Taxonomy of Aerobic Marine Eubacteria

29 Pages·2004·5.18 MB·English

JOURNAL OF BACTERIOLOGY, Apr. 1972, p. 402-429 Vol.110,No.1
Copyright01972 AmericanSocietyforMicrobiology PrintedinU.SA.
Taxonomy of Aerobic Marine Eubacteria
LINDA BAUMANN, PAUL BAUMANN, M. MANDEL, AND RICHARD D. ALLEN
DepartmentofMicrobiology, UniversityofHawaii,Honolulu,Hawaii96822,andDepartmentofBiology, The
University ofTexas, M.D.AndersonHospitaland ThmorInstituteatHouston, Houston, Texas 77025
Receivedforpublication 27December 1971
Two hundred and eighteen strains ofnonfermentative marine bacteria were
submitted toan extensive morphological, physiological, and nutritional charac-
terization. All the strains were gram-negative, straight or curved rods which
were motile by means ofpolar orperitrichous flagella. Awide variety oforganic
substrates served as sole sources ofcarbon and energy. The strains differed ex-
tensively in their nutritional versatility, being able to utilize from 11 to 85
DDD
carbon compounds. Some strains had an extracellular amylase, gelatinase, li- ooo
pase, or chitinase and were able to utilize n-hexadecane and to denitrify. None www
of the strains had a yellow, cell-associated pigment or a constitutive arginine nnn
dihydrolase system, nor were they able to hydrolyze cellulose or agar. The re- lololo
aaa
sults ofthe physiological and nutritional characterization were submitted to a ddd
numerical analysis which clustered the strains into 22 groups on the basis of eee
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phenotypic similarities. The majority ofthese groups were separable by a large
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number of unrelated phenotypic traits. Analysis ofthe moles per cent guanine rororo
plus cytosine (GC) content in the deoxyribonucleic acid of representative mmm
strains indicated that the peritrichously flagellated groups hada GC content of h h h
53.7 to 67.8 moles %; polarly flagellated strains had a GC content of 30.5 to tttttt
ppp
64.7 moles %. The peritrichously flagellated groups were assigned to the genus :::
Alcaligenes. The polarly flagellated groups, which had a GC content of43.2 to //jb//jb//jb
48.0 moles %, were placed into a newly created genus, Alteromonas; groups ...
aaa
which had a GC content of 57.8 to 64.7 moles % were placed into the genus sss
Pseudomonas; and the remaining groups were left unassigned. Twelve groups mmm
were given the following designations: Alteromonas communis, A. vaga, A. .o.o.o
macleodii, A. marinopraesens, Pseudomonas doudoroffi, P. marina, P. nautica, rrr
ggg
Alcaligenes pacificus, A. cupidus, A. venustus, and A. aestus. The problems of / / /
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assigning species ofaerobic marine bacteria to genera are discussed. nnn

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ppp
rrr
Gram-negative, heterotrophic bacteria which adequate characterization. The limited il il il
are motile by means offlagella are readily iso- number of traits used and their often dubious 333
,,,
lated from the ocean and appear to comprise a validity make it difficult if not impossible to 2 2 2
major component of the bacterial flora of the assign new strains to previously named spe- 000
111
sea (28, 32, 34, 35, 41). Most or all ofthese or- cies. In addition, type strains are not available 999
ganisms require sodium ion for growth (32, 34, for most species, so that further characteri- b b b
35, 41). On the basis oftheirability to ferment zation and comparison with new isolates is not yyy

