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Int J Syst Evol Microbiol 56 (2006), 237-241; DOI  10.1099/ijs.0.63948-0
© 2006 International Union of Microbiological Societies

Larkinella insperata gen. nov., sp. nov., a bacterium of the phylum ‘Bacteroidetes’ isolated from water of a steam generator

Marc Vancanneyt1, Olga I. Nedashkovskaya2, Cindy Snauwaert1, Stefanie Mortier1, Katrien Vandemeulebroecke1, Bart Hoste1, Peter Dawyndt3, Galina M. Frolova2, Danielle Janssens1 and Jean Swings1,3

1 BCCM/LMG Bacteria Collection, Ghent University, Ledeganckstraat 35, B-9000 Ghent, Belgium
2 Pacific Institute of Bioorganic Chemistry of the Far-Eastern Branch of the Russian Academy of Sciences, Pr. 100 Let Vladivostoku 159, 690022, Vladivostok, Russia
3 Laboratory of Microbiology, Ghent University, Ledeganckstraat 35, B-9000 Ghent, Belgium

Correspondence
Marc Vancanneyt
Marc.Vancanneyt{at}UGent.be


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A Gram-negative bacterium, designated strain LMG 22510T, was isolated from water of a pharmaceutical company steam generator. The cells had a ring-like and horseshoe-shaped morphology and possessed gliding motility. Phylogenetic analysis of the 16S rRNA gene sequence showed that the strain was a member of the Flexibacter group within the phylum ‘Bacteroidetes’; its nearest neighbour was Spirosoma linguale (88·8 % sequence similarity). DNA base content, fatty acid composition and biochemical characteristics were determined. Genotypic and phenotypic data indicated that strain LMG 22510T could not be assigned to any recognized genus; therefore, a novel genus and species is proposed, Larkinella insperata gen. nov., sp. nov., with LMG 22510T (=NCIMB 14103T) as the type strain.

Published online ahead of print on 16 September 2005 as DOI 10.1099/ijs.0.63948-0.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Larkinella insperata LMG 22510T and Spirosoma linguale LMG 10896T are AM000022 and AM000023, respectively.


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The Flexibacter group within the phylum ‘Bacteroidetes’ (previously Cytophaga–Flavobacterium–Bacteroides or CFB) is a phylogenetically diverse group of micro-organisms. One of the lineages in this group comprises the genera Spirosoma, Runella and Flectobacillus, currently classified in the family Spirosomaceae (Larkin & Borrall, 1984Go). The genera Spirosoma and Flectobacillus each comprise one species, Spirosoma linguale and Flectobacillus major, respectively. The genus Runella includes two species, Runella slithyformis (type species) and Runella zeae (Chelius et al., 2002Go). Other more recently discovered taxa related to species of the latter family are the distantly related genera Dyadobacter and Arcicella (Chelius & Triplett, 2000Go; Nikitin et al., 2004Go; Reddy & Garcia-Pichel, 2005Go). A characteristic morphological feature of the majority of members of the above-mentioned genera is the formation of ring-like or horseshoe-shaped cells, except for cells of R. zeae and Dyadobacter species, which are straight to curved rods. In the present report, the genotypic and phenotypic properties of strain LMG 22510T, which was isolated from water of a steam generator, are described; data show that the organism should be classified as a novel species in a new genus in the Flexibacter group of the phylum ‘Bacteroidetes’.

Strain LMG 22510T was isolated and purified from cooled water produced by a steam generator in a pharmaceutical company in Belgium in 2004. The Gram-negative cells have ring-like and horseshoe-shaped morphology and gliding motility. Colonies produce non-diffusible pale-pink pigments on tryptic soy agar (BBL) at 28 °C under aerobic conditions. The latter cultivation conditions were used for maintenance of the cultures and further experiments, unless indicated otherwise.

The phylogenetic position of strain LMG 22510T was determined by complete 16S rRNA gene sequence analysis. Genomic DNA was prepared according to the protocol of Niemann et al. (1997)Go. 16S rRNA gene amplification, purification and sequencing were performed as described by Vancanneyt et al. (2004)Go except that 16S rRNA gene amplicons were purified using a NucleoFast 96 PCR Clean-up kit (Macherey-Nagel). Sequencing reactions were performed using a BigDye Terminator Cycle Sequencing kit (Applied Biosystems) and purified using a Montage SEQ96 Sequencing Reaction Clean-up kit (Millipore). Electrophoresis of sequence reaction products was performed using an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Sequence assembly was performed using the program AutoAssembler (Applied Biosystems). The 16S rRNA gene sequences (continuous stretch of 1466 bp) and sequences retrieved from GenBank/EMBL of related reference strains were aligned and a phylogenetic tree was constructed by the neighbour-joining method using the BioNumerics software package (version 3.5; Applied Maths). Unknown bases were discarded for the analyses. Bootstrapping analysis was undertaken to test the statistical reliability of the topology of the neighbour-joining tree using 500 bootstrap resamplings of the data (Fig. 1Go). Comparison of the sequences revealed highest sequence similarity (88·1 %) between strain LMG 22510T and S. linguale ATCC 23276.



