Reduction of hexavalent chromium (VI) by indigenous alkaliphilic and halotolerant Microbacterium sp. M5: comparative studies under growth and nongrowth conditions.

AIMS: To evaluate hexavalent chromium (Cr (VI)) reduction potential of indigenous isolate M5, under growing and nongrowing conditions. METHODS AND RESULTS: Microbacterium sp. M5 was isolated from soil samples collected from a common effluent treatment plant, after enrichment of indigenous microbial diversity in the presence of 200 mg l-1 of Cr (VI). The isolate achieved complete reduction of 400 mg l-1 Cr (VI) supplement to Luria Bertani medium having initial pH of 9·0 after 48 h incubation. Furthermore, the reduction potential of resting and surfactant treated cell membrane compromised cells of M5 was evaluated. The control and biosurfactant treated cells achieved 22·71 ± 0·5% and 40·56 ± 0·5% reduction of 50 mg l-1 Cr (VI) in Tris-HCl buffer, under resting cells conditions. To the best of our knowledge, this is the first report where cells with compromised cell membrane obtained after exposure to biosurfactant have been evaluated for Cr (VI) reduction. CONCLUSION: The Cr (VI) reduction potential of Microbacterium sp. M5 could be effectively exploited for treatment of chromium-rich effluents, under nongrowing conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The isolate M5 could be a potential inoculum for effluent treatment plants as it is able to support Cr (VI) reduction under wide range of pH, salinity and in the presence of different metal ions.

Identification of cyclic lipopeptides produced by Bacillus vallismortis R2 and their antifungal activity against Alternaria alternata.

AIM: To identify the different antifungal biomolecules produced by isolate Bacillus vallismortis R2 and analyse their effect on Alternaria alternata, a common pathogen causing black point disease of wheat. METHODS AND RESULTS: The different antifungal molecules produced by isolate R2 were purified by column chromatography. The liquid chromatography-mass spectrometry of purified fractions confirmed the ability of R2 to produce biomolecules putatively similar to three different families of cyclic lipopeptides (CLPs) viz. surfactins, iturins and fengycins. The synergistic interaction among CLPs was evident from significant increase in the antifungal activity of mixture of purified fractions as compared to that of the individual fractions by agar well diffusion assay. The evaluation of antifungal activity of purified fractions by 96-well microtitre plate assay showed that the fengycin-like molecules supported significantly higher antifungal activity against A. alternata than iturin A and no antifungal activity was supported by surfactin. CONCLUSIONS: The isolate R2, producing different CLPs, can be used as an environmental friendly alternative to chemical fungicides. Among the three different CLPs viz. surfactin, iturin A and fengycin produced by R2, the fengycins were the most active lipopeptides. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on co-production of three different types of CLPs by the cells of B. vallismortis.

Effect of antibiotic on survival and development of Spodoptera litura (Lepidoptera: Noctuidae) and its gut microbial diversity.

Addition of antibiotics to artificial diets of insects is a key component in the rearing of insects in the laboratory. In the present study an antimicrobial agent, streptomycin sulphate was tested for its influence on survival and fitness of Spodoptera litura (Fabricus) (Lepidoptera: Noctuidae) as well as its gut microbial diversity. The antibiotic did not adversely affect the survival of S. litura. Faster growth of larvae was recorded on diet amended with different concentrations of streptomycin sulphate (0.03, 0.07 and 0.15%) as compared to diet without streptomycin sulphate. The overall activity of various digestives enzymes increased on S+ diet while the activity of detoxifying enzymes significantly decreased. In addition, alteration in microbial diversity was found in the gut of S. litura larvae fed on diet supplemented with antibiotic (S+) and without antibiotic (S-).

Metabolic profiles and phylogenetic diversity of microbial communities from chlorinated pesticides contaminated sites of different geographical habitats of India.

AIMS: To study the microbial communities in three sites contaminated with chlorinated pesticides and evaluation of dehydrodechlorinase (linA) gene variants involved in gamma-hexachlorocyclohexane (γ-HCH, lindane) degradation. METHODS AND RESULTS: Using a culture-independent method, 16S rRNA genes were amplified from microbial communities occurring in contaminated soils. From 375 clone libraries analysed, 55 different restriction fragment length polymorphism phylotypes were obtained. Dehydrodechlorinase (linA) gene, which initiates the γ-HCH degradation, was directly amplified by PCR from the DNA extracted from soils. Deduced amino acid sequences of eight variant genotypes of linA showed few amino acid changes. All the variants of linA had mutations of F151L and S154T, and one of the genotype carried 12 amino acid changes when compared to a linA of Sphingomonas sp. reported from the same soil. CONCLUSIONS: The microbial communities displayed complex and diverse groups similar to bacteria involved in biodegradation. The presence of biodegradative genes like linA indicates the presence of communities with capacity to biodegrade the persistent pesticide HCH. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides insights to evaluate the presence of catabolic genes and assessing the bioremediation potential of the industrial soils contaminated by chlorinated pesticides.