carbohydrates, these strains can be subdivided possible. The diversity of the aerobic marine ggg
uuu
into two large groups. The fermentative strains bacteria, which is suggested by the number of eee
have recently been the object of a taxonomic genera to which they have been assigned, is sss
ttt
study which has shown that most ofthese iso- confirmed by the wide range of the moles per
lates can be placed into the genusBeneckea (1, cent guanine plus cytosine (GC) content in
5). The nonfermentative strains have in the their deoxyribonucleic acid (DNA) contents. In
past been assigned to a large number of gen- a study of 57 marine isolates, Leifson and
era, including Pseudomonas, Achromobacter, Mandel (33) found that peritrichously flagel-
Alcaligenes, Vibrio, Flavobacterium (6,.21, 24, lated aerobes had DNA containing 49 to 67
33, 34, 41, 47, 55) and Arthrobacter (7); how- moles % GC and that aerobic, polarly flagel-
ever, the taxonomy ofthese isolates is unsatis- lated strains had GC contents of 40 to 67
factory since generic and specific assignments moles %.
have usually been made on the basis ofan in- The present study is concemed with the
402
VOL. 110, 1972 AEROBIC MARINE EUBACTERIA 403
application of the methods of Stanier et al. 186, 191, and 192 of group G-3. The mechanism of
(51) to the characterization of 218 nonfermen- cleavage ofdiphenolic intermediates by thesestrains
tative, flagellated, marine bacteria, none of was determined by spectrophotometric methods,
which produces a yellow, cell-associated pig- using cell-free extracts. Strains 66, 70, 74, and 76
from group C-3aswellasall thestrainsofgroupG-3
ment. The results indicate that this collection
which were able to grow on benzoate were grown on
of strains consisted of a number of well de-
BM containing 0.15% sodium benzoate. The cells
fined species and groups the majority ofwhich were centrifuged, washed twice in half-strength
could be assigned to Pseudomonas, Alcali- ASW containing 50 mM Tris-hydrochloride (pH 7.5),
genes, and a newly created genus, Altero- and suspended in 100 mm Tris-hydrochloride (pH
monas. 8.0) containing 0.1 mM ethylenediaminetetraacetic
acid. Following ultrasonic disruption ofthe cells, the
MATERIALS AND METHODS
suspension was centrifuged for30 minat 30,000 x g,
The methods as well as the composition of the and the supernatant fraction was assayed for proto- D
media used in this study have been previously de- catechuate 3,4 oxygenase (50), protocatechuate 4,5 o
scribed (5, 51). Only additional methods or signifi- oxygenase (53), catechol 1,2 oxygenase (25), and cat- w
cant modifications will be considered. Unless other- echol 2,3oxygenase (14). n
wise stated, all cultures were incubated at 25 C. The Oxidase test. The method described by Stanier lo
a
abbreviations used are the following: ASW (artificial et al. (51) was used inthis study. Inthecaseofsome d
sea water), BM (basal medium), BMA (basal me- strains, the oxidase reaction developed slowly, e
dium agar), YEB (yeast extract broth), YEA (yeast making interpretation difficult. The speed of the d

extract agar), MA(Difco MarineAgar), F-1 (fermen- color change was greatly accelerated by the addition fr
tation medium 1), F-2 (fermentation medium 2), ofadrop oftoluene priorto the addition of1%(w/v) o
m
PHB (poly-d-hydroxybutyrate), and Tris [tris(hy- N,N'-dimethyl-p-phenylenediamine. Addition of

droxymethyl)aminomethane]. These abbreviations toluene did not affect the oxidase reaction ofknown h
are the same as those used previously (5). The ma- oxidase-negative strains (Escherichia coli, Acineto- tt
p
jority of the strains were maintained on MA slants bactercalco-aceticus, andP. maltophilia). :
/
at 20 to 22 C and were transferred every 4 weeks. A Morphology and flagellation. Morphological /jb
few strains, however, rapidly lost their viability and examination of cells was performed as previously .
had to be transferred every 2 weeks. Some strains described (5). The medium used was YEB con- as
grew poorly on MA and were maintained on BMA taining 0.1% sodium succinate, 0.1% sodium lactate, m
containing 0.2% potassium lactate. Tests forsodium and 0.1% sodium acetate. Each strain was grown in .
o
and organic growth factor requirements were per- this medium, and its flagella were stained by the r
formed as previously described (5) with a mredium method ofLeifson(5, 31). Strains 51 to57 (groups B- g
/
containing 0.1% potassium succinate, 0.1% potas- 1 and -2), 139 to 144(groupF-2), 209to213(groupI- o
sium lactate, and 0.1% potassiumacetate. Strain 214 2), and two representative strains from each of the n
grew slowly in BM containing 0.2% D-glucose, potas- remaining groups were grown in the above medium, A
sium succinate, or potassium acetate. The results of negatively stained, and examined by means of the p
ptlhaetinnugtrwiteiroenadliffsiccrueletnitnoginotfertphriestsstirnacien obnylyresplliigchat estlreacitnrsonwemriecrgorsocwonpeon(1M).AInanadddsittaiionne,drfeoprrfelsaegnetlaltaibvye ril 3
growth occurred on BMA containing the tested theLeifson method. ,
2
carbon compound (5, 51). Growth was improved DNA basecompositions. The moles percent GC 0
when the solid medium was supplemented with 10 in the DNAofrepresentative strainswas determined 1
mg of L-methionine, L-arginine, L-histidine, L-leu- from buoyant density measurements in CsCl gra- 9

