Pannonibacter phragmitetus gen. nov., sp. nov., a novel alkalitolerant bacterium isolated from decomposing reed rhizomes in a Hungarian soda lake

In the past, the taxonomic status of the marine star-shaped-aggregate-forming bacteria has altered several times. In the early fifties, Stapp & Knösel (1954) proposed the species Agrobacterium stellulatum for these bacteria. Based on investigations of Baltic Sea isolates, six additional Agrobacterium species (Agrobacterium aggregatum, Agrobacterium agile, Agrobacterium ferrugineum, Agrobacterium gelatinovorum, Agrobacterium kieliense, Agrobacterium luteum and Agrobacterium sanguineum) were later described (Ahrens & Rheinheimer, 1967; Ahrens, 1968). On the basis of genotypic analysis of marine isolates from various origins, Rüger & Höfle (1992) recommended the dissection of the genus Agrobacterium into subdivision 1, including the terrestrial and plant-pathogenic species, and subdivision 2, including the marine star-shaped-aggregate-forming bacteria as Agrobacterium ferrugineum, Agrobacterium gelatinovorum and Agrobacterium stellulatum (with two more novel species, Agrobacterium atlanticum and Agrobacterium meteori). The high DNADNA similarity between Agrobacterium stellulatum ATCC 15215^T and Agrobacterium aggregatum ATCC 25650 served as evidence for the identity of these species, with the priority of Agrobacterium stellulatum. A reclassification of marine Agrobacterium species based on 16S rDNA sequence analysis was performed by Uchino et al. (1998). They demonstrated the heterogeneity of the species of the marine subdivision of the genus Agrobacterium and revealed that these organisms belong to distinct lineages within the α-subclass of the Proteobacteria. In the α-3 group of the Proteobacteria, Agrobacterium gelatinovorum, Agrobacterium atlanticum and Agrobacterium meteori were described as members of a new genus, Ruegeria, as Ruegeria gelatinovora and Ruegeria atlantica (Agrobacterium meteori was determined to be a synonym of R. atlantica), while Agrobacterium ferrugineum showed the highest similarity to Rhodobacter species. The species Agrobacterium aggregatum, Agrobacterium stellulata and Agrobacterium kieliense belonged to the α-2 group of Proteobacteria as members of the new genera Stappia and Ahrensia. According to the results of 16S rDNA sequence comparison of Agrobacterium stellulatum strains IAM 12621^T (=ATCC 15215^T) and IAM 12614 (=ATCC 25650), the separation of the species as Stappia stellulata and Stappia aggregata was proposed. Stappia stellulata-like α-Proteobacteria were successfully cultivated and characterized from samples of the external tissue and the inner shell surfaces of Crassostrea virginica by Boettcher et al. (2000), providing evidence that this microbe can be associated with animal hosts. A new cluster within the α-2 group of the Proteobacteria was established by Suzuki et al. (2000) with strains isolated from different marine environments (surfaces of Rhodophyta, sand and algal sand mat) and described as a new genus, Roseibium, with two species, Roseibium denhamense and Roseibium hamelinense.

An earlier study on the planktonic and reed biofilm bacterial communities of Lake Fert (Neusiedlersee), a shallow, alkaline soda lake in Hungary (mean depth, 1·3 m; pH, 7·810·0; conductivity, 15003000 µS cm^-1; dominant cations, Na⁺ and Mg²⁺; dominant anions, and ), which can be characterized by extremely extensive reed coverage (85 % of the Hungarian part), revealed the presence of a number of bacterial species adapted to this special aquatic environment (Borsodi et al., 1998).

This paper presents a polyphasic characterization of three alkalitolerant bacterial strains, C6/19^T, C6/8 and C6/17, isolated from the surface of decomposing reed rhizomes in Lake Fert. Based on the results of phenotypic and phylogenetic analyses, a new genus and species, Pannonibacter phragmitetus gen. nov., sp. nov., is proposed within the α-subclass of the Proteobacteria.

