Flectobacillus glomeratus sp. nov., a Curved, Nonmotile, Pigmented Bacterium Isolated from Antarctic Marine Environments

System. Appl. Microbiol. 9, 265-272 (1987)

Flectobacillus glomeratus sp. nov., a Curved, Nonmotile, Pigmented Bacterium Isolated from Antarctic Marine Environments A.

J. McGUIRE, P. D.

FRANZMANN, and T. A. McMEEKIN

Australian Collection of Antarctic Microorganisms, Department of Agricultural Science, University of Tasmania, Box 252C, Hobart 7001, Tasmania Australia

Received December 22, 1986

Summary A new species of the genus Flectobacillus, Flectobacillus glomeratus, is described from antarctic marine environments. Strain ACAM 171 was isolated from Burton Lake (68°37.5'S;78°05'E). Another strain, ACAM 111, was isolated from surface seawater collected near Davis Base (68°34.6'S;77°58'E), in Prydz Bay. Cells of the strains are vibrioid to highly coiled. Cells of similiar morphology were located in these envirionments by fluorescence microscopy. The cells are Gram-negative, nonmotile and become coccoid in old or ageing cultures. The strains do not produce acids from carbohydrates or alcohols and do not grow on organic acids or alcohols as sole sources of carbon. They are obligate aerobes and require seawater or marine salts for good growth. ACAM 171 is psychrophilic and ACAM 111 is psychrotrophic. The strains contain pigments, either yellow or tan, which are not flexirubins. The G+C content of their DNA is 33.0 and 33.2 mol %. The type strain is UQM 3055 (ACAM 171).

Key words: Flectobacillus - Flectobacillus glomeratus - Curved bacteria - Marine bacteria - Antarctic bacteria

Introduction The aerobic heterotrophic bacterial flora of Burton Lake (68°37.5'5; 78°05'E), an antarctic, meromicnc lake with a marine connection (Burton, 1981) was investigated in the austral spring of 1984. Part of the micro flora was composed of vibrioid to spirilled or coiled pigmented cells. A similiar strain was isolated from a water sample collected in nearby Prydz Bay. Of the four original strains collected in Antarctica, only two strains remain extant. Previous microbiological examinations of marine environments have encountered difficulties with the identification of strains with these general characteristics (Hauxhurst et al., 1980; Kaneko et al., 1979). Although one marine species, Flectobacillus marinus, is both coiled and pigmented (Larkin and Borrall, 1984a), many marine strains possess attributes which make placement in that species inappropriate. There is also a paucity of information on the taxonomy of bacteria indigenous to antarctic ecosystems. The characterisation of two strains of pigmented coiled bacteria collected in Antarctica is described herein.

Materials and Methods Bacterial strains. The bacterial strains used in this study are listed in Table 1. Media. The media used in this study were sterilized at 121°C for 15 min and the pH was unadjusted unless otherwise indicated. Ingredients used or the reference source for each medium: Sea water yeast agar (SWYA): yeast extract, 1 g; agar, 15 g; seawater, 11. Half strength seawater broth (1/2 SWB): glucose, 1 g; sodium acetate, 1 g; peptone, 1 g; yeast extract, 1 g; distilled water, 500 ml; seawater, 500 ml. Half strength seawater agar (1/2 SWA): agar, 15 g; 1/2 SWB 11. Zobell's 2216 broth (Zobell's broth): (ZoBell, 1946). Zobell's 2216 agar (Zobell's agar): (ZoBell, 1946). MicrocycluslSpirosoma Agar (MS): (Larkin and Borrall, 1984b). MicrocycluslSpirosoma Broth (MSB): as for MS, without agar. Peptone Succinate Salts (PSS): (Krieg, 1984). Modified PSS (MPSS): (Krieg, 1984). Nutrient Agar (NA): Bonox, 3 g; peptone,S g; agar, 15 g; distilled water, 1 I Blood Agar: (Cruickshank, 1965).

266

A. J. McGuire, P. D. Franzmann, and T. A. McMeekin Table 1. Strains used in this study

Collection No.

