Aquabacter spiritensis, gen. nov., sp. nov. an aerobic, gas-vacuolate aquatic bacterium

Arch Microbiol (1991) 155:137-142 030289339100007A Archives of Microbiology �9 Springer-Verlag 1991 Aquabacter spiritensis, gen. nov., sp. nov. an aerobic, gas-vacuolate aquatic bacterium Roar L. Irgens 1, Karel Kersters 2, Paul Segers 2, Monique Gillis 2, and James T. Staley 3 1 Department of Biology, Southwest Missouri State University, Springfield, MO 65804, USA 2 Laboratorium voor Microbiologie, en microbi~le Genetica, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium 3 Department of Microbiology, University of Washington, Seattle, WA 98195, USA Received April 18, 1990/Accepted October 1, 1990 Abstract. A gram-negative, rod-shaped, gas-vacuolate, aerobic, heterotrophic bacterium was isolated from Spirit Lake, Washington. It was further characterized by phase-contrast and electron microscopy and phenotypic tests. The isolate does not utilize carbohydrates. Deoxy- ribonucleic acid-r ibosomal ribonucleic acid ( rRNA) hy- bridizations showed that this bacterium is a member of rRNA superfamily IV where it occupies a separate pos- ition on the Azorhizobium caulinodans rRNA sub-branch which, is part of an rRNA cluster containing also the Beijerinckia, the Bradyrhizobium japonicum and the Rhodopseudomonas palustris sub-branches. Its relation- ship to other gas-vacuolated genera within rRNA superfamily IV is discussed. A new genus Aquabacter with one species Aquabacter spiritensis is proposed. The type strain is strain SPL-1 (= ATCC 43981 = LMG 8611). Key words: Aquabacter - Aquatic bacterium - Gas vacuoles Gas vesicles are found in prokaryot ic organisms primar- ily f rom aquatic habitats. They are especially common in phototrophic organisms such as the cyanobacteria and the green and purple sulfur bacteria (Cohen-Bazire 1969), but they have also been reported in a number of hetero- trophic bacteria such as Ancalomicrobium and Pro- sthecomicrobium (Staley 1968), Ancylobacter (Van Ert and Staley 1971 a; Rai 1983), Meniscus (Irgens 1977), Stella (Vasilyeva 1985), Clostridium (Krasi l 'nikov et al. 1971), and Enhydrobacter (Van Ert and Staley 1971b; Staley et al. 1987). A l though heterotrophic bacteria con- taining gas vesicles have been reported in lakes, particu- larly from the hypolimnetic layer (Caldwell and Tiedje 1975; Hirsch and Pankratz 1970; Walsby 1974), few of these have been successfully cultivated in the laboratory. We describe here a new gas-vacuolate, gram-negative, Offprint requests to. R. L. Irgens aerobic bacterium Aquabacter spiritensis, that was iso- lated from Spirit Lake, Washington. Materials and methods Source of sample The sample from which this bacterium was isolated was obtained from Spirit Lake, Washington, on April 10, 1981, 1 year after the eruption of Mount St. Helens. The sample, obtained at 5.0 m depth was diluted to 10 4 and plated out on dilute peptone (DP) medium. Out of three plates of this dilution, only one of the 83 colonies found had the chalky, white appearance typical of gas-vacuolate bacteria. This isolate was labeled as strain SPL-1. Media The DP medium from which this bacterium was originally isolated (Staley 1981) contained per liter: peptone, 100 rag, Hutner's salts solution, 20 ml, agar 15 g. SPL-I was restreaked on Super-MMB (S-MMB) with 0.2% sodium succinate as the carbon source (Van Neerven and Staley 1988). S-MMB with a selected carbon source and with peptone and yeast extract each reduced to 0.01% was used in all biochemical tests and growth experiments. Techniques of microscopic observation Phase contrast photomicrographs were taken on 35-mm film (Kodak Technical Pan) with a Canon FTb camera attached to a Zeiss RA 38 microscope. The presence of gas vacuoles was demon- strated by examination of whole cells in a JEOL-100 B electron microscope. The characteristic refractile appearance of gas vacuoles as observed by phase contrast microscopy was lost after freezing a wet mount for 3 - 5 min. Freezing apparently destroys the gas vesicle membrane. Methods of bacteriological characterization Unless otherwise specified, all inocula were taken from 4-to-6-day- old broth cultures. Test cultures were incubated from 7 - 14 days in liquid medium before a particular test was confirmed. Twenty-nine 138 carbon sources were tested for utilization at a concentration of 0.2% (wt/vol) in S-MMB broth. Additional