MIC 329
The Gram-positive World*
*(Well, really a talk about my favorite
Gram (+) organism, Bacillus subtilis)
The changing definition of Bacillus:
Any rod-shaped bacterium
then
Gram-positive rods
then
Aerobic Gram (+) rods
then along came 16S sequences…...
Sporosarcina
Listeria
Enterococcus
Sporolactobacillus
The ongoing schism of the genus Bacillus
•
•
•
•
•
•
Geobacillus
Thermobacillus
Virgibacillus
Salibacillus
Paenibacillus
Gracibacillus
Why study Bacillus subtilis?
• Best-characterized Gram-positive bacterium
– Biochem., metabolism well-studied
•
•
•
•
•
•
Good genetic system (transformation, transduction)
Advanced molecular biology techniques
Entire genome sequenced / annotated
Easy to grow, manipulate in culture
Widely used in industry, agriculture
“Simple” model for cellular differentiation
Bacillus subtilis differentiation cycle
*Repair
Germination
and Outgrowth
Exponential
Growth
Dormancy
*Protection
*Photochemistry
V
Postexponential
Phase
Sporulation
IV
III
II
B. subtilis spore anatomy
oc
ic
outer coat
inner coat
cortex
membranes
core
nucleoid
Endospores are resistant to:
•
•
•
•
•
Heat (both wet and dry)
Ultraviolet (UV) radiation
Extreme desiccation (including vacuum)
Lysozyme
Chemicals (organic solvents, oxidizing
agents, etc.)
Spore
Protective
Mechanisms
Environmental
Factors
Genetic
Factors
Spore
Resistance
Sporulation/
Germination
Physiology
Repair of
Damage
Abundance of spores in extreme locales
Sample site
3 desert soils
Nearsubsurface
granite
Painted metal
surface
(unshaded)
Location
(Date)
Tucson, AZ
(1996-7)
Catalina
Mtns., AZ
(2000)
Rooftop, Ft.
Worth, TX
(1995)
Number of
spores
~1 x 10 8 / g
2
~ 5 x 10 / g
3
~ 2 x 10 / m
2
Sonoran Desert Environment:
• Solar UV:
– ~10 J /m2 sec UV-B (noon)
– ~25 J/m2 sec UV-A (noon)
• Temperature extremes:
– Avg. -7 to +46 ˚C (air)
– ~70-80 ˚C (surfaces)
• Desiccation:
– Avg. 13%-30% RH
– Avg. 28 cm rainfall / year
Spores are 1-2 orders of magnitude more
UV resistant than vegetative cells
10 0
Spores
Percent survival
10
LD
90
1
Vegetative
cells
0.1
0.01
0
10
20
30
40
50
60
70
UV dose (Joules / square meter)
80
90
(254-nm
UV-C)
200
wavelength (nm)
600
1000
1400
1800
2200
Relative flux
100
in space
on Earth's s urface
50
UV
vis
vs.
IR
UV-C
200 nm
25 4 nm
us ed in
the lab
UVB
UV-A
300 nm
Solar UV
Spectrum
400 nm
>290 nm
reaches
the Earth's
s urface
Laboratory
UV
DNA Protective Factors in Spores
•
•
•
•
Spore coat proteins
Spore pigment in coat
Dipicolinic acid in core
SASP in core
The spore coat layers protect spores from
solar UV wavelengths
LD90(% of wild type)
w.t.
cotE gerE
100
*
*
0
UV-C
UV-B
UV-B+A
sun
Treatment
*
UV-A
sun
Riesenman and Nicholson. AEM 66: 620. 2000.
Spore pigment offers significant protection
against environmentally-relevant UV wavelengths
Wild-type (+) CuSO4
Wild-type (-) CuSO4
DcotA (+) CuSO4
Hullo, et al. J. Bacteriol. 183: 5426. 2001.
