INTERNATIONAL JOURNAL O F
SYSTEMATIC BACTERIOLOGY
Vol. 18, No. 3
July 1968
pp. 241-252
Copyright 1968, Iowa State University P r e s s
PROPOSAL O F A NEW SPECIES
PSEUDOMONAS BATHY C ETES
M.M. Quigley and R.R. Colwell
Department of Biology, Georgetown University
Washington, D. C. 20007
ABSTRACT.
T w e n t y - t h r e e s t r a i n s of b a c t e r i a
isolated f r o m mud samples taken in the Pacific Ocean a t depths ranging from 9,400 to
10, 400 m e t e r s have been found to be m e m b e r s
of t h e g e n u s P s e u d o m o n a s .
F r o m r e s u l t s of
A d a n s o n i a n a n a l y s i s t h e s t r a i n s a p p e a r to c o n Pseudomonas bathystitute a new species.
c etes n. sp. i s therefore proposed and the
m e d i a n o r g a n i s m f o r t h e group, s t r a i n C2M2
(ATCC 23597), i s d e s i g n a t e d a s t h e t y p e s t r a i n .
Deoxyribonucleic acid base composition determinations and electron micrographs provide confirmatory evidence for the identificabathycetes.
t i o n a n d c l a s s i f i c a t i o n of
2.
- - - - - - - - - INTRODUCTION
One of the first applications of n u m e r i c a l taxonomy t o the
identification and classification of b a c t e r i a was a study of
m a r i n e m i c r o o r g a n i s m s undertaken by Colwell and Liston
(1961). Other data have appeared since, notably the investigation of P f i s t e r and Burkholder (1965) who a l s o applied
computer taxonomic techniques t o t h e characterization of
unknown m a r i n e isolates.
Recently, m a r i n e microbiology
studies have been extended to include taxonomic analyses of
bacteriological i s o l a t e s f r o m t h e t r u e "deep sea,'' 5 5 ,
depths in e x c e s s of 6, 000 m e t e r s (Quigley 1967; Quigley and
Colwell 1968). The incentive for such studies was the r e l a tive lack of good information concerning deep- s e a f o r m s .
Other than quantitative e s t i m a t e s of t h e capabilities of m i c r o o r g a n i s m s p r e s e n t i n seawater o r m a r i n e sediments (Zobell
1954; ZoBell and Morita 1959), no species descriptions a r e
available in the l i t e r a t u r e f o r the t r u e deep-sea bacteria.
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The p r e s e n t study is intended a s a p r e c i s e description of
t h e s p e c i e s , Pseudomonas bathycetes n. s p . , which r e p r e sented t h e m a j o r phenetic c l u s t e r of a taxonomic a n a l y s i s of
deep- s e a bacteria. The b a c t e r i a w e r e isolated f r o m sedim e n t s collected f r o m t h e Philippine T r e n c h ( 9 , 8 5 4 and 9, 443
m e t e r s ) and t h e Challenger Deep (10, 373 m e t e r s ) i n t h e
M a r i a n a s T r e n c h of t h e Pacific Ocean.
MATERIALS AND METHODS
Twenty-three i s o l a t e s w e r e examined. All the i s o l a t e s
included in the study w e r e collected in November, 1964,
during t h e Dodo Expedition by R. Y. Morita, Oregon State
University, Corvallis, Oregon, and C. E. ZoBell, S c r i p p s
Institution of Oceanography, University of California a t San
Diego, La Jolla, California.
Sampling data a r e given i n
Table 1. Methods of sampling and c u l t u r e p r o c e d u r e s have
been d e s c r i b e d e l s e w h e r e (Quigley and Colwell 1968).
The maintenance m e d i u m employed throughout t h e studies
consisted of: Yeast e x t r a c t (Difco), 0. 370; P r o t e o s e peptone
(Difco), 1.070; and a salt solution consisting of, p e r l i t e r of
distilled w a t e r : sodium chloride, 2.470; p o t a s s i u m chloride,
0.0770;magnesium chloride, 0.53700; and magnesium sulfate,
0.770;adjusted t o pH 7. 2-7.4 with sodium hydroxide. IncuA t o t a l of 116
bation of i n o c u l a t e d - t e s t m e d i a was a t 25'C.
morphological, cultural, physiological and biochemical c h a r a c t e r i s t i c s was d e t e r m i n e d f o r each strain of t h e s p e c i e s
set. The t e s t s and t e s t p r o c e d u r e s followed have been cited
previously (Quigley and Colwell 1968).
