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Microbial Type Culture Collection and Gene bank (MTCC), Institute of Microbial Technology, Sector 39A, Chandigarh – 160 036, India
Correspondence
R. K. Jain
rkj{at}imtech.res.in
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 1DT is AM261414.
A table detailing the cellular fatty acid composition of strain 1DT and Clostridium aciditolerans is available with the online version of this paper.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Attempts have been made to exploit this property for the remediation of soil contaminated with polynitroaromatics such as 2,4-dinitrophenol, 2,4,6-trinitrotoluene, 1,3,5-trinitrohexahydro-1,3,5-triazine etc. (Gorontzy et al., 1993; Hughes et al., 1998, 1999; Lewis et al., 1996; Shin et al., 1997). However, little is known about the Clostridium species that degrade mononitrophenols such as p-nitrophenol (pNP) and m-nitrophenol. To our knowledge, there is only a single report available on pNP transformation by Clostridium pasteurianum and Clostridium sp. W1 under anaerobic conditions (Gorontzy et al., 1993). In the present study, by using the polyphasic approach, we determined the taxonomic position of the bacterial strain 1DT that is capable of degrading pNP under anaerobic conditions. The bacterium 1DT was isolated from a soil sample (red-sandy soil) in a pot that was obtained from the subsurface environment from a depth of about 3–4 m during an archaeological survey at Hyderabad, India located between longitude 7 °30' east and latitude 1 °23' north. The sample yielding strain 1DT was isolated by performing standard plate-dilution technique on tryptone soy agar (TSA; Himedia), with 0.01 % of resazurin as an indicator, at 30 °C under anaerobic conditions. The isolate was maintained as a glycerol stock at –70 °C until further characterization. The reference strain Clostridium aciditolerans DSM 17425T was obtained from DSMZ, Germany.
Strain 1DT was found to be an obligate anaerobe and it formed a cream coloured colony. Cell morphology was observed under a phase-contrast microscope (Zeiss, Axiophot) using an oil-immersion objective (x100) to ascertain the shape. Presence of spore formation was determined by staining with malachite green as described previously (Gerhardt et al., 1994). Motility was confirmed by using the hanging-drop method immediately after taking out the culture from anaerobic conditions. Physiological tests such as growth at different temperatures, pH and salt concentrations were performed using basal TSA medium with L-cysteine hydrochloride (0.05 %) as a reducing agent. Strain 1DT was found to grow between temperatures 20 and 45 °C with optimum growth at 30 °C. Biological buffers (pH 5.5–6.5 MES and 7.5–8.5 NaHCO3/Na2CO3 system) were used for pH adjustment. The efficiency of strain 1DT to degrade pNP was tested in tryptone soy broth (TSB) supplemented with 0.2–0.5 mM of pNP in serum, vials under anaerobic conditions. Analysis on quantification of pNP was done by using HPLC with a Waters 600 model equipped with a Waters 996 photodiode array detector operating at 315 nm. Separation was carried out with a Waters Spherisorb 5 µm C8 column and the mobile phase was 1 % acetic acid/methanol (solvent A) and 1 % acetic acid/water (solvent B). Compounds were eluted at a flow rate of 1.5 ml min–1 by using a gradient starting with 35 % of solvent A followed by a gradual increase to 68 % (0–10 min).
