Proposal of Cyclobacterium marinus gen. nov., comb. nov. for a

INTERNATIONALJOURNAL OF SY~TEMATIC
BACTERIOLOGY,
Oct. 1990, p. 337-347
OO20-7713/~/O40337-11$02
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Copyright 0 1990, International Union of Microbiological Societies
Vol. 40, No. 4
Proposal of Cyclobacterium marinus gen. nov., comb. nov. for a
Marine Bacterium Previously Assigned to the Genus Flectobacillus
H. D. RAJ1* A N D S. R. MALOY2
Department of Microbiology, California State University, Long Beach, California 90840,’ and Department of
Microbiology, University of Illinois, Urbana, Illinois 618012
The recent isolation and characterization of two marine strains of Flectobacillus marinus are significant for
precise definition of this species, which previously was based on a single strain. Polyphasic taxonomic studies
showed that this species is not closely related to the only other species, F . major, of the genus FZectobuciZZus.
Because F. marinus cannot be assigned to any other genus, a new genus, Cyclobacterium, in the family
Spirosomaceae is proposed. We recommend that F . marinus be transferred to the new genus, with
Cyclobacterium marinus comb. nov. as the type species. The type strain is C. marinus Raj (= ATCC 25205).
Descriptions of the new genus and its species, necessitated by this change, and a key for differentiation from
other family members are presented together with an emended description of the genus FbctobuciZZus.
and 5. F. major 3 and S. linguale lN,2, and 5 were obtained
from John Larkin; the rest of the cultures were obtained
from the American Type Culture Collection (ATCC), Rockville, Md. DSM strains were from the Deutsche Sammlung
von Mikroorganismen, Gottingen, Federal Republic of Germany.
In addition, two pink-pigmented marine ring-forming bacteria that were recently isolated by D. A. Bazylinski and
H. W. Jannasch at the Woods Hole Oceanographic Institute,
Woods Hole, Mass., were included in this study. These
organisms were obtained from the deep sea (about 2 m above
the benthic floor) near the thermal vents of the Guaymas
Basin (Gulf of California) tectonic spreading center. They
were obtained from hydrocarbon-containing seawater (4°C)
that was collected at a depth of 2,003 m with a Niskin
sampler. Primary isolation of these barotolerant ring-forming
bacteria was made at room temperature on a very-lownutrient artificial seawater medium containing (per liter) 0.05
g of casein hydrolysate, 0.05 g of peptone, and 5.0 ml of
Wolfe mineral elixir (23). These two isolates were identified
as F. marinus and were designated strains WH-A (= ATCC
43824) and WH-B (= ATCC 43825).
A pale pink-pigmented freshwater isolate was also included in this study. This organism was isolated from
sediments of Lake George near New York by D. L. Tison,
Rensselaer Polytechnic Institute, Troy, N.Y. Primary isolation was made by spread plating the sediments onto an agar
medium containing 0.1% peptone, 0.02% yeast extract, and
0.1% glucose at room temperature. This isolate was identified as R. slithyformis and was designated strain RPI (=
ATCC 49304).
Morphological and cultural characteristics. The morphological and cultural traits of all of the organisms except F.
marinus (Table 1) were determined by using young slant
cultures grown on tryptone-glucose extract agar (Difco Laboratories, Detroit, Mich.) made with distilled water and
fortified with 0.1% yeast extract (TGEY medium); F. marinus strains were grown on modified Zobell marine agar (11).
Electron microscopy. Exponentially grown cells of F.
major GromovT and F. marinus strains were fixed in glutaraldehyde for scanning electron microscopy or were negatively stained with 1% phosphotungstic acid (pH 7) for
transmission electron microscopy as described previously
(11).
Biochemical and physiological characteristics. The bio-
More than 60 years ago Orskov (10) created the genus
‘‘Microcyclus” for gram-negative, nonmotile, aerobic, vibrioid bacteria that exhibit a closed ringlike morphology. Since
then, several similar isolates have been readily identified as
members of this genus on the basis of their characteristic
morphology alone (3, 11, 13, 16). However, the systematics
of these ring-forming bacteria has been reexamined and
redefined during the last decade.