Profiling differential expression of cellulases and metabolite footprints in Aspergillus terreus.

This study reports differential expression of endoglucanase (EG) and beta-glucosidase (betaG) isoforms of Aspergillus terreus. Expression of multiple isoforms was observed, in presence of different carbon sources and culture conditions, by activity staining of poly acrylamide gel electrophoresis gels. Maximal expression of four EG isoforms was observed in presence of rice straw (28 U/g DW substrate) and corn cobs (1.147 U/ml) under solid substrate and shake flask culture, respectively. Furthermore, the sequential induction of EG isoforms was found to be associated with the presence of distinct metabolites (monosaccharides/oligosaccharides) i.e., xylose (X), G(1), G(3) and G(4) as well as putative positional isomers (G(1)/G(2), G(2)/G(3)) in the culture extracts sampled at different time intervals, indicating specific role of these metabolites in the sequential expression of multiple EGs. Addition of fructose and cellobiose to corn cobs containing medium during shake flask culture resulted in up-regulation of EG activity, whereas addition of mannitol, ethanol and glycerol selectively repressed the expression of three EG isoforms (Ia, Ic and Id). The observed regulation profile of betaG isoforms was distinct when compared to EG isoforms, and addition of glucose, fructose, sucrose, cellobiose, mannitol and glycerol resulted in down-regulation of one or more of the four betaG isoforms.

Diastematomyelia. A case report.

Diastematomyelia is a rare form of spinal dysraphism characterized by a sagittal cleft of varying extent in the spinal cord, conus medullaris or filum terminale with splaying of the posterior vertebral elements. This condition results from the presence of an osseous, cartilaginous or fibrous septum producing a complete or incomplete sagittal division of the spinal cord into two hemicords. It may be isolated or associated with other segmental anomalies of the vertebral bodies (1).

Biosurfactant production by Pseudomonas sp. and its role in aqueous phase partitioning and biodegradation of chlorpyrifos.

AIM: To study the effect of biosurfactant on aqueous phase solubility and biodegradation of chlorpyrifos. METHODS AND RESULTS: A Pseudomonas sp. (ChlD), isolated from agricultural soil by enrichment culture technique in the presence of chlorpyrifos, was capable of producing biosurfactant (rhamnolipids) and degrading chlorpyrifos (0.01 g l(-1)). The partially purified rhamnolipid biosurfactant preparation, having a CMC of 0.2 g l(-1), was evaluated for its ability to enhance aqueous phase partitioning and degradation of chlorpyrifos (0.01 g l(-1)) by ChlD strain. The best degradation efficiency was observed at 0.1 g l(-1) supplement of biosurfactant, as validated by GC and HPLC studies. CONCLUSION: The addition of biosurfactant at 0.1 g l(-1) resulted in more than 98% degradation of chlorpyrifos when compared to 84% in the absence of biosurfactant after 120-h incubation. SIGNIFICANCE AND IMPACT OF THE STUDY: This first report, to the best of our knowledge, on enhanced degradation of chlorpyrifos in the presence of biosurfactant(s), would help in developing bioremediation protocols to counter accumulation of organophosphates to toxic/carcinogenic levels in environment.

Screening and selection of lovastatin hyper-producing mutants of Aspergillus terreus using cyclic mutagenesis.

134 fungal cultures isolated from different soil samples were screened for lovastatin production. Of these, 38 isolates produced different levels of lovastatin. An Aspergillus terreus strain GD13, producing 190 mg/l of lovastatin was selected and subjected to a rational mutation-selection programme based on the resistance to lovastatin and fatty acid synthase (FAS) inhibitors, viz., iodoacetamide and N-ethylmaleimide. After three cycles of mutagenesis, a hyper-producing mutant (EM19) exhibiting 7.5-fold (1424 mg/l) higher levels of lovastatin when compared to wild type parent strain was obtained.

Molecular characterization of multiple xylanase producing thermophilic/thermotolerant fungi isolated from composting materials.