cine, and L-isoleucine per liter and 20 mg of DL-va- dients (36). Reference DNA from bacteriophage 2C b
line per liter. This mediumwas devisedbyJ. L. Rei- (Bacillus subtilis host) at a density of 1.742 g/cm3 y
chelt in the course ofa separate study involving the wasincluded ineachgradient. g
u
nutritional screening of growth factor requiring fac- Methods of isolation. The enrichment methods e
ultative anaerobes of marine origin. Strain 214 did used in thisstudyhavebeen previously described(5). s
t
not have growth factor requirements, however, since The strains were purified by streaking on BMA
it could be successively transferred in a minimal containing 0.1% of the same carbon compound as
medium. used in the enrichment or on BMA containing 0.1%
Fluorescin production. The ability to produce sodium succinate, 0.1% sodium lactate, and 0.1%
fluorescin was tested by inoculating strains onto sodium acetate. In the case of strains obtained by
medium B ofKinget al. (30), modified by the addi- direct isolation, sea water samples were collected
tion of half-strength ASW. Three strains of P. flu- asepticallyandfiltered through0.45am-poresizefil-
orescens gave good pigment production when grown ters (Millipore Corp.). The filters were placed onto
on this medium. MA or BMA containing 0.1% of the organic sub-
Aromatic ring cleavage. Strains capable of strate. Subsequent purification ofstrains isolated on
growth on benzoate, o-hydroxybenzoate, m-hydroxy- defined media was performed by streaking on ho-
benzoate, p-hydroxybenzoate, L-tryptophan, or mologous media. Strains obtained by directisolation
quinate were grown on these compounds and tested on MA were purified by streaking on BMA con-
for the mechanism ofaromatic ring cleavage (5, 51). taining 0.1% sodium succinate, 0.1% sodium lactate,
This test wasunsuccessful with benzoate-grown cells and0.1%sodiumacetate.
ofall the strains ofgroup C-3and strains 168, 177to Source ofstrains examined. The information in
404 BAUMANN ET AL. J. BACTERIOL.
parentheses following each strain number indicates 68 (creatine); 69 (creatine, NO3-); 70 (allantoin); 71
the method of isolation, the carbon compound sup- (allantoin); 72 (allantoin); 73 (allantoin); 74 (crea-
porting the initial growth, and the source of sea tine); 75 (caprylate*); and76(6-aminovalerate, DI).
water. A carbon compound in parentheses indicates Strains assigned to group D-1 were: 77 (L-valine,
that the strain was isolated by enrichment methods DI); and 78(isobutyrate, DI).
in which the designated carbon compound was the Strains assigned to Alcaligenes cupidus sp. nov.
sole source of carbon and energy. Enrichment cul- (group D-2) were: 79 (allantoin); 80 (glycolate); 81
tures which were incubated anaerobically with (glycolate); 82 (ethyleneglycol); and83(L-mandelate,
NaNO3 as the terminal electron acceptor are desig- DI).
nated by NO3- within the parentheses. A carbon Strains assigned to Alcaligenes venustus sp. nov.
compound followed by the letters DI indicates that (groups D-3 and D-4) were: 84 (L-histidine); 85 (L-
the strain was obtained by direct isolation in which lysine); 86 (o-hydroxybenzoate); 87 (o-hydroxyben-
the designated compound was the sole source of zoate); 88 (L-lysine); 89 (L-lysine); 90 (L-lysine); 91
carbon and energy. In the case ofsome ofthestrains (L-histidine); 92 (nicotinamide, DI); 93 (nicotin- D
obtained by direct isolation, MA was the medium amide); 94 (nicotinamide, DI); 95 (L-histidine); 96 o
supporting the initial growth. When depth (m) of (nicotinamide); and 97 (histamine). w
samplingisindicated, thestrainswereobtainedfrom Strains assigned to Alteromonas macleodii sp. n
samples taken at different stations 10to35 miles off nov. (group E-1) were: 98 (lactose, DI); 99 (lactose, lo
thecoast ofOahu, Hawaii. Strainsundesignatedwith DI); 100 (lactose, DI); 101 (lactose, DI); 102 (lactose, a
d
respect to the depth ofsamplingwereobtained from DI); 103 (caprylate, DI, 5 m); 104 (L-tyrosine, 10 m); e
samples of surface water off the immediate coast of 105 (butyrate, DI, 100 m); 106 (chitin*); 107 (lactose, d

