Proposal of Cyclobacterium Marinus Gen. Nov., Comb. Nov. for a Marine Bacterium Previously Assigned to the Genus Flectobacillus

INTERNATIONALJOURNAL OF SY~TEMATICBACTERIOLOGY, Oct. 1990, p. 337-347 Vol. 40, No. 4 OO20-7713/~/O40337-11$02.W/O Copyright 0 1990, International Union of Microbiological Societies

Proposal of Cyclobacterium marinus gen. nov., comb. nov. for a Marine Bacterium Previously Assigned to the Genus Flectobacillus

H. D. RAJ1* AND 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.

More than 60 years ago Orskov (10) created the genus and 5. F. major 3 and S. linguale lN,2, and 5 were obtained ‘‘Microcyclus” for gram-negative, nonmotile, aerobic, vibri- from John Larkin; the rest of the cultures were obtained oid bacteria that exhibit a closed ringlike morphology. Since from the American Type Culture Collection (ATCC), Rock- then, several similar isolates have been readily identified as ville, Md. DSM strains were from the Deutsche Sammlung members of this genus on the basis of their characteristic von Mikroorganismen, Gottingen, Federal Republic of Ger- morphology alone (3, 11, 13, 16). However, the systematics many. of these ring-forming bacteria has been reexamined and In addition, two pink-pigmented marine ring-forming bac- redefined during the last decade. teria that were recently isolated by D. A. Bazylinski and The name “Microcyclus” was found to be illegitimate by H. W. Jannasch at the Woods Hole Oceanographic Institute, Raj (15) because of the precedence of a fungal genus with the Woods Hole, Mass., were included in this study. These same name, and the members of the bacterial genus “Micro- organisms were obtained from the deep sea (about 2 m above cyclus” were transferred to the new genus Ancylobacter the benthic floor) near the thermal vents of the Guaymas (15). The ring-forming bacteria have been found to be a Basin (Gulf of California) tectonic spreading center. They heterogeneous group consisting of four genera, Ancylobac- were obtained from hydrocarbon-containing seawater (4°C) ter, Flectobacillus, Runella, and Spirosoma; the last three that was collected at a depth of 2,003 m with a Niskin genera are grouped in the family Spirosomaceae (6,8,9) and sampler. Primary isolation of these barotolerant ring-forming are separated from the genus Ancylobacter, which remains bacteria was made at room temperature on a very-low- unaifiliated with any family (6, 16, 19, 22). Because of these nutrient artificial seawater medium containing (per liter) 0.05 changes, “Microcyclus aquaticus” (Orskov), “Microcyclus g of casein hydrolysate, 0.05 g of peptone, and 5.0 ml of major” (Gromov), and “Microcyclus flavus” (Raj) became Wolfe mineral elixir (23). These two isolates were identified Ancylobacter aquaticus, Flectobacillus major, and as F. marinus and were designated strains WH-A (= ATCC Spirosoma linguale, respectively; each species had two or 43824) and WH-B (= ATCC 43825). more strains. “Microcyclus marinus” (Raj) with its single A pale pink-pigmented freshwater isolate was also in- strain (11) was placed as a second species in the genus cluded in this study. This organism was isolated from Flectobacillus, the type species of which is F. major (1, 6). sediments of Lake George near New York by D. L. Tison, In view of the two additional marine isolates recently Rensselaer Polytechnic Institute, Troy, N.Y. Primary isola- identified as Flectobacillus marinus (H. D. Raj, S. R. Maloy, tion was made by spread plating the sediments onto an agar D. A. Bazylinski, and H. W. Jannasch, Abstr. Annu. Meet. medium containing 0.1% peptone, 0.02% yeast extract, and Am. SOC.Microbiol. 1988, R-9, p. 23), we reassessed the 0.1% glucose at room temperature. This isolate was identi- systematics of the bacteria in the family Spirosomaceae, fied as R. slithyformis and was designated strain RPI (= placing particular emphasis on the taxonomic status of F. ATCC 49304). marinus and F. major and the levels of relatedness of these Morphological and cultural characteristics. The morpho- organisms with the other family members. logical and cultural traits of all of the organisms except F. marinus (Table 1) were determined by using young slant MATERIALS AND METHODS cultures grown on tryptone-glucose extract agar (Difco Lab- Ring-forming bacteria studied. The members of the family oratories, Detroit, Mich.) made with distilled water and Spirosomaceae (6,9) which were included in this study were fortified with 0.1% yeast extract (TGEY medium); F. mari- F. marinus RajT (= ATCC 25205=) (superscript T indicates nus strains were grown on modified Zobell marine agar (11). type strain), F. major GromovT (= ATCC 2949fiT) and 3, Electron microscopy. Exponentially grown cells of F. Runella slithyformis 4T (= ATCC 29530T) and 6 (= ATCC major GromovT and F. marinus strains were fixed in gluta- 29531), and S. linguale Raj (= ATCC 23276), lN(= DSM raldehyde for scanning electron microscopy or were nega- 74N) (superscript N indicates neotype strain), 2 (= DSM 79, tively stained with 1% phosphotungstic acid (pH 7) for transmission electron microscopy as described previously (11). * Corresponding author. Biochemical and physiological characteristics. The bio-

