International Journal of Systematic and Evolutionary Microbiology (2014), 64, 3553–3558
DOI 10.1099/ijs.0.061168-0
Alcanivorax xenomutans sp. nov., a hydrocarbonoclastic bacterium isolated from a shrimp cultivation pond K. Rahul,1 Ch. Sasikala,1 L. Tushar,2 R. Debadrita2 and Ch. V. Ramana2 Correspondence
1
Chintalapati Sasikala [email protected] or sasi449@
2
yahoo.ie
Bacterial Discovery Laboratory, Centre for Environment, Institute of Science and Technology, J.N.T. University Hyderabad, Kukatpally, Hyderabad 500 085, India Department of Plant Sciences, School of Life Sciences, University of Hyderabad, P.O. Central University, Hyderabad 500 046, India
Two bacterial strains (JC109T and JC261) were isolated from a sediment sample collected from a shrimp cultivation pond in Tamil Nadu (India). Cells were Gram-stain-negative, motile rods. Both strains were positive for catalase and oxidase, hydrolysed Tween 80, and grew chemoorganoheterotrophically with an optimal pH of 6 (range pH 4–9) and at 30 6C (range 25–40 6C). Based on 16S rRNA gene sequence analysis, strains JC109T and JC261 were identified as belonging to the genus Alcanivorax with Alcanivorax dieselolei B-5T (sequence similarity values of 99.3 and 99.7 %, respectively) and Alcanivorax balearicus MACL04T (sequence similarity values of 98.8 and 99.2 %, respectively) as their closest phylogenetic neighbours. The 16S rRNA gene sequence similarity between strains JC109T and JC261 was 99.6 %. The level of DNA–DNA relatedness between the two strains was 88 %. Strain JC109T showed 31±1 and 26±2 % DNA–DNA relatedness with A. dieselolei DSM 16502T and A. balearicus DSM 23776T, respectively. The DNA G+C content of strains JC109T and JC261 was 54.5 and 53.4 mol%, respectively. Polar lipids of strain JC109T included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminophospholipids, two unidentified phospholipids and two unidentified lipids. The major fatty acids were C10 : 0, C12 : 0, C16 : 0, C12 : 0 3-OH, C16 : 1v7c, C18 : 1v7c and C19 : 0 cyclo v8c. Both strains could utilize diesel oil and a variety of xenobiotics as carbon and energy sources. The results of physiological, biochemical, chemotaxonomic and molecular analyses allowed the clear differentiation of strains JC109T and JC261 from all other members of the genus Alcanivorax. Strains JC109T and JC261 are thus considered to represent a novel species, for which the name Alcanivorax xenomutans sp. nov. is proposed. The type strain is JC109T (5KCTC 23751T5NBRC 108843T).
Millions of litres of diesel and petroleum oil enter the environment from natural and, more prominently, via anthropogenic sources, which introduces problems for a diverse range of ecosystems (Ryerson et al., 2012). Unearthing novel hydrocarbon-degrading micro-organisms is imperative in the bioremediation processes of hydrocarbon-contaminated sites (Chandran & Das, 2010; Atlas & Hazen, 2011). In this paper, we report two novel strains belonging to the genus Alcanivorax which were Abbreviations: DPG, diphosphatidylglycerol; ME, minimum-evolution; ML, maximum-likelihood; NJ, neighbour-joining; PE, phosphatidylethanolamine; PG, phosphatidylglycerol. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains JC109T and JC261 are HE601937 and HG974551, respectively. One supplementary table and two supplementary figures are available with the online version of this paper.
061168 G 2014 IUMS
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isolated from a sediment sample of a shrimp cultivation pond and are able to degrade diesel oil. The genus Alcanivorax was first proposed by Yakimov et al. (1998) with Alcanivorax borkumensis as the type species and the description of the genus was later amended by Ferna´ndez-Martı´nez et al. (2003). At the time of writing, the genus Alcanivorax comprised eight recognized species (http://www.bacterio.net/a/alcanivorax.html): A. borkumensis (Yakimov et al., 1998), Alcanivorax jadensis (Bruns & BertheCorti, 1999), Alcanivorax venustensis (Ferna´ndez-Martı´nez et al., 2003), Alcanivorax dieselolei (Liu & Shao, 2005), Alcanivorax balearicus (Rivas et al., 2007), Alcanivorax hongdengensis (Wu et al., 2009), Alcanivorax pacificus (Lai et al., 2011) and Alcanivorax marinus (Lai et al., 2013) were isolated from marine sediment/water samples. Alcanivorax dieselolei was specifically isolated from a hydrocarboncontaminated site (Liu & Shao, 2005). Members of the genus 3553
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Alcanivorax are Gram-stain-negative, aerobic, motile or non-motile rods, chemo-organotrophic, tolerate high salt concentrations and can thrive in the presence of hydrocarbons (Harayama et al., 2004; Lai et al., 2013). The DNA G+C content of members of the genus ranges from 53 to 66 mol%. They are also characterized by the presence of C16 : 0, C18 : 1v7c and C16 : 1v7c as predominant cellular fatty acids (Ferna´ndez-Martı´nez et al., 2003; Lai et al., 2013). Polar lipid profiles of the genus Alcanivorax have not been extensively studied. However, strain JC109T, A. dieselolei DSM 16502T, A. balearicus DSM 23776T, A. borkumensis CIP 105606T and A. marinus R8-12T (Lai et al., 2013) contained diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), unidentified phospholipids, unidentified aminolipids and unidentified lipids. Strains JC109T and JC261 were isolated from a sediment sample collected from a shrimp cultivation pond in Ramnad, Tamil Nadu, India (9u 169 N 79u 69 E) during September 2011. Physicochemical parameters including temperature, salinity and pH of the sample were analysed immediately after sample collection. The temperature at the site of sample collection was 29 uC. Salinity and pH of the sample were 2.8 % (w/v) and pH 8. One gram of air-dried sediment was serially diluted up to 1024 and 100 ml was spread on a growth medium (pH 7.5) containing (per litre): 0.2 g KH2PO4, 0.25 g NH4Cl, 0.5 g KCl, 0.15 g CaCl2 . 2H2O, 20.0 g NaCl, 0.62 g MgCl2 . 