Name

Source

ACAM 111 ACAM 171 UQM 1803

Original isolate Original isolate Escherichia coli Flectobacillus major Runella slithyformis strain 2 Runella slithyformis strain 4 Spirosoma linguale strain 2

Prydz Bay, Antarctica Burton Lake, Antarctica UQM collection Dr. ]. Larkin Dr. j. Larkin Dr. j. Larkin Dr. j. Larkin

ACAM = Australian Collection of Antarctic Microorganism, Department of Argicultural Science, University of Tasmania, GPO Box 252C, Hobart, Tasmania 7001 Australia UQM = University of Queensland Collection,Department of Microbiology,University of Queensland, St.Lucia, Queensland 4067 Australia Dr.]. Larkin, Department of Microbiology, Louisiana State University, Baton Rouge, Louisiana 70803 USA.

Chocolate Agar: (Cruickshank, 1965). Tryptone Soy Agar (TSA): Trypticase soy broth (Oxoid), 3 g; yeast extract, 0.1 g; agar, 15 g, distilled water, 1 I. TSA with 3% sucrose (TSAS). TSA with 3% glucose (TSAG). Phenol red mannitol salts (PRMS): Lab-lernco powder, 1 g; peptone, 10 g; mannitol, 10 g; sodium chloride, 7.5 g; phenol red, 0.025 g; agar, 15 g; distilled water, 11; pH adjusted to 7.5. Eosin methylene blue agar (Levine) (EMB): EMB agar (Oxoid), 37.5 g; distilled water, 1 I. Peptonized milk agar (PMA): peptonized milk agar (Oxoid), 2 g; agar, 15 g; distilled water, 1 I. McConkey agar: McConkey agar (Oxoid), 52 g; distilled water, 1 I. Plate Count agar (PCA): Plate count agar (Oxoid), 17.5 g; distilled water, 1 I. Sensitest agar: Sensitest agar (Oxoid), 32 g; distilled water, 11. Marine agar: Marine agar (Difco), 55.1 g; distilled water, 11. C.L.E.D. agar (CLED): CLED agar (Oxoid), 36 g; distilled water, 1 I. Brain Heart Infusion agar (BHI): BHI agar (Oxoid), 37 g; distilled water, 1 I. Tryptone glucose extract agar (TGEY): TGEY (Difco), 24 g; yeast extract, 1 g; distilled water, 1 I. Isolation. Samples of Burton Lake water were collected with a Kemmerer water sampler and a close interval sampler similiar to that described by Croome and Tyler (1984). Strain ACAM 171 was isolated on SWYA at 10°C and was maintained in pure culture by monthly transfer on 1/2 SWA. Strain ACAM 111 was isolated on Zobell's agar from surface water collected in Prydz Bay. ACAM 111 was originally designated strain 611 and was discussed by McMeekin (1987) in his preliminary observations on psychrotrophic and psychrophilic bacteria from Antarctic water samples. Both strains were lyophilized as a suspension in halfstrength sea-water which contained 10% skim milk, however, ACAM 111 was slow to recover from lyophilized preparations. Samples of Burton Lake water were fixed at the collection site with formalin added until its final concentration reached 1.0%. The samples were examined by fluorescence microscopy after staining with acridine orange (Zimmermann, 1977), for the presence of morphologically distinctive bacterial cells. Environmental parameters were determined for Burton Lake as previously described (Franzmann et aI., 1987). Electron microscopy. Cells were stained with phosphotungstic acid (0.5%) for 15 seconds and were examined with an Hitachi H-300 or a ]EOL-1200 EX transmission electron microscope. For the preparation of thin sections, strain ACAM 111 was