337 TABLE 1. Morphological and cultural characteristicsa S. linguale C. marinus RajT, F. major R. slithyformis 2,5, and Trait no. Characteristic WH-A, and WH-B GromovT and 3 6, and RPI 4=, lN, RG Gram-negative unicells having: 1 Round ends 2 Tapered ends 3 Vibriod, horseshoe, and ringlike shapes 4 Coils 5 Spiral forms 6 Cell width (pm) 7 Outer ring diam (pm) 8 Filaments 9 Short rods 10 Pleomorphic 11 Encapsulated 12 Branching 13 Gas vacuoles 14 Flagellar motility 15 Sheaths 16 Spores or cysts 17 Zoogloea Growth in broth cultures 18 Aerobic 19 Anaerobic 20 Facultative 21 Pellicle 22 Ring 23 Flaky sediment 24 Viscid sediment 25 Even turbidity 26 Flocculent turbidity Appearance of colonies 27 Mucoid 28 Dull 29 Opaque 30 Transparent or translucent 31 Smooth 32 Contoured Elevation of colonies 3? Convex 34 Concave 35 Flat 36 Pulvinate 37 Raised 38 Umbonate Form of colonies 39 Circular 40 Filamentous 41 Irregular 42 Rhizoid 43 Spindle Margins of colonies 44 Entire 45 Curled 46 Erose 47 Filamentous 48 Granular 49 Lobate 50 Undulate Pigment (nondifhsible) 51 Yellowish 52 Pinkish or pale rose 53 Grey or off-white Colony size 54 Punctiform (1 mm) 55 Small (1-2 mm) 56 Medium (24 mm) 57 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 VOL.40, 1990 CYCLOBACTERIUM MARINUS GEN. NOV., COMB. NOV. 339