carbon source assimilation tests were performed using API 50CH, 50AO and 50AA galleries (API System, Montalieu Vercieu, France) and the methods described previously (De Ley et al. 1986). Growth at various temperatures was performed in a gradient temperature block (Matches and Liston 1973) from 14 to 44~ Anaerobic growth was tested on S-MMB agar plus 0.3 % pyruvate in anaerobic jars (BBL). Nitrogen fixation ability was tested by attempting to grow the isolate in S-MMB broth without any source of combined nitrogen. Macromolecular hydrolysis was tested on S-MMB agar plus the specified carbon source. The following macromolecules were tested: starch (1.0%), tributyrin (1.0%), and gelatin (1.0%). Hydrolysis of tributyrin was indicated by a clear zone around the colony. Starch hydrolysis was demonstrated with Lugol's iodine and gelatin hy- drolysis was demonstrated with a solution of mercuric chloride (15% HgC12 in 20% HC1). Catalase was tested by dropping 3.0% H202 on colonies on S-MMB plus 0.3% succinate, and oxidase was tested by swabbing growth off a plate and dropping oxidase reagent (N,N, Nt,N1, - tetramethyl-p-phenylene-diamine dihydrochloride, Difco 3550-38) onto the sample. Amino acid deaminases were tested by growing the organism in S-MMB with the specified amino acid added in place of Nt-I4C1. Formation of NH3 was checked with Nessler reagent. Urease was tested with a heavy suspension of cells in 2.0% urea buffered to pH 7.0 with 0.05 M phosphate and using 0.001% phenol red as indicator. Nitrate reduction was tested in S-MMB containing 0.3% succi- nate and 0.5% KNO3. The formation of nitrite was checked by the addition of sulfanilic acid (8.0 g in 1,000 ml of 5.0 N acetic acid) and dimethyl-alpha-naphthylamine (5.0 g in 1,000 ml of 5.0 N acetic acid). Indole production was determined after growth in S-MMB plus 0.5% tryptophan by adding Kovac's reagent (5.0 g ofp-dimethyl- aminobenzaldehyde in 75 ml of amyl alcohol and 25 ml of concen- trated HC1). The production of HzS was tested using SIM medium (Difco) plus 0.1% yeast extract. DNA was extracted by the method of Marmur (1961) and the molar % G + C was determined by the thermal denaturation method (De Ley and Van Muylem 1963) and was calculated by using the equation of Marmur and Dory (1962) as modified by De Ley (1970). DNA: rRNA hybridizations were carried out as described pre- viously (De Ley and De Smedt 1977; Dreyfus et al. 1988; De Vos et al. 1989). The following parameter was measured: Tm(o), which is the temperature at which 50% of the hybrid was denatured. Results Morphological characteristics Figures 1 and 2 show the appearance of the bacter ium under the l ight (phase-contrast) and electron microscope, respectively. F igure 1 shows the bright, refracti le regions of the gas vacuole of the rod-shaped bacter ium and Fig. 2 shows the indiv idual vesicles of the gas vacuole. The bacter ium measures approx imate ly 0 .5 -1 .0 by 1 .5 - 3.0 gm. It is encapsulated and flagella have been reported when using a direct selection method on semisol id med ium as descr ibed by Lara and Konopka (1987). Cultural characteristics On agar plates the organism produces colonies with a circular form, entire margin, sl ightly convex elevation Fig. 1. Phase contrast photomicrograph showing Aquabacter spiritensis cells with gas vacuoles (bar = 10 gm) Fig. 2. Transmission electron micrograph of Aquabacter spiritensis cells stained with phosphotungstic acid. The gas vesicles appear as small transparent inclusions (bar = 2.0 gm) and a smooth, gl istening surface. The colonies appear translucent or opaque and chalky white. As previously observed by Van Ert and Staley (1971 a), and by Irgens (1977), the var iat ion in colony opacity is apparent ly due to var iat ion in the number of gas vacuoles within the indiv idual cells of the colony. The translucent colonies contain fewer cells with gas vacuoles. In stat ionary cul- tures contain ing 0.1% agar, the cells accumulate in the upper 1.0 cm of the tube. The isolate grows rather slowly. A culture volume of 100 ml of S -MMB in a 500-ml Er lenmeyer f lask incubated at 35~ and 250 rpm on a rotary shaker requires two to three days to reach max imum yield. Gas vacuoles were more common when the peptone concentrat ion of S -MMB was increased to 0.4 g/1 and the succinate concentrat ion was decreased to 0.4 g/1. Growth characteristics The bacter ium grew