The name “Microcyclus” was found to be illegitimate by
Raj (15) because of the precedence of a fungal genus with the
same name, and the members of the bacterial genus “Microcyclus” were transferred to the new genus Ancylobacter
(15). The ring-forming bacteria have been found to be a
heterogeneous group consisting of four genera, Ancylobacter, Flectobacillus, Runella, and Spirosoma; the last three
genera are grouped in the family Spirosomaceae (6,8,9) and
are separated from the genus Ancylobacter, which remains
unaifiliated with any family (6, 16, 19, 22). Because of these
changes, “Microcyclus aquaticus” (Orskov), “Microcyclus
major” (Gromov), and “Microcyclus flavus” (Raj) became
Ancylobacter aquaticus, Flectobacillus major, and
Spirosoma linguale, respectively; each species had two or
more strains. “Microcyclus marinus” (Raj) with its single
strain (11) was placed as a second species in the genus
Flectobacillus, the type species of which is F. major (1, 6).
In view of the two additional marine isolates recently
identified as Flectobacillus marinus (H. D. Raj, S. R. Maloy,
D. A. Bazylinski, and H. W. Jannasch, Abstr. Annu. Meet.
Am. SOC.Microbiol. 1988, R-9, p. 23), we reassessed the
systematics of the bacteria in the family Spirosomaceae,
placing particular emphasis on the taxonomic status of F.
marinus and F. major and the levels of relatedness of these
organisms with the other family members.
MATERIALS AND METHODS
Ring-forming bacteria studied. The members of the family
Spirosomaceae (6,9) which were included in this study were
F . marinus RajT (= ATCC 25205=) (superscript T indicates
type strain), F. major GromovT (= ATCC 2949fiT) and 3,
Runella slithyformis 4T (= ATCC 29530T) and 6 (= ATCC
29531), and S. linguale Raj (= ATCC 23276), lN(= DSM
74N) (superscript N indicates neotype strain), 2 (= DSM 7 9 ,
* Corresponding author.
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TABLE 1. Morphological and cultural characteristicsa
S . linguale
Trait no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
3?
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
C . marinus RajT,
WH-A, and WH-B
Characteristic
F. major
GromovT and 3
R. slithyformis 4=,
6, and RPI
lN, 2 , 5 , and
RG
Gram-negative unicells having:
Round ends
Tapered ends
Vibriod, horseshoe, and ringlike shapes
Coils
Spiral forms
Cell width (pm)
Outer ring diam (pm)
Filaments
Short rods
Pleomorphic
Encapsulated
Branching
Gas vacuoles
Flagellar motility
Sheaths
Spores or cysts
Zoogloea
Growth in broth cultures
Aerobic
Anaerobic
Facultative
Pellicle
Ring
Flaky sediment
Viscid sediment
Even turbidity
Flocculent turbidity
Appearance of colonies
Mucoid
Dull
Opaque
Transparent or translucent
Smooth
Contoured
Elevation of colonies
Convex
Concave
Flat
Pulvinate
Raised
Umbonate
Form of colonies
Circular
Filamentous
Irregular
Rhizoid
Spindle
Margins of colonies
Entire
Curled
Erose
Filamentous
Granular
Lobate
Undulate
Pigment (nondifhsible)
Yellowish
Pinkish or pale rose
Grey or off-white
Colony size
Punctiform (1 mm)
Small (1-2 mm)
Medium ( 2 4 mm)
Large (4-6 mm)
a +, Most common feature present; L, less common feature present; -, feature absent. All bacteria except C.marinus were grown on TGEY medium (14)at
room temperature; C. marinus strains were grown on modified Zobell marine medium (12).
Unlike the type strain, strain RPI produced intracellular volutin deposits in older pleomorphic cultures.
In contrast to previous findings (11, 12, 14), all of the C. marinus strains formed capsules. Also, lack of capsule formation by R. slithyformis has not been
unequivocally established.
Strains WH-A and WH-B exhibited slightly more intense pinkish pigmentation than the type strain.
338
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CYCLOBACTERIUM MARINUS GEN. NOV., COMB. NOV.
339
TABLE 2. Physiological characteristic9
Trait no.