Oahu. Anasterisk(*) followingthecarboncompound DI); 108 (lactose, DI); 109 (MA, DI, 750 m); 110 fr
indicates thattheisolatedstrainwasunabletoutilize (MA, DI, 750m); 111 (MA, DI, 750 m); 112(MA, DI, o
m
the carbon compound used for its initial isolation. 100 m); 113 (MA, DI, 750 m); 114 (MA, DI, 500 m);
These strains, which were obtained from enrich- 115 (MA, DI, 1100 m); 116 (MA, DI, 750 m); 117 h
ments, werepurifiedonmediawhichdifferedincom- (MA, DI, 1100 m); and 118(MA, DI, 750 m). tt
p
position from the enrichment medium and probably Strains assigned to Alteromonas marinopraesens :
rreipcrhemseennttefdloraa.minority component of the total en- c12o0mb(.L-nseorvi.ne(,grNoOu3p-)E;-2)12w1er(eL:-ly1s1i9ne(*L,-lNy0si3n-e)*;, N1O2,2-()L;- //jb.
Strain assignment. Strains assigned to Altero- serine); 123 (L-serine); 124(L-tyrosine); 125(glycine); a
s
monas communis sp. nov. (group A-1) were: 1 126 (L-histidine); 127 (L-serine, DI); 128 m
(chitin*); 2 (chitin*); 3 (betaine); 4 (chitin*); 5 (be- (laevulinate*); 129 (laevulinate*); 130 (L-valine, DI); .
taine, DI); 6 (ethanol); 7 (B-alanine); 8 (DL-B-hy- 131 (L-serine); 132 (laevulinate*); 214 (B-16 ofR. A. or
droxybutyrate); 9 (p-hydroxybenzoate); 10 (m-hy- MacLeod, ATCC 19855); 215 (Vibrio haloplanktis, g
/
droxybenzoate); 11 [L-(+)-tartrate]; 12 (acetate); 13 ATCC 14393); and 216 (Vibrio marinopraesens, o
(benzoate*); 14 (L-lysine); 15 (citrate); 16 (L-gluta- ATCC 19648). n
mate); 17 (glycerol); 18 (ethanol); 19 (benzoate*); 20 Strains assigned to Alcaligenes aestus sp. nov. A
(betaine); 21 (L-glutamate); 22 (p-hydroxybenzoate); (group F-1) were: 133 (meso-inositol, DI, 100 m); 134 p
a2l3an(iLn-ep)r;oli2n7e)(;ace2t4at(eL)-;pr2o8li(neb)e;nzo2a5te(*b)u;ty2ra9te(*m)-;hy2d6ro(x,B-- (1m3e6so(-Li-nvoasliitnoe,l,DDII,,620000mm));;113375((acdriepataitnee,,DDII,, 125000 mm));; ril 3
ybenzoate); 30 [L-(+)-tartrate]; 31 (L-lysine); 32 (o- and 138(,3-alanine, DI, 600 m). ,
hydroxybenzoate*); and33(citrate). Strains assigned to Pseudomonas marina comb. 2
0
Strains assigned to Alteromonas vaga sp. nov. nov. (group F-2) were: 139 (sarcosine, DI); 140 (D- 1
(group A-2) were: 34 (m-hydroxybenzoate); 35 (ace- galactose, DI); 141 (caprylate, DI); 142 (meso-inosi- 9
tate); 36 (m-hydroxybenzoate); 37 (betaine); 38 (im- tol, DI); 143 (2,3-butyleneglycol, DI); 144(sarcosine, b
hydroxybenzoate, DI); 39(L-lysine); 40(glycerol); 41 DI); and 219(Arthrobacter marinus, ATCC 25374). y