Dipicolinic acid (pyridine-2,6-dicarboxylate)
Ca
O-
+
N
C
O
+
C
C
C
DPA
C
C
C
-O
O
• Unique to spore core
• Exists as Ca+2-chelate
• Abundant (up to 10%
of dry weight
• Important in heat
resistance
25
20
UV
resis tance 15
relative
10
to FB108
5
PS832
0
FB7 2
UV-C
UV-B
FB1 08
Full s un
UV-A
sun
DPA is especially important for spore
resistance to UV-B radiation
Slieman and Nicholson. AEM 67: 1274. 2001.
Spore photochemistry is due to
SASP-DNA interaction
• SASP are Small, Acidsoluble Spore Proteins
• SASP are synthesized at
Stage III of sporulation
• SASP bind to DNA and shift
its conformation from B to A
• UV irradiation of SASPDNA complexes results in
formation of SP and not
T<>T
Bacillus subtilis differentiation cycle
SP Repair
Germination
and Outgrowth
UV-->
SP produced
in DNA
Exponential
Growth
Dormancy
V
Postexponential
Phase
Sporulation
IV
III
II
SASP production
The UV photochemistry of DNA in
vegetative cells and spores is different
H
O
C
SUGAR
N
1
N
2
3
6
4
5
H
PHOSPHATE
H
O
C
SUGAR
N1
N
3
2
C
vegetative cells
DNA in solution
(B-DNA)
C
SUGAR
N1
2
3
6
4
5
H
2
6
H
5
4
C
O
C
CH 3
spores
dehydrated DNA
(A-DNA)
+UV
O
H
C
SUGAR
N1
N
2
3
5
H
PHOSPHATE
C
O
H
C
C
SUGAR
N1
N
3
2
C
CH 3
cis-syn cyclobutyl thymine dimer (csTT)
H
C
O
C
C
4
5
4
6
O
N3
C
O
CH 3
H
C
N1
C
C
O
SUGAR
6
CH 3
N
C
PHOSPHATE
O
adjacent thymines
+UV
H
O
H
C
C
C
6
H
5
CH 2
4
C
O
C
CH3
spore photoproduct (SP)
30
80
Temperature (ÞC)
Polystyrene
1/2”
Plate
glass
UV flux (J / m2 sec)
Saran Wrap
20
70
10
60
0
A. Filter lid
B. 3x dried spore spots
C. Microscope slide
D. Platform
E. Box
-1
0
1
2
Hours relative to solar noon
3
Solar UV, not heat or desiccation, determines spore survival
100
10
%S
w.t
.
uvrB42
1
same
strains,
shielded
.1
splB1
.01
.001
uvrB42, splB1
0
1
2
Solar UV-A dos e (MJ/m )2
3
SP is repaired in germinating
spores by SP lyase and NER
wild-type
uvrB42
splB1
uvrB42,splB1
% Survival
100
LD90
10
1
(254-nm
UV-C)
vegetative cells
0.1
0
100
200
UV dose ( J / m 2 )
300
Spores of B. subtilis DNA repair mutants
respond differently to lab UV and Solar UV
w.t.
uvrB42
splB1
Yaming Xue
Appl. Environ. Microbiol.
62: 2221-2227. 1996.
Do spores exposed to solar UV accumulate
different types of DNA damage(s)?