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
C . marinus
RajT, WH-A,
and WH-B
Characteristicb
Growth at:
67°C
20-30°C
3140°C
4145°C
PH 5
PH 6
PH 7
PH 8
PH 9
pH 10
Growth in the presence Of
0% NaCl
1.5% (wthol) NaCl
3% (wt/vol) NaCl
5% (wthol) NaCl
10% (wthol) NaCl
15% (wthol) NaCl
20% (wt/vol) NaCl
5% (wt/vol) sucrose
10% (wt/vol) sucrose
15% (wthol) sucrose
20% (wthol) sucrose
Growth after 10 min of exposure to:
55°C
80°C
95°C
Growth after 2.5 min of exposure to:
70% Ethanol
3% Hydrogen peroxide
0.1% Zepharine chloride
Growth on the following nonselective and selective media:
Blood agar
Brock synthetic seawater medium
Eosin methylene blue agar
Marine agar 2216
Microcyclus-spirosoma agar
Modified Zobell2216 marine agar
Nutrient agar
Trypticase soy agar
Tryptone-glucose extract agar containing 0.1% yeast extract
F . major
GromovT
and 3
R . slithyformis
4T, 6,
and RPI
S . linguale
lN, 2, 5 ,
and Raj
W
+ (25)'
W
W
+
+
W
-
+
+
+
W
W
-
-
+
-
+
+
+
+
+
+
W
+
+
+, Good growth; w, weak growth; v, variable results; -, no growth after incubation for 35 days at room temperature.
Media other than the seawater media used for C. marinus were fortified with 3.0% NaCl and 0.03% K,HPO, (12).
The numbers in parentheses are optimum temperatures (in degrees Celsius).
Strain 2 did not grow on blood agar.
Strain RPI grew on eosin methylene blue agar as small (diameter, ca. 1 mm) colonies.
Strain 3 did not grow on Trypticase soy agar.
chemical and physiological traits (Tables 2 through 4) were
determined by using the procedures described previously
(8, 9, 11, 13). In the case of F. marinus, all media other
than seawater media were fortified with 3.0% NaCl and
0.03% K,HP04. Antibacterial agents were tested on Mueller-Hinton agar (BBL Microbiology Systems, Cockeysville,
Md.) or microcyclus-spirosoma agar. Sole carbon sources
were tested (in amounts having carbon contents equivalent
to the carbon content of 0.5% glucose) by using the following two media for comparison: Simmons citrate agar containing bromthymol blue indicator (13) and modified Koser
citrate agar having phenol red indicator (9) with the agar
deleted and the citrate replaced with the sole carbon source
being tested (Table 4). Compared with the former medium,
the latter medium was found more sensitive and yielded
better results.
Jaccard coefficient. A total of 183 traits (17 morphological,
40 cultural, 52 physiological, and 74 biochemical traits;
Tables 1 through 4) was analyzed to determine the Jaccard
coefficient of similarity (18) for each pair of bacteria studied.
RESULTS AND DISCUSSION
New freshwater isolate RPI was found to be almost
identical to the type strain of R . slithyformis (Tables 1
through 4). It differed from the type strain in having intracellular volutin deposits in older pleomorphic cultures and in
growing on eosin methylene blue agar (Difco) as small
colonies (diameter, ca. 1 mm).
The two recent marine ring-forming isolates (strains
WH-A and WH-B) resembled F . marinus type strain Raj (=
ATCC 25205) not only morphologically (Fig. 1) but also
biochemically, culturally, and physiologically (Tables 1
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RAJ AND MALOY
INT. J. SYST.BACTERIOL.
TABLE 3. Susceptibilities to antibiotics and sulfonamides"
Trait no.
Antimicrobial agent
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
Ampicillin (10 pg)
Aureomycin (15 pg)
Cephalothin (30 pg)
Colistin (10 pg)
Erythromycin (15 pg)
Furadantin + macrodantin (300 pg)
Kanamycin (30 pg)
Neomycin (30 pg)
Nitrofurantoin (300 pg)
Penicillin G (10 U)
Polymyxin B (50 pg)
Streptomycin (10 pg)
Sulfamethoxazole/trimethoprim (25 pg)
Sulfathiazole (300 pg)
Tetracycline (30 pg)
Triple sulfa (250 pg)
C . marinus Raj',
WH-A, and WH-B
F . major
Gromov' and 3
R . slithyformis
4T, 6, and RPI
S . IinguaIe IN,
2, 5, and Raj
S
R
S
R
S
S
S
S
S
S
NT
S
R
S
NT
Vb
V"
S
S
R
S
S
NT
S
R
S
S
S
R
S
S
S
V"
S
S
R
Sd
S
S
S
R
R
R
S
R
R
R
R
S
S
R
R
S
S
S
V"
S
S
R
S
S
S
S
R
a All antimicrobial agents were tested on Mueller-Hinton agar or microcyclus-spirosoma agar for most bacteria; for C. marinus Mueller-Hinton agar was
fortified with 3.0% NaCl and 0.03%K,HP04. The cultures were incubated at room temperature. S, Susceptible; R, resistant; V, variable results; NT, not tested.
