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International Journal of Systematic and Evolutionary Microbiology (2011), 61, 295–298 Nitratireductor indicus sp. nov., isolated fromdeep-sea water Qiliang Lai, Zhiwei Yu, Jun Yuan, Fengqin Sun and Zongze Shao Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen, People’s Republic of China A taxonomic study was carried out on a novel bacterial strain, designated C115T, isolated from acrude-oil-degrading consortium, enriched from deep-sea water of the Indian Ocean. Cells wereGram-negative short rods, mobile by means of a monopolar flagellum. Growth was observed atsalinities of 0–7 % and at 10–43 6C. It was unable to degrade Tween 80 or gelatin. 16S rRNAgene sequence analysis showed that strain C115T was related most closely to Nitratireductoraquibiodomus NL21T (96.5 % similarity), Nitratireductor kimnyeongensis KY 101T (96.4 %) andNitratireductor basaltis J3T (96.2 %). The predominant fatty acids were summed feature 8(C18 : 1v7c and/or C18 : 1v6c, 81.8 %) and C18 : 0 (7.0 %). The G+C content of the chromosomalDNA of strain C115T was 59 mol%. Based on its morphology, physiology and fatty acidcomposition together with 16S rRNA gene sequence comparisons, the novel strain mostappropriately belongs to the genus Nitratireductor, but can be distinguished readily fromrecognized species of the genus. Strain C115T is therefore considered to represent a novelspecies of the genus Nitratireductor, for which the name Nitratireductor indicus sp. nov. isproposed. The type strain is C115T (5RC92-7T 5CCTCC AB 209298T 5LMG 25540T5MCCC 1A01260T).
The genus Nitratireductor, belonging to the family cultivated for morphological and biochemical character- and, at the time of writing, comprises three recognized Genomic DNA was prepared according to the method of by PCR by using the primers described previously Nitratireductor basaltis ). In this study, we Sequences of related taxa were obtained from isolated a novel strain, designated C115T, from a crude-oil- the GenBank database. Phylogenetic analysis was per- degrading consortium enriched from a deep-sea water sample collected from the Indian Ocean. The strain was multiple alignment of the data by DNAMAN (version 5.1; found to be related phylogenetically to members of the Lynnon Biosoft). Distances (distance options according to genus Nitratireductor. Characterization and classification of Kimura’s two-parameter model) and clustering with the strain C115T were based on data from the present Deep-sea water was sampled with Niskin bottles attached were determined by using bootstrap values based on to a CTD (conductivity, temperature and depth) sampler in December 2005 during cruise DY-105A of R/V Da-Yang A nearly full-length 16S rRNA gene sequence (1448 nt) of Yi-Hao. The sampling site was located at 25.3217u S strain C115T was obtained. Phylogenetic analysis of strain 70.0405u E in the south-western part of the Indian Ridge, at a water depth of 2488 m. The seawater was enriched with Phyllobacteriaceae, forming a robust clade with members crude oil, and bacteria in the community were isolated on of the genus Nitratireductor in the neighbour-joining tree a similar tree topology was obtained with the C115T was obtained as a novel bacterium. It was further minimum-evolution method. Strain C115T was relatedmost closely to N. aquibiodomus NL21T (96.5 % 16S rRNA The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene gene sequence similarity), N. kimnyeongensis KY 101T sequence of strain C115T is GU447302.
(96.4 %) and N. basaltis J3T (96.2 %). Levels of 16S rRNAgene sequence divergence between strain C115T and the A supplementary figure and a supplementary table are available with theonline version of this paper.
type strains of recognized Nitratireductor species were Fig. 1. Neighbour-joining tree showing thephylogenetic positions of strain C115T andrepresentatives of some other related taxa,based Bootstrap values (expressed as percentagesof 1000 replications) are shown at branchpoints. Bar, 0.005 nucleotide substitution rate(Knuc) units.
¢3.5 %, and thus the data support the view that strain followed by GC analysis of the fatty acid methyl esters according to the instructions of the MIDI system (The fatty acid profiles of N. aquibiodomus DSM Gram staining, catalase, oxidase and lipase (Tween 80) 15645T, N. kimnyeongensis KCTC 22759T and N. basaltis activities, hydrolysis of aesculin and starch, optimal growth KCTC 22119T were determined in parallel with that for temperature and pH, tolerance of NaCl and general cell strain C115T in this study. The detailed results are shown morphology were studied as described previously in Supplementary Table S1, available in IJSEM Online.