6H2O, 2.84 g Na2SO4, 2.83 g HEPES, 3.0 g yeast extract, 3.0 g peptone, 0.5 g Casamino acids, 0.5 g glucose and 3.0 g sodium pyruvate. Purification of the bacteria was achieved by repeated streaking on nutrient agar (HiMedia) containing 2 % (w/v) NaCl. Strains JC109T and JC261 were preserved as glycerol stocks and by lyophilization. Unless otherwise mentioned, both strains were grown in nutrient broth or on nutrient agar (HiMedia) with 2 % NaCl. Unless otherwise mentioned, the reference strains A. dieselolei DSM 16502T and A. borkumensis CIP 105606T were cultured in marine broth (HiMedia) supplemented with 1 % sodium pyruvate (pH 7.6 at 28 uC) and A. balearicus DSM 23776T was grown in nutrient broth or on nutrient agar with 1.5 % (w/v) NaCl (pH 7.2 at 28 uC) as recommended by the culture collection. Genomic DNA was extracted and purified from strains JC109T and JC261 according to the method of Marmur (1961) and the G+C of the DNA was 54.5 and 53.4 mol%, respectively, as determined by HPLC (Mesbah et al., 1989). Cell material for 16S rRNA gene sequencing was taken from a colony. DNA was extracted and purified by using a Qiagen genomic DNA extraction kit. Recombinant Taq polymerase (Genei) was used for PCR. The almostcomplete sequence of the 16S rRNA gene was obtained by sequencing with primers F27 (59-GTTTGATCCTGGCTCAG-39) and R1489 (59-TACCTTGTTACGACTTCA-39) [positions 11–27 and 1489–1506, respectively, according to the Escherichia coli 16S rRNA numbering system of the International Union of Biochemistry (Brosius et al., 1978; Lane et al., 1985)]. PCR amplification was done as described 3554
by Imhoff et al. (1998) and Imhoff & Pfennig (2001). 16S rRNA gene sequencing was performed using the BigDye Terminator v1.1 Sequencing kit (Applied Biosystems) in a 3730-DNA-Analyser (Applied Biosystems) as specified by the manufacturer. For sequencing, the primers F27, F790 (59-GATACCCTGGTAGTCC-39) and R1489 were used. Identification of phylogenetic neighbours and calculation of pairwise 16S rRNA gene sequence similarity were achieved using the EzTaxon-e server (Kim et al., 2012). The CLUSTAL W algorithm of MEGA 5.2 was used for sequence alignments and MEGA 5.2 (Tamura et al., 2011) software was used for phylogenetic analysis of the individual sequences. Distances were calculated by using the Kimura correction in a pairwise deletion manner (Kimura, 1980). Neighbour-joining (NJ), maximum-likelihood (ML) and minimum-evolution (ME) methods in the MEGA 5.2 software were used to reconstruct phylogenetic trees. Percentage support values were obtained using a bootstrap procedure based on 1000 replications. The results of phylogenetic analysis of the 16S rRNA gene sequences (1416 bp for strain JC109T and 1409 bp for strain JC261) suggested that strains JC109T and JC261 formed a clade that is positioned distinctly outside the clades formed by the next most closely related species of the genus Alcanivorax in the family Alcanivoracaceae (Fig. 1; nodes that were obtained by all treeing methods are represented by filled circles and open circles represent nodes that were recovered by the NJ and ME methods) and sequence similarities with the nearest phylogenetic members were in agreement with the EzTaxon-e server result. EzTaxon-e server search analysis revealed that strains JC109T and JC261 were related most closely to members of the genus Alcanivorax, and highest sequence similarity was observed with A. dieselolei B-5T (99.3 and 99.7 %, respectively), A. balearicus MACL04T (98.8 and 99.2 %, respectively) and other members of the genus Alcanivorax (,95 %). The 16S rRNA gene sequence similarity between strains JC109T and JC261 was 99.6 %. The taxonomic relationship between strains JC109T, JC261, A. dieselolei DSM 16502T and A. balearicus DSM 23776T was examined using DNA–DNA hybridization studies. Genomic relatedness was determined by the membrane filter technique (Seldin & Dubnau, 1985; Tourova & Antonov, 1988) using a DIG High Prime DNA labelling and Detection Starter kit II (Roche). Hybridization was performed with three replications for each sample (control: reversal of strains was used for binding and labelling), and the results are presented as means±SD. The DNA–DNA reassociation value between strains JC109T and JC261 was 88 %, while the level of relatedness between strain JC109T and A. dieselolei DSM 16502T and A. balearicus DSM 23776T was only 31±1 and 26±2 % (based on DNA–DNA hybridization), respectively; these hybridization values are within the recommended standards to delineate a bacterial species (Stackebrandt & Goebel, 1994). Cells of strains JC109T, JC261, A. dieselolei DSM 16502T, A. balearicus DSM 23776T and A. borkumensis CIP 105606T International Journal of Systematic and Evolutionary Microbiology 64
Alcanivorax xenomutans sp. nov.
Alcanivorax venustensis ISO4T (AF328762)
100
Alcanivorax marinus R8-12T (KC415169)
0.02
Alcanivorax pacificus W11-5T (DQ659451) Alcanivorax hongdengensis A-11-3T (EU438901)
100 99
100
Alcanivorax borkumensis SK2T (AM286690) Alcanivorax jadensis T9T (AJ001150)
96
Alcanivorax xenomutans JC109T (HE601937) Alcanivorax xenomutans JC261 (HG974551)
100
Alcanivorax dieselolei B-5T (AY683537) 90
Alcanivorax balearicus MACL04T (AY686709) Halomonas ventosae Al12T (AY268080) Marinobacter zhejiangensis CN74T (EU293413) Hahella chejuensis KCTC 2396T (CP000155) Microbulbifer thermotolerans JAMB A94T (AB124836)
92 99
‘Marinimicrobium haloxylanilyticum’ SX15 (GQ920839) ‘Gilvimarinus agarilyticus’ M5c (GQ872424)
100
Azotobacter beijerinckii ATCC 19360T (AJ308319) Pseudomonas segetis FR1439T (AY770691) Rhodospirillum sulfurexigens JA143T (AM710622)
Fig. 1. Phylogenetic tree based on 16S rRNA gene sequences showing the relationship of strains JC109T and JC261 within the family Alcanivoracaceae. The tree was reconstructed by the NJ method using the MEGA 5.2 software and rooted by using Rhodospirillum sulfurexigens JA143T as the outgroup. Numbers at nodes are the percentage of bootstrap support (based on 1000 resamplings). GenBank accession numbers for 16S rRNA gene sequences are shown in parentheses. Bar, 2 nucleotide substitutions per 100 nt. Filled circles indicate that the corresponding nodes were obtained by all treeing methods (NJ, ME and ML). Open circles indicate that the corresponding nodes were obtained by the NJ and ME methods.
