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1 Department of Biological Sciences, 331 Life Sciences Building, Louisiana State University, Baton Rouge, LA 70803, USA
2 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Korea
Correspondence
Sung-Taik Lee
e_stlee{at}kaist.ac.kr
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9c and C11 : 0 iso 3OH, predominantly appeared in strain Ko07T as well as in all type strains of the recognized Lysobacter species. The DNA–DNA hybridization values of strain Ko07T with those of recognized Lysobacter species were estimated to be 2–20 %. Despite sharing common taxonomic features in important phenotypic characteristics, such as gliding movement, long-rod shape and proteolytic activity, strain Ko07T could be distinguished from the Lysobacter species with validly published names by its low DNA–DNA hybridization value, a comparatively low DNA G+C content (63·8 mol%), substrate utilization and some physiochemical characteristics. On the basis of the results obtained in this study, it is proposed that strain Ko07T should be classified as representing a novel member of the genus Lysobacter, for which the name Lysobacter concretionis sp. nov. is proposed. The type strain is Ko07T (=KCTC 12205T=DSM 16239T).
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strain Ko07T, Lysobacter gummosus ATCC 29489T and Lysobacter brunescens ATCC 29482T are AB161359, AB161361 and AB161360, respectively.
16S rRNA gene sequence identity data for strain Ko07T and closely related bacteria are available as supplementary material in IJSEM Online.
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for non-fruiting-body, gliding bacteria with high G+C content. The Lysobacter species with validly published names are Lysobacter antibioticus, Lysobacter brunescens, Lysobacter enzymogenes and Lysobacter gummosus. Although the description of an antibiotic-producing species, ‘Lysobacter lactamgenus’, has been published (Ono et al., 1984; Kimura et al., 1996), its taxonomic position has not yet been confirmed and its name has not been validated; thus, no novel species of the genus Lysobacter has been proposed since 1978. Fatty acid profiles for two type strains, L. enzymogenes ATCC 29487T and L. antibioticus ATCC 29479T, are available from the MIDI database, but identifying bacterial strains as belonging to the genus Lysobacter by using the MIDI system only is insufficient. Furthermore, as the Lysobacter species were classified originally on the basis of only a few phenotypic characteristics (Christensen & Cook, 1978), it was unclear whether they could still retain their taxonomic positions if phylogenetic and/or chemotaxonomic features were considered.
Anaerobic granules are bacterial aggregates that result from the flocculation of sludge in an upflow anaerobic sludge blanket (UASB) reactor (de Zeeuw & Lettinga, 1980). They are thought to be composed of micro-organisms, inorganic nuclei and extracellular polymers (Fukuzaki et al., 1991; Shen et al., 1993). Great attention has been paid to the internal structure and catalytic activities of the granules (MacLeod et al., 1990; Schmidt & Ahring, 1996). In our laboratory, the relationship between the structure and resistance to toxic chemicals in anaerobic granules from a brewery wastewater-treatment UASB reactor was studied (Bae & Lee, 1999; Bae et al., 2000). In a series of studies, we attempted to isolate micro-organisms from the anaerobic granules in order to investigate the community structure based on a culture system. Interestingly, the granules contained aerobic bacteria, even though they had been kept under anaerobic conditions for 2 years. Strain Ko07T was one of the dominant bacterial isolates to grow under aerobic conditions. In this study, a polyphasic approach, including phylogenetic analysis based on 16S rRNA gene sequences, DNA–DNA relatedness, and chemotaxonomic and phenotypic properties, was used to determine the precise taxonomic position of strain Ko07T. The results obtained indicate that strain Ko07T should be classified as representing a novel species of the genus Lysobacter, and that it can be clearly distinguished from Lysobacter species with validly published names. In this report, we propose Ko07T as the type strain of a novel species, for which the name Lysobacter concretionis sp. nov. is proposed. We also present the interspecies relationships of Lysobacter species newly inferred by the polyphasic approach used.
For the isolation of aerobic bacteria, brownish-black granules (around 2 mm in diameter) from a brewery wastewater-treatment UASB reactor, which had been operated anaerobically for 2 years, were homogenized by using an Ace Homogenizer (Nihonseiki Kaisha). The suspension was spread on R2A agar plates (Difco), after being serially diluted with 50 mM phosphate buffer (pH 7·0). The plates were incubated at 30 °C for 2 weeks. Single colonies were purified by transfer onto fresh plates, followed by a second incubation under the same conditions. The purified colonies were tentatively identified by partial 16S rRNA gene sequencing. Ko07T was one of the dominant isolates to grow on the plates under aerobic conditions. The isolate was deposited in the Korean Collection for Type Cultures as KCTC 12205T (=DSM 16239T). Lysobacter species with validly published names were obtained from BCCMTM/LMG (Belgian Co-ordinated Collections of Microorganisms/Laboratorium voor Microbiologie).