The taxonomic data w e r e coded and t r a n s f e r r e d t o IBM
punch c a r d s . S i m i l a r i t y values w e r e computed and phenetic
c l u s t e r s obtained following t h e method of Colwell and Liston
(1961). F e a t u r e frequency (Colwell 1964) and median organi s m calculations (Liston, Wiebe and Colwell 1963) w e r e det e r m i n e d f o r t h e c l u s t e r output i n t h e c o u r s e of t h e computer
analysis. The c o m p u t e r s u s e d i n t h e study w e r e t h e IBM
1620 Model 11 computer with 131 1 Disk P a c k S y s t e m and t h e
IBM 360/40 System. P r o g r a m s w r i t t e n f o r taxonomic analy s i s and used in t h i s study w e r e G T P - 2 (Georgetown Taxonomy P r o g r a m No. 2), GTP-3,G T P - 4 and GTP-5. G T P - 2 is
a p r o g r a m employing a modification of t h e highest-link s o r t ing method of Sne?th (19 57) whereby t h e affinities amongst
s t r a i n s of a n a n a l y s i s set a r e employed both t o d e t e r m i n e
group saltations and t o position strains within a group.
G T P - 3 provides f e a t u r e frequencyof o c c u r r e n c e f o r c l u s t e r s
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SYSTEMATIC
Table 1.
Sampling data for Pseudomonas bathycetes s t r a i n s .
Station
CUre
No.
-
Date
-
No.
-
Depth
loo
25, 1964
9,443 CI
loo 3.8'
28, 1964
10,373 m
2m
NOV.
25, 1964
B
281
NOY.
C
282
IJOV.
Longitude
Letitude
9,854 rn
A
Table 2.
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101 N
:>J
nC201 N
126"
401 E
i26O
40' E
142'
191 E
Frequency of o c c u r r e n c e of morphological f e a t u r e s
for P seudomona s bathycet e s ,
Feature
Singles
Width 0.2-0.6 p
G r a m negative
Rods
Pairs
Length 0.2-0.6 p
Round end
Motile
Polar f l a g e l l a
Tapered end
Chains
Filaments
Curved rods
Length 0.6-1.2 p
Peritrichous f l a g e l l a
S p i r a l rods
Oval (spheres)
Ref r a c t i l e
Frequency
+ (1.0)
+ (1.0)
+ (1.0)
+ (1.0)
+
+
(0.91
(0.9)
(0.7)
+, (0.7)
f (0.7)
+_
k (0.4)
2. (0.3 j
- (0.2)
-
-
-
(0.1)
(0.1)
(0.1)
(0.0)
(0.0)
(0.0)
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INTERNATIONAL JOURNAL
and GTP-4 computes for each cluster the hypothetical median organism. The comparison of each s t r a i n of the cluster
with the hypothetical median permits selection of a strain
with the highest similarity to the computed median a s the
"typical" o r type strain. GTP-5 output i s the full S-value
matrix ordered by GTP-2. The programs have been documented for the IBM 1620 Computer U s e r s ' Group and a r e
available from the authors (RRC) o r directly from the
Georgetown University Computation Center, Washington, D.
C. 20007, U . S . A .
RESULTS AND DISCUSSION
In the initial analysis, 38 strains were examined. Seven
phenetic clusters were distinct at 6570 S (similarity level)
and these have been described elsewhere (Quigley and Colwell 1968). Clusters I and I1 were found in the e a r l i e r stages
to merge a t 76% S and have, therefore, been treated a s a
species cluster ( F i g . 1). A value of S ??570 has been suggested a s a possible species level of similarity (Colwell and
Liston 1961; Liston, Wiebe and Colwell 1963).
Feature frequencies computed for the set of 23 strains
a r e presented in Tables 2, 3 , 4 and 5. In general, the strains
a r e Gram-negative, nonpigmented, short, slender rods occurring a s singles and pairs, oxidase positive, capable of
growth a t temperatures ranging from 0-37"C, and at salt
concentrations of 0- 10% NaCl. The strains were oxidative
o r not reactive in glucose, and were capable of growth on a
vitamin-free casamino acids medium, with basal salts solution a s diluent. The s t r a i n s did not demonstrate a requirement for natural o r synthetic seawater for growth but growth
was stimulated when a seasalts medium was provided. The
arginine, ornithine and lysine decarboxylase reactions were
negative. Only glucose and sucrose, of the carbohydrates
tested, were utilized (oxidatively) by the 2 3 strains.