Strain isolation and cultivation.
Strains C6/19^T, C6/8 and C6/17 were isolated in 1998 from uprooted reed rhizomes [Phragmites australis (Cav.) Trin. et Steudel] subjected to biodegradation (using the litter-bag technique) in the water body of Lake Fert (47°45' N, 16°50' E). Reed rhizome parts were serially washed with sterile water and then serial dilutions were made from scrapings taken from both the inner and outer surfaces. A modified Horikoshi alkaline medium (Horikoshi, 1996) consisting of 10·0 g cellulose MN300 (Macherey Nagel) (instead of the originally described D-glucose), 5·0 g peptone, 5·0 yeast extract, 1·0 g KH₂PO₄, 0·2 g MgSO₄.7H₂O, 5·0 g Na₂CO₃ and 15·0 g agar l^-1, adjusted to pH 9·0, was used for plating and isolation (following a 714 day incubation period at 28 °C) and maintenance of all bacterial strains.

Morphology.
Morphological observations of single colonies developed on the isolation medium were made by stereo-microscopy. Morphology and motility of live cells were investigated by phase-contrast microscopy of hanging-drop preparations and by observing bacterial cells growing in nutrient broth (DSMZ medium 1) and synthetic broth (Szabó, 1974) using a light microscope. Gram type was determined according to Claus (1992). Spore staining was carried out as described by Murray et al. (1994). Poly-β-hydroxyalkanoate (PHA) inclusion bodies were visualized by the staining method of Murray et al. (1994). For electron microscopy, cells developed on nutrient agar plates after 48 h incubation at 28 °C were pre-fixed in 1 % (v/v) glutaraldehyde buffered with sodium cacodylate (0·1 M, pH 7·2) for 2 h at room temperature. The pre-fixed samples were embedded in 2 % agar and washed three times in sodium cacodylate. Post-fixation was carried out in cacodylate-buffered 0·5 % (w/v) OsO₄ solution for 1 h. Subsequent to staining with uranyl acetate (2 % in distilled water) for 30 min, the samples were dehydrated in a graded series of ethanol (50, 70, 90, 96 and 100 %), transferred to propylene oxide and embedded in Durcupan (Fluka). Ultrathin sections were cut with a Reichert-Ultracut ultramicrotome and stained with lead citrate. The specimens were examined using a JEM100CX II electron microscope (JEOL).

Physiological and biochemical characterization.
Production of bacteriochlorophyll (Bchl) a was detected by fluorescent spectroscopy. Cultures were grown in liquid DSMZ medium 607 and modified Horikoshi alkaline medium for 4 days at 25 °C. Methanolic extracts were prepared from dense suspensions (10⁹10¹⁰ c.f.u. ml^-1) of intact cells. Fluorescence emission spectra of the cultures and methanolic extracts were measured with a Spex FluoroMax-2 spectrofluorometer (Jobin Yvon) at room temperature. The excitation wavelength was 500 nm (with a 10 nm slit) and spectra were recorded between 600 and 900 nm (with a 2 nm slit). Oxidase activity, production of catalase and acetoin, reduction of nitrate to nitrite and nitrogen, methyl red reaction, aesculin hydrolysis, citrate utilization, production of H₂S from cysteine and indole from tryptophan and phenylalanine deamination were tested following the standard methods of Cowan & Steel (1974). D-Glucose oxidation and fermentation were tested by the method of Hugh & Leifson (1953). Urease and phosphatase activities, hydrolysis of casein, gelatin, Tween 80, starch and hippurate were examined according to Smibert & Krieg (1994). Cellulase activity was examined by testing disintegration of Whatman no. 1 chromatography paper strips placed in peptone broth after 68 weeks of incubation at 28 °C. Acid production from different carbohydrates was determined by employing API CH50 test strips and CHE inoculation fluid (bioMérieux). To test the sole carbon source utilization spectra of the strains, Biolog GN2 microtitre plates were used. The influence of environmental factors (temperature, pH and salt) on growth was studied by incubation of inoculated nutrient broth from 4 to 40 °C, at pH 7·011·0 and at salt concentrations from 0 to 10·0 % (w/v) NaCl for 7 days. All tests were performed in duplicate.

Analysis of chemotaxonomic characteristics.
For chemotaxonomic analysis, cells were cultivated in liquid Rich medium (Yamada & Komagata, 1972) to mid-exponential phase in a rotary shaker at 28 °C. meso-Diaminopimelic acid (DAP) in the cell wall was determined by TLC according to Yamada & Komagata (1970). Isoprenoid quinones were extracted as described by Collins et al. (1977) and the profile was analysed by HPLC following the method of Groth et al. (1997). Cellular fatty acid methyl esters were prepared as described by Stead et al. (1992) and analysed by GC (Groth et al., 1996). Polar lipids were determined by the method of Minnikin et al. (1979).