(L-glutamate); 42 (m-hydroxybenzoate); 43 (sarco- Strains assigned to group G-1 were: 145 (n-bu- g
u
sine); 44 (sarcosine); 45 (L-lysine); 46 (m-hydroxy- tanol); 146 (n-propanol); 147 (malonate*); 148 (L- e
benzoate); 47 (L-histidine); 48 (sarcosine); 49 (ci- valine); 149 (n-butanol); 150 (D-mandelate); 151 s
trate); and 50(succinate). (2,3-butyleneglycol); 152 (histamine); 153 (L-valine, t
Strains assignedto groupB-1 were: 51 (nicotinate, DI); 154(histamine, DI); and 155(isobutanol, DI).
DI); 52(L-isoleucine); and53(L-lysine). Strains assigned to group G-2 were: 156 (laevuli-
Strains assigned to group B-2 were: 54 (betaine, nate); 157 (caprylate, N03-); 158 (glycine); 159 (fl-
DI, 10 m); 55 (L-tyrosine, 5 m); 56 (L-tyrosine, DI); alanine, DI); and 160(,B-alanine, DI).
57 (histamine, DI, 300 m). Strains assigned to Pseudomonas nautica sp. nov.
Strains assigned to group C-1 were: 58 (creatine, (group G-3) were: 161 (chitin*, 600 m); 162 (butyr-
DI); and59(creatine, DI). ate, NO3-); 163 (butyrate, NO3-); 164 (butyrate,
Strains assigned to Alcaligenes pacificus sp. nov. NO3-); 165 (butyrate, NO3-); 166(caprylate, NO3-);
(group C-2) were: 60 (L-histidine); 61 (L-lysine); 62 167 (valerate, N03-); 168 (acetate, NO3-); 169 (bu-
(L-lysine); 63 (laevulinate); 64(histamine); and65(L- tyrate, NO3-); 170(butyrate, NO3-); 171 (propylene-
glutamate). glycol); 172 (adipate, DI); 173 (adipate, DI); 174 (bu-
Strains assigned to Pseudomonas doudoroffii sp. tyrate, NO3-); 175 (butyrate, N031); 176 (butyrate,
nov. (group C-3) were: 66 (allantoin); 67 (benzoate); NO3-); 177 (adipate); 178 (lactate, NO3-); 179 (ace-
VOL. 110, 1972 AEROBIC MARINE EUBACTERIA 405
tate, NO3-); 180 (caprylate, N03-); 181 (MA, DI, nority of the cell population. Nonstalked, ro-
750 m); 182 (MA, DI, 750 m); 183 (MA, DI, 750 m); sette-forming marine bacteria have been pre-
184 (MA, DI, 1100 m); 185 (MA, DI, 750 m); 186 viously described by Leifson et al. (32). Strains
(MA, DI, 500 m); 187 (butyrate, DI); 188 (caprylate, of groups B-2 and I-2 often gave rise to involu-
NO3-); 189 (butyrate, N03-); 190 (butyrate, N03-); tion forms whose extent and occurrence varied
191 (laevulinate, 600 m); 192 (adipate, 600 m); 193 with the phase of growth and the composition
(adipate); and 194(butyrate, DI).
of the medium. Involution forms were more
Strains assigned to group H-1 were: 195 (malo- frequent in YEB supplemented with 0.1% so-
nate); 196(betaine); 197(sorbitol,DI);and198(meso-
inositol, DI). dium succinate, 0.1% sodium lactate, and 0.1%
Strains assigned to group H-2 were: 199 (succi- sodium acetate than in BM containing the
nate, N03-); 200 (propionate, N03-); 201 (acetate, latter three substrates. In general, fewer invo-
NO3-); 202 (malonate*); 203 (succinate); and 204 L- lution forms were observed in exponential than
citrulline, DI, 600 m). in early stationary phase ofgrowth.
Strains assignedtogroupI-1 were: 205(L-proline); Pigment production. None of the strains
206 (,B-alanine); 207 (L-leucine); and 208 (L-serine,
made fluorescin or had a yellow, cell-asso-
DI).
Strains assigned to group I-2 were: 209 (creatine, ciated pigment. Strains 68, 70, 71, and 72
DI); 210 (creatine, DI); 211 (allantoin, DI, 150 m); (group C-3) produced a soluble, light brown D
212 (betaine, NO3-); 213(sarcosine, DI). pigment on MA after 6 to 14 days of incuba- o
Other strains were: 214, 215, 216 (see group E-2); tion at 20 to 22 C. No pigment was produced w
n
2m1a7ri(nPu.s,niAgrTiCfaCcie1n4s4,05A);TC21C9(1s9e3e75g)r;ou2p18F-(2P)..perfecto- b20y9,th2e1s0e,satnradin2s13on(agrmoiupniIm-a2)lgmaevdeiruims.e tSotrcaoilnos- loa
nies containing a blue-black pigment which d
e
was not soluble in water. Pigment production d
RESULTS
was more intense on BMA containing 0.2% f
r
Sodium and organic growth factor re- sodium lactate than on YEA. The remaining om
quirements. All the strains in our collection strains were nonpigmented.
were able to grow in BM (which contained 0.2 Tests negative for ali strains. None of the ht
sodium) supplemented with potassium strains was able to fix molecular nitrogen or tp
M 0.1%
succinate, 0.1% potassium lactate, and 0.1% grow chemolithotrophically with molecular :/
/
potassium acetate, to a final turbidity of160 to hydrogen as the sole source of energy and jb
340 Klett units. When the sodium salts in BM carbon dioxide as the sole source of carbon. .a
were replaced with equimolar amounts of po- None ofthe strains luminesced or made slime sm
tassium salts, the medium did not support the on BMA containing 5% (w/v) sucrose. The fol- .
o
growth of our isolates (final turbidity readings lowing strains were tested for a constitutive r
g
0 to 15 Klett units). These results indicated arginine dihydrolase system and were found to /

that the strains had no organic growth factor be negative: 2, 8, 19, 32 (group A-1); 40, 44, 47, o
n
requirements but required sodium ion for 50 (group A-2); 51, 52 (group B-1); 54, 57,
growth. (group B-2); 58, 59 (group C-1); 60, 62, 64 A
p
forFetrhmeierntaabtiiliotny.toAllfetrhmeensttrDa-ignslucwoesreebtyesttheed ((ggrroouupp DC--12));; 7696,,8170,(gr7o4up(gDr-o2u)p; 8C4-,3)8;6 7(7g,rou7p8 ril 3
use of two media, F-1 and F-2 (5). Strains 67 D-3); 89, 93, 96 (group D-4); 99, 101, 107, 113, ,
to 76, which were able to grow on D-fructose 117 (group E-1); 121, 124, 130 (group E-2); 133, 20
but not D-glucose, were also tested on media 135 (group F-1); 139, 141, 143 (group F-2); 146, 1
which differed from F-1 and F-2 in containing 149, 153, 155 (group G-1); 156, 158 (group G-2); 9