Strain 6 was susceptible to kanamycin.
F. major 3 was susceptible to neomycin, and R . slithyformis 6 and S . linguale 2 and 5 were resistant to neomycin.
Strain 2 was resistant to streptomycin.
through 4). They exhibited a very high similarity coefficient
(97%) with F. marinus type strain Raj, as reported previously (Raj et al., Abstr. Annu. Meet. Am. SOC.Microbiol.
1988). However, there were a few differences among the
strains. Compared with the type strain, new strains WH-A
and WH-B had somewhat more intense pink pigmentation
(trait 52). Also, unlike the type strain, strain WH-A did not
utilize L-glutamate as a sole carbon source (trait 165), and
strain WH-B did not produce any reaction in litmus milk
(trait 116) and produced a small amount of ammonia from
peptone water (trait 123) and weak acidity from raffinose and
rhamnose (traits 130 and 131). Strain WH-A was susceptible
to neomycin (trait 101), and strain WH-B was resistant to
this drug.
F. marinus and F. major GromovT were morphologically
distinct. The latter was considerably larger (as much as 50
times or more larger in cell volume) with wide open ringlike
cells and some straight or slightly curved rods that had
tapered andlor uniformly rounded ends (Fig. 2). Also, under
certain cultural conditions, F . major Gromov' formed long
filaments (>50 bm) with bulbous and involuted shapes but
rarely ringlike cells. When a subculture of F . major GromovT was grown in MR-VP medium (Difco) or its lyophile
was grown in TGEY broth at room temperature on a shaker
at <60rpm, pleomorphic cells were produced. The bizarre
shapes and sizes of this bacterium were originally observed
in old cultures and in cultures grown on rich nutrient media
(3). Such morphological variations of F . major are shown in
Fig. 3. The pointed ends seen in some cells (Fig. 3) were also
observed previously (2, 3). However, electron microscopy
revealed that these cells with pointed ends actually had
tapering rounded ends (with somewhat declining cell width
at the termini instead of uniformly rounded ends with the
same cell width), resulting from a deep constriction during
cell division (Fig. 2). Such morphological variations have
never been observed in F . marinus subcultures.
Culturally, unlike F . marinus, F . major formed flocculent
growth and a pellicle in broth cultures and large (5-mmdiameter), smooth, slimy, glistening, transparent, pinkish
colonies on agar media (Table 1). Physiologically and biochemically (Tables 2 through 4), F . marinus was clearly
differentiated from F . major by its inability to hydrolyze
gelatin, starch, tributyrin, and urea and its inability to
produce acid from cellobiose and dextrin; also, it was
resistant to aureomycin, kanamycin, penicillin G, streptomycin, and sulfamethoxazole/trimethoprim. In addition, unlike F . major, F . marinus grew in or on media containing
seawater or 3% NaCl and utilized acetate, citrate, fumarate,
malate, malonate, and tartrate as single carbon sources.
Metabolic studies to elucidate the primary and secondary
pathways for carbohydrate catabolism showed that both F .
marinus Raj' and F . major Gromov' oxidized glucose and
gluconate primarily via the Embden-Meyerhof and EntnerDoudoroff pathways, respectively, with some concurrent
participation of the pentose phosphate pathway, in conjunction with the tricarboxylic acid (TCA) cycle. However, in
contrast to F. marinus, which catabolized each of these
substrates by the three primary pathways described above
concurrently with a strong amphibolic TCA cycle (11, 12,
14), F . major seemed to oxidize the same substrates by
mutually exclusive operation of the Embden-Meyerhof or
Entner-Doudoroff pathway and did not seem to have an
active TCA cycle for utilization of acetate, citrate, and other
TCA intermediates (Raj, Abstr. Annu. Meet. Am. SOC.