grown in Zobell's 2216 broth and strain ACAM 171 was grown in 1/2 SWB for 7 days at 15°C. An equal volume of 2% glutaraldehyde in filtered and sterile seawater was added to the broths which were allowed to stand for 24 h at 4°C. The cells were treated with 2% osmium tetroxide in distilled water and embedded in 3% agar. The agar blocks were dehydrated through a series of alcohol solutions of increasing concentration and embedded in Spurr's medium. The blocks were sectioned on a LKB Ultratome III (LKB Products, Sweden). The sections were collected on copper grids and stained with lead citrate for 8 min and uranyl acetate for 10 min. Thin sections were examined with an Hitachi H-300 transmission electron microscope. For scanning electron microscopy, several drops of the fixed cell suspension used for the preparation of thin sections were placed on a 1 em! polycarbonate Nuclepore filter (pore size 0.2 urn) and vapour fixed with 10% acrolein. The filter was washed twice with sterile seawater and twice with sterile distilled water before passage through an alcohol dehydration series. The filter was transferred to 100% dry acetone and critical point dryed in a Balzers Union critical point dryer 11 120A (Balzers, Union, Leichstenstein). The filter was mounted on a stub, gold coated, and examined with a JEOL JSM-840 scanning electron microscope (JEOL Ltd., Tokyo, Japan). Cellular morphology. Cellular morphology and size were determined using phase contrast light microscopy and electron microscopy. The Gram-reaction was determined using the Gram stain (Skerman, 1967). The proportions of cells of strain ACAM 111 at different stages of its developmental cycle were determined at time intevals of 1, 5 and 8 d by preparation of negative stains from broth cultures (Zebell's broth) which had been incubated at 25°C. Biochemical and Physiological tests. Unless otherwise stated tests performed on ACAM 111 were incubated at 25°C and tests performed on ACAM 171 were incubated at 15°C. The ability to produce acid from sugars or alcohols was tested on media based on Leifson's oxidationlfermentation media for marine organisms (Leifson, 1963). However, for these tests, aged seawater was used in preference to artifical seawater, bromocresol purple was used in place of phenol red, and the concentration of carbon source was 0.5% (wt:vol). Tubes were stab inoculated and incubated for 5 weeks. The utilization of organic acids or alcohols as sole sources of carbon was tested with the media of Gordon and Mihm (1957) but the sodium chloride concentration was increased to 15 gil. The plates were incubated for a minimum of three weeks. Hydrolysis of starch and gelatin were tested using 2% starch

Flectobacillus glomeratus sp. nov. or 4% gelatin incorporated in 1/2 SWA. After inoculation and incubation for three weeks, hydrolysis was indicated by zones of clearing when the plates were flooded with aqueous iodine or 15% HgCI in 20% HCI respectively. Hydrolysis of cellulose, casein and chitin were tested by the method of Skerman (1967) using double layer plates and 1/2 SWA as the basal medium. Esculin hydrolysis was tested by the method of Holding and Collee (1971) but 50% seawater was used instead of distilled water. Growth in litmus milk broth was tested by the method of Cruick-

shank (1965). Ability to grow on different media was tested by examination for bacterial growth after inoculation and incubation for three weeks. The media tested were MicrocycluslSpirosoma broth (MSB), MSB + 1.5% NaCl, MSB + 3.0% NaCl, Peptone Succinate Salts Broth (PSS), Modified PSS, Zobell's 2216e Broth, 1/2 SWB, Nutrient Agar (NA), NA + 5% Sucrose, NA + 2.5% NaCl, Blood Agar, Chocolate Agar, Trypticase Soy Agar (TSA), TSA + 3% Glucose (TSAG), TSA + 3% Sucrose (TSAS), TSA + 2.5% NaCI, Peptonized Milk Agar, MicrocycluslSpirosoma Agar + 3.0% NaCl, MacConkey Agar, Plate Count Agar, 1/2 SWA, Zobell's 2216e Agar, Sensitest Agar, Marine Agar, C.L.E.D. Agar, Brain Heart Infusion Agar, Mannitol Salts Agar and Eosin Methylene Blue Agar. The strains were tested with API 20 Enterobacteriaceae strips although the use of the API system had to be adapted for these slow growing environmental isolates. Cells grown on 1/2 SWA for 10 days at 15°C were suspended in sterile seawater to the recommended turbidity and used to inoculate the strips. The strips were incubated (five days to two weeks) until at least two tests were positive or until growth could be observed in the wells. Susceptibility to antibiotics was tested using the minimum inhibitory concentration method (Washington and Sutter, 1980) but the antibiotics were diluted in 1/2 SWA. The strains were suspended in 50% sterile seawater and inoculated onto the antibiotic plates with a multipoint inoculator.Control media without added antibiotics were inoculated between each set of antibiotic plates. The plates were incubated at 15°C. Susceptibility for each strain was determined when the strain showed macroscopic growth on the control media. The media of lowest antibiotic concentration without visible growth was recorded as the minimum inhibitory concentration. The time required for growth to occur on the control media varied between strains from seven

