Editor: Aharon Oren
‘Lysobacter enzymogenes ssp. cookii ’ Christensen 1978 should be recognized as an independent species, Lysobacter cookii sp. nov.
Version of Record online: 25 JUN 2009
© 2009 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved
FEMS Microbiology Letters
Volume 298, Issue 1, pages 118–123, September 2009
How to Cite
Kawamura, Y., Tomida, J., Morita, Y., Naka, T., Mizuno, S. and Fujiwara, N. (2009), ‘Lysobacter enzymogenes ssp. cookii ’ Christensen 1978 should be recognized as an independent species, Lysobacter cookii sp. nov. FEMS Microbiology Letters, 298: 118–123. doi: 10.1111/j.1574-6968.2009.01703.x
- Issue online: 20 JUL 2009
- Version of Record online: 25 JUN 2009
- Received 9 June 2009; accepted 18 June 2009.Final version published online 16 July 2009.
- gram-negative bacteria;
- gliding bacteria;
- Lysobacter cookii;
- ‘Lysobacter enzymogenes ssp. cookii ’
‘Lysobacter enzymogenes ssp. cookii’ was proposed by Christensen and Cook in 1978; however, this subspecies name has not been cited in the Approved Lists of Bacterial Names and therefore the nomenclature has not been validated. In our genetic approach to clarify the relationships of the designated type strain of ‘L. enzymogenes ssp. cookii’ PAGU 1119 (GenBank accession number ATCC29488) within the genus Lysobacter revealed that the strain was closely related to Lysobacter capsiciYC5194T (99.4%) rather than L. enzymogenesDSM2043T (97.2%). The value for whole genome DNA–DNA relatedness between strain PAGU 1119 and L. enzymogenes DSM 2043T or L. capsiciYC5194T was 20.7–26.1% or 60.9–62.0%, respectively. Although PAGU 1119 and L. capsiciYC5194T showed relatively high DNA relationships, the fatty acid profiles and some phenotypic characteristics were different, and we concluded that PAGU 1119 should be placed in a new species. We therefore propose a new species with the name Lysobacter cookii sp. nov. The type strain is PAGU 1119T (ATCC29488).
The genus Lysobacter was established by Christensen & Cook (1978) for gliding bacteria with high G+C contents that do not produce fruiting bodies, with Lysobacter enzymogenes as the type species. These authors mainly used phenotypic characteristics to establish this genus; the taxonomic position and phylogenetic features of the organisms were confirmed by Bae et al. (2005). Since then, 11 species have been proposed as a new species within Lysobacter (Bae et al., 2005; Lee et al., 2006, Weon et al., 2006, 2007; Yassin et al., 2007; Park et al., 2008; Romanenko et al., 2008; Aslam et al., 2009; Wang et al., 2009). To date, 15 species have been recognized as members of the genus Lysobacter.
In 1978, Christensen & Cook also proposed two subspecies: ‘Lysobacter enzymogenes ssp. enzymogenes’ and ‘Lysobacter enzymogenes ssp. cookii.’ These subspecies were not cited in the Skerman et al. (1980 and 1989), even though they were listed in the Index of Bacterial and Yeast Nomenclatural Changes (Moore & Moore, 1989), and the nomenclature has therefore not been validated. In 2006, Tindall & Euzéby requested that the Judicial Commission rule that these names be treated as having been included on the approved lists, on the amended edition of the lists. Up to now, no opinion related to this issue has been announced, and the nomenclatural status for these subspecies is still not fixed.
During our investigation of the taxonomic relationships of L. enzymogenes strains, we found that ‘L. enzymogenes ssp. cookii’ were actually not closely related to L. enzymogenes and should be recognized as an independent species within the genus Lysobacter, namely Lysobacter cookii sp. nov.
Materials and methods
Strains used in this study
We used the following type strains: L. enzymogenes (PAGU 1067T=DSM2043T), Lysobacter antibioticus (PAGU 1068T=DSM2044T), Lysobacter capsici (PAGU 1064T=YC5194T), Lysobacter gummosus (PAGU 1069T=DSM6980T), Lysobacter koreensis (PAGU 1128T=NBRC101156T), Lysobacter niastensis (PAGU 1071T=DSM18481T), and Lysobacter yangpyeongensis (PAGU 1070T=DSM17635T). All strains were purchased directly from each culture collection, except L. capsiciYC5194T, which was kindly provided by Dr J.H. Park (Park et al., 2008). We also used the strain PAGU 1119 (ATCC29488), purchased direct from American Type Culture Collection (ATCC), which was designated as the type strain of ‘Lysobacter enzymogenes ssp. cookii.’ All strains were grown on R2A agar (Wako Pure Chemical Ltd, Osaka, Japan) plates or 2% trypticase soy agar at 30 °C under aerobic conditions.