DNA base composition and DNADNA hybridization.
DNA extraction from cells and analysis of the G+C content by HPLC were carried out by the method of Groth et al. (1996). DNADNA hybridization was performed as described by De Ley et al. (1970), with the modifications of Escara & Hutton (1980) and Huß et al. (1983), using a Gilford System 2600 spectrometer equipped with a Gilford 2527-R thermoprogrammer and plotter. Renaturation rates were computed with the program TRANSFER.BAS (Jahnke, 1992).

16S rDNA sequence determination and phylogenetic analysis.
The 16S rRNA gene was amplified according to the method of Rainey et al. (1996). The PCR product was purified by using the Prep-A-Gene kit (Bio-Rad). Sequencing of the PCR product was done by using a Big Dye Terminator cycle sequencing kit (Applied Biosystems), in a Gen-Amp 2400 PCR machine (Perkin Elmer) according to the procedure provided by the manufacturer. An Applied Biosystems model 310 Genetic Analyzer was employed for automated sequencing. Alignment of the sequences was performed against the ARB-formatted RDP database release 7.1 (Maidak et al., 1996) using the ARB program package (Strunk & Ludwig, 1995). A supplementary BLAST search (Altschul et al., 1997; ) was also carried out to update the sequence results. Evolutionary distances were calculated using Kimura's two-parameter and JukesCantor methods (Kimura, 1980; Jukes & Cantor, 1969). Phylogenetic trees were constructed according to the neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981) and least-squares (De Soete, 1983) treeing algorithms.

Morphological and physiological characteristics
Colonies of strains C6/19^T, C6/8 and C6/17 developed on the surface of the isolation medium were circular, entire, smooth, convex, opaque and whitish-beige in colour. Cells of the strains stained Gram-negative, were motile with polar flagella, contained PHA and did not produce endospores. The cells occurred as straight or slightly curved rods (2·04·0x0·30·6 µm in size), generally singly, and showed typical Gram-negative ultrastructure in thin sections under TEM (Fig. 1). Cells growing in synthetic liquid broth formed rosette-like or star-shaped aggregates consisting of four to six cells.

(190K): Fig. 1. (a) Longitudinal section showing a rod-shaped cell of Pannonibacter phragmitetum C6/19T with Gram-negative cell wall structure and a cap-like structure on one pole (arrowhead). The roundish bright area marked with a triangle in one of the cross-sections may represent a PHA inclusion body. Bar, 0·5 µm. (b) Longitudinal section of a dividing cell with characteristic cap-like structures (arrowheads) at the polar ends of the daughter cells. Bar, 0·5 µm.

Strains C6/19^T, C6/8 and C6/17 were isolated and maintained on cellulose-containing Horikoshi alkaline medium, but cultures of the strains also developed on peptone-based nutrient agar medium. No signals were found at the highest sensitivity of the spectrofluorometer, indicating that the strains did not synthesize Bchl a under aerobic conditions. All three strains exhibited positive oxidase and catalase reactions and produced acid from D-glucose under both aerobic and anaerobic conditions. Reduction of nitrate to nitrogen was positive for all strains but nitrites were only detectable with strain C6/17. The VogesProskauer and methyl red tests were negative. Hydrolysis of aesculin, casein, gelatin, Tween 80, starch and hippurate, deamination of phenylalanine and production of indole from tryptophan and H₂S from cysteine were also negative for all strains. Arginine hydrolysis, urease and phosphatase activities and the utilization of citrate were positive. Cellulase activity was not detected. All strains grew at 1037 °C, pH 7·011·0 and 05 % (w/v) NaCl. Optimum growth occurred between 22 and 28 °C and at pH 9·010·0. Of the carbohydrates of the API 50CH test strip, all three strains produced acid from L-arabinose, aesculin and D-fucose after 24 h incubation and D-xylose and L-fucose after 48 h; the remaining tests were negative. According to the Biolog GN2 test results, of the 95 different carbon sources, 35 were oxidized by all strains, 25 gave strain-dependent results and the remaining 35 substrates were not utilized. Full details of the API 50CH and Biolog GN2 results are available as supplementary material in IJSEM Online (http://ijs.sgmjournals.org).