D-fructose. Only those strains which were un- 171, 175, 181, 185, 190, 193 (group G-3); 196, by
able to ferment D-glucose or D-fructose were 197 (group H-1); 201, 202 (group H-2); 205, 207 g
included in this study. (group I-1); 210, 213 (group I-2); and strains u
Gram stain, cell shape, and motility. All 214-219. e
s
the cells were gram-negative when stained in Oxidase test. Strains 34 to 50 (group A-2), t
exponential phase of growth. Strains 1 to 33 79 to 83 (group D-2), and 139 to 144 (group F-
(Fig. 1), 199, 200, and 201 were curved rods; 2) were oxidase negative. The remaining
the remainingstrains were straight rod (Fig. 2- strains were oxidase positive.
19). With the exception of strains 142, 191, Range of organic compounds utilized. A
192, 218, and 219, all the strains were motile. total of 149 organic compounds were tested for
Strains of groups B-1, B-2 and 1-2 had a tend- their ability to serve as sole sources of carbon
ency to aggregate into rosettes (Fig. 5 and 6). and energy. Of these, 127 were utilized by one
The aggregated cells always comprised a mi- or more strains in our collection (Table 1).
406 BAUMANN ET AL. J. BACTERIOL.
...%Oii,lWje J
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FIG. 1-16. Phase-contrast micrographs ofcells inexponentialphase ofgrowth(x 2,000). MarkerinFig. 16
represents 5 Mm. Fig. 1, Alteromonas communis, strain 8; Fig. 2, A. vaga, strain 47; Fig. 3, A. macleodii,
strain 107; Fig. 4,A. marinopraesens, strain 121;Fig.5,groupB-i, strain51;Fig. 6,groupB-2, strain55;Fig.
7, Pseudomonas doudoroffii, strain 70; Fig. 8, P. marina, strain 140; Fig. 9, group G-1, strain 146; Fig. 10,
group G-2, strain 160; Fig. 11, P. nautica, strain 179; Fig. 12,group H-1, strain 197; Fig. 13,groupI-1, strain
207; Fig. 14,groupI-2, strain210;Fig. 15,Alcaligenespacificus, strain62;andFig. 16,A. cupidus, strain 79.
VOL. 110, 1972 AEROBIC MARINE EUBACTERIA 407
F,eva 'A"'&a
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FIG. 17-19. Phase-contrast microgrphs ofcells in exponentialphase ofgrowth (x 2,000). Markerin Fig. s
19represents 5fim; Fig. 17,AlIcaligenes venustus, strain86; Fig. 18,A.-aestus, strain 134;-andFig. 19,group m
H-2, strain202. .o
FIG. 20-24. Variation in Leifson flagella stains characteristic of strains in groups B-l, B-2, I-2, and P. r
g
marina (group F-2). Photomicrographs illustrating single polar flagella have not been included. (x 2,500). /

MarkerinFig. 24represents 5Aim; Fig. 20, P. marina,strain 143; Fig.21,groupB-1, strain52;Fig. 22,group o
I-2,strain211;Fig.23,P. marina,strain 140;andFig.24,P. marina,strain 143. n

A
p
Only acetate and pyruvate served as universal ters of strains within the data was by a com- ril
carbon sources. The compounds which were plete-linkage method (manuscript in prepara- 3
,
not utilized by any of the strains were: cellu- tion). On the basis of the numerical analysis, 2
lose, agar, inulin, mucate, formate, oxalate, the strains were separated into major groups 0
1
methanol, isopropanol, geraniol, phenylethane- (designated by letters) having S-values of66% 9
diol, naphthalene, DL-norleucine, DL-a-ami- or less (see Fig. 45). Further subdivisions were b
nobutyrate, D-tryptophan, m-aminobenzoate, made at the 68 to 76% S-values. The resulting y