(representing the type species of the genus Alcanivorax) were grown in 250 ml conical flasks containing 100 ml mineral salts medium [consisting of (per litre): 0.5 g KH2PO4, 0.2 g MgSO4 . 7H2O, 20.0 g NaCl, 0.6 g NH4Cl, 0.05 g CaCl2 . 2H2O and 5 ml of ferric citrate solution (0.1 %, w/v)] with pyruvate (22 mM) as carbon source at 28 uC under shaking at 100 r.p.m. Cells were harvested by centrifugation (10 000 g for 15 min at 4 uC) on reaching a cell density of 70 % of the maximum optical density and the pellet was used for fatty acid and polar lipid analysis. Cellular fatty acids were methylated, separated and identified according to the instructions for the Microbial Identification System [Microbial ID; MIDI 6.0 version; Agilent: 6850; peak identification was done based on the RTSBA6 database (Sasser, 1990); http://www.midi-inc.com/] which was outsourced to Royal Research Laboratories, Secunderabad, India. Whole-cell fatty acid analysis of strain JC109T revealed that C10 : 0, C12 : 0, C16 : 0, C12 : 0 3-OH, C16 : 1v7c, C18 : 1v7c and C19 : 0 cyclo v8c were the major fatty acids with minor amounts of C14 : 0, C12 : 0 2-OH and C17 : 0 cyclo (Table S1, available in the online Supplementary Material). The fatty acid profile of strain JC261 was http://ijs.sgmjournals.org
qualitatively identical to that of strain JC109T. Strains JC109T and JC261 shared the presence of major fatty acids with the closely related type strains of A. dieselolei and A. balearicus. However, significant differences in the relative amounts of C18 : 1v7c were found between strains JC109T and JC261 and the type strains of A. dieselolei and A. balearicus. Polar lipids were extracted from 1 g freeze-dried cells with methanol/chloroform/saline (2 : 1 : 0.8, by vol.) as described by Kates (1986). The lipids were separated using silica gel TLC (Kieselgel 60 F254; Merck) by two-dimensional chromatography using chloroform/methanol/water (65 : 25 : 4, by vol.) in the first dimension and chloroform/methanol/ acetic acid/water (80 : 12 : 15 : 4, by vol.) in the second dimension (Tindall et al., 1987; Tindall, 1990; Oren et al., 1996). Total polar lipids were detected by spraying with 5 % ethanolic molybdophosphoric acid and further characterized by spraying with ninhydrin (specific for amino groups), molybdenum blue (specific for phosphates), Dragendorff reagent (quaternary nitrogen) or a-naphthol (specific for sugars) (Kates, 1972; Oren et al., 1996). Polar lipids of strain JC109T included DPG, PG, PE, two unknown 3555
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aminophospholipids (APL1, APL2), two unknown phospholipids (PL1, PL2) and two unknown lipids (L2, L3) (Fig. S1a). The polar lipid profile of strain JC109T was similar to that of the type species of the genus Alcanivorax, A. borkumensis CIP 105606T, which contained DPG, PG and PE as major compounds along with minor amounts of PL2, L1 and L3 (Fig. S1d). Differences were the presence of APL1, APL2, PL1 and L2 in strain JC109T and the absence of L1. Polar lipids of strain JC109T differ from those of A. dieselolei DSM 16502T in the presence of APL1, APL2 and L2 (Fig. S1b). Polar lipids of strain JC109T also differ from A. balearicus DSM 23776T in the presence of APL2 (Fig. S1c). Colonies of strains JC109T and JC261 grown on nutrient agar (HiMedia) were creamish white, circular and convex with entire margin. Morphological properties such as cell shape, cell size and motility were observed by phase-contrast light microscopy (Olympus BH-2). Cells of strain JC109T were rod-shaped (1.860.3 mm), motile and divided by binary fission. For scanning electron microscopy, cells were prepared by fixing with 2 % (v/v) glutaraldehyde and 1 % (w/v) osmium tetroxide (OsO4) as previously described (Priester et al., 2007). The dry specimen was mounted on a specimen stub and fixed using an electrically conductive double-sided adhesive carbon tape and sputter-coated with gold/palladium alloy before examination in the field emission scanning electron microscope (Zeiss Ultra 55). Cells of strain JC109T were rod-shaped and showed pilus formation (Fig. S2). Cells of strain JC109T were larger than those of A. dieselolei DSM 16502T and A. balearicus DSM 23776T. The pH range for growth was tested using nutrient broth, adjusted to different pH values (pH 4.0–11.0, intervals of 0.5 units) by using the appropriate biological buffers as described by Xu et al. (2005). The buffer systems used were: pH 4.0–5.0, 0.1 M citric acid/0.1 M sodium citrate; pH 6.0– 8.0, 0.1 M KH2PO4/0.1 M NaOH; pH 9.0–10.0, 0.1 M NaHCO3/0.1 M Na2CO3; pH 11.0, 0.05 M Na2HPO4/ 0.1 M NaOH. Final pH was determined by using a pH indicator (Fisher Scientific). NaCl [0.5–22 % (w/v) at 0.5 % intervals] and temperature (4, 10, 15, 25, 30, 37, 40 and 45 uC) ranges for growth were examined in nutrient broth and growth was measured turbidometrically at 540 nm in a colorimeter (Systronics). Strains JC109T and JC261 grew at a pH range of 4–9 with optimum at pH 6 and differed from A. dieselolei DSM 16502T and A. balearicus DSM 23776T which have a pH range of 6–9. NaCl was required for growth of the two strains and they were able to tolerate up to 20 % (w/v), while A. borkumensis CIP 105606T, A. dieselolei DSM 16502T and A. balearicus DSM 23776T are less tolerant (Table 1). The temperature range for growth further differentiated strains JC109T and JC261 from their closest phylogenetic neighbours (Table 1).
Table 1. Differential characteristics between strains JC109T and JC261 and their closest phylogenetic neighbours in the genus Alcanivorax Strains: 1, JC109T; 2, JC261; 3, A. dieselolei DSM 16502T; 4, A. balearicus DSM 23776T; 5, A. borkumensis CIP 105606T. All data from this study unless otherwise indicated. Common to all: cells are rodshaped and Gram-stain-negative; all strains were positive for oxidase and catalase; negative for nitrate and nitrite reduction, H2S production, urease, methyl red and Voges–Proskauer tests, and hydrolysis of casein, gelatin and starch. Arginine dihydrolase, phenylalanine deaminase, ornithine decarboxylase and lysine decarboxylase activities were absent. Negative for acid production from D-glucose, mannose, galactose, arabinose, cellobiose and salicin. Glutamate, glutamine, methionine, aspartate, peptone, Casamino acids and urea could not be used as sole source of carbon, nitrogen and energy. +, Positive; W, weakly positive; 2, negative. Characteristic
1
2
3
4
5
Motility + + + + 2 Temperature 25–40 25–40 15–45 4–37 4–37 range (uC) NaCl range for 0.5–20 0.5–20 1–15 0–15 1.0–15 growth (%, w/v) Na+ requirement + + + 2 + Hydrolysis of + + + 2 + Tween 80 Utilization of compounds as sole source of carbon and energy Sucrose + + + 2 2 Mannitol + + 2 + 2 W + Acetate + + + Citrate + + + + 2 Adipic acid + + + + 2 W 2 Glucose 2 2 2 Malic acid + + + + 2 Valerate + + 2 2 + L-Arabinose + + 2 W 2 Lactate 2 2 + 2 2 W Thioglycolate + + 2 2 Phenylacetic + + + + 2 acid DNA G+C 54.5 53.4 62.1 62.8 53.4 content (mol%)* *Data for strains 3, 4 and 5 were from Liu & Shao (2005), Rivas et al. (2007) and Yakimov et al. (1998), respectively.
