U n reso lv ed Issues
CTC/INT R eduction
CTC a p p e a rs to h a v e a n o n flu o re sc e n t fo rm azan of som e ty p e w hich
m ay b e d u e to a m o re stable, cationic re d u c tio n in term ed iate. T he chem ical
p ro cess of CTC re d u c tio n sh o u ld b e in v e stig a te d flu o ro m etrically , allowing a
d e te rm in a tio n o f re d o x -p o te n tia l v ersu s m ax im al flu o rescen ce
T he u se o f a single te tra zo liu m s a lt o n m ix ed p o p u la tio n s o f b a c te ria
carry in g o u t d iffe re n t form s o f m etab o lism m a y b e ex p ected to y ield
130
v a ria b le resu lts. B acterial m e ta b o lism is d iv e rse a n d tra n sm e m b ra n e red o x
p o te n tia l is g o v e rn e d b y sev e ra l p rocesses, in clu d in g ATP h y d ro ly sis,
p y ro p h o sp h a ta se activity, e n d p ro d u c t efflux (lactate, etc.),
extracytoplasm ic oxidation, in a d d itio n to e le c tro n tra n sp o rt. Also, b acteria
such as th e S treptococci a n d Lactobacilli lack cytochrom es. Investigations
o f h e te ro tro p h ic p la te c o u n ts v e rsu s CTC/INT (+) cells in e n v iro n m e n ta l
sam ples a m e n d e d w ith v a rio u s n u trie n ts w ould p ro v id e im p o rta n t
in fo rm a tio n o n th e g e n eral u se o f te tra z o liu m salts fo r m icro b ial viability.
Enteric B acteria a n d Cold M arine Exposure
T he p a th o g en icity a n d v iru len c e o f th e e n teric b a c te ria exposed to
th e p o la r m a rin e e n v iro n m e n t w as n o t in v estig ated . W hile it is know n th a t
v ia b le -b u t-n o n c u ltu ra b le , a n d in ju re d p a th o g e n ic b a c te ria m a in ta in th e ir
p ath o g en icity in n u m e ro u s o th e r en v iro n m e n ts (4, 16, 30, 33) it rem ain s to
b e seen w h e th e r th is is th e case in th is e n v iro n m e n t o v e r p ro lo n g e d
ex posure.
T h e m a rk e d th e rm o se n sitiv ity o f se v e ra l G ram -negative b a c te ria
a fte r p o la r m a rin e exposure in d icates a p o ssib le ad ap tiv e re sp o n se to lowte m p e ra tu re s. In creases in p ro p o rtio n s o f u n s a tu ra te d fa tty acid s in cell
m e m b ra n es o f E. coli, S. typhim urium , a n d Vibrio Cholerae in re sp o n se to
lo w ered e n v iro n m e n ta l a n d g ro w th te m p e ra tu re s a re w ell rec o g n ize d (5)
(c h a p te r I) . W h eth er this ty p e o f re sp o n se is re la te d to th erm o sen sitiv ity
w ith re s p e c t to p la te c o u n ts aw aits fu r th e r in v estig atio n .
T he use of 2 -dim ensional gel e lectro p h o resis to elu cid ate p ro te in
ex pression p a tte rn s in re s p o n se to cold sh o ck is a p ro m isin g ap p licatio n .
Several cold-shock in d u cib le p ro m o te rs h a v e b e e n id e n tifie d (6, 10)
131
o f h e te ro tro p h ic p la te c o u n ts v ersu s CTC/INT (+) cells in e n v iro n m e n ta l
sam ples a m e n d e d w ith v a rio u s n u trie n ts w ould p ro v id e im p o rta n t
in fo rm atio n o n th e g e n eral use o f te tra zo liu m salts fo r m icro b ial viability.
C o n c lu d in g
R e m a rk s
T he significance o f e n v iro n m e n ta l issu es is gaining re c o g n itio n
w orldw ide. Of all th e reso u rces o n e a rth , w a te r is b y fa r th e m o st essential
to m o d e rn life. My w ork in A n tarctica o n ly re in fo rc e d m y im p ressio n of
th e in d isp en sab le n a tu re o f w ater in o u r ev ery d ay lives, as well as th e
in te rn a tio n a l n a tu re o f m a n y w ater re so u rce decisions. M ean estim ates o f
crew a t M cM urdo Station, a n d w ater usage valu es fo r Nov. 1992 w ere 1,150,
a n d 38,000 g a l/day, respectively. T h ere was a full tim e d esalin izatio n p lan t,
d rin k in g w a ter p la n t, as w ell as h e a te d a n d in su la te d d istrib u tio n lines. A
source o f p o ta b le fresh w ate r was essen tial to m y lab w ork, th e galley,
survival o n th e sea ice, b a th in g , a n d statio n w aste disposal.
Sewage was, a n d still is d isch arg ed u n tre a te d a t M cM urdo Station,
how ever, galley solid w astes a re n o lo n g e r p u t in to th e sew er. In lig h t of
th e fin d in g th a t re s p ira to ry activ ity o f sev e ra l e n teric b a c te ria was lim ited
b y n u trie n t av ailab ility r a th e r th a n te m p e ra tu re , th is p ra c tic e m a y h e lp
re d u c e th e p ersisten ce o f th e sew age b a cte ria . In a d d itio n , th e N ational
Science F o und ation D ivision o f Polar P rogram s p la n s to b u ild a w astew ater
tre a tm e n t p lan t, a n d re lo c a te th e o u tfall (to d e e p e r w ater a n d h ig h e r
c u rre n t speed) a t M cM urdo S tation in th e n e a r fu tu re.
132
Biological p ro d u c tiv ity in p o la r reg io n s is im m ense, as is th e ir
iso latio n fro m h u m a n settlem en t. Polar re g io n s also c o n tain som e o f th e
m o st p ristin e a n d stable ecosystem s o n e a rth . As it d id fo r m a n y o f m y
scientific colleges o n th e ice, th e A ntarctic p ro v id e d m e w ith a n
o p p o rtu n ity to experience a n u tte rly u n iq u e w o rld w h ile c a rry in g o u t m y
scientific studies, a n d fo r th a t I a m grateful..
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