Morphological characteristics and motility were observed by using differential interference contrast microscopy. Gram staining and tests for catalase and oxidase were performed according to protocols outlined by Smibert & Krieg (1981). Some physiological properties and substrate utilizations were determined by using API 20 NE, API ID 32 GN and API 50 CHB kits (bioMérieux). Degradation of chitin, starch and Tween 80 was also investigated, by using the protocols outlined by Atlas (1993). Cells grown on R2A agar plates at 30 °C for 2 days were used for the experiments mentioned above. To determine whether strain Ko07T had nitrite reductase genes (nirK and nirS), PCR was performed by using two different primer systems developed by Braker et al. (1998), nirK1F/nirK5R for nirK and nirS1F/nirS6R for nirS, under various thermal profiles.
Cells grown on R2A agar plates for 2 days were used for the analysis of cellular fatty acid composition. The cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI). The fatty acids were analysed by using a gas chromatograph (6890; Hewlett Packard), equipped with the Microbial Identification software package (Sasser, 1990). Ubiquinones were extracted from cells grown in liquid medium with the same composition as R2A but without the agar, according to the protocol described by Komagata & Suzuki (1987). The ubiquinones were analysed by HPLC using a C18 reversed-phase column at 254 nm, and a mobile phase comprising acetonitrile and iso-propanol (65 : 35, v/v) at 1 ml min–1.
Genomic DNA was isolated by using the protocol described by Schmidt et al. (1991). RNA was removed by treatment with a mixture of RNase A and T1 (each at 20 U ml–1), at 30 °C for 1 h. The DNA extract was used for the genomic and the phylogenetic studies. The genomic DNA G+C content was determined as described by Mesbah et al. (1989). The DNA–DNA hybridization value was estimated fluorometrically by using photobiotin-labelled DNA probes and a microdilution well, according to the method of Ezaki et al. (1989).
The 16S rRNA gene was amplified by using the bacterial universal primer set 9F (5'-GAG TTT GAT CCT GGC TCA G-3') and 1512R [5'-ACG G(A/T/C)T ACC TTG TTA CGA CTT-3']. The PCR product was purified with a GFX PCR DNA and a Gel Band Purification kit (Amersham Biosciences). It was sequenced by using an ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems) and an automatic DNA sequencer (model 310; Applied Biosystems). For full sequencing, the following primers were also used: 519F (5'-CAG CAG CCG CGG TAA TAC-3'), 907F [5'-AAA CTC AAA (G/T)GA ATT GAC GG-3'], 536R (5'-GTA TTA CCG CGG CTG CTG-3') and 1100R (5'-GGG TTG CGC TCG TTG-3'). The partial sequences were aligned and combined by using the BioEdit program (Hall, 1999). A BLAST search was performed to obtain the 16S rRNA gene sequences from related taxa in GenBank. These collected sequences were aligned and edited with the CLUSTAL_X program (Thompson et al., 1997) and the BioEdit program (Hall, 1999), respectively. The evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1983), and the phylogenetic tree was constructed by using the neighbour-joining method (Saitou & Nei, 1987) in the MEGA 2 program (Kumar et al., 2001). A bootstrap method was used to obtain the confidence levels for the neighbour-joining analysis, with a 1000 bootstrap data set (Felsenstein, 1985).
Strain Ko07T is aerobic, Gram-negative and rod-shaped (0·7–1·0x1·0–13·5 µm). The size of the cells changed with time. Cells grown on R2A agar plates for 5 days appeared as curved rods longer than 5 µm, whereas cells incubated for 30 days were shorter, as shown in Fig. 1. Flagella movement was not observed by microscopy. The colonies grown on R2A agar plates for 2 days were yellowish in colour, smooth, circular, convex and non-glossy, and 2–4 mm in diameter. The colour changed to a weak brown on longer incubation (
3 weeks). Spreading and swarming colonies, indicating gliding movement, were observed on an R2A agar plate that was incubated for 1 month, and on YA [0·5 % (w/v) yeast extract plus 1·5 % Bacto agar] and CCA (0·2 % tryptone plus 1·5 % Bacto agar) plates that had been incubated for 2 weeks. Tests for catalase and oxidase were positive. Gelatin was liquefied within 1 day, indicating proteolytic activity; this was also observed for all the type strains of species of the genus Lysobacter. The capability to degrade polysaccharide on chitin and starch that is present in some Lysobacter species (Christensen & Cook, 1978) was not observed in strain Ko07T. Lipase activity on Tween 80 was also not detected, whereas most Lysobacter species have this activity (Christensen & Cook, 1978). Differences in phenotypic characteristics including substrate utilization between Ko07T and type strains of some Lysobacter species are summarized in Table 1; the results suggest that Ko07T is different from the other type strains in phenotypic aspects. Nitrate was not reduced to nitrite. The nitrite reductase genes nirK and nirS were not amplified by the primer systems nirK1F/nirK5R and nirS1F/nirS6R, respectively. Ko07T grew well at 25–30 °C and pH 6·8–7·5, but not at 4 or 45 °C.