TABLE 2. Physiological characteristic9 C. marinus F. major R. slithyformis S. linguale Trait no. Characteristicb RajT, WH-A, GromovT 4T, 6, lN, 2, 5, and WH-B and 3 and RPI and Raj Growth at: 58 67°C W 59 20-30°C + (25)' 60 3 140°C W 61 4145°C - 62 PH 5 - 63 PH 6 W 64 PH 7 + 65 PH 8 + 66 PH 9 W 67 pH 10 - Growth in the presence Of 68 0% NaCl - 69 1.5% (wthol) NaCl + 70 3% (wt/vol) NaCl + 71 5% (wthol) NaCl + 72 10% (wthol) NaCl W 73 15% (wthol) NaCl W 74 20% (wt/vol) NaCl - 75 5% (wt/vol) sucrose - 76 10% (wt/vol) sucrose - 77 15% (wthol) sucrose - 78 20% (wthol) sucrose - Growth after 10 min of exposure to: 79 55°C + 80 80°C - 81 95°C - Growth after 2.5 min of exposure to: 82 70% Ethanol - 83 3% Hydrogen peroxide - 84 0.1% Zepharine chloride - Growth on the following nonselective and selective media: 85 Blood agar + 86 Brock synthetic seawater medium + 87 Eosin methylene blue agar + 88 Marine agar 2216 + 89 Microcyclus-spirosoma agar + 90 Modified Zobell2216 marine agar + 91 Nutrient agar W 92 Trypticase soy agar + 93 Tryptone-glucose extract agar containing 0.1% yeast extract + +, 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 40 cultural, 52 physiological, and 74 biochemical traits; determined by using the procedures described previously Tables 1 through 4) was analyzed to determine the Jaccard (8, 9, 11, 13). In the case of F. marinus, all media other coefficient of similarity (18) for each pair of bacteria studied. than seawater media were fortified with 3.0% NaCl and 0.03% K,HP04. Antibacterial agents were tested on Muel- ler-Hinton agar (BBL Microbiology Systems, Cockeysville, RESULTS AND DISCUSSION Md.) or microcyclus-spirosoma agar. Sole carbon sources were tested (in amounts having carbon contents equivalent New freshwater isolate RPI was found to be almost to the carbon content of 0.5% glucose) by using the follow- identical to the type strain of R. slithyformis (Tables 1 ing two media for comparison: Simmons citrate agar con- through 4). It differed from the type strain in having intrac- taining bromthymol blue indicator (13) and modified Koser ellular volutin deposits in older pleomorphic cultures and in citrate agar having phenol red indicator (9) with the agar growing on eosin methylene blue agar (Difco) as small deleted and the citrate replaced with the sole carbon source colonies (diameter, ca. 1 mm). being tested (Table 4). Compared with the former medium, The two recent marine ring-forming isolates (strains the latter medium was found more sensitive and yielded WH-A and WH-B) resembled F. marinus type strain Raj (= better results. ATCC 25205) not only morphologically (Fig. 1) but also Jaccard coefficient. A total of 183 traits (17 morphological, biochemically, culturally, and physiologically (Tables 1 340 RAJ AND MALOY INT. J. SYST.BACTERIOL.

TABLE 3. Susceptibilities to antibiotics and sulfonamides" C. marinus Raj', F. major R. slithyformis S. IinguaIe Trait no. Antimicrobial agent IN, WH-A, and WH-B Gromov' and 3 4T, 6, and RPI 2, 5, and Raj