Various biochemical tests [on specified media containing 2 % (w/v) NaCl] such as hydrolysis of starch, casein, gelatin and Tween 80, oxidase, catalase and urease, nitrate
reduction, nitrite reduction, H2S production, acid production from carbohydrates, and methyl red and Voges– Proskauer tests were performed by the procedures as outlined by Cappuccino & Sherman (1999). Arginine dihydrolase, phenylalanine deaminase, ornithine decarboxylase, lysine decarboxylase and aesculin hydrolysis were determined as described by Smibert & Krieg (1981). Strains JC109T and JC261 grew chemo-organoheterotrophically. Oxidation of various organic substrates for growth was tested using GN2 MicroPlates (Biolog) in accordance with the manufacturer’s instructions. Utilization of organic
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Alcanivorax xenomutans sp. nov.
carbon compounds as carbon and energy source for organoheterotrophic growth was also tested in a mineral medium as previously described (Lakshmi et al., 2011) containing specific organic compounds (0.35 %, w/v or v/v) replacing sodium pyruvate. Growth was measured turbidometrically (OD540) after 48 h. Nitrogen source utilization was tested by replacing ammonium chloride with different nitrogen sources (NaNO3, NaNO2, glutamate, aspartate, glutamine and urea). Utilization of glutamate, glutamine, methionine, aspartate, peptone, Casamino acids and urea as sole source of carbon, nitrogen and energy was also determined. The results are given in the species description and Table 1. Strains JC109T, JC261, A. borkumensis CIP 105606T and A. dieselolei DSM 16502T all grew well in a minimal medium containing diesel oil, octane or xylene as sole source of carbon and energy. In addition, strains JC109T and JC261 grew in the presence of hexane, benzene and toluene as sole source of carbon, while nitrogenated (nitrobenzene, nitrobenzoic acid) and halogenated aromatic hydrocarbons (chlorobenzene, 2,4dichlorophenoxy acetic acid) did not support growth. None of the compounds tested above supported the growth of A. balearicus DSM 23776T. Strains JC109T and JC261 showed phenotypic and genotypic distinctiveness from their closest phylogenetic neighbours, A. dieselolei DSM 16502T, A. balearicus DSM 23776T and other members of the genus Alcanivorax (Table 1) and hence they are considered to represent a novel species of the genus Alcanivorax, for which we propose the name Alcanivorax xenomutans sp. nov.
thioglycolate and phenylacetate as sole carbon and energy source. Glycerol, proline, ethanol, glucose and lactate are not used as sole source of carbon and energy. Ammonium chloride is used as sole nitrogen source; cannot use glutamine or glutamate as sole source of carbon, nitrogen and energy. The polar lipid profile includes DPG, PG, PE, two unknown aminophospholipids, two unknown phospholipids and two unknown lipids. Major fatty acids include C10 : 0, C12 : 0, C16 : 0, C12 : 0 3-OH, C16 : 1v7c, C18 : 1v7c and C19 : 0 cyclo v8c. Minor amounts of C14 : 0, C12 : 0 2-OH and C17 : 0 cyclo are present. The type strain is JC109T (5KCTC 23751T5NBRC 108843T), which was isolated from a sediment sample of a shrimp cultivation pond, Ramnad, Tamil Nadu, India. JC261, isolated from the same pond, is a second strain of the species. The DNA G+C content of the type strain is 54.5 mol% and of strain JC261 is 53.4 mol%.
Acknowledgements We thank Professor J. Euze´by for his expert suggestion regarding the species epithet and Latin etymology. K. R. thanks the CSIR, New Delhi, for the award of SRF. L. T. thanks the UGC, New Delhi, for the award of JRF/SRF. We thank CIP for providing A. borkumensis CIP 105606T in exchange.
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Description of Alcanivorax xenomutans sp. nov.
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Cells are short rods, 1.860.3 mm, motile, non-endosporeforming and Gram-stain-negative. Forms bright creamishwhite colonies on nutrient agar. Hydrolyses Tween 80; oxidase activity is weak at 2 % but positive at 5 % NaCl concentration; catalase-positive. Hydrolysis of casein, gelatin, aesculin and starch are negative. Nitrate and nitrite reduction, urease, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, methyl red and Voges– Proskauer tests, citrate utilization and production of H2S are negative. Acid is not produced from D-glucose, mannose, galactose, arabinose, cellobiose or salicin. Facultatively anaerobic. Chemo-organoheterotrophy is the only growth mode. Temperature range for growth is 25–40 uC (optimum 30 uC). Growth occurs with 0.5–20 % (w/v) NaCl (optimum 2–5 %) and at pH 4–9 (optimum pH 6). Able to degrade diesel oil and other hydrocarbons. Oxidizes cis-aconitic acid, methyl pyruvate, monomethyl succinate, sebacic acid, c-hydroxybutyric acid, b-hydroxybutyric acid, 2,3-butanediol, Tween 40 and Tween 80. Can grow using sucrose, mannitol, citrate, adipate, malate, valerate, arabinose, http://ijs.sgmjournals.org
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marinus sp. nov., isolated from deep-sea water. Int J Syst Evol Microbiol 63, 4428–4432.
Stackebrandt, E. & Goebel, B. M. (1994). A place for DNA–DNA
Lakshmi, K. V. N. S., Sasikala, Ch., Ashok Kumar, G. V., Chandrasekaran, R. & Ramana, ChV. (2011). Phaeovibrio sulfidiphilus
reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
gen. nov., sp. nov., phototrophic alphaproteobacteria isolated from brackish water. Int J Syst Evol Microbiol 61, 828–833.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using
Lane, D. J., Pace, B., Olsen, G. J., Stahl, D. A., Sogin, M. L. & Pace, N. R. (1985). Rapid determination of 16S ribosomal RNA sequences
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739.
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Tindall, B. J. (1990). Lipid composition of Halobacterium lacuspro-
Liu, C. & Shao, Z. (2005). Alcanivorax dieselolei sp. nov., a novel
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Tindall, B. J., Tomlinson, G. A. & Hochstein, L. I. (1987). Polar lipid
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acid from microorganisms. J Mol Biol 3, 208–218. Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise
measurement of the G+C content of deoxyribonucleic acid by highperformance liquid chromatography. Int J Syst Bacteriol 39, 159–167. Oren, A., Duker, S. & Ritter, S. (1996). The polar lipid composition of
Walsby’s square bacterium. FEMS Microbiol Lett 138, 135–140. Priester, J. H., Horst, A. M., Van de Werfhorst, L. C., Saleta, J. L., Mertes, L. A. & Holden, P. A. (2007). Enhanced visualization of
microbial biofilms by staining and environmental scanning electron microscopy. J Microbiol Methods 68, 577–587. Rivas, R., Garcı´a-Fraile, P., Peix, A., Mateos, P. F., Martı´nez-Molina, E. & Vela´zquez, E. (2007). Alcanivorax balearicus sp. nov., isolated
composition of a new halobacterium. Syst Appl Microbiol 9, 6–8. isms by rapid DNA-DNA hybridization. Meth Microbiol 19, 333– 355. Wu, Y., Lai, Q., Zhou, Z., Qiao, N., Liu, C. & Shao, Z. (2009).