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  		Fig. 1. Phylogenetic tree based on 16S rRNA gene sequences of Larkinella insperata LMG 22510T and Spirosoma linguale LMG 10896T and representative members of related genera within the Flexibacter group. Rhodothermus marinus was used as outgroup. Numbers at nodes indicate bootstrap values (%).

 
Because no 16S rRNA gene sequence was publicly available for the type strain of S. linguale, the nearly complete gene sequence (continuous stretch of 1460 bp) was determined for S. linguale LMG 10896T using the same methodology and approach as described above. A similarity of 97·8 % was calculated between the sequences of S. linguale strains LMG 10896T and ATCC 23276. S. linguale LMG 10896T and strain LMG 22510T shared a similarity of 88·8 %. Similarity values with the type strains of the type species of the genera Arcicella, Dyadobacter, Flectobacillus and Runella, more distantly related taxa, were lower (83·6–86·3 %).

For determination of DNA base content, DNA was extracted following the protocol of Pitcher et al. (1989)Go as modified by Leisner et al. (2002)Go. DNA was enzymically degraded into nucleosides as described by Mesbah et al. (1989)Go. The nucleoside mixture obtained was separated by HPLC using a Waters Symmetry Shield C8 column maintained at 37 °C. The solvent was 0·02 M NH4H2PO4 (pH 4·0) with 1·5 % acetonitrile. Non-methylated phage lambda DNA (Sigma) was used as the calibration reference. The DNA G+C content of strain LMG 22510T was 53 mol%.

Determination of the fatty acid methyl ester composition of strain LMG 22510T and its nearest neighbour S. linguale LMG 10896T was performed using the standard protocol of the Sherlock Microbial Identification System (version 5.0; MIDI). Dominant fatty acids for both strains studied are summarized in Table 1Go and included 15 : 0 iso, 16 : 1{omega}5c, 17 : 0 iso 3-OH and summed feature 3 (comprising 15 : 0 iso 2-OH, 16 : 1{omega}7c and/or 16 : 1{omega}7t; cf. Table 1Go). Major quantitative differences between LMG 22510T and S. linguale LMG 10896T were found in the amounts of the above-mentioned fatty acids. Apart from the latter differences, strain LMG 22510T can be distinguished from S. linguale by the presence of small amounts of 14 : 0 and 16 : 0 iso 3-OH and by the absence of 13 : 0 iso.


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  		Table 1. Fatty acid content (mean percentages of total) of Larkinella insperata LMG 22510T and S. linguale LMG 10896T

Strains: 1, L. insperata LMG 22510T; 2, S. linguale LMG 10896T. Those fatty acids for which the amount for both taxa is less than 1 % are not given; –, not detected; tr, less than 1 %.

 
Isoprenoid quinones were extracted from lyophilized cells and analysed as described by Akagawa-Matsushita et al. (1992)Go. Menaquinones were detected by monitoring at 270 nm and were identified by comparison with known quinones from the reference strain, Cyclobacterium marinum LMG 13164T. The main isoprenoid quinone was MK-7.

Analysis of polar lipids was carried out as described previously (Ivanova et al., 2000Go). Phospholipids of LMG 22510T were phosphatidylethanolamine and phosphatidylserine. Also, one glycolipid and four unidentified lipids were detected.

Phenotypic analysis was performed using previously described methods (Nedashkovskaya et al., 2003Go, 2004Go). The physiological, biochemical and morphological characteristics of strain LMG 22510T are given in the species description and in Tables 2 and 3GoGo. Strain LMG 22510T and its nearest neighbour, S. linguale, have many similar properties, but also a significant number of distinct features, e.g. differences in hydrolysis of gelatin, starch, Tweens 20 and 80, utilization and acid production from particular carbohydrates and susceptibility to antibiotics (Table 2Go). Phenotypic features, summarized in Table 3Go, demonstrate that the novel taxon also differs from other more distantly related genera.


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  		Table 2. Phenotypic properties of L. insperata LMG 22510T and its nearest phylogenetic neighbour S. linguale LMG 10896T

Strains: 1, L. insperata LMG 22510T; 2, S. linguale LMG 10896T. Both strains produce ring-like and horseshoe-shaped cells. Both strains are positive for respiratory type of metabolism, motility by gliding, oxidase,catalase, {beta}-galactosidase and alkaline phosphatase activities, hydrolysis of Tween 40, utilization of mannose and sucrose, susceptibility to ampicillin, carbenicillin and doxycycline and resistance to benzylpenicillin, chloramphenicol, gentamicin, neomycin and polymyxin B. Both strains are negative for nitrate reduction, flexirubin and H2S production, NaCl requirement for growth, indole and acetoin(Voges–Proskauer reaction) production, hydrolysis of agar, casein, DNA, urea, cellulose (CM-cellulose and filter paper) and chitin, acid production from adonitol, L-arabinose, dulcitol, L-fucose, N-acetylglucosamine, D-glucose, glycerol, inositol, D-lactose, D-maltose, mannitol, D-melibiose, L-raffinose, L-rhamnose, sorbitol, L-sorbose, D-xylose and L-xylose and utilization of citrate, inositol, malonate, mannitol and sorbitol. W, Weakly positive.

