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Bowmanella Pacifica Sp. Nov., Isolated from a Pyrene-Degrading Consortium

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Bowmanella Pacifica Sp. Nov., Isolated from a Pyrene-Degrading Consortium International Journal of Systematic and Evolutionary Microbiology (2009), 59, 1579–1582 DOI 10.1099/ijs.0.001826-0 Bowmanella pacifica sp. nov., isolated from a pyrene-degrading consortium Qiliang Lai,13 Jun Yuan,1,23 Baojiang Wang,1 Fengqin Sun,1 Nan Qiao,1 Tianling Zheng2 and Zongze Shao1 Correspondence 1Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Zongze Shao Administration, Xiamen 361005, PR China [email protected] 2MOE of Key Lab for Coast and Wetland Ecosystem, School of Life Sciences, Xiamen University, Tianling Zheng Xiamen 361005, PR China [email protected] A taxonomic study was carried out on a strain, designated W3-3AT, which was isolated from a pyrene-degrading consortium, enriched from sediment of the Pacific Ocean. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain W3-3AT belonged to the genus Bowmanella, with the highest sequence similarity (99.0 %) with Bowmanella denitrificans BD1T, whereas sequence similarities with other species were less than 93 %. The nucleotide sequence similarity of both gyrB and rpoD genes of strain W3-3AT and B. denitrificans BD1T was 81.1 %. However, the protein sequence similarities of the gyrB and rpoD genes of strain W3-3AT and B. denitrificans BD1T were 96.1 % and 91.0 %, respectively. Phylogenetic trees based on these housekeeping genes showed that strain W3-3AT and B. denitrificans BD1T formed a distinct lineage in the Gammaproteobacteria. The DNA–DNA hybridization value between strain W3-3AT and B. denitrificans BD1T was 43 %. Strain W3-3AT could also be differentiated from B. denitrificans BD1T based on the repetitive extragenic palindromic DNA-PCR fingerprint. The G+C content of the chromosomal DNA of strain W3-3AT was 49 mol%. The combined genotypic and phenotypic data showed that strain W3-3AT represents a novel species of the genus Bowmanella, for which the name Bowmanella pacifica sp. nov. is proposed, with the type strain W3-3AT (5CGMCC 1.7086T5LMG 24568T5MCCC 1A01018T). During the screening of pyrene-degrading bacteria, an taxonomic and classical phenotypic characteristics. Based Alteromonas-like bacterium, designated strain W3-3AT, was on these results, we consider that strain W3-3AT should be isolated from a pyrene-degrading consortium, enriched classified as representing a novel species of the genus from sediment of the Pacific Ocean in 2002 (Wang et al., Bowmanella, which was proposed by Jean et al. (2006). 2008), and was selected for further characterization by Strains isolated in this study were stored at 280 uCinM2 21 using a polyphasic approach, including genotypic, chemo- medium (l seawater: CH3COONa, 5.0 g; tryptone, 0.5 g; yeast extract, 0.5 g; glucose, 0.5 g; sucrose, 0.5 g; sodium citrate, 0.05 g; malic acid, 0.05 g; NH4NO3, 1.0 g; NH4Cl, 3These authors contributed equally to this work. 0.2 g; KH2PO4, 0.5 g; adjusted to pH 7.6) (Wang et al., Abbreviations: gyrB, DNA gyrase subunit B gene; rpoD, DNA-directed 2008), supplemented with 16 % (v/v) glycerol for main- RNA polymerase subunit D gene; rep-PCR, repetitive extragenic tenance. M2 medium was used for routine cultivation of palindromic DNA-PCR. the isolates and most phenotypic tests. All cultures were The GenBank/EMBL/DDBJ accession numbers for the nucleotide incubated at 28 uC unless noted otherwise. sequences reported in this study are EU440951 (Bowmanella pacifica W3-3AT, 16S rRNA gene), EU441038 (B. pacifica W3-3AT, partial gyrB Genomic DNA was prepared according to the method of gene), EU441040 (B. pacifica W3-3AT, partial rpoD gene), EU441037 Ausubel et al. (1995) and the 16S rRNA gene was amplified (Bowmanella denitrificans BD1T, partial gyrB gene) and EU441039 (B. by PCR using primers that have been described previously T denitrificans BD1 , partial rpoD gene). (Liu & Shao, 2005). Sequences of related taxa were Transmission electron micrographs of cells of strain W3-3AT, dendro- obtained from the GenBank database. Phylogenetic analysis T grams showing the phylogenetic positions of strain W3-3A and related was performed using MEGA version 4 (Tamura et al., 2007) species based on gyrB and rpoD gene sequences, and results of rep- after multiple alignments of data by using DNAMAN (version PCR and polar lipid analysis of strain W3-3AT and B. denitrificans BD1T and a table showing the cellular fatty acid contents of strain W3-3AT and 5.1; Lynnon Biosoft). Distances (distance options accord- B. denitrificans BD1T are available as supplementary material with the ing to the Kimura two-parameter model) and clustering online version of this paper. with the neighbour-joining method of Saitou & Nei (1987) Downloaded from www.microbiologyresearch.org by 001826 G 2009 IUMS Printed in Great Britain 