Alcanivorax hongdengensis sp. nov., an alkane-degrading bacterium isolated from surface seawater of the straits of Malacca and Singapore, producing a lipopeptide as its biosurfactant. Int J Syst Evol Microbiol 59, 1474–1479. Xu, P., Li, W. J., Tang, S. K., Zhang, Y. Q., Chen, G. Z., Chen, H. H., Xu, L. H. & Jiang, C. L. (2005). Naxibacter alkalitolerans gen. nov., sp. nov.,
a novel member of the family ‘Oxalobacteraceae’ isolated from China. Int J Syst Evol Microbiol 55, 1149–1153.
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Yakimov, M. M., Golyshin, P. N., Lang, S., Moore, E. R. B., Abraham, W. R., Lu¨nsdorf, H. & Timmis, K. N. (1998). Alcanivorax borkumensis
Ryerson, T. B., Camilli, R., Kessler, J. D., Kujawinski, E. B., Reddy, C. M., Valentine, D. L., Atlas, E. L., Blake, D. R., de Gouw, J. A. & other
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DOI 10.1099/ijs.0.061168-0
Alcanivorax xenomutans sp. nov., a hydrocarbonoclastic bacterium isolated from a shrimp cultivation pond K. Rahul,1 Ch. Sasikala,1 L. Tushar,2 R. Debadrita2 and Ch. V. Ramana2 Correspondence
1
Chintalapati Sasikala [email protected] or sasi449@
2
yahoo.ie
Bacterial Discovery Laboratory, Centre for Environment, Institute of Science and Technology, J.N.T. University Hyderabad, Kukatpally, Hyderabad 500 085, India Department of Plant Sciences, School of Life Sciences, University of Hyderabad, P.O. Central University, Hyderabad 500 046, India
Two bacterial strains (JC109T and JC261) were isolated from a sediment sample collected from a shrimp cultivation pond in Tamil Nadu (India). Cells were Gram-stain-negative, motile rods. Both strains were positive for catalase and oxidase, hydrolysed Tween 80, and grew chemoorganoheterotrophically with an optimal pH of 6 (range pH 4–9) and at 30 6C (range 25–40 6C). Based on 16S rRNA gene sequence analysis, strains JC109T and JC261 were identified as belonging to the genus Alcanivorax with Alcanivorax dieselolei B-5T (sequence similarity values of 99.3 and 99.7 %, respectively) and Alcanivorax balearicus MACL04T (sequence similarity values of 98.8 and 99.2 %, respectively) as their closest phylogenetic neighbours. The 16S rRNA gene sequence similarity between strains JC109T and JC261 was 99.6 %. The level of DNA–DNA relatedness between the two strains was 88 %. Strain JC109T showed 31±1 and 26±2 % DNA–DNA relatedness with A. dieselolei DSM 16502T and A. balearicus DSM 23776T, respectively. The DNA G+C content of strains JC109T and JC261 was 54.5 and 53.4 mol%, respectively. Polar lipids of strain JC109T included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminophospholipids, two unidentified phospholipids and two unidentified lipids. The major fatty acids were C10 : 0, C12 : 0, C16 : 0, C12 : 0 3-OH, C16 : 1v7c, C18 : 1v7c and C19 : 0 cyclo v8c. Both strains could utilize diesel oil and a variety of xenobiotics as carbon and energy sources. The results of physiological, biochemical, chemotaxonomic and molecular analyses allowed the clear differentiation of strains JC109T and JC261 from all other members of the genus Alcanivorax. Strains JC109T and JC261 are thus considered to represent a novel species, for which the name Alcanivorax xenomutans sp. nov. is proposed. The type strain is JC109T (5KCTC 23751T5NBRC 108843T).
Millions of litres of diesel and petroleum oil enter the environment from natural and, more prominently, via anthropogenic sources, which introduces problems for a diverse range of ecosystems (Ryerson et al., 2012). Unearthing novel hydrocarbon-degrading micro-organisms is imperative in the bioremediation processes of hydrocarbon-contaminated sites (Chandran & Das, 2010; Atlas & Hazen, 2011). In this paper, we report two novel strains belonging to the genus Alcanivorax which were Abbreviations: DPG, diphosphatidylglycerol; ME, minimum-evolution; ML, maximum-likelihood; NJ, neighbour-joining; PE, phosphatidylethanolamine; PG, phosphatidylglycerol. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains JC109T and JC261 are HE601937 and HG974551, respectively. One supplementary table and two supplementary figures are available with the online version of this paper.
061168 G 2014 IUMS
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isolated from a sediment sample of a shrimp cultivation pond and are able to degrade diesel oil. The genus Alcanivorax was first proposed by Yakimov et al. (1998) with Alcanivorax borkumensis as the type species and the description of the genus was later amended by Ferna´ndez-Martı´nez et al. (2003). At the time of writing, the genus Alcanivorax comprised eight recognized species (http://www.bacterio.net/a/alcanivorax.html): A. borkumensis (Yakimov et al., 1998), Alcanivorax jadensis (Bruns & BertheCorti, 1999), Alcanivorax venustensis (Ferna´ndez-Martı´nez et al., 2003), Alcanivorax dieselolei (Liu & Shao, 2005), Alcanivorax balearicus (Rivas et al., 2007), Alcanivorax hongdengensis (Wu et al., 2009), Alcanivorax pacificus (Lai et al., 2011) and Alcanivorax marinus (Lai et al., 2013) were isolated from marine sediment/water samples. Alcanivorax dieselolei was specifically isolated from a hydrocarboncontaminated site (Liu & Shao, 2005). Members of the genus 3553
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Alcanivorax are Gram-stain-negative, aerobic, motile or non-motile rods, chemo-organotrophic, tolerate high salt concentrations and can thrive in the presence of hydrocarbons (Harayama et al., 2004; Lai et al., 2013). The DNA G+C content of members of the genus ranges from 53 to 66 mol%. They are also characterized by the presence of C16 : 0, C18 : 1v7c and C16 : 1v7c as predominant cellular fatty acids (Ferna´ndez-Martı´nez et al., 2003; Lai et al., 2013). Polar lipid profiles of the genus Alcanivorax have not been extensively studied. However, strain JC109T, A. dieselolei DSM 16502T, A. balearicus DSM 23776T, A. borkumensis CIP 105606T and A. marinus R8-12T (Lai et al., 2013) contained diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), unidentified phospholipids, unidentified aminolipids and unidentified lipids. Strains JC109T and JC261 were isolated from a sediment sample collected from a shrimp cultivation pond in Ramnad, Tamil Nadu, India (9u 169 N 79u 69 E) during September 2011. Physicochemical parameters including temperature, salinity and pH of the sample were analysed immediately after sample collection. The temperature at the site of sample collection was 29 uC. Salinity and pH of the sample were 2.8 % (w/v) and pH 8. One gram of air-dried sediment was serially diluted up to 1024 and 100 ml was spread on a growth medium (pH 7.5) containing (per litre): 0.2 g KH2PO4, 0.25 g NH4Cl, 0.5 g KCl, 0.15 g CaCl2 . 