94 Ampicillin (10 pg) S S S S 95 Aureomycin (15 pg) R S NT S 96 Cephalothin (30 pg) S S S S 97 Colistin (10 pg) R R R R 98 Erythromycin (15 pg) S S S S 99 Furadantin + macrodantin (300 pg) S S NT S 100 Kanamycin (30 pg) R S Vb S 101 Neomycin (30 pg) R V" V" V" 102 Nitrofurantoin (300 pg) S S S S 103 Penicillin G (10 U) R S S S 104 Polymyxin B (50 pg) R R R R 105 Streptomycin (10 pg) R S S Sd 106 Sulfamethoxazole/trimethoprim (25 pg) R S S S 107 Sulfathiazole (300 pg) S S NT S 108 Tetracycline (30 pg) S S S S 109 Triple sulfa (250 pg) R R R 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 differentiated from F. major by its inability to hydrolyze (97%) with F. marinus type strain Raj, as reported previ- gelatin, starch, tributyrin, and urea and its inability to ously (Raj et al., Abstr. Annu. Meet. Am. SOC.Microbiol. produce acid from cellobiose and dextrin; also, it was 1988). However, there were a few differences among the resistant to aureomycin, kanamycin, penicillin G, strepto- strains. Compared with the type strain, new strains WH-A mycin, and sulfame thoxazole/trimethoprim. In addition, un- and WH-B had somewhat more intense pink pigmentation like F. major, F. marinus grew in or on media containing (trait 52). Also, unlike the type strain, strain WH-A did not seawater or 3% NaCl and utilized acetate, citrate, fumarate, utilize L-glutamate as a sole carbon source (trait 165), and malate, malonate, and tartrate as single carbon sources. strain WH-B did not produce any reaction in litmus milk Metabolic studies to elucidate the primary and secondary (trait 116) and produced a small amount of ammonia from pathways for carbohydrate catabolism showed that both F. peptone water (trait 123) and weak acidity from raffinose and marinus Raj' and F. major Gromov' oxidized glucose and rhamnose (traits 130 and 131). Strain WH-A was susceptible gluconate primarily via the Embden-Meyerhof and Entner- to neomycin (trait 101), and strain WH-B was resistant to Doudoroff pathways, respectively, with some concurrent this drug. participation of the pentose phosphate pathway, in conjunc- F. marinus and F. major GromovT were morphologically tion with the tricarboxylic acid (TCA) cycle. However, in distinct. The latter was considerably larger (as much as 50 contrast to F. marinus, which catabolized each of these times or more larger in cell volume) with wide open ringlike substrates by the three primary pathways described above cells and some straight or slightly curved rods that had concurrently with a strong amphibolic TCA cycle (11, 12, tapered andlor uniformly rounded ends (Fig. 2). Also, under 14), F. major seemed to oxidize the same substrates by certain cultural conditions, F. major Gromov' formed long mutually exclusive operation of the Embden-Meyerhof or filaments (>50 bm) with bulbous and involuted shapes but Entner-Doudoroff pathway and did not seem to have an rarely ringlike cells. When a subculture of F. major Gro- active TCA cycle for utilization of acetate, citrate, and other movT was grown in MR-VP medium (Difco) or its lyophile TCA intermediates (Raj, Abstr. Annu. Meet. Am. SOC. was grown in TGEY broth at room temperature on a shaker Microbiol. 1987, K-166, p. 230; Raj, Abstr. Annu. Meet. at <60rpm, pleomorphic cells were produced. The bizarre Am. SOC.Microbiol. 1989, R-23, p. 284). shapes and sizes of this bacterium were originally observed The Jaccard coefficient, which indicates the unbiased in old cultures and in cultures grown on rich nutrient media percentage of similarity based on the number of positive (3). Such morphological variations of F. major are shown in features (N,) and number of dissimilarities (Nd) shared by Fig. 3. The pointed ends seen in some cells (Fig. 3) were also each pair of organisms (18), was computed from more than observed previously (2, 3). However, electron microscopy 180 traits (Tables 1 through 4). F. major shared only 41 N, revealed that these cells with pointed ends actually had and 31 Nd traits with R. slithyformis, 56 N, and 33 Nd traits tapering rounded ends (with somewhat declining cell width with S. linguale, and 48 N, and 48 Nd traits with F. marinus. at the termini instead of uniformly rounded ends with the Similarly, R. slithyformis shared only 27 N, and 62 Nd traits same cell width), resulting from a deep constriction during with F. marinus and 35 N, and 46 Nd traits with S. linguale, cell division (Fig. 2). Such morphological variations have which shared 54 N, and 39 Nd traits with F. marinus. Thus, never been observed in F. marinus subcultures. we calculated that F. major has Jaccard similarity coeffi- Culturally, unlike F. marinus, F. major formed flocculent cients of about 57, 63, and 50% with R. slithyformis, S. growth and a pellicle in broth cultures and large (5-mm- linguale, and F. marinus, respectively. While R. slithyformis diameter), smooth, slimy, glistening, transparent, pinkish exhibits about 43 and 30% similarity with S. linguale and F. colonies on agar media (Table 1). Physiologically and bio- marinus, respectively, S. linguale exhibits only 58% similar- chemically (Tables 2 through 4), F. marinus was clearly ity with F. marinus. Surprisingly, F. major exhibits the VOL. 40, 1990 CYCLOBACTERIUM MARINUS GEN. NOV., COMB. NOV. 341

TABLE 4. Biochemical characteristics"