Microbiol. 1987, K-166, p. 230; Raj, Abstr. Annu. Meet.
Am. SOC.Microbiol. 1989, R-23, p. 284).
The Jaccard coefficient, which indicates the unbiased
percentage of similarity based on the number of positive
features (N,) and number of dissimilarities ( N d ) shared by
each pair of organisms (18), was computed from more than
180 traits (Tables 1 through 4). F . major shared only 41 N ,
and 31 Nd traits with R . slithyformis, 56 N , and 33 Nd traits
with S . linguale, and 48 N , and 48 Nd traits with F . marinus.
Similarly, R . slithyformis shared only 27 N , and 62 Nd traits
with F. marinus and 35 N , and 46 Nd traits with S. linguale,
which shared 54 N , and 39 Nd traits with F . marinus. Thus,
we calculated that F . major has Jaccard similarity coefficients of about 57, 63, and 50% with R . slithyformis, S .
linguale, and F . marinus, respectively. While R . slithyformis
exhibits about 43 and 30% similarity with S. linguale and F .
marinus, respectively, S . linguale exhibits only 58% similarity with F . marinus. Surprisingly, F . major exhibits the
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VOL.40, 1990
341
TABLE 4. Biochemical characteristics"
~~
~
Trait no.
Characteristic
110-112
113
114
115
116
117
118-122
Agar, cellulose, and chitin digestion
Casein hydrolysis
Gelatin liquefaction
Starch hydrolysis
Litmus milk: reduction, acid, or soft curd
Litmus milk: alkaline, hard curd, or peptonization
Indole, methyl red, Voges-Proskauer, HZS,and NO, reduction tests
NH, produced from peptone
Acid production from carbohydrates
Glucose, inulin, maltose, and sucrose
Galactose, mannose, r a n o s e , and rhamnose
Arabinose, fructose, lactose, melibiose, a-methyl glucoside, salicin, trehlose, and xylose
Cellobiose and dextrin
Ribose
Sorbose
Acid production from the sugar alcohols
Adonitol, dulcitol, erythritol, glycerol, inositol, mannitol, and sorbitol
Carbon sources utilized
Acetate
Benzoate, formate, glycine, glyoxylate, a-ketoglutarate,
lactate, methanol, methylamine, and propionate
Citrate
Fumarate and malate
Gluconate, L-glutamate, and pyruvate
Glycerol phosphate
L-Serine
Malonate and tartrate
Oxalacetate
Succinate
Enzyme activities
Catalase
P-Galactosidase
Hemoly sins, lecithinase, lysine decarboxylase, ornithine
decarboxylase, and phenylalanine deaminase
Lipase (tributyrin)
Oxidase
Phosphatase
Urease
DNA G+C content (mol%)k
123
124-127
128-131
132-139
140-141
142
143
144-150
151
152-160
161
162-163
164-166
167
168
169-170
171
172
173
174
175-178
179
180
181
182
183
C . marinus RajT,
WH-A, and WH-B
F. major
GromovT and 3
R. slithyformis
4T, 6, and RPI
S . linguale lN,
2, 5 , and Raj
Wb
+
+
W
-
-
+
+
+
+g
+h
+-
-
-
-
-
+
+
+
+
+
+
+
-
+
W
-
51.0-52.9
+, Positive reaction; w, weak reaction; v, variable reaction; -, negative reaction; NT, not tested.
Strain 2 did not hydrolyze casein, and strain 5 hydrolyzed casein very slowly.
Strain WH-B did not produce any reaction in litmus milk.
Strain WH-B produced a small amount of NH, from peptone water.
R. slithyformis 6 and RPI utilized glucose very slowly, and F. major 3 produced very weak acidity from inulin.
R. slithyformis 6 did not produce acid from galactose and mannose. C. marinus WH-B, F . major 3, and R . slithyformis 6 produced weak acidity from raffinose
and rhamnose, but R. slithyformis 4 did not produce acid from these two sugars.
Strain 2 produced acid from arabinose and fructose very slowly.