1579 IP: 137.108.70.6 On: Sun, 19 Jun 2016 18:21:53 Q. Lai and others and minimum-evolution methods of Rzhetsky & Nei W3-3AT and B. denitrificans BD1T had low DNA–DNA (1992, 1993) were determined by using bootstrap values relatedness (43 %), demonstrating their affiliation to based on 1000 replications. As the topology of the different species in accordance with the cut-off value of minimum-evolution tree was similar to that obtained 70 % recognized by Wayne et al. (1987) for discrimination using the neighbour-joining method, the data generated of bacterial species. Strain W3-3AT and B. denitrificans using minimum-evolution are not shown. BD1T were compared further by using repetitive extragenic palindromic DNA-PCR fingerprint (rep-PCR). The primer A nearly full-length 16S rRNA gene sequence (1496 nt) of T BOX-A1R (59-CTACGGCAAGGCGACGCTGACG-39) was strain W3-3A was determined. As shown in Fig. 1, the used for rep-PCR fingerprint analysis (Versalovic et al. phylogenetic tree based on 16S rRNA gene sequences 1991). PCR was carried out with the following cycle showed that strain W3-3AT and Bowmanella denitrificans T conditions: denaturation for 5 min at 94 uC; 35 cycles of BD1 formed an independent monophyletic cluster, with a 15 s at 94 uC, 30 s at 53 uC and 8 min at 65 uC and a final high level of similarity (99.0 %); similarity values between T extension at 65 uC for 8 min. The PCR products were the sequence of strain W3-3A and those of other related separated by using agarose (2 %, w/v) gel electrophoresis. taxa were all less than 93 %. The high level of 16S rRNA T The rep-PCR result is shown in Supplementary Fig. S3 (in gene similarity confirmed that strain W3-3A belonged to IJSEM Online). Strain W3-3AT showed a rep-PCR the genus Bowmanella. fingerprint pattern that was different from that of B. T For further comparison of strain W3-3AT with B. denitrificans BD1 . These results further confirmed the denitrificans BD1T, two housekeeping genes, the DNA results of the DNA–DNA hybridization. gyrase subunit B gene (gyrB) and the DNA-directed RNA General cell morphology was studied under an Olympus polymerase subunit D gene (rpoD) of the two strains were inverted microscope (Olympus IX70) using 1 day-old sequenced using the method described by Yamamoto et al. cultures of strain W3-3AT grown on M2 agar medium. (2000). The similarity values between the gyrB and rpoD For electron microscopy, exponential-phase cells were T T gene sequences of strain W3-3A and B. denitrificans BD1 harvested, suspended and absorbed on a Formvar– were both 81.1 %. In addition, the protein sequence carbon-coated grid, and stained with phosphotungstic T similarities of the gyrB and rpoD genes of strain W3-3A acid. The Gram reaction and catalase and oxidase activities T and B. denitrificans BD1 were 96.1 and 91.0 %, respect- were carried out according to Dong & Cai (2001). The ively. As shown in Supplementary Figs S1 and S2 (available optimal temperature for growth (using 4, 10, 20, 25, 37, 42 in IJSEM Online), phylogenetic trees based on the and 55 uC) and pH (from pH 3 to 12) were determined in sequences of the two housekeeping genes showed that M2 medium. Tolerance of NaCl was tested by using Luria– strain W3-3AT and B. denitrificans BD1T formed an Bertani (LB) medium (10 g peptone l21 and 5 g yeast l21), independent monophyletic cluster, similar to that obtained supplemented with NaCl concentrations of 0, 0.5, 1, 3, 5, 7, based on the 16S rRNA gene sequences. Strain W3-3AT 8, 9, 10, 12 and 15 % (w/v). Other biochemical tests were could be differentiated from B. denitrificans BD1T based on carried out in duplicate using API 20NE and API ZYM these results. strips (bioMe´rieux) and the Biolog GN2 MicroPlate panel, according to the manufacturers’ instructions, with the DNA–DNA hybridization experiments were performed T adjustment that the NaCl concentration was 3.0 %. With with genomic DNA of strain W3-3A and B. denitrificans the exception of the Biolog test, B. denitrificans BD1T was BD1T using a method that was described previously (Liu & tested at the same time as strain W3-3AT for comparison. Shao, 2005). Genomic DNA from Escherichia coli DH5a was used as an outgroup sample. Salmon sperm DNA was Strain W3-3AT was found to be a Gram-negative, non- used as a negative control. The results showed that strain pigmented, rod-shaped bacterium that was motile by Fig. 1. Neighbour-joining tree showing the phylogenetic positions of strain W3-3AT and representatives of some other related taxa, based on 16S rRNA gene sequences. Bootstrap values (expressed as percentages of 1000 replications) are shown at branch points. Bar, 0.01 nucleotide substitution rate (Knuc) units. Downloaded from www.microbiologyresearch.org by 1580 International Journal of Systematic and Evolutionary Microbiology 59 IP: 137.108.70.6 On: Sun, 19 Jun 2016 18:21:53 Bowmanella pacifica sp.
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