First, we determined the 16S rRNA gene sequences of strain PAGU 1119, and investigated the genetic position of the strain within the genus Lysobacter. The PCR primers used for amplification of 16S rRNA gene were as described previously (Kawamura et al., 1999, 2003). After confirming single amplification products on 1% agarose gels, sequences were determined with an automatic sequencer (Model 3130, Applied Biosystems) using a dye-terminator reaction kit (Applied Biosystems). The clustal-x software originally described by Thompson et al. (1997) was used to align sequences, and phylogenetic distances were calculated by the neighbor-joining method. Phylogenetic trees were drawn using treeview software (Page, 1996).
To clarify the exact genomic relationships of PAGU 1119 strain, we decided to measure the whole genomic DNA reassociation rate. DNA from each strain was prepared by the standard procedure of Marmur (1961). We also used the silica–guanidinium thiocyanate DNA purification method described previously (Boom et al., 1990). Quantitative microplate DNA–DNA hybridization was carried out as described previously (Ezaki et al., 1989). Hybridization experiments were carried out at 42 °C (optimal conditions) and 52 °C (stringent conditions) using 2 × SSC and 50% formamide. The optimal temperature was 55 °C below the thermal denaturation temperature, because formamide lowered the hybridization temperature (Meinkoth & Wahl, 1984).
Cellular lipids and fatty acids were analyzed as described previously (Naka et al., 2000; Li et al., 2003). Briefly, bacterial cells grown on R2A medium were harvested, and cellular lipids were extracted twice with chloroform : methanol (2 : 1, v/v). The cellular lipids were analyzed using two-dimensional TLC. For the fatty acids, the harvested cells were hydrolyzed with 3.75 M NaOH in methanol : water (1 : 1, v/v) at 100 °C for 30 min. After neutralization with 6 N HCl, the fatty acids were extracted twice with n-hexane. Methyl ester derivatives of fatty acids were performed by the treatment of 10% trimethylsilyldiazomethane in n-hexane (Nacalai Tesque Inc., Kyoto, Japan), and analyzed by GC/MS.
Biochemical traits were determined with API20NE, API ZYM (bioMérieux) and Nonfergram (Wako Pure Chemical Ltd) according to the manufacturers' recommendations. All phenotypic characterization experiments were performed in duplicate.
Results and discussion
On the phylogenetic tree based on 16S rRNA gene sequences, three species groups were formed: L. capsici, L. gummosus and L. antibioticus formed one cluster; L. yangpyeongensis, Lysobacter niabensis, L. koreensis and Lysobacter oryzae formed another cluster; Lysobacter defluvii, Lysobacter spongiicola, Lysobacter concretionis and Lysobacter daejeonensis also formed a different cluster. PAGU 1119 strain formed a cluster with L. capsici, L. antibioticus and L. gummosus with high similarity values (99.4%, 99.2%, and 99.1%, respectively) but the strain was somewhat remote from L. enzymogenes (97.2%) and other members of the genus Lysobacter (Fig. 1).
The DNA–DNA hybridization values are shown in Table 1. Surprisingly, <27% DNA relatedness was shown between strain PAGU 1119 and L. enzymogenes PAGU 1067T, whereas >50% reassociation values were observed with L. capsici PAGU 1064T (62.0% and 53.3% under the optimal and stringent conditions, respectively). From these data, we further confirmed that PAGU 1119 (‘L. enzymogenes ssp. cookii’) was genetically close to L. capsici but not to L. enzymogenes.
|Strains||DNA-hybridization (%) with biotin-labeled DNA from|
|L. cookii PAGU 1119T||L. enzymogenes PAGU 1067T||L. capsici PAGU 1064T|
|L. cookii PAGU 1119T||100.0||100.0||26.1 ± 2.6||26.6 ± 0.9||62.0 ± 0.1||53.3 ± 2.2|
|L. enzymogenes PAGU 1067T||20.7 ± 0.3||20.8 ± 1.0||100.0||100.0||36.0 ± 2.3||18.9 ± 1.4|
|L. capsici PAGU 1064T||60.9 ± 2.2||60.6 ± 2.9||28.7 ± 0.9||28.8 ± 1.4||100.0||100.0|
|L. antibioticus PAGU 1068T||24.9 ± 0.3||23.9 ± 0.4||23.6 ± 2.6||25.2 ± 1.0||31.2 ± 2.0||19.7 ± 0.7|
|L. gummosus PAGU 1069T||35.9 ± 7.4||35.6 ± 9.0||33.1 ± 3.1||32.8 ± 4.1||45.3 ± 6.5||31.0 ± 6.9|
|L. yangpyeongensis PAGU 1070T||11.6 ± 0.4||11.3 ± 0.7||14.0 ± 1.2||14.3 ± 2.0||18.8 ± 1.3||9.2 ± 0.8|
|L. niastensis PAGU 1071T||13.5 ± 2.0||13.4 ± 2.5||15.4 ± 0.9||15.4 ± 1.8||20.1 ± 1.5||10.2 ± 1.1|
|L. koreensis PAGU 1128T||14.9 ± 0.1||15.2 ± 1.1||18.7 ± 2.4||18.0 ± 2.6||26.2 ± 0.9||12.3 ± 1.4|
The cellular fatty acid profiles of PAGU 1119 and related species are shown in Table 2. The major cellular fatty acids in PAGU 1119 were 15:0 iso (18.4%), 16:0 (11.4%), 17:1 iso (10.5%), 16:1ω7c/15:0 iso 2-OH (10.8%), 17:0 cyclo (8.5%), 16:1 (6.7%), 17:0 iso (6.6%), 18:1 (5.9%), 16:0 iso (5.5%), 15:0 anteiso (4.0%), 11:0 iso 3-OH (2.1%), and 15:1 iso (1.8%). No significant distinctive features were found in the fatty acid profiles of strain PAGU 1119 compared with the profiles of Lysobacter species. The presence of 16:1 could distinguish PAGU 1119 (6.7%) from L. enzymogenes (undetected). The presence of somewhat large amounts of 18:1 branched and 19:0 cyclo could also distinguish PAGU 1119 (0.6% and 0.8%, respectively) from L. capsici (6.3% and 4.6%, respectively).