H
O
C
SUGAR
N
1
N
2
3
6
4
5
C
H
PHOSPHATE
H
O
C
SUGAR
N1
N
3
2
C
vegetative cells
DNA in solution
(B-DNA)
C
SUGAR
N1
2
3
6
4
5
H
2
6
H
5
4
C
O
C
CH 3
spores
dehydrated DNA
(A-DNA)
+UV
O
H
C
SUGAR
N1
N
2
3
5
H
PHOSPHATE
C
O
H
C
C
SUGAR
N1
N
3
2
C
CH 3
cis-syn cyclobutyl thymine dimer (csTT)
H
C
O
C
C
4
5
4
6
O
N3
C
O
CH 3
H
C
N1
C
C
O
SUGAR
6
CH 3
N
C
PHOSPHATE
O
adjacent thymines
+UV
H
O
H
C
C
6
H
5
CH 2
4
C
O
C
CH3
spore photoproduct (SP)
Probing DNA damages with EndoV and alkali
10 kb
DNA
UV
ss break
ds break
10 kb
4 kb
CPD
6 kb
T<>T
10 kb
Endo V
High pH
High pH
T<>T
10 kb
High pH
6 kb
10 kb
4 kb
4 kb
4 kb
4 kb
6 kb
10 kb
6 kb
6 kb
B. subtilis spore DNA
exposed to sunlight
accumulates ss breaks,
ds breaks and cyclobutane
dimers in addition to SP.
0.8% neutral agarose
0.8% alkaline agarose
A
UV wavelength (nm)
250
300
UV-C
400
350
UV-A
UV-B
2
1
Summary of DNA
Damage in solar
UV-irradiated
spores.
3
4
B
SP*
Py<>Py
SS
DS
AP
UV treatment:
1
2
+
+
+
+
-
+
+
-
3
4
+
+
+
+/-
+
+
-
Appl. Environ. Microbiol. 66:199-205. 2000.
Tony Slieman
SP is repaired in germinating
spores by SP lyase and NER
wild-type
uvrB42
splB1
uvrB42,splB1
% Survival
100
LD90
10
1
(254-nm
UV-C)
vegetative cells
0.1
0
100
200
UV dose ( J / m 2 )
300
SP lyase-mediated DNA repair in
B. subtilis
• Encoded by splB gene.
• Synthesized at Stage III of sporulation,
packaged in the dormant spore.
• Active during spore germination.
• Direct reversal of SP to thymines in situ.
• “Dark repair” process.
Organization and expression of
the splAB operon in B. subtilis
ptsI
splA
?
splB
P1--EsigG
UUUU
Enz I
of PTS
UUUU
P3--EsigG
SplA
9.2 kD
TRAP-like
J. Bact. 175:1735.1993.
J.Bact. 176: 3983.1994.
J.Bact.177: 4402. 1995.
UUUU
Patricia Fajardo
SplB
40 kD
SP lyase
Curr. Micro.34:133.1997.
MGG 255:587.1997.
J.Bact. 182:555.2000.
Mario Pedraza
“Radical SAM” Model for SP repair
Sp lB
Sp lB
4 Fe
4S
1
Sp lB
Sp lB
3'
5'
TT
2
1. SplB dimerizes via a [4Fe-4S] center.
2. Specific binding to SP in DNA.
3. SAM split by electron donation from Fe-S
center, producing 5’-adenosyl radical.
4. Radical abstracts proton from C-6 of SP,
reverses SP back to 2 T’s.
5'
Sp lB
Sp lB
TT
3'
S-AdoMet
3
met
4
5'-Ade
Sp lB
5'
Sp lB
TT
3'
Tony Slieman
Roberto Rebeil
J.Bact. 180:4879. 1998. PNAS 98: 9038. 2001.
J.Bact. 182: 6412. 2000.
CONCLUSIONS
• In the laboratory:
–
–
–
–
Spores are highly UV resistant.
SP is the major DNA damage.
CPD, ss, ds breaks negligible at biol. relevant UV doses.
SP lyase > NER during germination.
• In the environment:
–
–
–
–
–
Spores are highly UV resistant.
SP is still the major DNA damage.
CPD, ss, ds breaks are significant at biol. relevant UV doses.
SP lyase = NER during germination.
Heat not a significant lethal component of sunlight.
Survival and persistence of
bacterial endospores in extreme
environments
Patricia Fajardo
Lilian Chooback
Heather Glanzberg
Jocelyn Law
Rachel Mastrapa
Heather Maughan
Mario Pedraza
Roberto Rebeil
Paul Riesenman
Tony Slieman
Yubo Sun
Yaming Xue