p-aminobenzoate, methylamine, tryptamine, a- groups were designated by the major group g
u
amylamine, 2-amylamine, pantothenate, and letter and a numeral. The phenotypic charac- e
guanine. teristics of the groups, expressed as the s
t
Numerical analysis. With the exception of number of strains having a given trait, are
flagellation, cell shape, PHB accumulation, given in Table 1. From these data it can be
arginine dihydrolase, mechanisms of aromatic seen that the majority of the groups can be
ring cleavage, and reduction of nitrate to ni- distinguished on the basis ofa large number of
trite, all the nutritional and physiological data nutritional and physiological traits. Figure 46
for each strain were submitted to a numerical gives the range ofthe number oforganic com-
analysis by Eleanora Szabo (Department of pounds utilized by each group as sole sources
Microbiology, University of Queensland, Bris- ofcarbon and energy.
bane, Australia) with programs for a GE 225 Flagellation, PHB accumulation, and
computer. The estimation of similarity be- mechanism of aromatic ring cleavage. As
tween strains was based on the inclusion of seen in Fig. 45, flagellation, PHB accumula-
both positive and negative characters, using tion, and the mechanism of aromatic ring
the simple similarity coefficient (S) described cleavage, traits which were not included in the
by Sokal and Sneath (49). The search for clus- numerical analysis, support the groupings es-
D
_,+ + + g+
,+
408 BAUMANN ET AL. J. BACTERIOL.
++ + I 1I I+ +II I + +
- --I + I++ III II I+I++ i+I++ I+I
24-. +P'+ -+ 11 1 1 I 1 1 11 1
e + v+ + + 1++ 1 1 l
-t + ++ +I 1 Il i li i
n vclo+ + + D
L. gs -.n.--.-IIIIIIII ..- I 1 1 lIi o
C? + 4q+nl+lllc w
n
lo
a
III P .I ± d
r0A c-4 ++;a + + +++ +I I I+ cI I ~I I+I++ ±++I I + I IIIIIIIII ed

f
r
o
+ ;4 + + I + + 111+11 m

L. h
N t +_ +1 k+ IIC+II0UIZ+U ia + i tt
C4 +a4 + + s + + cel 4 +to +Lo ++a+ p
:
/
/
jb
.
a
Q s
L d > + ,_+ + S+ C4 ob M + + + + + + + + m
.
4Q o
0. +X,x + + o + oo IIL"OIo Lo rg
/

o
n
I.. _+ $, + + O + +IIIIIIIIIIII+II- A
p
r
il
3
,

a4 2
0
0. 1
cs ;I+ 4+ I++I_ Iz+ + + + + ++ 9

b
S.. y

g
0. u
H e
s
t
E-
cs
4EHH *CDO*C* * -.- ). . . . . . . ..
*O. )o *---.....-...........o.......
*-a -
UcbZ,vg,bcE ¢o$-4Z4¢co 43sd-dconC) d; O0 ;C.-~ co~ ~ 3H~1~z vX ;x sO
co .cC* ooo
~~~~~~~~~~~~~~.~~~~~~~.
VOL. 110, 1972 AEROBIC MARINE EUBACTERIA 409
II ++++++ ++++++ +I++I I III+I
II +++++I ±IIIII +I+II I IIIII
I+++ +±C +1 + II+III
I + + + + + + Nq CS 1 1n + : gS_
a)~~~~~~~~4
ii ++_ s-l l1 C I I+II
++ Cl N 11 + 1+1111
+ i C4~ C4 + +1 1 1 + 1
+ + +Csf+ + + +C4- - -_4.. -4rCD t 1- 00 O a) =~~~~~~~a
~~~0
o
+ + + + t + + I+ +
I + + + +oC t- CD rCc e + +I+CD LO IeI D
02~~ ~ ~ o
w
I ++CD+to L +CD + +U +I+II II I+I n
0
lo
I+++I-+± +++t ++ + + ++I+ dQo~~~~~,~W ~cc~;;~~~~~~~~~~~~~~ ade
- + +CCLi- r +I + I+ d

f
r
XC + + +++ e + + + + I i i o
m
0,5
+Cd + + + + + + ++ + 1+ ++I h
t
+ ±-++ +++ _I +I I _1 + 0 tp:
/
~ /jb
4 C.) .a
++ +I+++++ +±+++± +I+III III+I± c sm
cd .o
II+C+I I+i+I'I + + oI II r
~ ~ ~ ~ c g
)CD /

I + I II +I o
n

+I+ ++ +I++I + A
p
a) t *^-W^ o r
I+ + + -- cq + + sI III +cq + _ INq C14Cf + c C il 3
,