~~ ~ C. marinus RajT, F. major R. slithyformis S. linguale Trait no. Characteristic lN, WH-A, and WH-B GromovT and 3 4T, 6, and RPI 2, 5, and Raj 110-112 Agar, cellulose, and chitin digestion - 113 Casein hydrolysis Wb 114 Gelatin liquefaction + 115 Starch hydrolysis W 116 Litmus milk: reduction, acid, or soft curd + 117 Litmus milk: alkaline, hard curd, or peptonization - 118-122 Indole, methyl red, Voges-Proskauer, HZS,and NO, re- - duction tests 123 NH, produced from peptone + Acid production from carbohydrates 124-127 Glucose, inulin, maltose, and sucrose + 128-131 Galactose, mannose, ranose, and rhamnose + 132-139 Arabinose, fructose, lactose, melibiose, a-methyl gluco- +g side, salicin, trehlose, and xylose 140-141 Cellobiose and dextrin +h 142 Ribose + 143 Sorbose - Acid production from the sugar alcohols 144-150 Adonitol, dulcitol, erythritol, glycerol, inositol, manni- - tol, and sorbitol Carbon sources utilized 151 Acetate - 152-160 Benzoate, formate, glycine, glyoxylate, a-ketoglutarate, - lactate, methanol, methylamine, and propionate 161 Citrate - 162-163 Fumarate and malate - 164-166 Gluconate, L-glutamate, and pyruvate + 167 Glycerol phosphate + 168 L-Serine + 169-170 Malonate and tartrate + 171 Oxalacetate - 172 Succinate + Enzyme activities 173 Catalase + 174 P-Galactosidase + 175-178 Hemoly sins, lecithinase, lysine decarboxylase, ornithine - decarboxylase, and phenylalanine deaminase 179 Lipase (tributyrin) - 180 Oxidase + 181 Phosphatase W 182 Urease - 183 DNA G+C content (mol%)k 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.

J 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%.

highest Jaccard similarity value with S. linguale, not with F. nent of the profile of F. marinus and is absent from the F. marinus. These findings not only confirmed the current major profile. Also, iso-C,,,, and n-C,,,, acids and the separate genus status for F. major, S. linguale, and R. hydroxy fatty acids are absent from F. marinus; these slithyformis but also supported the proposal that F. marinus findings contrast with the fatty acid profile of F. major (20). should be reassigned to a separate genus (14, 22). On the basis of these data, F. marinus can be clearly Cellular fatty acid composition has been used to differen- distinguished from F. major. However, like the other family tiate the ring-forming bacteria chemotaxonomically (20). The members tested, both of these organisms possess a mena- cellular fatty acid profile of F. marinus differs from the quinone system with MK-7 as the major component and profiles of F. major and other members of the family MK-6 and MK-8 as very minor components (20). Spirosomaceae in that anteiso-C,,,, acid is a major compo- DNA-DNA homology studies (7) in which the type strains 342 RAJ AND MALOY INT. J. SYST.BACTERIOL.

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. VOL. 40, 1990 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. 344 VOL.40, 1990 CYCLOBACTERIUM MARINUS GEN. NOV.. COMB. NOV. 345