267

to ten days. The reference strains, Flectobacillus major, Spirosoma linguaIe strain 2 and Runella slithyformis strains 2 and 4 (Table 1) were similarly tested for susceptibility to antibiotics, however the antibiotics were added to MS agar which was incubated at 25°C. The ability to grow anaerobically was tested on 1/2 SWA with a BBL Anaerobic GasPak System. Pigments. Strains ACAM 111 and ACAM 171 were grown on 1/2 SWA for 10 days at 15°C. The pigments were extracted with cold ethanol and the absorption spectrum of each pigment was determined between 300 and 500 nm (Pye Unicam SP8-200 spectrophotometer, Pye Unicam Ltd., Cambridge, England.). Pigments were tested for bathochromic shift, characteristic of flexirubins (Weeks, 1981), after alkalization with potassium hydroxide. DNA base composition. DNA was extracted as detailed by Blackall et al. (1985), however, the concentration of sodium lauryl sulphate was increased to 25% w/v. A Pye Unicam SP8-200 UVNis thermoprogramed spectrophotometer (Pye Unicam Ltd., Cambridge, England) was used for the determination of the thermal denaturation profile. The type strain of Escherichia coli, UQM 1803, with a mol % G+C of 51.7 (Blackall et al., 1985) was used as a reference strain. The mol % G+C was calculated with the formula of Mandel and Marmur (1968): %G+C test = %G+Cstandard + 2.08 (Tmtest - Tmsrandard)'

Results Characteristics for the strains isolated from Burton Lake and published data for Flectobacillus marinus and Flectobacillus major are given in Table 2. The isolates were polymorphic, with vibrioid, coiled, spirilled and coccoid forms. The various morphological forms are shown in scanning electronmicrographs of the strains (Figs. 1,2 and 3). The examination of thin sections of the strains showed a cell wall structure typical of Gramnegative cells. An examination of strain ACAM 111 revealed a pro-

Fig. 1-3. Scanning electronmicrograph (Bar = 1.0 urn) of cells of Flectobacillus glomeratus. Strain ACAM 171 T grown in half-strength seawater broth for 7 d at 15°C (1); strain ACAM 111 grown in Zobell's 2216 broth for 7 d at 25°C (2); strain ACAM 111 grown in Zobell's 2216 broth for 7 d at 25°C (3).

268

A. J. McGuire, P. D. Franzmann, and T. A. McMeekin

Table 2. Some characteristics of two strains of coiled and pigmented antarctic bacteria (ACAM 111 and ACAM 171) compared with published data for Flectobaeillus major and Flee-

tobaeillus marinus Character

Gram Reaction Pigmentation Fluorescence Coil formation Coccoid cells Anaerobic growth Growth at 25°C Motility mol % G+C Cell size Length (urn) Diameter (urn) Ring Diameter (urn) Acid from: Arabinose Xylose Rhamnose Fructose Glactose Glucose Mannose Sorbose Salicin Cellobiose Lactose Maltose Melibiose Sucrose Trehalose Raffinose Glycerol Mannitol Sorbitol Adonitol Inositol Sole carbon source Formate Acetate Methanol Benzoate Succinate Malonate Pyruvate Oxaloacetate Citrate Tartate Malate Propionate