2H2O, 20.0 g NaCl, 0.62 g MgCl2 . 6H2O, 2.84 g Na2SO4, 2.83 g HEPES, 3.0 g yeast extract, 3.0 g peptone, 0.5 g Casamino acids, 0.5 g glucose and 3.0 g sodium pyruvate. Purification of the bacteria was achieved by repeated streaking on nutrient agar (HiMedia) containing 2 % (w/v) NaCl. Strains JC109T and JC261 were preserved as glycerol stocks and by lyophilization. Unless otherwise mentioned, both strains were grown in nutrient broth or on nutrient agar (HiMedia) with 2 % NaCl. Unless otherwise mentioned, the reference strains A. dieselolei DSM 16502T and A. borkumensis CIP 105606T were cultured in marine broth (HiMedia) supplemented with 1 % sodium pyruvate (pH 7.6 at 28 uC) and A. balearicus DSM 23776T was grown in nutrient broth or on nutrient agar with 1.5 % (w/v) NaCl (pH 7.2 at 28 uC) as recommended by the culture collection. Genomic DNA was extracted and purified from strains JC109T and JC261 according to the method of Marmur (1961) and the G+C of the DNA was 54.5 and 53.4 mol%, respectively, as determined by HPLC (Mesbah et al., 1989). Cell material for 16S rRNA gene sequencing was taken from a colony. DNA was extracted and purified by using a Qiagen genomic DNA extraction kit. Recombinant Taq polymerase (Genei) was used for PCR. The almostcomplete sequence of the 16S rRNA gene was obtained by sequencing with primers F27 (59-GTTTGATCCTGGCTCAG-39) and R1489 (59-TACCTTGTTACGACTTCA-39) [positions 11–27 and 1489–1506, respectively, according to the Escherichia coli 16S rRNA numbering system of the International Union of Biochemistry (Brosius et al., 1978; Lane et al., 1985)]. PCR amplification was done as described 3554
by Imhoff et al. (1998) and Imhoff & Pfennig (2001). 16S rRNA gene sequencing was performed using the BigDye Terminator v1.1 Sequencing kit (Applied Biosystems) in a 3730-DNA-Analyser (Applied Biosystems) as specified by the manufacturer. For sequencing, the primers F27, F790 (59-GATACCCTGGTAGTCC-39) and R1489 were used. Identification of phylogenetic neighbours and calculation of pairwise 16S rRNA gene sequence similarity were achieved using the EzTaxon-e server (Kim et al., 2012). The CLUSTAL W algorithm of MEGA 5.2 was used for sequence alignments and MEGA 5.2 (Tamura et al., 2011) software was used for phylogenetic analysis of the individual sequences. Distances were calculated by using the Kimura correction in a pairwise deletion manner (Kimura, 1980). Neighbour-joining (NJ), maximum-likelihood (ML) and minimum-evolution (ME) methods in the MEGA 5.2 software were used to reconstruct phylogenetic trees. Percentage support values were obtained using a bootstrap procedure based on 1000 replications. The results of phylogenetic analysis of the 16S rRNA gene sequences (1416 bp for strain JC109T and 1409 bp for strain JC261) suggested that strains JC109T and JC261 formed a clade that is positioned distinctly outside the clades formed by the next most closely related species of the genus Alcanivorax in the family Alcanivoracaceae (Fig. 1; nodes that were obtained by all treeing methods are represented by filled circles and open circles represent nodes that were recovered by the NJ and ME methods) and sequence similarities with the nearest phylogenetic members were in agreement with the EzTaxon-e server result. EzTaxon-e server search analysis revealed that strains JC109T and JC261 were related most closely to members of the genus Alcanivorax, and highest sequence similarity was observed with A. dieselolei B-5T (99.3 and 99.7 %, respectively), A. balearicus MACL04T (98.8 and 99.2 %, respectively) and other members of the genus Alcanivorax (,95 %). The 16S rRNA gene sequence similarity between strains JC109T and JC261 was 99.6 %. The taxonomic relationship between strains JC109T, JC261, A. dieselolei DSM 16502T and A. balearicus DSM 23776T was examined using DNA–DNA hybridization studies. Genomic relatedness was determined by the membrane filter technique (Seldin & Dubnau, 1985; Tourova & Antonov, 1988) using a DIG High Prime DNA labelling and Detection Starter kit II (Roche). Hybridization was performed with three replications for each sample (control: reversal of strains was used for binding and labelling), and the results are presented as means±SD. The DNA–DNA reassociation value between strains JC109T and JC261 was 88 %, while the level of relatedness between strain JC109T and A. dieselolei DSM 16502T and A. balearicus DSM 23776T was only 31±1 and 26±2 % (based on DNA–DNA hybridization), respectively; these hybridization values are within the recommended standards to delineate a bacterial species (Stackebrandt & Goebel, 1994). Cells of strains JC109T, JC261, A. dieselolei DSM 16502T, A. balearicus DSM 23776T and A. borkumensis CIP 105606T International Journal of Systematic and Evolutionary Microbiology 64
Alcanivorax xenomutans sp. nov.
Alcanivorax venustensis ISO4T (AF328762)
100
Alcanivorax marinus R8-12T (KC415169)
0.02
Alcanivorax pacificus W11-5T (DQ659451) Alcanivorax hongdengensis A-11-3T (EU438901)
100 99
100
Alcanivorax borkumensis SK2T (AM286690) Alcanivorax jadensis T9T (AJ001150)
96
Alcanivorax xenomutans JC109T (HE601937) Alcanivorax xenomutans JC261 (HG974551)
100
Alcanivorax dieselolei B-5T (AY683537) 90
Alcanivorax balearicus MACL04T (AY686709) Halomonas ventosae Al12T (AY268080) Marinobacter zhejiangensis CN74T (EU293413) Hahella chejuensis KCTC 2396T (CP000155) Microbulbifer thermotolerans JAMB A94T (AB124836)
92 99
‘Marinimicrobium haloxylanilyticum’ SX15 (GQ920839) ‘Gilvimarinus agarilyticus’ M5c (GQ872424)
100
Azotobacter beijerinckii ATCC 19360T (AJ308319) Pseudomonas segetis FR1439T (AY770691) Rhodospirillum sulfurexigens JA143T (AM710622)
Fig. 1. Phylogenetic tree based on 16S rRNA gene sequences showing the relationship of strains JC109T and JC261 within the family Alcanivoracaceae. The tree was reconstructed by the NJ method using the MEGA 5.2 software and rooted by using Rhodospirillum sulfurexigens JA143T as the outgroup. Numbers at nodes are the percentage of bootstrap support (based on 1000 resamplings). GenBank accession numbers for 16S rRNA gene sequences are shown in parentheses. Bar, 2 nucleotide substitutions per 100 nt. Filled circles indicate that the corresponding nodes were obtained by all treeing methods (NJ, ME and ML). Open circles indicate that the corresponding nodes were obtained by the NJ and ME methods.