For biochemical tests mentioned in Table 1 and species description, strain 1DT was grown at 30 °C on TSA medium with L-cysteine hydrochloride (0.05 %) as a reducing agent and under anaerobic conditions. Catalase and oxidase tests were carried out as described by Cowan & Steel (1965). Indole test, Voges–Proskauer test, methyl red test, H2S production and nitrate reduction were performed as described by Lanyi (1987). Casein, gelatin, starch and Tween 20 hydrolysis were determined as described by Smibert & Krieg (1994). Assimilation of various substrates by strain 1DT was determined by using the Biolog system according to the manufacturer's instructions except that reinforced clostridial agar (RCA, Himedia) was used instead of the Biolog Universal agar with 5 % blood. Furthermore, the ability of strain 1DT to produce acid from sugar was checked in medium containing (g 100 ml–1) 0.01 KCl, 0.01 yeast extract, 0.025 K2HPO4, 0.025 MgSO4, 0.2 (NH4)2SO4, 0.01 bromocresol purple supplemented with 1.0 % of each filter-sterilized carbon compound. Fatty acid methyl esters (Sato & Murata, 1988) were extracted from cells grown on TSA with L-cysteine hydrochloride (0.05 %) at 30 °C under anaerobic conditions and analysed by Microbial Identification System (MIDI) as described by Pandey et al. (2002). Peptidoglycan type was analysed according to Komagata & Suzuki (1987) and menaquinones were extracted and analysed as described by Minnikin et al. (1984). Polar lipids were analysed as described by Suresh et al. (2004). Fermentation end products of glucose were determined by GC with a flame-ionization detector (Shimadzu) as described by Mountfort & Rhodes (1991). DNA was isolated as described by Shivaji et al. (1989). The G+C content of genomic DNA was determined as described by Mesbah et al. (1989). The enzymically degraded nucleosides were separated by HPLC using a reverse-phase column (C18; Phenomenex); non-methylated 
DNA (49.85 mol% G+C) was used as the calibration reference. DNA–DNA hybridization was performed by the membrane filter method (Tourova & Antonov, 1987) as described by Shivaji et al. (1992).
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and the species description. They grew well in medium containing pNP at a concentration of 0.5 mM. HPLC analysis revealed that strain 1DT is capable of degrading pNP and the complete degradation of pNP was observed within 24 h as there was no pNP spectrum observed through HPLC analysis. Neither intermediate(s) nor end product(s) was observed during the anaerobic degradation of pNP, suggesting complete degradation by strain 1DT. However, further characterization of the biochemical steps involved in the degradation is the subject of future studies.
The 16S rRNA gene was amplified by PCR using primers 8-27f (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492r (5'-TACGGYTACCTTGTTACGACTT-3') as described by Pandey et al. (2002). Amplified PCR product was purified using QIAquick PCR purification kit (Qiagen), sequenced using the Big Dye Terminator kit (Applied Biosystems) and an ABI Prism model 3130xl automatic DNA sequencer. The almost-complete 16S rRNA gene sequence (1461 bp continuous stretch) was obtained and used for the initial BLAST search. Closely related sequences were retrieved from the EMBL database. For alignment, CLUSTAL_X was used (Thompson et al., 1997) and the results obtained were edited manually. Pair-wise evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1980). A neighbour-joining phylogenetic tree was constructed using TREECON (version 1.3b) (Van de Peer & De Wachter, 1997). The stability among the groupings of the phylogenetic tree was assessed based on 1000 replicates. Sporacetigenium mesophilum DSM 16796T (AY682207) was used as the outgroup.
Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain 1DT represents a novel species of the genus Clostridium. It exhibited highest similarity to C. aciditolerans JW/YJL-B3T (98.2 %) followed by Clostridium scatologenes ATCC 25775T (95.1 %), Clostridium drakei SL1T (95.0 %), Clostridium carboxidivorans P7T (95.0 %) and much lower similarity to other species of the genus with validly published names. Although Clostridium magnum ATCC 49199T showed 95.3 % similarity in pair-wise sequence analysis, there was complete dissimilarity observed between nt 980 and 1022 (according to Escherichia coli number) that was not taken into account. The neighbour-joining phylogenetic analysis revealed that strain 1DT formed a clade with the type strain of C. aciditolerans, with 100 % bootstrap value (Fig. 1), and formed a coherent cluster with other members of cluster I of the genus Clostridium (Collins et al., 1994). Strain 1DT showed highest similarity to C. aciditolerans at 16S rRNA gene sequence level; however, it exhibited only 36.4 % mean value of DNA–DNA hybridization at whole genome level, which is significantly below the recommended threshold value of less than 70 % for the delineation of a new species (Wayne et al., 1987). There were several phenotypic properties as well as end products of glucose fermentation to distinguish strain 1DT from C. aciditolerans (Table 1). The fatty acid analysis of strains 1DT and C. aciditolerans indicated that they had C16 : 0, iso-C17 : 1I/anteiso B and C14 : 0 as major components; however, fatty acids iso-C13 : 0, C13 : 0, iso-C15 : 0, anteiso-C15 : 0, C15 : 1
8c, C15 : 16c and iso-C18 : 1 H were present in C. aciditolerans in significant quantities but absent in strain 1DT (see Supplementary Table S1 available in IJSEM Online). Presence of C16 : 0 as a major fatty acid, which is also reported in other species (Wilde et al., 1997; Broda et al., 2000a, b; Spring et al., 2003), further affiliates strain 1DT to the genus Clostridium. The polar lipid analysis revealed the presence of diphosphatidylglycerol (DPG), as a major lipid and other lipids include phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and two unknown phospholipids. Based on these data presented, strain 1DT represents a novel species in the genus Clostridium, for which the name Clostridium nitrophenolicum sp. nov. is proposed.
