Arthrobacter nitrophenolicus sp. nov. a new 2-chloro-4-nitrophenol degrading bacterium isolated from contaminated soil

Strain SJConT, a 2-chloro-4-nitrophenol (2C4NP) degrading bacterium, was isolated from soil collected from a pesticide-contaminated site in Punjab, India. The strain, which stained Gram positive, displayed a rod-coccus life cycle, and possessed a type A3α peptidoglycan (l-Lys–l-Ala3), MK-9(H2) as the major menaquinone, anteiso-C15 and iso-C15:0 as the major cellular fatty acids, and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and a glycolipid as the major polar lipids, showed morphological and chemotaxonomic properties consistent with those reported for members of the genus Arthrobacter. Phylogenetic analysis of the 16S rRNA gene sequence of strain SJConT confirmed that it was a member of this genus with Arthrobacter globiformis DSM 20124T being the closest relative (sequence similarity of 97 %). The DNA G + C content of strain SJConT was 69 ± 1 mol% and DNA homology with A. globiformis DSM 20124T was 45 %, suggesting that strain SJConT represented a novel species of the genus Arthrobacter, which we have named Arthrobacter nitrophenolicus sp. nov The type strain is SJConT (=MTCC 10104T =DSM 23165T).

Genus Arthrobacter was first proposed by Conn and Dimmick (1947) with the description of the type species Arthrobacter globiformis. Subsequently, Koch et al. (1995) emended the description with the reclassification of Micrococcus agilis as Arthrobacter agilis. The currently validated 78 members of genus Arthrobacter are members of phylum Actinobacteria, order Actinomycetales, family Micrococcaceae, and are characterized by the presence of a rod-coccus growth cycle and genomes with high G ? C content (59-66 mol%) (Keddie et al. 1986). Members of genus Arthrobacter stain Gram positive are catalase positive and are sub-divided on the basis of the lysine-containing peptidoglycan into two groups, A3a and A4a (Schleifer and Kandler 1972;Stackebrandt et al. 1983;Keddie et al. 1986;Koch et al. 1995).
The primary habitat of Arthrobacter is soil and interestingly Arthrobacter strains with the ability to degrade nitrophenols and/or chlorophenols which include Arthrobacter chlorophonolicus A6 (Westerberg et al. 2000), Arthrobacter ureafaciens, strain CPR706 (Bae et al. 1996), Arthrobacter citrus (Karigar et al. 2006), Arthrobacter protophormae strain RKJ100 (Chauhan et al. 2000), Arthrobacter sp. strain JS443 (Jain et al. 1994) and Arthrobacter aurescens TW17 (Hanne et al. 1993) have all been isolated from pesticide-contaminated soil. We had previously reported for the first time on the isolation of Arthrobacter strain SJCon that degraded 2-chloro-4-nitrophenol (2C4NP) . Chlorohydroquinone was identified as a major intermediate product which was further degraded via formation of maleylacetate . As strain SJCon T is a potential degrader of This article is dedicated in the memory of Dr Rakesh Jain for his excellent contributions to the field of microbial sciences and biodegradation.
various nitrophenolic compounds including 2-chloro-4nitrophenol, 4-nitrophenol and 3-methyl-4-nitrophenol, and it has the potential for use in the bioremediation of nitrophenolic contaminated sites. In this communication, we report on the chemotaxonomic and genotypic properties of the strain and designate it as a new species of genus, A. nitrophenolicus sp nov. (The type strain is SJCon T =MTCC 10104 T =DSM 23165 T ).
The method for isolating strain SJCon T from a pesticidecontaminated soil by an enrichment method using 2-chloro-4-nitrophenol as sole carbon source (Sigma-Aldrich, GmbH, Steinheim, Germany) has been reported previously .
Colony morphology was examined on Nutrient agar plates after incubation at 30°C for 24 h. Cell morphology was examined by light microscopy (Zeiss) at 91000 and motility was checked using the method described by Skerman (1967). Gram staining was performed using Hi-Media Gram Staining kit (HiMedia, India) according to the manufacturer's instructions. Strain SJCon T had morphological characteristics consistent with members of the genus Arthrobacter. Cells exhibited a rod-coccus growth cycle, were non-motile, did not form spores and stained Gram positive.
Growth at different temperatures (between 4 and 50°C) was determined using Nutrient agar plates. Strain SJCon T was streaked on nutrient agar plates, incubated at different temperatures and the growth on the plate scored. The growth in nutrient broth containing different NaCl concentrations (0.5-7 %) was monitored by measuring optical density at 600 nm in a Lambda 35 spectrophotometer (Perkin Elmer). Growth at different pH (pH 4.5-12) was tested in nutrient broth after the pH had been adjusted using appropriate buffers as described previously . Growth occurred between 10 and 40°C with the optimum temperature for growth at 30°C. The optimum pH for growth was 7 and no growth occurred below pH 6 or above pH 10. NaCl was not required for growth, but was tolerated up to 4 %.
Hydrolysis of gelatin, casein, starch, Voges-Proskauer, methyl red, oxidation-fermentation tests, catalase and oxidase activities, growth on Simmon's citrate and Mac-Conkey agar, production of H 2 S and indole, reduction of nitrate and acid production from carbohydrates were determined as described previously .
A summary of the results from the phenotypic tests is presented in Table 1 and listed in the species description.
The utilization of carbon and energy sources was tested using Biolog GP2 Microplates (Hayward, CA). For this, the inoculum was prepared by re-suspending Nutrient agar grown colonies to a turbidity equivalent to 0Á5 McFarland units and the GP2 Microplates inoculated following the manufacturer's instructions. The plates were incubated at 30°C for 24 h and the results read using a MicroPlate Reader equipped with Microlog 4.2 software. The results are listed in the species description.
DNA extraction and purification have been described previously . The 16S rRNA gene was

3-Methyl-4-nitrophenol
? -amplified using universal bacterial primers 8F (5 0 -AGA GTT TGA TCC TGG CTC AG-3 0 ) and 1492R (5 0 -GGT TAC CTT GTT ACG ACT T-3 0 ) ) and sequenced using n ABI automated sequencer (Applied Biosystems, USA). The sequence (1,446 bp with GenBank accession number GQ927310) was compared against sequences available in the GenBank database (version 188.0) using EzTaxon server 2.1, the sequences of the nearest phylogentic members downloaded, aligned with those of related Arthrobacter species and a phylogenetic tree constructed using the neighbour-joining method as implemented in MEGA (Tamura et al. 2007) and Bioedit (Hall 1999). During phylogenetic reconstruction, all ambiguous nucleotides were excluded from the analysis and a total of 1,270 nucleotides were used in the final analysis. Phylogenetic analysis showed that strain SJCon T was a member of the genus Arthrobacter and showed the highest similarity to A. globiformis DSM 20124 T and related members of the 'globiformis' group (average sequence similarity of 97%) (Fig. 1).
The G ? C mol% content of the genomic DNA was determined in a Lambda 35 spectrophotometer (Perkin Elmer, Waltham, MA, USA) using the thermal denaturation (Tm) method and determined to be 69 ± 1 mol%.
DNA hybridization was performed using Biotin DecaLa-belTM Kit and Biotin Chromogenic Detection Kit (Fermentas Life Sciences) following the manufacturer's instruction. The DNA-DNA relatedness value of strain SJCon T with A. globiformis DSM 20124 T was found to be 45 %.
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