(representing the type species of the genus Alcanivorax) were grown in 250 ml conical flasks containing 100 ml mineral salts medium [consisting of (per litre): 0.5 g KH2PO4, 0.2 g MgSO4 . 7H2O, 20.0 g NaCl, 0.6 g NH4Cl, 0.05 g CaCl2 . 2H2O and 5 ml of ferric citrate solution (0.1 %, w/v)] with pyruvate (22 mM) as carbon source at 28 uC under shaking at 100 r.p.m. Cells were harvested by centrifugation (10 000 g for 15 min at 4 uC) on reaching a cell density of 70 % of the maximum optical density and the pellet was used for fatty acid and polar lipid analysis. Cellular fatty acids were methylated, separated and identified according to the instructions for the Microbial Identification System [Microbial ID; MIDI 6.0 version; Agilent: 6850; peak identification was done based on the RTSBA6 database (Sasser, 1990); http://www.midi-inc.com/] which was outsourced to Royal Research Laboratories, Secunderabad, India. Whole-cell fatty acid analysis of strain JC109T revealed that C10 : 0, C12 : 0, C16 : 0, C12 : 0 3-OH, C16 : 1v7c, C18 : 1v7c and C19 : 0 cyclo v8c were the major fatty acids with minor amounts of C14 : 0, C12 : 0 2-OH and C17 : 0 cyclo (Table S1, available in the online Supplementary Material). The fatty acid profile of strain JC261 was http://ijs.sgmjournals.org
qualitatively identical to that of strain JC109T. Strains JC109T and JC261 shared the presence of major fatty acids with the closely related type strains of A. dieselolei and A. balearicus. However, significant differences in the relative amounts of C18 : 1v7c were found between strains JC109T and JC261 and the type strains of A. dieselolei and A. balearicus. Polar lipids were extracted from 1 g freeze-dried cells with methanol/chloroform/saline (2 : 1 : 0.8, by vol.) as described by Kates (1986). The lipids were separated using silica gel TLC (Kieselgel 60 F254; Merck) by two-dimensional chromatography using chloroform/methanol/water (65 : 25 : 4, by vol.) in the first dimension and chloroform/methanol/ acetic acid/water (80 : 12 : 15 : 4, by vol.) in the second dimension (Tindall et al., 1987; Tindall, 1990; Oren et al., 1996). Total polar lipids were detected by spraying with 5 % ethanolic molybdophosphoric acid and further characterized by spraying with ninhydrin (specific for amino groups), molybdenum blue (specific for phosphates), Dragendorff reagent (quaternary nitrogen) or a-naphthol (specific for sugars) (Kates, 1972; Oren et al., 1996). Polar lipids of strain JC109T included DPG, PG, PE, two unknown 3555
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aminophospholipids (APL1, APL2), two unknown phospholipids (PL1, PL2) and two unknown lipids (L2, L3) (Fig. S1a). The polar lipid profile of strain JC109T was similar to that of the type species of the genus Alcanivorax, A. borkumensis CIP 105606T, which contained DPG, PG and PE as major compounds along with minor amounts of PL2, L1 and L3 (Fig. S1d). Differences were the presence of APL1, APL2, PL1 and L2 in strain JC109T and the absence of L1. Polar lipids of strain JC109T differ from those of A. dieselolei DSM 16502T in the presence of APL1, APL2 and L2 (Fig. S1b). Polar lipids of strain JC109T also differ from A. balearicus DSM 23776T in the presence of APL2 (Fig. S1c). Colonies of strains JC109T and JC261 grown on nutrient agar (HiMedia) were creamish white, circular and convex with entire margin. Morphological properties such as cell shape, cell size and motility were observed by phase-contrast light microscopy (Olympus BH-2). Cells of strain JC109T were rod-shaped (1.860.3 mm), motile and divided by binary fission. For scanning electron microscopy, cells were prepared by fixing with 2 % (v/v) glutaraldehyde and 1 % (w/v) osmium tetroxide (OsO4) as previously described (Priester et al., 2007). The dry specimen was mounted on a specimen stub and fixed using an electrically conductive double-sided adhesive carbon tape and sputter-coated with gold/palladium alloy before examination in the field emission scanning electron microscope (Zeiss Ultra 55). Cells of strain JC109T were rod-shaped and showed pilus formation (Fig. S2). Cells of strain JC109T were larger than those of A. dieselolei DSM 16502T and A. balearicus DSM 23776T. The pH range for growth was tested using nutrient broth, adjusted to different pH values (pH 4.0–11.0, intervals of 0.5 units) by using the appropriate biological buffers as described by Xu et al. (2005). The buffer systems used were: pH 4.0–5.0, 0.1 M citric acid/0.1 M sodium citrate; pH 6.0– 8.0, 0.1 M KH2PO4/0.1 M NaOH; pH 9.0–10.0, 0.1 M NaHCO3/0.1 M Na2CO3; pH 11.0, 0.05 M Na2HPO4/ 0.1 M NaOH. Final pH was determined by using a pH indicator (Fisher Scientific). NaCl [0.5–22 % (w/v) at 0.5 % intervals] and temperature (4, 10, 15, 25, 30, 37, 40 and 45 uC) ranges for growth were examined in nutrient broth and growth was measured turbidometrically at 540 nm in a colorimeter (Systronics). Strains JC109T and JC261 grew at a pH range of 4–9 with optimum at pH 6 and differed from A. dieselolei DSM 16502T and A. balearicus DSM 23776T which have a pH range of 6–9. NaCl was required for growth of the two strains and they were able to tolerate up to 20 % (w/v), while A. borkumensis CIP 105606T, A. dieselolei DSM 16502T and A. balearicus DSM 23776T are less tolerant (Table 1). The temperature range for growth further differentiated strains JC109T and JC261 from their closest phylogenetic neighbours (Table 1).