 

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  		Table 3. Phenotypic properties that distinguish the genus Larkinella from related taxa

Data from Larkin & Borrall (1984)Go, Chelius & Triplett (2000)Go, Chelius et al. (2002)Go, Reddy & Garcia-Pichel (2005)Go and this study. W, Weakly positive.

 
Phylogenetic, genomic and phenotypic data clearly indicate that LMG 22510T represents a novel genus and species within the Flexibacter group, for which the name Larkinella insperata gen. nov., sp. nov. is proposed.

Description of Larkinella gen. nov.
Larkinella (Lar.ki.nel'la. N.L. fem. n. Larkinella named in honour of the American microbiologist John M. Larkin, who described the family Spirosomaceae in co-authorship with Renée Borrall).

Ring-like and horseshoe-shaped cells, motile by gliding. Gram-negative. Do not form endospores. Strictly aerobic. Produce non-diffusible pale-pink pigments. Chemo-organotroph. Cytochrome oxidase-, catalase- and alkaline phosphatase-positive. Dominant cellular fatty acids are 15 : 0 iso, 16 : 1{omega}5c, 17 : 0 iso 3-OH and summed feature 3 (comprising 15 : 0 iso 2-OH, 16 : 1{omega}7c and/or 16 : 1{omega}7t). The main isoprenoid quinone is MK-7. The genus is a member of the phylum ‘Bacteroidetes’. The type species is Larkinella insperata.

Description of Larkinella insperata sp. nov.
Larkinella insperata (in.spe.ra'ta. L. fem. adj. insperata unexpected, referring to the unexpected source from which the bacterium was isolated).

Main characteristics are the same as those given for the genus. In addition, cells have an outer diameter of 1·5–3·0 µm and width of 0·5–0·9 µm. Colonies are 1–2 mm in diameter, circular and shiny with entire edges. Growth is observed at 10–40 °C and with 0–2 % NaCl. {beta}-Galactosidase-positive. Decomposes gelatin and Tween 40. Does not degrade agar, casein, starch, DNA, Tweens 20 or 80, urea, cellulose (CM-cellulose or filter paper) or chitin. Does not produce acid from adonitol, L-arabinose, D-cellobiose, dulcitol, D-fructose, L-fucose, D-galactose, N-acetylglucosamine, D-glucose, glycerol, inositol, D-lactose, D-maltose, mannitol, D-melibiose, L-raffinose, L-rhamnose, sorbitol, L-sorbose, D-sucrose, D-xylose or L-xylose. Utilizes L-arabinose, D-glucose, D-lactose, D-mannose and D-sucrose as sole carbon sources for growth, but not citrate, inositol, malonate, mannitol or sorbitol. Flexirubin-type pigments are absent. Does not reduce nitrate. No production of H2S, indole or acetoin (Voges–Proskauer reaction). Susceptible to ampicillin, carbenicillin and doxycycline; resistant to benzylpenicillin, chloramphenicol, erythromycin, gentamicin, kanamycin, lincomycin, neomycin, oleandomycin, polymyxin B, streptomycin and tetracycline. Major fatty acid components (>1·0 %) include 14 : 0, 15 : 0 iso, 15 : 0 anteiso, 15 : 0 iso 3-OH, 16 : 0, 16 : 1{omega}5c, 16 : 0 3-OH, 16 : 0 iso 3-OH, 17 : 0 iso 3-OH and summed feature 3 (comprising 15 : 0 iso 2-OH, 16 : 1{omega}7c and/or 16 : 1{omega}7t). The G+C content of the DNA is 53·0 mol%.

The type strain is LMG 22510T (=NCIMB 14103T), isolated from water produced by a steam generator in a pharmaceutical company in Belgium.


   ACKNOWLEDGEMENTS
 
This research was supported by the Belgian Federal Public Planning Service – Science Policy; O. I. N. was supported by grants of the Federal Agency for Sciences and Innovations of the Ministry for Education and Sciences of the Russian Federation no. 2-2.16, the Russian Foundation for Basic Research no. 05-04-48211 and the Program of Fundamental Investigations of the Presidium of the Russian Academy of Sciences ‘Molecular and Cell Biology’.


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Larkin, J. M. & Borrall, R. (1984). Family I. Spirosomaceae Larkin and Borrall 1978, 595AL. In Bergey's Manual of Systematic Bacteriology, vol. 1, pp. 125–126. Edited by N. R. Krieg & J. G. Holt. Baltimore: Williams & Wilkins.

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