Phenotypic characteristics that distinguish strain C6/19^T from Stappia stellulata and Roseibium denhamense are presented in Table 1. Although several features of the strains isolated from the decomposing reed rhizomes in Lake Fert were the same as those of Stappia and Roseibium published earlier (Uchino et al., 1998; Suzuki et al., 2000; Hiraishi & Shimada, 2001), the most characteristic phenotypic discrepancy was that no Bchl a production was detected under aerobic conditions in strains C6/19^T, C6/8 and C6/17. Moreover, all three isolates showed more intensive (after 24 to 48 h) acid production than Stappia species, but Roseibium species utilized more carbohydrates than strain C6/19^T (Table 1). The broad sole carbon source utilization spectrum (mostly carbohydrates and carbonic acids) of strains C6/19^T, C6/8 and C6/17 in the Biolog test, despite their lack of direct cellulolytic activity, confirms the important role of these microbes in the biodegradation of dead organic material.

Table 1. Differential phenotypic properties of strain C6/19T and its closest phylogenetic neighbours Data for reference taxa were taken from Uchino et al. (1998) and Hiraishi & Shimada (2001) (Stappia stellulata) and Suzuki et al. (2000) (Roseibium denhamense). Characters are scored as: +, positive; -, negative; W, weakly positive; ND, no data available. All three taxa are catalase and oxidase positive and reduce nitrate to nitrogen. All three taxa are negative for production of H2S from cysteine and hydrolysis of starch.

In contrast to Stappia and Roseibium species, which require sodium ions for growth, all of the strains isolated from Lake Fert were able to grow equally well in the absence of Na⁺ and with up to 5 % (w/v) NaCl. Unlike Roseibium denhamense, strains C6/19^T, C6/8 and C6/17 showed a wide pH tolerance (from pH 7·0 to 11·0), with an optimum at pH 9·010·0. Unfortunately, no data are available on the pH tolerance of Stappia species. The growth characteristics of the Lake Fert strains can be related to the special chemical features of their aquatic habitat.

Chemotaxonomic properties
The cell wall of the type strain contained meso-DAP in the peptidoglycan. The major isoprenoid quinone detected in strain C6/19^T was Q-10 and minor amounts of Q-7 and Q-9 were also present. Polar lipids of strain C6/19^T included phosphatidyl glycerol, diphosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl serine and one unknown phospholipid. The cellular fatty acid composition of strain C6/19^T was dominated by C_{18 : 1}ω7c (75·8 %). Among the other fatty acids detected were C_{16 : 0} (6·5 %), C_{18 : 0} (1·6 %), C_{16 : 1}ω7c (0·7 %), C_{20 : 1}ω7c (0·2 %), C_{14 : 0} 3-OH (1·3 %), C_{16 : 0} 3-OH (0·7 %), C_{18 : 0} 3-OH (2·2 %), 10-Me C_{19 : 0} (0·6 %) and 11-Me C_{18 : 1} (9·9 %).

DNA base composition and DNADNA hybridization
The DNA G+C content of strain C6/19^T was 64·6 mol%. DNADNA similarity values among strains C6/19^T, C6/8 and C6/17 determined by hybridization ranged between 105·7 and 94·9 %. Strain C6/19^T showed DNADNA hybridization of 37·1 % with Stappia stellulata.

Phylogenetic analysis
The phylogenetic positions of the nearly complete (1475, 1454 and 1472 bp) 16S rDNA sequences of strains C6/19^T, C6/8 and C6/17 are shown in Fig. 2. The phylogenetic dendrogram was constructed from evolutionary distances of Kimura's two-correction parameter by the neighbour-joining method and the stability of the groupings was estimated by bootstrap analysis (1000 replications). Maximum-likelihood and least-squares tree generations resulted in the same phylogenetic placement of the strains. Strains C6/8 and C6/17 have sequence similarities of 100 and 99·9 % to strain C6/19^T. The closest relatives of strain C6/19^T, C6/8 and C6/17 were Stappia aggregata, Stappia stellulata, Roseibium denhamense and a Crassostrea virginica symbiont, with 95·8, 94·2, 93·6 and 95·6 % sequence similarity, respectively. The 16S rDNA sequence similarity between the group of strains C6/19^T, C6/8 and C6/17 and the other α-Proteobacteria reference strains was less than 91·7 %.

(72K): Fig. 2. Neighbour-joining phylogenetic tree of Pannonibacter phragmitetus C6/19T and related members of the α-subclass of the Proteobacteria based on 16S rDNA sequence analysis. Bootstrap values greater than 50 % are given at branch-points. Bar, 10 nucleotide substitutions per 100 nucleotides.