ACAM 111

ACAM 171

F.major F.marinus

Yellow

Tan

Pink

Pink

+ +

+

+

+ +

+ 33.0

33.2

1-5 4-15 0.3-0.6 0.4-0.6 NA NA

NG

NG

NG

39.5-40.3 34-38 1.5-5 1.5-5 0.6-1.0 0.3-0.7 5.0-10.0 0.8-2.0 + + + + + + +

+ + + + + + +

+

+ + + + + + + +

+ + + + + +

NA NA

+ NA + NA NA

NA

~-glycerophosphate

Hydrolysis of: Cellulose Chitin Gelatin Starch Casein

+

+ + + + + +

W + NG

+ + +

Esculin

API 20£ strip tests ~-galactosidase

Arginine decarboxylase

+ NA

ACAM 111

Lysine decarboxylase Ornithine decarboxylaseCitrate H 2S production Urease Tryptophane deaminase Indole Voges-Proskauer Glucose Mannose Inositol Sorbitol Rhamnose Sucrose Melibiose Arabinose Oxidase + W Catalase Nitrate reduction Gelatin Growth in liquid media MSB+ 1.5% NaCI MBS+3%NaCI PSS MPSS Zobell's ++++ 1/2 SWB ++++ Growth on solid media NA NA+2.5% NaCI Blood Agar + Chocolate Agar + TSA TSAG TSAS + TSA+2.5% NaCI ++ PMA TSA+3% NaCl ++ MacConkey Agar Plate Count Agar 1/2 SWA +++ Zobell's ++++ Sensitest Agar Marine Agar ++++ CLED + PRMS + EMB +

ACAM 171

F. major F.marinus NA

+ NA

+ W

NA NA +

NA

+ + NA

+ +

+ + + + + W

+ + + + + +

+

++++ ++++

+ + +

NA NA NA NA NA NA

NA NA NA NA NA NA

++++

NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

++ ++ ++/-

+ ++++

++++ ++++

++++ ++++ ++++

++++

++++

NA = results not available. NG = no growth. Data for F. major and F. marinus from Larkin and Borrall (1984c) and Larkin et al. (1977).

NA

W

Character

+ NA

gression from coiled and vibrioid forms in young cultures to coccoid forms in ageing cultures. After incubation for 24 h, 72% of the cells were coiled or vibrioid and 20% were coccoid and 8% were in transition between the two forms. After 5 d this ratio had changed to 62%, 34% and 4% respectively and after 8 days the proportions were 24%, 69% and 7% respectively.

Flectobacillus glomeratus sp. nov.

Fig. 4. Photomicrograph of fluorescing bacterial cells collected from a depth of 11.2 m in Burton Lake and which had been stained with acridine o range. (Bar = 10 urn, arrow sho ws cells with Flectobacillus-Microcyclus-like morphology).

Fig. 4 shows some bacterial cells from a depth of 11.2 m in Burton Lake which were stained with acridine or ange and examine d by fluorescence microscopy . Some cells are very similar in morphology to the "S" and "0" shape d cells as seen in Figs. 2 and 3. The distrib ution of the " Microcyclus-Flectobacillus" morphotypes in Burton Lake in December 1984, determine d by fluorescent counts, is shown in Fig. 5. Cells with this morphology were distributed thro ughout the entire depth of the lake but concentrated at a density grad ient between depths of 11 and 12 m (Fig. 5). This was also the region of the redoxcline within the lake (Fig. 5). ACAM 171 was isolated from water collected from a depth of 11.0 m. Tabl e 3. Mini mum inh ibitory concentrations of antibiotics for two strains of coiled pigment antarctic bacteria and for four reference strains

Antibiotic

Penicillin Amoxycillin Cloxacillin Piperacillin Cotrimoxalose Tetracycline Deoxycycline

Chloramphenicol Erythromycin Lincomycin Nitrofuran toin Nalidixic Acid Cephradine Cefoxitin Streptomycin Gcnramycin Tobr arnycin Neomycin Vancomycin a

18

System. Appl. Mic ro biol. VoL 9/3

(- l . ;:00

6

concentration in Ilg/1.

6

+300 Eh rrW

20 'Cells 104m, -1<_l

1 1-031 Density 9 m1- (o-<»

1028

Fig. 5. Variations in numbers of cells with Flectobacillus-Microcyclus-like morphology, Eh, and wat er density, with depth in Burton Lake , Antarctica (October, 1984 ).