Table 1. Differential characteristics between strains JC109T and JC261 and their closest phylogenetic neighbours in the genus Alcanivorax Strains: 1, JC109T; 2, JC261; 3, A. dieselolei DSM 16502T; 4, A. balearicus DSM 23776T; 5, A. borkumensis CIP 105606T. All data from this study unless otherwise indicated. Common to all: cells are rodshaped and Gram-stain-negative; all strains were positive for oxidase and catalase; negative for nitrate and nitrite reduction, H2S production, urease, methyl red and Voges–Proskauer tests, and hydrolysis of casein, gelatin and starch. Arginine dihydrolase, phenylalanine deaminase, ornithine decarboxylase and lysine decarboxylase activities were absent. Negative for acid production from D-glucose, mannose, galactose, arabinose, cellobiose and salicin. Glutamate, glutamine, methionine, aspartate, peptone, Casamino acids and urea could not be used as sole source of carbon, nitrogen and energy. +, Positive; W, weakly positive; 2, negative. Characteristic
1
2
3
4
5
Motility + + + + 2 Temperature 25–40 25–40 15–45 4–37 4–37 range (uC) NaCl range for 0.5–20 0.5–20 1–15 0–15 1.0–15 growth (%, w/v) Na+ requirement + + + 2 + Hydrolysis of + + + 2 + Tween 80 Utilization of compounds as sole source of carbon and energy Sucrose + + + 2 2 Mannitol + + 2 + 2 W + Acetate + + + Citrate + + + + 2 Adipic acid + + + + 2 W 2 Glucose 2 2 2 Malic acid + + + + 2 Valerate + + 2 2 + L-Arabinose + + 2 W 2 Lactate 2 2 + 2 2 W Thioglycolate + + 2 2 Phenylacetic + + + + 2 acid DNA G+C 54.5 53.4 62.1 62.8 53.4 content (mol%)* *Data for strains 3, 4 and 5 were from Liu & Shao (2005), Rivas et al. (2007) and Yakimov et al. (1998), respectively.
Various biochemical tests [on specified media containing 2 % (w/v) NaCl] such as hydrolysis of starch, casein, gelatin and Tween 80, oxidase, catalase and urease, nitrate
reduction, nitrite reduction, H2S production, acid production from carbohydrates, and methyl red and Voges– Proskauer tests were performed by the procedures as outlined by Cappuccino & Sherman (1999). Arginine dihydrolase, phenylalanine deaminase, ornithine decarboxylase, lysine decarboxylase and aesculin hydrolysis were determined as described by Smibert & Krieg (1981). Strains JC109T and JC261 grew chemo-organoheterotrophically. Oxidation of various organic substrates for growth was tested using GN2 MicroPlates (Biolog) in accordance with the manufacturer’s instructions. Utilization of organic
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Alcanivorax xenomutans sp. nov.
carbon compounds as carbon and energy source for organoheterotrophic growth was also tested in a mineral medium as previously described (Lakshmi et al., 2011) containing specific organic compounds (0.35 %, w/v or v/v) replacing sodium pyruvate. Growth was measured turbidometrically (OD540) after 48 h. Nitrogen source utilization was tested by replacing ammonium chloride with different nitrogen sources (NaNO3, NaNO2, glutamate, aspartate, glutamine and urea). Utilization of glutamate, glutamine, methionine, aspartate, peptone, Casamino acids and urea as sole source of carbon, nitrogen and energy was also determined. The results are given in the species description and Table 1. Strains JC109T, JC261, A. borkumensis CIP 105606T and A. dieselolei DSM 16502T all grew well in a minimal medium containing diesel oil, octane or xylene as sole source of carbon and energy. In addition, strains JC109T and JC261 grew in the presence of hexane, benzene and toluene as sole source of carbon, while nitrogenated (nitrobenzene, nitrobenzoic acid) and halogenated aromatic hydrocarbons (chlorobenzene, 2,4dichlorophenoxy acetic acid) did not support growth. None of the compounds tested above supported the growth of A. balearicus DSM 23776T. Strains JC109T and JC261 showed phenotypic and genotypic distinctiveness from their closest phylogenetic neighbours, A. dieselolei DSM 16502T, A. balearicus DSM 23776T and other members of the genus Alcanivorax (Table 1) and hence they are considered to represent a novel species of the genus Alcanivorax, for which we propose the name Alcanivorax xenomutans sp. nov.
thioglycolate and phenylacetate as sole carbon and energy source. Glycerol, proline, ethanol, glucose and lactate are not used as sole source of carbon and energy. Ammonium chloride is used as sole nitrogen source; cannot use glutamine or glutamate as sole source of carbon, nitrogen and energy. The polar lipid profile includes DPG, PG, PE, two unknown aminophospholipids, two unknown phospholipids and two unknown lipids. Major fatty acids include C10 : 0, C12 : 0, C16 : 0, C12 : 0 3-OH, C16 : 1v7c, C18 : 1v7c and C19 : 0 cyclo v8c. Minor amounts of C14 : 0, C12 : 0 2-OH and C17 : 0 cyclo are present. The type strain is JC109T (5KCTC 23751T5NBRC 108843T), which was isolated from a sediment sample of a shrimp cultivation pond, Ramnad, Tamil Nadu, India. JC261, isolated from the same pond, is a second strain of the species. The DNA G+C content of the type strain is 54.5 mol% and of strain JC261 is 53.4 mol%.
Acknowledgements We thank Professor J. Euze´by for his expert suggestion regarding the species epithet and Latin etymology. K. R. thanks the CSIR, New Delhi, for the award of SRF. L. T. thanks the UGC, New Delhi, for the award of JRF/SRF. We thank CIP for providing A. borkumensis CIP 105606T in exchange.
References Atlas, R. M. & Hazen, T. C. (2011). Oil biodegradation and bioremediation: a tale of the two worst spills in U.S. history. Environ Sci Technol 45, 6709–6715.
Description of Alcanivorax xenomutans sp. nov.
Brosius, J., Palmer, M. L., Kennedy, P. J. & Noller, H. F. (1978).
Alcanivorax xenomutans [xe.no.mu9tans. Gr. adj. xenos foreign; L. part. adj. mutans transforming, converting; N.L. part. adj. xenomutans transforming foreign (xenobiotic) compounds].
Bruns, A. & Berthe-Corti, L. (1999). Fundibacter jadensis gen. nov., sp.
Cells are short rods, 1.860.3 mm, motile, non-endosporeforming and Gram-stain-negative. Forms bright creamishwhite colonies on nutrient agar. Hydrolyses Tween 80; oxidase activity is weak at 2 % but positive at 5 % NaCl concentration; catalase-positive. Hydrolysis of casein, gelatin, aesculin and starch are negative. Nitrate and nitrite reduction, urease, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, methyl red and Voges– Proskauer tests, citrate utilization and production of H2S are negative. Acid is not produced from D-glucose, mannose, galactose, arabinose, cellobiose or salicin. Facultatively anaerobic. Chemo-organoheterotrophy is the only growth mode. Temperature range for growth is 25–40 uC (optimum 30 uC). Growth occurs with 0.5–20 % (w/v) NaCl (optimum 2–5 %) and at pH 4–9 (optimum pH 6). Able to degrade diesel oil and other hydrocarbons. Oxidizes cis-aconitic acid, methyl pyruvate, monomethyl succinate, sebacic acid, c-hydroxybutyric acid, b-hydroxybutyric acid, 2,3-butanediol, Tween 40 and Tween 80. Can grow using sucrose, mannitol, citrate, adipate, malate, valerate, arabinose, http://ijs.sgmjournals.org
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