of the family members were used showed that R. slithy- only. However, yellow-pigmented, marine, ring-forming iso- formis formed 42% interspecific heteroduplexes with S. lates (4, 5) that morphologically resembled the bacteria linguale, 50% interspecific heteroduplexes with F. major, included in this study were found to be physiologically as and 45% interspecific heteroduplexes with F. marinus, well as biochemically different from any previously de- whereas F. major exhibited 50% genetic relatedness with S. scribed marine or nonmarine ring-forming bacteria, includ- linguale. We believe that these homology values support the ing yellow-pigmented Spirosoma isolates. Since pigment current separate genus status for each of the family mem- production is also a function of the cultural conditions bers. In the same study, F. marinus Raj’ was paired with provided, it cannot be a stable criterion for differentiation F. major GromovT, LAH, S-1, and 014, and DNA-DNA among related phenotypes. Therefore, a better classificatory homology values of 71, 50, 40, and 63%, respectively, were scheme (16a) should be based on stable and reliable bio- obtained. Estimates of genetic relatedness based on levels of chemical parameters (Table 4) that also allow distinct differ- DNA homology are meaningful only when closely related entiation between the freshwater pink-pigmented Runella bacteria which possess genomes of the same size are com- and Flectobacillus isolates and the marine pink-pigmented pared; however, such estimates were not made (7) even Cyclobacterium isolates, as follows: though F. major can be more than 50-fold larger in cell Key to the genera of the family Spirosomaceae: volume than F. marinus (16a). Nevertheless, on the basis of the level of DNA homology between F. major GromovT and I. Nonmethylotrophic freshwater bacteria; no growth in F. marinus RajT (71%) and the similar guanine-plus-cytosine media containing seawater or 3% NaCl; sugar alcohols (G+C) contents of these organisms (38 to 40 mol%), Larkin (glycerol , mannitol, sorbitol) not oxidized. and Borrall placed both species in the genus Flectobacillus (6, 7). Since the other three strains of F. major exhibited A. Acid not produced from most carbohydrates, in- levels of DNA homology of 82 to 97% with type strain cluding ribose; lipase (tributyrin) but not urease pro- Gromov, they are recognized as legitimate strains of this duced. species (7). However, because of the wide range of homology values (40 to 71%) obtained when the four F. major Genus I. Runella strains were paired with the type strain of F. marinus (7), these results do not validate the current placement of both AA. Acid produced oxidatively from most carbohy- organisms in the same genus. Furthermore, since G+C drates but not from ribose; lipase (tributyrin) and ure- contents of taxonomically diverse bacteria can be very ase produced. similar, this characteristic is not a sufficient criterion for Genus 11. Flectobacillus 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 AAA. Acid produced oxidatively from most carbohy- mol%) . drates, including ribose; lipase (tributyrin) and urease The results of recent molecular studies (22) based on not produced. sequence catalogs of 16s rRNAs of F. major, F. marinus, R. Genus 111. Spirosoma slithyformis, and S. linguale indicate that these ring-forming spiroids form a cluster deep within the Flavobacterium- Bacteroides phylum (21). Furthermore, the data clearly 11. Nonmethylotrophic marine bacteria; no growth in media show relatively large evolutionary distances (ca. 20%) lacking seawater or 3% NaCl; glycerol, mannitol, sorbi- among the four members of the Spirosomaceae (22). These to1 not oxidized. findings suggest that these four species have diverged suffi- ciently to be classified in four distinct genera; thus, F. A. Acid produced oxidatively from most carbohydrates marinus should be separated from F. major at the genus but not from ribose; lipase (tributyrin) and urease not level. This molecular differentiation is consistent with the produced. findings of the numerical studies, as well as the chemotaxo- Genus IV. Cyclobacterium nomic studies described above. In view of the evidence obtained from the polyphasic taxonomic studies cited above, we propose that F. marinus A description of the proposed new genus Cyclobacterium should be separated from F. major at the genus level. is given below. Because it cannot be assigned to any other taxon, we Cyclobacterium gen. nov. Cyclobacterium (Cy.clo.bac. propose that F. marinus should be reclassified as the type ter’i.um. Gr. n. cyclos, a circle; Gr. n. bakterion, a small species of a new genus, Cyclobacterium, in the family rod; M. L. neut. n. Cyclobacterium, a circle-shaped bacte- Spirosomaceae. rium). Mostly circle-shaped (ringlike) and horseshoe-shaped The current determinative scheme for classification of cells with an outer diameter of 0.8 to 1.5 pm and a cell width members of the Spirosomaceae (6) is essentially based on of 0.3 to 0.7 pm. The cells have rounded (never tapered) the pigmentation produced by these bacteria; for example, ends. Coils, spiral forms, and some straight rods occur less members of the genus Spirosoma are differentiated from the frequently. Filamentous or pleomorphic cells are rare. other members of the family on the basis of yellow pigment Gram negative. Encapsulated. Nonflagellated and non-

FIG. 3. Photomicrographs showing sequential morphological variations of F. major GromovT when 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. 346 RAJ AND MALOY INT.J. SYST.BACTERIOL.