The absor ption spectra of the pigments extracted from ACAM 171 showed peaks at 508 nm and 477 nm with a spur at 454 nm. The pigment fro m ACAM 111 showe d peaks at 480 nm and 455 nm with a spur at 430 nm. The pigment s maintained the same characteristics after alkalization with potassium hydroxide. The antib iotic susceptibilities of the original isolates and the reference strains are given in Table 3. ACAM 111 and ACAM 171 had DNA of 33 .0 and 33 .2 mol% G+C respectively. Two further antarctic strains

ACAM 111

2' 2 64 2 <4 8 8 >32 < 0.1 1 64 >32 4 <8 < 0.1 8 8 32 1

10

269

ACAM 171

1 I

64 1 <4 > 16 16 > 32 <0.1 1 64 >3 2 8 <8 < 0.1 4 >8 >32 8

Flectobacillus

Spirosom a

Runella

major

linquale

slith yfo rm is

1 0.1 > 64 2 <4 1 <1 > 32 1 8 >64 <4 2 8 4 >8 >8 >32 4

1 I

64 8 <4 4 1 >32 1 1 >64 16 4 > 128 8 >8 >8 >32 >8

2

4

0.1 1 4 4 4 <1 < 1 > 32 1 1 >64 16 2 <8 0.1 >8 >8 > 32 1

0.1 1 4 8 4 < 1 <1 > 32 1 1 > 64 16 2 <8 0.1 >8 >8 > 32 4

270

A. J. McGuire, P. D. Franzmann, and T. A. McMeekin

which were of similar morpholology but which are no longer extant had DNA of mol% G+C of 32.8 and 33.4.

Discussion The strains described in this study were pigmented, nonmotile, Gram-negative, vibrioid to spiral, and produced coccoid forms in ageing cultues. Table 4 compares some characteristics of the strains with those of related genera. The strains possess characteristics sufficiently different from existing species to warrant the creation of a new species. The strains are not members of the genus Flavobacterium as their pigments show no bathochromic shift (Weeks, 1981) and their morphology is inconsistent with the morphology of members of that genus. The production of coccoid cells or spherical bodies in old or ageing cultures occurs in strains of the genus Oceanospirillum (Krieg, 1984). Also like members of the genus Oceanspirillum the isolates failed to produce acid from carbohydrates and alcohols. Placement of the isolates in this genus is opposed by their possession of pigments, their non-motility and the mol% G+C of their DNA in the range 32.0-32.2%. As the strains are strictly aerobic, curved, and pigmented, they show greatest affinity with members of Spirosomaceae. The strains vary in mol% G+C and pigmentation from members of the genus Spirosoma. Members of the genus Runella produce acid from a very limited range of carbohydrates similiar to our isolates but there is a large variation in mol% G+C and pigmentation between these isolates and members of the genus Runella. On the basis of morphology and mol% G+C of their DNA,the isolates show the greatest affinity with members of the genus Flectobacillus, particularly the salt-requiring

Characteristics

Original isolates

Flectobacillus

Runella

Spirosoma

Microcyclus

Curved Cells Rings formed Coils & helices formed Coccoid cells formed Pigmentation White to cream Yellow Pink Orange Tan Ability to produce acid from CHO's. Motility Mol. % G+C

+ v v

+ + +

+ + +

+ + +

+

species, Flectobacillus marinus (Larkin and Borrall, 1984c). The strains show differences from Flectobacillus marinus, as they do not produce acids from sugars, do not use organic acids, form coccoid cells, are not pink pigmented and are biochemically less reactive (Table 2). Antibiotic susceptibility tests showed the isolates were resistant to the aminoglycosides, gentamycin, tobramycin and neomycin, although the organisms were susceptible to the other aminoglycoside tested, streptomycin. The reference strains, Flectobacillus major, Spirosoma linguale and Runella slithyformis strains 2 and 4 showed resistance to the same aminoglycosides. However, direct comparison of the minimum inhibitory concentrations of the reference strains and the new strains is not possible as they could not be grown on the same basal media. The isolates and reference strains showed very similiar minimum inhibitory concentrations to most of the antibiotics tested. The morphological forms of bacteria in laboratory cultures can vary from those found in the aquatic environment (Stevenson, 1978). The examination of water samples from the lake by fluorescence microscopy showed that organisms of the same morphology as the isolates (Fig. 4) occurred throughout the water column and particularily in the area of the density gradient and the redoxcline (Fig. 5). As the in situ cells are not in their coccoid morphological form, they are probably involved in active growth and are autochthonous in the lake. Immediately below the redoxcline is a population of photosynthetic bacteria which is dominated by members of the genus Chlorobium (Burke and Burton, 1987). Photosynthetic production in this region may support the higher numbers of heterotrophs at depths associated with the redoxcline. The presence of large numbers of cells of similar morphology in the anaerobic hypolimnion of the lake may be a result of cell sedimentation. Attempts to isolate anaerobic heterotrophs from the hypolimnion of the lake were largely unsuccessful.