well at a temperature range of 27- 37 ~ C. Broth cultures grown at temperatures below 35~ tended to become stringy. Good growth was obta ined in the pH range of 7.0 to 9.0. The culture tested positive for the following enzymes: catalase, oxidase, alanine and glutamate deaminases, urease, and nitrate reductase. The test for tryptophanase was negative. Nutrition The following carbon sources were utilized: lactate, pyruvate, acetate, malate, succinate, propionate, buty- rate, glutamate, and alanine. Tests with API galleries indicated that maleate, glutarate, DL-glycerate, DL-/% hydroxybutyrate and c~-ketoglutarate were also utilized as organic compounds. The following carbon sources were not utilized: glucose, malonate, DL-proline, glu- conate, meso-tartrate, L-lysine, fructose, maltose, sucrose, lactose, rhamnose, arabinose, mannitol, sorbitol, glycerol, ethanol, methanol, fumarate, formate and ci- trate. With the API method, the following compounds were not utilized: ribose, D- and L-xylose, adonitol, D-galactose, D-mannose, L-sorbose, meso-erythritol, xylitol, dulcitol, inositol, methyl-D-xyloside, methyl-D- mannoside, methyl-D-glucoside, glucosamine, N-acetyl- glucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, melibiose, trehalose, inulin, melezitose, raf- finose, turanose, starch, glycogen, /%gentiobiose, D- lyxose, D-tagatose, D- and L-fucose, D- and L-arabitol, gluconate, 2-ketogluconate, 5-ketogluconate, iso- butyrate, n-valerate, isovalerate, n-caproate, heptanoate, caprylate, pelargonate, caprate, oxalate, adipate, pimelate, suberate, azelate, sebacate, D- and L-tartrate, levulinate, citraconate, itaconate, mesaconate, aconitate, phenylacetate, benzoate, o-hydroxybenzoate, m-hy- droxybenzoate, p-hydroxybenzoate, D- and L-mandelate, phthalate, isophthalate, terephthalate, glycine, L-leucine, L-isoleucine, L-norleucine, L-valine, DL-norvaline, L-ser- ine, L-threonine, L-cysteine, L-methionine, L-phenylala- nine, L-tyrosine, L-histidine, D- and L-tryptophan, trigonelline, L-aspartate, L-ornithine, L-citrulline, L-argi- nine, DL-kynurenine, betaine, creatine, DL-2-amino- butyrate, DL-3-aminobutyrate, DL-4-aminobutyrate, DL- 5-aminovalerate, 2-aminobenzoate, 3-aminobenzoate, 4-aminobenzoate, acetamide, sarcosine, ethylamine, bu- tylamine, amylamine, ethanolamine, benzylamine, di- aminobutane, spermine, histamine, and tryptamine. Growth occurs when ammonium chloride (NH4C1) is used as the only nitrogen source in place of peptone and yeast extract, along with an 8-vitamin solution (Staley 1968). There is no growth in a medium without added nitrogen; N2-fixation in microaerophilic conditions has not been tested. The organism does not hydrolyze starch or gelatin. The lipid, tributyrin, is hydrolyzed. The G + C content of the DNA is 67.3 tool%. DNA : rRNA hybridization studies Table 1 shows the results of DNA:rRNA hybridizations between DNA of the gas-vacuolated strain SPL-1 and rRNA from Alcaligenes xylosoxidans subsp, denitrificans ATCC 15173 T (rRNA superfamily III) and several 139 Table 1. Tin(e) values of hybrids of Aquabacter spiritensis SPL-1T DNA versus labeled rRNA of Alcaligenes xylosoxidans subsp. denitrifieans ATCC 15173 T and type strains of various taxa ofrRNA superfamily IV rRNA from" Tin(e) in ~ Alcaligenes xylosoxidans subsp, denitrificans ATCC 15173 T 61.4 [FIavobacterium] capsulatum ATCC 14666 T 65.0 Brucella abortus ATCC 23448 T 66.2 Glueonobaeter oxydans LMG 1408 r 66.3 Bradyrhizobiumjaponicum LMG 6138 T 70.1 Azorhizobium caulinodans LMG 6465 f 72.9 a Abbreviations: ATCC = American Type Culture Collection, Rockville, MD, USA; LMG = Culture Collection, Laboratorium voor Microbiologie, State University of Gent, Gent, Belgium representatives of rRNA superfamily IV (De Vos et al. 1989). Discussion Nutrition The gas-vacuolated strain SPL-1 is limited in the number of carbon sources that it can utilize. It is entirely limited to the use of a few short chain organic acids, including a few amino acids and tributyrin. It is a strict aerobe. Morphological characteristics The bacterium is encapsulated, rod-shaped, stains gram negatively, and measures 0.5 - 1.0 pm by 1.5 - 3 gm. For- mation of gas vacuoles appears to be a constitutive characteristic, present in young as well as in old cultures on any medium. For the purpose of classification it is of interest to compare the isolate to other gram-negative rods that are