Strain 2 produced acid from dextrin very slowly.
C. marinus WH-A did not utilize L-glutamate as a sole carbon source, and R. slithyformis 4 utilized L-glutamate very slowly.
Strain 3 utilized oxalacetate.
G+C content determined by the thermal denaturation method. The G+C content of C. marinus WH-A was 38.7 mol%; the G +C content of strain WH-B was
not determined. Other data were obtained from references 6 and 14. The G+C content of C. marinus determined by the buoyant density method was 38.3 to 38.7
mol%.
J
highest Jaccard similarity value with S . linguale, not with F .
marinus. These findings not only confirmed the current
separate genus status for F. major, S . linguale, and R .
slithyformis but also supported the proposal that F. marinus
should be reassigned to a separate genus (14, 22).
Cellular fatty acid composition has been used to differentiate the ring-forming bacteria chemotaxonomically (20). The
cellular fatty acid profile of F. marinus differs from the
profiles of F. major and other members of the family
Spirosomaceae in that anteiso-C,,,, acid is a major compo-
nent of the profile of F. marinus and is absent from the F.
major profile. Also, iso-C,,,, and n-C,,,, acids and the
hydroxy fatty acids are absent from F. marinus; these
findings contrast with the fatty acid profile of F. major (20).
On the basis of these data, F. marinus can be clearly
distinguished from F. major. However, like the other family
members tested, both of these organisms possess a menaquinone system with MK-7 as the major component and
MK-6 and MK-8 as very minor components (20).
DNA-DNA homology studies (7) in which the type strains
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INT. J. SYST.BACTERIOL.
RAJ AND MALOY
FIG. 1. Scanning electron micrographs of C. marinus RajT (A) and strains WH-A and WH-B (B and C). Reprinted from Critical Reviews
in Microbiology (16a) with permission of the publisher.
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CYCLOBACTERIUM MARINUS GEN. NOV., COMB. NOV.
343
FIG. 2. Transmission electron micrographs of negatively stained cells (A and B) and scanning electron micrographs (C and D) of F. major
GromovT, showing ringlike shapes with tapered ends (arrows). Bars = 1 km. Panels A and B are reprinted from Critical Reviews in
Microbiology (16a) with permission of the publisher.
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CYCLOBACTERIUM MARINUS GEN. NOV.. COMB. NOV.
of the family members were used showed that R . slithyformis formed 42% interspecific heteroduplexes with S .
linguale, 50% interspecific heteroduplexes with F. major,
and 45% interspecific heteroduplexes with F. marinus,
whereas F. major exhibited 50% genetic relatedness with S .
linguale. We believe that these homology values support the
current separate genus status for each of the family members. In the same study, F. marinus Raj’ was paired with
F. major GromovT, LAH, S-1, and 014, and DNA-DNA
homology values of 71, 50, 40, and 63%, respectively, were
obtained. Estimates of genetic relatedness based on levels of
DNA homology are meaningful only when closely related
bacteria which possess genomes of the same size are compared; however, such estimates were not made (7) even
though F. major can be more than 50-fold larger in cell
volume than F. marinus (16a). Nevertheless, on the basis of
the level of DNA homology between F. major GromovT and
F . marinus RajT (71%) and the similar guanine-plus-cytosine
(G+C) contents of these organisms (38 to 40 mol%), Larkin
and Borrall placed both species in the genus Flectobacillus
(6, 7). Since the other three strains of F . major exhibited
levels of DNA homology of 82 to 97% with type strain
Gromov, they are recognized as legitimate strains of this
species (7). However, because of the wide range of homology values (40 to 71%) obtained when the four F . major
strains were paired with the type strain of F. marinus (7),
these results do not validate the current placement of both
organisms in the same genus. Furthermore, since G+C
contents of taxonomically diverse bacteria can be very
similar, this characteristic is not a sufficient criterion for
grouping these bacteria in a single genus (16). Thus, R .
slithyformis and S . linguale are placed in different genera (6,
7) even though they have the same G+C content (49 to 51
mol%).