FlavoOceanobacterium spirillum + + +

+

+

+ v

+

v +

+ v

v

32-34

+

+

+

+

+

34---40

49-50

51-53

v 66-69

31-42

+ 42-51

data for related genera from Larkin and Borrall (1984a, 1984b, 1984c), Krieg (1984), Staley and Konopka (1984), and Holmes et al. (1984). v = variable result. a

Table 4. A comparison of the characteristics of two strains of coiled pigmented antarctic bacteria with related genera"

Flectobacillus glomeratus sp. nov.

The taxon Flectobacillus marinus originated with the combination of "Microcyclus marinus" into the genus Flectobacillus (Borrall and Larkin, 1978). In a numerical taxonomic study of bacteria from the Arctic Beaufort Sea, Kaneko et al., (1979) determined three phenons of Gramnegative ring-forming bacteria which were morphologically similar to members of the genus Microcyclus. The strains grew at 5°C and did not produce acids from carbohydrates. The strains had different physiological and nutritional characteristics from previously described species (Kaneko et al., 1979). One group of strains (phenon L5) consisted of yellow or orange pigmented curved rods which were psychrotrophic and biochemically unreactive (Kaneko et al., 1979). Similar strains isolated from the Gulf of Alaska (Hauxhurst et al., 1980) could not be identified with currently described species. There was an obvious need to examine the taxonomy of the nonmotile, pigmented, marine bacteria and to describe new species to represent these groups. Description of Flectobacillus glomeratus sp. nov.

tglo-mer-a'tus L.v. glomero- to form into a ball) Cells are vibrioid to highly spirilled and may form rings with an outer diameter of 1.0 to 2.0 urn. Cell diameters vary from 0.3 to 0.6 urn and cell length varies from 1.0 to 15.0 urn. Coccoid cells are produced in ageing cultures and predominate in old cultures. Cells are Gram-negative, non-motile and non-flexible. Pigments, yellow or tan, which are not flexirubins, are produced. Oxidase positive and weakly catalse positive. Metabolism is strictly respiratory. Biochemical and physiological characters are listed in Table 2. Growth occurs on a range of media and is stimulated by the incorporation of NaCI or seawater. The mol% G+C of the DNA ranges from 33.0 to 33.2 (thermal denaturation). The species was isolated from antarctic marine environments and is probably indigenous to marine environments of low temperature. Type strain: Strain ACAM 171T has been deposited in The Culture Collection, Department of Microbiology, University of Queensland, St. Lucia 4067, Australia, as strain UQM 3055. The acronym ACAM denotes strains held in Australian Collection of Antarctic Microorganisms, Faculty of Agricultural Science, University of Tasmania, GPO Box 262C, Hobart 7001 Australia. Acknowledgements. The Australian Collection of Antarctic Microorganisms is funded by The Australien Research Grants Scheme and the University of Tasmania. We thank H. R. Burton of the Antarctic Division for his continued assistance and interest. This work was supported by the Australian Research Grants Scheme and reference strains were kindly supplied by Dr. J. M. Larkin.

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c,

Sly, L. I.: Cellulolytic and dextranolytic gram-negative bacteria: revival of the genus Cel/vibrio. J. Appl, Bact. 59, 81-97 (1985)

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Dr. T. A. McMeekin, Australian Collection of Antarctic Microorganisms, Dept. of Agricultural Science, University of Tasmania, Box 252C, Hobart 7001, Tasmania, Australia