strict aerobes, catalase and oxidase positive, have a tool% G + C ratio of about 67.0 and do not assimilate carbo- hydrates. Alcaligenes is one genus that satisfies these requirements, and a possible relationship to Alcaligenes was investigated by performing DNA:rRNA hybridiza- tion with SPL-1 DNA versus a rRNA probe of A. xylo- soxidans subsp, denitrificans, ATCC 15173 ~r. The low Tin(e) value of 61.4~ demonstrates that strain SPL-1 does not belong to the authentic Alcaligenaceae (De Ley et al. 1986) and is not even a member of rRNA superfamily III (De Vos etal. 1989). Carbon assimilation studies also revealed clear differences between Alcaligenes and strain SPL-I: of 147 organic compounds (API method), only 13 served as carbon source for strain SPL-1, whereas authentic Alcaligenes strains utilize an average of 55 of these compounds. Another notable difference is, of course, the ability of SPL-1 to form gas vesicles. DNA: rRNA hybridizations with other rRNA probes (Table 1) showed that strain SPL-1 belongs in rRNA superfamily IV sensu De Ley (see De Vos et al. 1989), 140 Tm(e) ~ 80 78 76 74 72 70 u) o O, -b , -vmm a i- .~ I lID o , -o _z o e., NO ~_z re ....i o" N,r 141 strain SPL-I . However, strain SPL- I differs phylogeneti- cally (A Tm(e) of 4 - 7 ~ C) and phenotypical ly considerably from these two genera, implying that it deserves a separ- ate generic status. Therefore, we propose the name Aquabacter gen. nov., containing one species Aquabacter spiritensis sp. nov. Differentiating features between Aquabacter spiritensis, Azorhizobium and Yanthobacter are given in Table 2. Description of Aquabacter gen. nov. Aquabacter (Aq. ua. bac'ter. L. n. aqua, water; M.L . masc. bacter equivalent of Gr. neut. n. bacterion, a rod; M.L . masc. n. Aquabacter, a water rod) cells are straight rods, 0 .5 - 1.0 by 1 .5 -3 lain. Usual ly non-motile. May be motile under certain specified conditions. Resting stages not known. Gas vacuoles arranged at random within the cells. Gram-negative. Colonies chalky white. Catalase and oxi- dase positive. Chemoorganotrophic , aerobic metabolism. Will grow in chemically defined medium using am- monium as a nitrogen source. Growth occurs at 25 to 37~ but not at 20 or at 40~ The tool% G + C of the DNA is 67% (one strain). Aquabacter belongs to the Azorhizobium rRNA branch within rRNA superfamily VI of the Proteobacteria. At the suprageneric level, Aquabacter is related to Azorhizobium and J(anthobacter. The type species is Aquabacter spiritensis sp. nov. Aquabacter spiritensis sp. nov. Aquabacter spiritensis (spi'ri. ten. sis. M.L . adj. spiritensis, named after Spirit Lake, Washington, USA from which it was isolated). The description of Aquabacter spiritensis is the same as that for the genus. Cultures usually show single encapsulated cells. Colonies are circular, slightly convex in elevation, with entire margin and smooth, glistening surface. The colonies may appear translucent or opaque, chalky white. The larger the number of gas vacuoles within the cells of the colony, the whiter the colony. Old agar slant cultures become rubbery. Nitrate is reduced, but not nitrite. Opti- mum growth occurs at 35~ at pH 7.0 to 9.0. Does not fix nitrogen. The following characteristics are negative: amylase, protease, tryptophanase (indole), nitrite reductase, nitro- genase, and phenylalanine deaminase. Carbon sources not utilized are: formate, citrate, fumarate, aspartate, arginine, methanol, ethanol, glycerol, malonate, DL-pro- line, gluconate, meso-tartrate, L-lysine and numerous carbohydrates. The following enzymes test positive: nitrate reductase, urease, and lipase. Organic compounds utilized as sources of carbon and energy are: acetate, lactate, pyruvate, succinate, malate, alanine, glutamate, propionate, butyrate, tributyrin, maleate, glutarate, DL-glycerate, DL-/%hydroxybutyrate and e-ketoglutarate. The G+C content of the DNA is 67.3 tool%. Isolated from Spirit Lake, near Mount St. Helens, Wash- ington, 5 m depth, April 10, 1981. Type strain. SPL-1, ATCC 43981, LMG 8611. Acknowledgements. This investigation was in part performed in the lab of James T. Staley, Department of Microbiology, University of Washington, Seattle, while the primary investigator was on sabbati- cal leave from the Department of Biology, Southwest Missouri State University, Springfield, Missouri. 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