Other biochemical tests were carried out by using The major fatty acids of strain C115T were summed API 20NE and API ZYM strips (bioMe´rieux) and Biolog GN2 microplates according to the manufacturers’ instruc- 18 : 1v7c and/or C18 : 1v6c; 81.8 %) and C18 : 0 (7.0 %), similar to the data for N. basaltis KCTC 22119T.
tions, except that the NaCl concentration was adjusted in Strain C115T could be differentiated from the type all tests to 3.0 %. N. aquibiodomus DSM 15645T, N.
strains of N. aquibiodomus and N. kimnyeongensis based kimnyeongensis KCTC 22759T and N. basaltis KCTC on the proportion of summed feature 8 and C 22119T were tested at the same time for comparison. The v8c (.15 % difference). The proportions of the indi- results of these tests are given in the species description and vidual fatty acids of the three reference type strains were slightly different from those given in the original The G+C content of the chromosomal DNA was determined according to the methods described by Antibiotic susceptibility tests were performed by the disc HPLC. The DNA G+C content of strain C115T was discs were obtained from Oxoid. Strain C115T and the type 59 mol%, a value close to those given for the type strains of strains of N. aquibiodomus, N. kimnyeongensis and N.
basaltis were tested at the same time in this study. All four strains were sensitive to (mg per disc) cefalexin (30), Fatty acids in whole cells grown on marine agar 2216 at cefobid (30), chloromycetin (30), erythromycin (15), 28 uC for 48 h were extracted, saponified and esterified, rifampicin (5) and rocephin (30), but resistant to International Journal of Systematic and Evolutionary Microbiology 61 Table 1. Differential characteristics between strain C115T and the type strains of recognized Nitratireductor species Strains: 1, C115T; 2, N. aquibiodomus NL21T (unless indicated, data from 3, N. kimnyeongensis KY 101T 4, N.
basaltis J3T In API 20NE tests in our study, all strains were negative for indole production, D-glucose fermentation and gelatinhydrolysis. In API ZYM tests in our study, all strains were positive for acid phosphatase, alkaline phosphatase, esterase (C4), leucineaminopeptidase, trypsin and valine aminopeptidase and negative for a-fucosidase, a-mannosidase, b-glucosidase and b-glucuronidase. +, Positive;W, weakly positive; 2, negative. ND, No data available.
phosphoamidase, a-chymotrypsin, a-glucosidase Clindamycin, co-trimoxazole, gentamicin, norfloxacin, ofloxacin, oxacillin, polymyxin B, vancomycin Cephradin, streptomycin, tetracycline, vibramycin *Data from this study using the strains listed in the text.
kanamycin (30), lincomycin (2) and metronidazole (5).
Nitratireductor, for which the name Nitratireductor indicus Detailed results of antibiotic susceptibility tests are given in Description of Nitratireductor indicus sp. nov.
On the basis of morphological, physiological and chemo-taxonomic characteristics, together with data from 16S Nitratireductor indicus (in9di.cus. L. masc. adj. indicus rRNA gene sequence comparisons, strain C115T is Indian, referring to the Indian Ocean, from where the type considered to represent a novel species of the genus Cells are rods, about 3.0 mm long and 1.3 mm wide, mobile the characteristics used to distinguish the type strain from by means of at least one polar flagellum (Supplementary Fig. S1). Positive for catalase, oxidase, urease, arginine The type strain, C115T (5RC92-7T 5CCTCC AB 209298T 5LMG 25540T 5MCCC 1A01260T), was isolated from hydrolysis) and nitrate reduction, but negative for the Gram reaction, indole production, D-glucose fermentation,lipase (Tween 80), amylase, gelatinase and denitrification.
After 3 days of incubation at 28 uC on marine agar, produces smooth grey colonies with regular edges that are This work was supported financially by the Scientific Research 2–3 mm in diameter, non-pigmented and slightly raised in Foundation of Third Institute of Oceanography, SOA (no. 2009065), the centre. Grows in the presence of 0–7 % NaCl (optimum the COMRA program (no. DYXM115-02-2-05) and the National 3 %) and at 10–43 uC (optimum 25–30 uC); no growth is Infrastructure of Natural Resources for Science and Technology observed at 45 uC after 1 week. The major fatty acids are Program of China (no. 2005DKA21209).
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