In terms of chemotaxonomic markers, apart from the major Q-10 respiratory quinone characteristic to all members of the α-2 group of the Proteobacteria, strain C6/19^T contained Q-7 and Q-9 as minor components. In addition to the physiological and chemotaxonomic characteristics, the genotypic properties of these strains also differed from those of Stappia and Roseibium species (e.g. the DNA G+C content of strain C6/19^T was higher).

The low 16S rDNA sequence similarity of strains C6/19^T, C6/8 and C6/17 to their nearest phylogenetic relatives, Stappia and Roseibium species, indicates that these isolates represent a new genus within the α-subclass of the Proteobacteria. Owing to their almost identical 16S rDNA sequences, >94 % DNADNA similarity and identical morphological and physiological features, strains C6/19^T, C6/8 and C6/17 correspond to a single species, for which the name Pannonibacter phragmitetus gen. nov., sp. nov. is proposed.

Description of Pannonibacter gen. nov.
Pannonibacter (Pan.no.ni.bac'ter. L. n. Pannonia Roman province in what is now Hungary, also referring to Pannon lakes, shallow soda lakes found in the western part of Hungary; N.L. masc. n. bacter from Gr. n. baktron rod; N.L. masc. n. Pannonibacter rod-shaped microbe from a Hungarian soda lake).

Colonies on alkaline agar medium are whitish-beige, circular, convex, smooth and shiny with entire edges. Cells are non-spore-forming rods, motile with polar flagella, contain PHA and stain Gram-negative. Facultatively anaerobic, chemo-organotrophic. Bchl a is not synthesized under aerobic conditions. D-Glucose is fermented with acid but no gas production. Oxidase and catalase are positive. Nitrate is reduced to nitrogen. Major cellular fatty acid is C_{18 : 1}ω7c and the main polar lipids are phosphatidyl glycerol, diphosphatidyl glycerol, phosphatidyl ethanolamine and phosphatidyl serine. Q-10 is the predominant respiratory quinone. The G+C content is 64·6 mol%. The genus is a member of the α-subclass of the Proteobacteria. The type species is Pannonibacter phragmitetus.

Description of Pannonibacter phragmitetus sp. nov.
Pannonibacter phragmitetus (phrag.mi'te.tus. N.L. masc. adj. phragmitetus of the plant association Scirpo-Phragmitetum, the habitat of the micro-organism).

Cells are motile, Gram-negative, straight to slightly curved rods (2·04·0x0·30·6 µm). On Horikoshi alkaline agar medium, colonies are small (24 mm in diameter), whitish-beige coloured, circular, entire, smooth and convex. No methyl-red or VogesProskauer reactions occur. Aesculin, casein, cellulose, gelatin, hippurate, starch and Tween 80 hydrolysis, phenylalanine deamination and production of H₂S and indole are negative. Arginine is hydrolysed and citrate is utilized. Urease and phosphatase activities are positive. In Biolog GN microplates, strains oxidize dextrin, glycogen, L-arabinose, cellobiose, D-fructose, L-fucose, D-galactose, gentiobiose, α-D-glucose, meso-inositol, lactulose, maltose, D-melibiose, L-rhamnose, sucrose, D-trehalose, turanose, monomethyl succinate, cis-aconitic acid, citric acid, formic acid, D-glucuronic acid, β-hydroxybutyric acid, γ-hydroxybutyric acid, α-ketobutyric acid, α-ketoglutaric acid, DL-lactic acid, quinic acid, succinic acid, bromosuccinic acid, L-asparagine, L-glutamic acid, L-pyroglutamic acid, urocanic acid and glycerol. In API CH50 test strips, acid is produced from L-arabinose, D-xylose, aesculin, D-fucose and L-fucose. Conditions for growth are 1037 °C, pH 7·011·0 and up to 5 % (w/v) NaCl. Optimum growth occurs between 22 and 28 °C and at pH 9·010·0.

The type strain, C6/19^T (=DSM 14782^T =NCAIM B02025^T), and reference strains C6/8 (=DSM 14780 =NCAIM B02027) and C6/17 (=DSM 14781 =NCAIM B02026) were isolated from the surface of decomposing reed rhizomes from Lake Fert, Hungary.

The authors wish to thank Mária Dinka for conducting the field experiments and for her help in collecting the reed rhizome samples and Katalin Szabó for her help in the bacteriochlorophyll investigation. This work was supported by a grant from the Hungarian National Science Foundation (OTKA T038021).