The strains were polarly flagellated when studied by flagella staining technique. However, when examined under the
electron microscope, some of the strains were found to be
polarly flagellated after 24hours growth in a seasalts-based
medium (Fig. 2) and peritrichously flagellated i f examined
after 48 hours o r later (Fig. 3 ) . Other investigators have
reported similar .observations for marine pseudomonads
(Buttiaux and Voisin 1958/1959; Leifson 1963).
The median organism calculated for the strain s e t was
C2M2 (ATCC 23597), which demonstrated a value of 9570 S
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SYSTEMATIC BACTERIOLOGY
Table 3.
2 45
F r e q u e n c y of o c c u r r e n c e of c u l t u r a l f e a t u r e s f o r
Pseudomonas bathycetes.
Feature
Convex colony
Opaque colony
Entire edge
O f f -white colony
Even t u r b i d i t y i n broth
Medium colony (2-5 mm)
Moderate t u r b i d i t y i n broth
small colony (1-2 mm)
P e l l i c l e i n broth
S l i g h t t u r b i d i t y i n broth
Heavy t u r b i d i t y i n b r c t h
Granular t u r b i d i t y i n broth
Rough cclcny
Translucent colony
Spreading on agar
Diffusible pigment prcduced
Visible inscluable pigment
Fluorescence under U/V l i g h t
Frequency
+ (1.0)
+ (1.0)
3- (1.0)
+ (1.0)
+ (0.9)
k (0.6)
+_ (0.6)
1- (0.3)
-
(0.2)
- (0.2)
- (0.2j
- (0.1)
- (0.0)
- (0.0)
- (0.0)
- (0.0)
- (0.0)
- (0.0)
Table 4. Sensitivity of P s e u d o m o n a s bathycetes t o antibiotics
and a n t i b a c t e r i a l agents.
Feature ( S e n s i t i v i t y )
Dihydrcstreptcmycin 10 ug
Chlorcmycetin 30 ug
Kanamycin 30 ug
Polymyxin 3 300 units
Terramycin 30 ug
Novobiocin 30 ug
P e n i c i l l i n 10 units
Erythromycin 15 ug
Tetracycline 30 ug
Aureornycin 30 ug
Pteridine O / U 9
Frequency
+ (1.0)
+ (1.0)
+ (1.0)
-I-, ( 0 . 7 )
k (0.7)
+, (0.7)
t (0.5)
- (0.1)
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Table 5.
F r e q u e n c y of o c c u r r e n c e of biochemical f e a t u r e s
f o r P s e u d o m o n a s bathycetes.
Feature
Oxichse p c s i t i v e
C-lucosc + iodoacetate -$ GrosrLh
Lipase ?resent: Ttreen 60
Lipase present: Tveen 20
Lipase Ijrescnt: %Teen 40
Catalrse p o s i t i v e
Glucose a c i u aercbic
Sucrcse a c i d aerobic
Glucose 4- iodcacetate -+ Acid
i i t n u s nil:: alkaline
Litnus nil!< reduced
Lipasc present: w e e n 80
Lecithinase present
TIC3 reduced
Li-mus m i l k a c i d
Galactcse a c i d aercbic
I h l t c s e a c i d aerobic
Starch hydrolyzed
Citrate pcsitive
Gelatin l i q u e f i e d
Glucose a c i d anaerobic
Gluccse gas
Lactose a c i d
ihnnitol acid
I n d o l e prcduced
I'Ieth:Fl red
Vcges-Proskauer
IICs reduced
Litmus mill: peptcnized
A r gin ine decarbcsyla se
Lysine d c c a r b o q l a s e
Orxithine d e c a r b o q l a s e
Flucrescence in Patcn's medium
Frequency
+ (1.0)
+ (0.9)
+ (0.9)
-I-(0.8)
-
-
-
-
-
-
(0.2)
(0.1)
(0.1)
(0.1)
(0.1)
(0.1)
(0.0)
(0.0)
(3.0)
(0.0)
(0.0)
(0.0)
(0.0)
(0.0)
(0.0)
(0.0)
(0.0)
(0.0)
(0.0)
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SYSTEMATIC
BACTERIOLOGY
2 47
95 90-
85-
>
- --
5- 80z :
3753
70-
6560-
F i g u r e 1.