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Cells are rod shaped measuring 3.5–5.0 µm in length and 0.6–0.9 µm in width and strictly anaerobic. They form spores that are oval and central in position. Colonies grown on TSA are circular, smooth and convex with wavy margin. Growth occurs between pH 6.5 and 8.0 and no growth is observed below pH 6.5 and above 8.0. Growth occurs between 20 and 45 °C and does not grow at 15 or 50 °C. Optimum growth is observed at pH 7.2 and temperature 30 °C. Growth occurs between 0 and 1 % NaCl and does not grow with 2.0 % NaCl. Strain 1DT is negative for catalase and oxidase, and positive for indole and methyl red test. Starch and urea are not hydrolysed. Does not reduce nitrate to nitrite and is negative for Voges–Proskauer test, citrate utilization and H2S production. Using Biolog MicroPlates (AN), the strain showed a positive reaction for the assimilation of D-fructose, L-fucose, D-galactose, D-galacturonic acid, palatinose and L-rhamnose and a negative reaction for acetic acid, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, N-acetyl-
-D-mannosamine adonitol, L-alaninamide, L-alanine, L-alanyl L-glutamine, L-alanyl L-histidine, L-alanyl L-threonine, amygdalin, D-arabitol, arbutin, L-asparagine, D-cellobiose, 
-cyclodextrin, -cyclodextrin, dextrin, dulcitol, i-erythritol, formic acid, D-gluconic acid, -D-glucose 1-phosphate, -D-glucose 6-phosphate, L-glutamic acid, L-glutamine, glycerol, glyoxylic acid, -hydroxybutyric acid, -hydroxybutyric acid, inosine, myo-inositol, itaconic acid, -ketobutyric acid, -ketobutyric acid, lactic acid, lactulose, malic acid, maltose, maltotriose, D-mannitol, mannose, D-melibiose, L-methionine, methyl -D-galactoside, methyl -D-glucoside, methyl -D-glucoside, 3-methyl-D-glucose, L-phenylalanine, propionic acid, pyruvic acid, D-raffinose, L-rhamnose, D-saccharic acid, salicin, L-serine, D-sorbitol, stachylose, succinic acid, sucrose, m-tartaric acid, L-threonine, thymidine, trehalose, turanose, uridine, urocanic acid and L-valine. It produced acid from glucose, dulicitol, fructose, galactose, maltose, mannose, sucrose and trehalose, but not from adonitol, arabinose, cellobiose, inositol, mannitol, melibiose, salicin or sorbitol. Fermentation end products from glucose include acetate, formate and pyruvate. Menaquinone present is MK-7 and the cell wall amino acid is meso-diaminopimelic acid. Polar lipids present are DPG, PG, PE and two unknown phospholipids (UKP1 and UKP2). The major cellular fatty acids are (%); C16 : 0 (28.02), iso-C17 : 1I/anteiso B (23.05), C16 : 17c/iso-C15 : 0 2-OH (10.82) and C14 : 0 (10.02). The DNA G+C content is 35.5 mol%.
The type strain, 1DT (=MTCC 7832T=JCM 14030T), was isolated from a subsurface soil sample from a depth of about 3–4 m.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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