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9c and summed feature 4 (C15 : 0 iso 2OH/C16 : 17c), although their relative amounts were slightly different (Table 1
). The fatty acid profiles obtained matched well those of L. antibioticus ATCC 29479T and L. enzymogenes ATCC 29487T from MIDI, and also those of strains N4-7 and C3, which have been identified as belonging to the genus Lysobacter (Sullivan et al., 2003). The results indicate that strain Ko07T has chemotaxonomic features in common with the recognized Lysobacter species. The genomic DNA G+C content of strain Ko07T was 63·8 mol%, which is lower than those of the type strains of recognized Lysobacter species, 65·7–69·2 mol% (Table 2), indicating that strain Ko07T should be considered as representing a novel species of the genus Lysobacter.
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), strain Ko07T is closely clustered with the type strains of Lysobacter species with validly published names and unnamed Lysobacter species, in an independent phylogenetic branch within the class ‘Gammaproteobacteria’ that is clearly distinct from the neighbouring genera, Xanthomonas and Thermomonas. The sequence similarity of strain Ko07T to those organisms in the cluster was 94·9–96·7 % (see Supplementary Table in IJSEM Online). The high bootstrap value (98 %) provides strong support for the inclusion of strain Ko07T in the genus Lysobacter. This was also supported by the aforementioned taxonomic characteristics and the typical phenotypic properties described originally by Christensen & Cook (1978), e.g. gliding movement, long-rod shape, proteolytic activity and the high G+C content of genomic DNA. L. brunescens ATCC 29482T was also placed in the Lysobacter cluster, but in a branch with a low bootstrap value, 42 % (Fig. 2). This organism has a sequence similarity of 94·1–96·1 % with some Thermomonas species and Xanthomonas melonis, of which some values are higher than those for type strains of the recognized Lysobacter species (95·2–95·6 %) (see Supplementary Table in IJSEM Online). In spite of the weak phylogenetic relationship with other Lysobacter species, strain Ko07T still shares the common features of members of the genus Lysobacter, with respect to the presented phenotypic and chemotaxonomic aspects (Table 1).
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), which are low enough to confirm that Ko07T should be classified as representing a novel Lysobacter species. Its lower DNA G+C content (63·8 mol%) and the differences in phenotypic characteristics (Table 1) also suggest that Ko07T should be considered as a novel species. Therefore, on the basis of the results obtained in this study, we propose that strain Ko07T should be classified as a novel species within the genus Lysobacter, for which the name Lysobacter concretionis sp. nov. is proposed.
Description of Lysobacter concretionis sp. nov.
Lysobacter concretionis (con.cret.i.on'is. L. gen. n. concretionis of a compacting, condensing, congealing).
Gram-negative, aerobic rod or filamentous shape, of various sizes (0·7–1·0x1·0–13·5 µm), after growth on R2A agar plates at 25 °C for 10 days. Does not move by means of flagella, but by gliding on the surface of the agar. Colonies grown on an R2A agar plate for 2 days are yellow and circular, but become a weak brownish-yellow-coloured swarming form after 1 month. Optimum growth temperature and pH are 25–30 °C and 6·8–7·5, respectively. The DNA G+C content is 63·8 mol%, as determined by HPLC. Q-8 is the predominant quinone. The major cellular fatty acids are C16 : 0 (2·4 %), C11 : 0 iso (6·42 %), C15 : 0 iso (36·1 %), C16 : 0 iso (19·9 %), iso C17 : 19c (13·9 %) and C11 : 0 iso 3OH (5·6 %). Does not reduce nitrate. Can liquefy gelatin, but cannot degrade chitin, Tween 80 or starch. Other phenotypic characteristics, such as substrate utilization and enzyme production, are summarized in Table 1.
The type strain is Ko07T (=KCTC 12205T=DSM 16239T), isolated from a UASB reactor treating brewery wastewater.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bae, J.-W. & Lee, S.-T. (1999). Layered structure of UASB granules gives microbial population resistance to toxic chemicals. Biotechnol Lett 21, 159–162.
Bae, J.-W., Rhee, S.-K., Hyun, S.-H., Kim, I. S. & Lee, S.-T. (2000). Layered structure of granules in upflow anaerobic sludge blanket reactor gives microbial populations resistance to metal ions. Biotechnol Lett 22, 1935–1940.[CrossRef]
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Christensen, P. & Cook, F. D. (1978). Lysobacter, a new genus of nonfruiting, gliding bacteria with a high base ratio. Int J Syst Bacteriol 28, 367–393.
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Felsenstein, J. (1985). Confidence limit on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
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