Phenetic clustering of t h e 2 3 s t r a i n s of Pseudomonas bathycetes by unweighted-pair, highest
linkage sorting of s i m i l a r i t y values.
when compared with t h e hypothetical median organism. The
o v e r - a l l guanine t cytosine content of the DNA was d e t e r mined by both buoyant density m e a s u r e m e n t s i n c e s i u m
chloride and t h e r m a l denaturation ( C i t a r e l l a and Colwell
1966). G t C value for C2M2, the type strain, was found t o
be 57'70 ( p = 1.716, T m = 9 3 ° C ) .
Consultation of the l i t e r a t u r e f o r published descriptions
t o f i t t h e deepsea pseudomonads was unsuccessful. P f i s t e r
and Burkholder (1965), who applied computer techniques to
t h e analysis of 151 b a c t e r i a l s t r a i n s isolated f r o m t h e Anta r c t i c Sea and f r o m w a t e r s in t h e vicinity of P u e r t o Rico,
demonstrated that o r g a n i s m s occurring i n different a r e a s of
t h e open ocean m a y be profitably studied by m e a n s of computer analysis. The 151 m a r i n e s t r a i n s of the analysis c a r r i e d out by P f i s t e r and Burkholder f o r m e d nine distinct
c l u s t e r s , not identified explicitly by them, but designated
a s Groups I-IX. None of t h e s e groups corresponded t o ,P.
bathycete s.
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Figure 2 .
Electron micrograph of a chromium shadowed
preparation of strain C2M2, the median organism
of Pseudomonas bathycetes, exhibiting polar
flagellation. 24 hour seawater broth culture,
(X40, 5 0 0 ) .
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SYSTEMATIC
Figure 3.
BAC T E R I O LOGY
2 49
Electron micrograph of a chromium shadowed
preparation of strain C2M2, the median organism
of Pseudomonas bathycetes, exhibiting peritrichous flagellation. 48 hour seawater broth culture.
(X16, 500).
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The 2 3 strains of _P. bathycetes were compared by computer with a set of 132 previously analyzed marine strains
(Colwell and Gochnauer 1963; Colwell, Gochnauer and Quigley 1967). Similarity values and phenetic clusters were calculated thus for a total of 170 isolates. At S 27570 (species
level of similarity) the 2 3 ,P. bathycetes strains clustered
separately f r o m the 147 other strains. However a t S values
between 7070 and 7470, the P. bathycetes strains clustered
with several known Pseudogonas strains, including
- named,
culture collection strains, thus providing strong evidence of
membership within the genus Pseudomonas.
Very few descriptions of marine species of bacteria a r e
available in the literature and from the available descriptions little information can be gleaned for identification and
classification purposes. Either the descriptions a r e scanty
o r the media employed cannot be duplicated. Comparison
of data for 2. bathycetes with the descriptions of marine
Pseudomonas spp. cited in Bergey' s Manual of Determinative Bacteriology (Breed et al., 1957), by ZoBell and Upham
(1944) and by Shewan (1963) do not reveal species level of
similarity with the deep-sea forms. Since the strains of P.
bathycetes a r e barotolerant, i. e . , able to grow in 1100 at&.
(in situ p r e s s u r e s ) , a p r o p e r 6 Kot shared by t e r r e s t r i a l and
shallow water species, and since a s a species group, P.
bathycetes does not appear to be similar t o any species described in the literature, it appears justifiable to propose
P. bathycetes a s a new species.
ACKNOWLEDGMENT
The authors wish to express their gratitude to Dr. G. B.
Chapmen who s o kindly provided advice and assistance with
the electron microscopy, to Rev. Stephen Winters, S. J. for
helpful discussion of questions pertaining to nomenclature,
and to Miss T. E. Lovelace for her excellent technical a s sistance. Also, the collaboration of Drs. C. E. ZoBell and
R. Y . Norita in providing the cultures i s gratefully acknowledged.
This investigation was supported by Contract Nonr-48 10
(00) (NR 103-667) between the Office of Naval Research, Department of the Navy, and Georgetown University.
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BACTERIOLOGY
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