motile. No resting or life cycle stages. No sheath or zoog- for lipase, oxidase, and urease are positive. Weakly positive loea. No gas vacuolation. for catalase. Optimum growth occurs at 20 to 25°C in media containing The G+C content of the DNA is 39.5 to 40.3 mol% seawater or 3.0% NaC1. Convex, mucoid, opaque, smooth, (thermal denaturation method). small (<2-mm) colonies grow on modified Zobell marine The natural habitat is freshwater lakes. agar or TGYE agar containing 3% NaCl. The type species is F. major; the type strain of this species No pellicle or ring formation occurs in broth cultures. is strain Gromov (= ATCC 29496). Strictly aerobic. Chemoorganotrophic but not rhethylo- Flectobacillus major comb. nov. Flectobacillus major (ma’ trophic. Metabolism is oxidative, never fermentative. Acids jor. L. adj. major, larger). (Basonym: “Microcyclus major” are produced from most carbohydrates but not from ribose, Gromov 1963). In addition to the generic characteristics sorbose, and sugar alcohols. given above, the original definition (6, 9) of this species Malonate and tartrate but not glycerol phosphate are remains valid. utilized as single carbon sources. Positive for oxidase and catalase but not for lipase and ACKNOWLEDGMENTS urease. We are grateful to D. A. Bazylinski, D. Frank, and D. Distel The G+C content of the DNA is 33.7 mol% (thermal (Oceanographic Institute, Woods Hole, Mass.) for the determina- denaturation method) or 38.3 to 38.7 mol% (bouyant density tion of the DNA base composition of C. marinus WH-A; to R. L. method). Weiss (San Diego State University, San Diego, Calif.) for Fig. lA, The natural habitat is marine environments. D. A. Bazylinski for Fig. 1B and C, and S. S. Sekhon (Veterans The type species is Cyclobacterium marinus comb. nov. Administration Medical Center, Long Beach, Calif.) for Fig. 2A and Cyclobacterium marinus comb. nov. Cyclobacterium mari- B; to R. J. Freligh for help with the photomicrography; and to nus (ma. ri’ nus. L. adj. marinus, of the sea, marine). Ngocdiep Le for laboratory assistance. (Basonyms: “Microcyclus marinus” Raj 1976 and Flectoba- cillus marinus Borrall and Larkin 1978.) LITERATURE CITED In addition to the characteristics given above for the genus 1. Borrall, R., and J. M. Larkin. 1978. Flectobacillus marinus description, gelatin and starch are not hydrolyzed. (Raj) comb. nov., a marine bacterium previously assigned to Acid is not produced from cellobiose or dextrin. Microcycfus. Int. J. Syst. Bacteriol. 28:341-343. Acetate, citrate, fumarate, and malate are utilized as 2. Claus, D. 1967. Taxonomy of some highly pleornorphic bacteria. Spisy Priorodoved. Fak. Univ. J. E. Purkyne Brno K40:254- single carbon sources. 257. Resistant to aueromycin, kanamycin, penicillin G, strep- 3. Gromov, B. V. 1963. A new bacterium of the genus Microcyclus. tomycin, and sulfamethoxazole/trimethoprim. Dokl. Akad. Nauk. SSSR 152:733-734. The type strain is strain C. marinus Raj (= ATCC 25205). 4. Hauxhurst, J. D., M. I. Krichevsky, and R. M. Atlas. 1980. Other strains are strains WH-A (= ATCC 43824) and WH-B Numerical taxonomy of bacteria from the Gulf of Alaska. J. (= ATCC 43825). Gen. Microbiol. 120:131-148. 5. Kaneko, T., M. I. Krichevsky, andR. M. Atlas. 1979. Numerical The removal of F. marinus from the genus Flectobacillus taxonomy of bacteria from the Beaufort Sea. J. Gen. Microbiol. necessitates reverting the emended definition of this genus 110:111-125. (1, 6) to the original description as first proposed (9), with 6. Larkin, J. M., and R. Borrall. 1984. Family I. Spirosornaceace, some slight modifications based on the additional differences p. 125-132. In N. A. Krieg and J. G. Holt (ed.), Bergey’s manual described in this paper. , of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Genus FZectobacillus Larkin et al. 1977 emended. (Flec.to. Baltimore. ba.cil’ lus. L. v.flecto, to curve; L. n. bacillus, a little staff, 7. Larkin, J. M., and R. Borrall. 1984. Deoxyribonucleic acid base rod; M. L. masc. n. Flectobacillus, curved rod.) Gram- composition and homology of Microcycfus, Spirosoma, and similar organisms. Int. J. Syst. Bacteriol. 34:211-215. negative curved rods with variable degrees of curvature from 8. Larkin, J. M., and P. M. Williams. 1978. Runella sfithyformis n. cell to cell. Most cells are shaped like the letter C (wide open gen., n. sp., a curved, non-flexible, pink bacterium. Int. J. Syst. rings), and sometimes cell ends touch or overlap (closed Bacteriol. 28: 32-36. rings). The outer ring diameter is 5 to 10 pm, and the cell 9. Larkin, J. M., P. M. Williams, and R. Taylor. 1977. Taxonomy width is 0.6 to 2.0 pm. of the genus Microcyclus grskov 1928: reintroduction and The cell termini are tapered or rounded or both. Coils are emendation of the genus Spirosoma Migula 1894 and proposal of less common, and spiral forms are rare. a new genus, Flectobacillus. Int. J. Syst. Bacteriol. 27:147-156. Bizarre shapes and sizes (filaments >50 Fm long with 10. 0rskov, J. 1928. Beschreibung eines neuen Mikroben, Microcy- bulbous structures, swollen involuted cells, and short rods) clus aquaticus rnit eigentumlicher Morphologie. Zentralbl. Bak- teriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 107:180- occur under certain cultural conditions. 184. Nonflagellated, nonmotile, and nonflexible. 11. Raj, H. D. 1976. A new species-Microcyclus marinus. Int. J. Optimum growth occurs at 20 to 25°C in or on freshwater Syst. Bacteriol. 26528-544. media but not in or on media containing seawater or 3.0% 12. Raj, H. D. 1977. Microcyclus and related ring-forming bacteria. NaCl. Colonies on such media are large (diameter, 5 mm) Crit. Rev. Microbiol. 5243-269. with a pinkish or pale rose pigment. A cottony pellicle but no 13. Raj, H. D. 1970. A new species-Microcycluspavus. Int. J. ring is formed in broth cultures. Syst. Bacteriol. 20:61-81. Strict aerobe. Chemoorganotrophic but not methylo- 14. Raj, H. D. 1981. The genus Microcyclus and related bacteria, p. trophic. 630-644. In M. P. Stan-, H. Stolp, H. G. Triiper, A. Balows, and H. G. Schlegel (ed.), The prokarotes. A handbook on habitats, Metabolism is oxidative, never fermentative. Acid is pro- isolation and identification of bacteria, vol. 1. Springer-Verlag, duced from most carbohydrates, including cellobiose and New York. dextrin, but not from ribose, sorbose, and sugar alcohols. 15. Raj, H. D. 1983. Proposal of Ancylobacter gen. nov. as a Glycerol phosphate, malonate, tartrate, and many TCA substitute for the bacterial genus Microcyclus grskov 1928. Int. intermediates are not utilized as single carbon sources. Tests J. Syst. Bacteriol. 33:397-398. VOL. 40, 1990 CYCLOBACTERZUM MARZNUS GEN. NOV., COMB. NOV. 347