The results of recent molecular studies (22) based on
sequence catalogs of 16s rRNAs of F . major, F. marinus, R .
slithyformis, and S . linguale indicate that these ring-forming
spiroids form a cluster deep within the FlavobacteriumBacteroides phylum (21). Furthermore, the data clearly
show relatively large evolutionary distances (ca. 20%)
among the four members of the Spirosomaceae (22). These
findings suggest that these four species have diverged sufficiently to be classified in four distinct genera; thus, F.
marinus should be separated from F. major at the genus
level. This molecular differentiation is consistent with the
findings of the numerical studies, as well as the chemotaxonomic studies described above.
In view of the evidence obtained from the polyphasic
taxonomic studies cited above, we propose that F. marinus
should be separated from F . major at the genus level.
Because it cannot be assigned to any other taxon, we
propose that F. marinus should be reclassified as the type
species of a new genus, Cyclobacterium, in the family
Spirosomaceae.
The current determinative scheme for classification of
members of the Spirosomaceae (6) is essentially based on
the pigmentation produced by these bacteria; for example,
members of the genus Spirosoma are differentiated from the
other members of the family on the basis of yellow pigment
345
only. However, yellow-pigmented, marine, ring-forming isolates (4, 5) that morphologically resembled the bacteria
included in this study were found to be physiologically as
well as biochemically different from any previously described marine or nonmarine ring-forming bacteria, including yellow-pigmented Spirosoma isolates. Since pigment
production is also a function of the cultural conditions
provided, it cannot be a stable criterion for differentiation
among related phenotypes. Therefore, a better classificatory
scheme (16a) should be based on stable and reliable biochemical parameters (Table 4) that also allow distinct differentiation between the freshwater pink-pigmented Runella
and Flectobacillus isolates and the marine pink-pigmented
Cyclobacterium isolates, as follows:
Key to the genera of the family Spirosomaceae:
I. Nonmethylotrophic freshwater bacteria; no growth in
media containing seawater or 3% NaCl; sugar alcohols
(glycerol, mannitol, sorbitol) not oxidized.
A. Acid not produced from most carbohydrates, including ribose; lipase (tributyrin) but not urease produced.
Genus I. Runella
AA. Acid produced oxidatively from most carbohydrates but not from ribose; lipase (tributyrin) and urease produced.
Genus 11. Flectobacillus
AAA. Acid produced oxidatively from most carbohydrates, including ribose; lipase (tributyrin) and urease
not produced.
Genus 111. Spirosoma
11. Nonmethylotrophic marine bacteria; no growth in media
lacking seawater or 3% NaCl; glycerol, mannitol, sorbito1 not oxidized.
A. Acid produced oxidatively from most carbohydrates
but not from ribose; lipase (tributyrin) and urease not
produced.
Genus IV. Cyclobacterium
A description of the proposed new genus Cyclobacterium
is given below.
Cyclobacterium gen. nov. Cyclobacterium (Cy.clo.bac.
ter’i.um. Gr. n. cyclos, a circle; Gr. n. bakterion, a small
rod; M. L. neut. n. Cyclobacterium, a circle-shaped bacterium). Mostly circle-shaped (ringlike) and horseshoe-shaped
cells with an outer diameter of 0.8 to 1.5 pm and a cell width
of 0.3 to 0.7 pm. The cells have rounded (never tapered)
ends. Coils, spiral forms, and some straight rods occur less
frequently. Filamentous or pleomorphic cells are rare.
Gram negative. Encapsulated. Nonflagellated and non-
FIG. 3. Photomicrographs showing sequential morphological variations of F . major GromovTwhen it was subcultured from a lyophile in
TGEY broth at room temperature on a shaker after overnight growth (l), intermediate growth (3 through 8), and 3 days of growth (9 through
11). The arrow in photograph 2 indicates an enlarged bulbous cell structures shown in photograph 1, and the arrows in photographs 4, 6, and
10 indicate either swollen ends or pointed ends of cells. Magnification, x 1,200 to x 1,500. Reprinted from Critical Reviews in Microbiology
(16a) with permission of the publisher.
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346
RAJ AND MALOY
INT.J. SYST.BACTERIOL.
motile. No resting or life cycle stages. No sheath or zoogloea. No gas vacuolation.
Optimum growth occurs at 20 to 25°C in media containing
seawater or 3.0% NaC1. Convex, mucoid, opaque, smooth,
small (<2-mm) colonies grow on modified Zobell marine
agar or TGYE agar containing 3% NaCl.