+I+ +±+1+ _q++IIcI+ic±C?liN C-1 + +3 ~~~~S- 20
1
9
C1± + ± O±±CDC 00 0 +±I+II I+ IC b
+c I +I+ II I+I COCrO- X CZ EU0CO y g
CDCW) b( Cco u
(D 0n L6 *_ _ bwb0 A es
.. .. .. .... O4)~. t
.. .. .......
> 8) E.. = E -;+ >l 0)0
0
4. .....4..........CD................... ¢ Q v4 t Wzc-1
C. .aj .. .=..................-. ............... +.oo z cq : n1l.6 =;C
,.|CZs ~<!~4*F..~;~O14.@>t~~a>)O ;.~A.z.,3* ; .-..5.z..lEaa.~-. .a..........
CO% .:< m° O0 CrOOCC :r. ,
410 BAUMANN ET AL. J. BACTERIOL.
CIO I-t+++± ++lIII+ ±11111 + + + +-- +
l
I {III- -iI -Ii-1II1-I111 1
2Cf I-± +I ++I I 111 [phone]
Cl4
I++l+ + ±+CC I III + 11 1 + +
I++I i ,++-1ic11 lI C i CI I II1CID1
I-I-I-l+ +++ I I I+Ico-I +1 i + I I li
D
o
cq CI±±I±CC + -,±- + IDIC+± +'CIDi mi w
n
ir~i I~ I+~-I II IID co +II i lo
a
~ I~~I-*I-*I I-I**- III I+ M L1O11LO de
d
-q + +±o+±iI±-TII ±I ± IC++ ++ILa ~ Cl+I fr
o
m
CCj h
1e:33w "' m Ii±++it+ +~+ +++±Ci 0+C++C-C±I+i++ + +±± ~4C4l+~cq -lI' ttp:
z //
4z.4 'C +±+ I-++ + ± + +I+ + +±+I I' e Y Y jb
c0i .a
6 vD + +++ II Il I I I11 L sm
w4 I±i+ I +++± ++ I C4 Ii ieD +1c I I I I+ICICc+I+ .o
m r
E- ~~~Cl~~ ++~~~~ ii i i ~~I~~~~ +1111 l+iii + I +i 1+ g
/

o
n
'cC + +++ +I +1 11 i IC +
A
p
C?II+ + +++I I+I ++ 1 1 II I + + ril
3
C +±±I+ +++I' i± ~ + I + I I ± + ,
2
<CC -l-- 0
1
9

b
y

g
u
e
s
t
~~ ~ ~ ~ :-
4-1
-040c-.0*01
0 IC)
....
.....~~~~~.
....
~~~~~~~+
VOL. 110, 1972 AEROBIC MARINE EUBACTERIA 411
++ I+ I-++- 1111I1I1 I+I +I I + lI i 111111 II
+1.-.I I-+I + I I+1 1 1 1 1 1
+C1+ +++ -41+C -+t+ - + +I t+ I++++ +
+ + +ccqJc C CO Cq CO + + + + + I+ I_I + + +cJ C'] + +C
CID --4 1 C + I
+ + + I-++i + +
D
U±0+I++ i+I 10+0 11C1_4 I Ii o
w
++ - 4I C N + 1 11 1 1 m II I I n
lo
+t+i I I ID CIDc+o+I+ II DI 1 I 1 I ad
e
+ + + + I+++ts -o tUI + + +iT4L+ O++ 1 d

f
r
o
+ + CID ++ + +et 1I+++ _ I I m

h
+ III -I+ I- II 0 +u 1I +I I+ I I I++++I I I t
4 t
p
1 ++ +I++++ III I II I+I +++ I III :/
/
jb
++ MC'+ 00 cqID + ++t- +t- + + ++++ I+ t- ++c + I .a
s
+++++ +++++ ++m+ + + + + + + c + m
.
o
+ + t+ - c>I + + + + + + NIIC_ +C C CqI+ ++I I rg
/

+ + + _ + _- _ II + + +_1- _ + + + + I o
n

+ +O= +00 11+ + I+ + I+ + + + _I II+ULoI + + +%0 A
p
r
++"++ + + + + + + + ~~~+~~~~+~++ + LoU + + + + _+ + 4N il 3
,

++++ + + + + + + II I I+ + + + + I +cq+ +-I 20
1
+ + + + + + CID +- Cs CD CO C + + + + IIII + i+ M 1-4 + + + II II 9
b
y
+ + + +++c+ + + + + + +C'cI+ + + +I III + I+c +I I+ + +I + g
u
e
+ +0 C t + +± 4 l I e + IC I I I I +I II II s
t
........~~~~~~~e.. . .......... ................................................
-4 - -4 P. . ...... ................................."..........
..... ........ ..... ......... .......................................................4..
co... ................... ........................................................c.o....c..o...... ....... ... ....... ....
Z ........<..14 < . . ..a.. 0. w. o =......a. )..1............Q....
|>ce8 0 0 a 0X E E eEEI e E = |
> V-4 1 t;; ;=.a- _4;; t; tco<-14,;C$xmP-4eSmvvvuSZ ZZ