16. R@, H. D. 1989. Oligotrophic methylotrophs: Ancylobacter Bergey’s manual of systematic bacteriology, vol. 1. The (basonym “Microcyclus” P)rskov) Raj gen. nov. Crit. Rev. Williams & Wilkins Co., Baltimore. Microbiol. 17:8%106. 20. Urakami, T., and K. Komagata. 1986. Methane-utilizingAmy- 16a.Rqj, H. D., and S. R. Maloy. 1990. Family Spirosomaceae: clobacter strains and comparison of their cellular fatty acid gram-negative,ring-forming, aerobic bacteria. Crit. Rev. Micro- compositions and quinone systems with those of Spirosoma, biol. 17:329-361. Flectobacillus, and Runella species. Int. J. Syst. Bacteriol. 17. Rrlj, H. D., and K. A. Paveglio. 1983. Contributing carbohydrate 36:415-421. catabolic pathways in Cyclobacterium marinus. J. Bacteriol. 21. Woese, C. R. 1987. Bacterial evolution. Microbiol. Rev. 51:221- 271. 153:335-339. 22. Woese, C. R., S. R. Maloy, L. Mandelco, and H. D. Raj. 1990. 18. Sneath, P. H. A. 1957. The applications of computers to taxon- Phylogenetic placement of the Spirosomaceae. Syst. Appl. omy. J. Gen. Microbiol. 17:201-226. Microbiol. 13:19-23. 19. Staley, J. T., and A. E. Konopka. 1984. Genus Microcyclus 23. Wolin, E. A., M. J. Wolin, and R. S. Wolfe. 1963. Formation of Orskov 1928, p. 133-139. In N. R. Krieg and J. G. Holt (ed.), methane by bacterial extracts. J. Biol. Chem. 238:2882-2885.