No pellicle or ring formation occurs in broth cultures.
Strictly aerobic. Chemoorganotrophic but not rhethylotrophic. Metabolism is oxidative, never fermentative. Acids
are produced from most carbohydrates but not from ribose,
sorbose, and sugar alcohols.
Malonate and tartrate but not glycerol phosphate are
utilized as single carbon sources.
Positive for oxidase and catalase but not for lipase and
urease.
The G+C content of the DNA is 33.7 mol% (thermal
denaturation method) or 38.3 to 38.7 mol% (bouyant density
method).
The natural habitat is marine environments.
The type species is Cyclobacterium marinus comb. nov.
Cyclobacterium marinus comb. nov. Cyclobacterium marinus (ma. ri’ nus. L. adj. marinus, of the sea, marine).
(Basonyms: “Microcyclus marinus” Raj 1976 and Flectobacillus marinus Borrall and Larkin 1978.)
In addition to the characteristics given above for the genus
description, gelatin and starch are not hydrolyzed.
Acid is not produced from cellobiose or dextrin.
Acetate, citrate, fumarate, and malate are utilized as
single carbon sources.
Resistant to aueromycin, kanamycin, penicillin G, streptomycin, and sulfamethoxazole/trimethoprim.
The type strain is strain C . marinus Raj (= ATCC 25205).
Other strains are strains WH-A (= ATCC 43824) and WH-B
(= ATCC 43825).
The removal of F . marinus from the genus Flectobacillus
necessitates reverting the emended definition of this genus
(1, 6) to the original description as first proposed (9), with
some slight modifications based on the additional differences
described in this paper.
,
Genus FZectobacillus Larkin et al. 1977 emended. (Flec.to.
ba.cil’ lus. L. v.flecto, to curve; L. n. bacillus, a little staff,
rod; M. L. masc. n. Flectobacillus, curved rod.) Gramnegative curved rods with variable degrees of curvature from
cell to cell. Most cells are shaped like the letter C (wide open
rings), and sometimes cell ends touch or overlap (closed
rings). The outer ring diameter is 5 to 10 pm, and the cell
width is 0.6 to 2.0 pm.
The cell termini are tapered or rounded or both. Coils are
less common, and spiral forms are rare.
Bizarre shapes and sizes (filaments >50 Fm long with
bulbous structures, swollen involuted cells, and short rods)
occur under certain cultural conditions.
Nonflagellated, nonmotile, and nonflexible.
Optimum growth occurs at 20 to 25°C in or on freshwater
media but not in or on media containing seawater or 3.0%
NaCl. Colonies on such media are large (diameter, 5 mm)
with a pinkish or pale rose pigment. A cottony pellicle but no
ring is formed in broth cultures.
Strict aerobe. Chemoorganotrophic but not methylotrophic.
Metabolism is oxidative, never fermentative. Acid is produced from most carbohydrates, including cellobiose and
dextrin, but not from ribose, sorbose, and sugar alcohols.
Glycerol phosphate, malonate, tartrate, and many TCA
intermediates are not utilized as single carbon sources. Tests
for lipase, oxidase, and urease are positive. Weakly positive
for catalase.
The G+C content of the DNA is 39.5 to 40.3 mol%
(thermal denaturation method).
The natural habitat is freshwater lakes.
The type species is F . major; the type strain of this species
is strain Gromov (= ATCC 29496).
Flectobacillus major comb. nov. Flectobacillus major (ma’
jor. L. adj. major, larger). (Basonym: “Microcyclus major”
Gromov 1963). In addition to the generic characteristics
given above, the original definition ( 6 , 9) of this species
remains valid.
ACKNOWLEDGMENTS
We are grateful to D. A. Bazylinski, D. Frank, and D. Distel
(Oceanographic Institute, Woods Hole, Mass.) for the determination of the DNA base composition of C. marinus WH-A; to R. L.
Weiss (San Diego State University, San Diego, Calif.) for Fig. l A ,
D. A. Bazylinski for Fig. 1B and C, and S. S. Sekhon (Veterans
Administration Medical Center, Long Beach, Calif.) for Fig. 2A and
B; to R. J. Freligh for help with the photomicrography; and to
Ngocdiep Le for laboratory assistance.
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