|11:0 iso 3-OH||2.1||4.4||3.0||8.0||9.7||9.3||8.0||5.5||9.0||7.2||7.2||6.9||6.0||15.5||3.2||5.2|
|16:1ω7c/15:0 iso 2-OH||10.8||3.0||13.9||8.3||6.4||2.0||6.5||3.3||1.4||9.5||–||–||6.1||–||1.1||2.9|
|Unknown (ECL 11.799)||–||–||–||2.0||1.8||–||1.4||–||–||–||–||–||–||–||–||–|
The polar lipids of PAGU 1119 strain and the two closely related type strains, L. enzymogenes (PAGU 1067T) and L. capsici (PAGU 1064T) were determined. The major polar lipids, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, and diphosphatidylglycerol, were same in these three strains (data not shown).
The summary of some biochemical and phenotypic characteristics are shown in Table 3. Colonies grown on an R2A agar plate after 2 days at 30 °C are creamy white to light brown. The DNA G+C content is 66.2 ± 0.4 mol% as determined by HPLC methods (Kawamura et al., 1998). PAGU 1119 strain could be differentiated from other members of the genus Lysobacter by many biochemical traits, for example nitrate reduction, aesculin hydrolysis, assimilation of d-glucose, d-mannose, malic acid, and others. Some enzyme activities such as α-chemotrypsin, α-galactosidase, β-galactosidase and N-acetyl-β-glucosaminidase were useful as characteristics differentiating PAGU 1119 from genetically closely related species (L. enzymogenes, L. capsici, and L. antibioticus).
|Cell size (μm)||0.3–0.5 × 4–50||0.3–0.5 × 2.0–20||0.4 × 2.0||0.4 × 6.5||0.5 × 38.0||0.5–0.6 × 2.0–4.0||0.5 × 2.0–5.0||0.4–0.6 × 3.0–4.0||0.3–0.5 × 1.8–2.0||0.5–0.8 × 1.5–2.0||0.5 × 1–3||0.3 × 11.0||1–2 (length)||0.7 × 1.0–13.5||0.4–0.6 × 3.0–4.0||0.5–0.6 × 1.3–1.5|
|DNA G+C content (mol%)||66.2||65.4||65.7||69.2||69.0||66.6||62.5||67.3||67.4||68.9||63.5||67.7||67.1||63.8||61.7||69.0|
|API 20NE tests for assimilation of|
|Enzyme activities (API ZYM)|
Although PAGU 1119 and L. capsiciYC5194T showed relatively high DNA relationships, the fatty acid profiles and some phenotypic characteristics were different. We therefore conclude that PAGU 1119 should be placed in a new species rather than a subspecies of L. capsici. We propose the name Lysobacter cookii sp. nov. for this new species.
Description of Lysobacter cookii sp. nov.
Lysobacter cookii (coo'ki.i. N.L. gen. n. cookii of Cook; named from F.D. Cook, the microbiologist who first isolated lysobacters).
Cells are aerobic, gram-negative, rod or filamentous shaped, of various sizes (0.3–0.5 × 4–50 μm), non-spore-forming, and nonmotile, but having gliding activity. Colonies grown on an R2A agar plate after 2 days at 30 °C are creamy white to light brown. No growth on MacConkey agar. The DNA G+C content is 66.2 ± 0.4 mol% as determined by HPLC. The major cellular fatty acids data are shown in Table 2. Catalase and oxidase positive. Does not reduce nitrate. Can hydrolyze aesculin but not arginine and starch. Liquidize gelatin. Does not produce indole. Cannot produce acid from xylitol, lactose or mannitol. Negative for lysine and ornithine decarboxylase. Positive for alkaline phosphatase, C4 and C8 esterase, and naphthol hydrase, but negative for urease, cystine aryl amidase, β-glucuronidase, α-mannosidase, and α-fucosidase. Other phenotypic characteristics are described in Table 1.
The type strain is PAGU 1119 (ATCC29488), isolated from soil in Ottawa, Canada.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequences of the strains PAGU 1119T is AB485771T.
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