AIMS: Molecular characterization of commercially important group of xylanase producing thermophilic/thermotolerant fungi. METHODS AND RESULTS: DNA from 16 thermophilic/thermotolerant fungal isolates was amplified by PCR using three sets of primers: (i) internal transcribed spacer sequence (ITSI-5.8S-ITSII), (ii) D1/D2 hyper variable region of 26S rDNA and (iii) 18S rDNA region. The amplified products of internal transcribed spacers (ITS) and D1/D2 region were sequenced and analysed using CLUSTALX, whereas, amplified 18S rDNA region was subjected to RFLP analysis based on restriction digestion with RsaI, MboI and Hinf I. CONCLUSIONS: The sequence based analyses of ITSI-5.8S-ITSII as compared with D1/D2 region of 26-28S rDNA was found to be a better tool for phylogenetic resolution of thermophilic/thermotolerant fungi. The ITSI-5.8S-ITSII sequence-based dendrogram indicates an early divergence of the alkaline active xylanase producing thermophilic fungal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was the first report on phylogenetic characterization of thermophilic/thermotolerant fungi.

Isolation of hexachlorocyclohexane-degrading Sphingomonas sp. by dehalogenase assay and characterization of genes involved in gamma-HCH degradation.

AIM: To screen and identify bacteria from contaminated soil samples which can degrade hexachlorocyclohexane (HCH)-isomers based on dechlorinase enzyme activity and characterize genes and metabolites. METHODS AND RESULTS: Dechlorinase activity assays were used to screen bacteria from contaminated soil samples for HCH-degrading activity. A bacterium able to grow on alpha-, beta-, gamma- and delta-HCH as the sole carbon and energy source was identified. This bacterium was a novel species belonging to the Sphingomonas and harbour linABCDE genes similar to those found in other HCH degraders. Gamma-pentachlorocyclohexene 1,2,4-trichlorobenzene and chlorohydroquinone were identified as metabolites. CONCLUSIONS: The study demonstrates that HCH-degrading bacteria can be identified from large environmental sample-based dehalogenase enzyme assay. This kind of screening is more advantageous compared to selective enrichment as it is specific and rapid and can be performed in a high-throughput manner to screen bacteria for chlorinated compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: The chlorinated pesticide HCH is a persistent and toxic environmental pollutant which needs to be remediated. Isolation of diverse bacterial species capable of degrading all the isomers of HCH will help in large-scale bioremediation in various parts of the world.

Production, purification and characterization of thermostable phytase from thermophilic fungus Thermomyces lanuginosus TL-7.

Ten different strains of Thermomyces lanuginosus, isolated from composting soils were found to produce phytase when grown on PSM medium. The wild type strain CM was found to produce maximum amount ofphytase (4.33 units/g DW substrate). Culturing T. lanuginosus strain CM on medium containing wheat bran and optimizing other culture conditions (carbon source, media type, nitrogen source, level of nitrogen, temperature, pH, inoculum age, inoculum level and moisture), increased the phytase yield to 13.26 units/g substrate. This culture was further subjected to UV mutagenesis for developing phytase hyperproducing mutants. The mutant (TL-7) showed 2.29-fold increase in phytase activity as compared to the parental strain. Employing Box-Behnken factor factorial design of response surface methodology resulted in optimized phytase production (32.19 units/g of substrate) by mutant TL-7. A simple two-step purification (40.75-folds) ofphytase from mutant TL-7 was achieved by anion exchange and gel filtration chromatography. The purified phytase (approximately 54 kDa) was characterized to be optimally active at pH 5.0 and temperature 70 degrees C, though the enzyme showed approximately 70% activity over a wide pH and temperature range (2.0-10.0 and 30-90 degrees C, respectively). The phytase showed broad substrate specificity with activity against sodium phytate, ADP and riboflavin phosphate. The phytase from T. lanuginosus was thermoacidstable as it showed up to 70% residual activity after exposure to 70 degrees C at pH 3.0 for 120 min. The enzyme showed Km 4.55 microM and Vmax 0.833 microM/min/mg against sodium phytate as substrate.

Role of transglycosylation products in the expression of multiple xylanases in Myceliophthora sp. IMI 387099.

This study reports the regulation of multiple xylanases produced by Myceliophthora sp. IMI 387099. Fructose was found to positively regulate the expression of multiple xylanase when used as sole carbon source. The xylanases (EX(1 )and EX(2)) of acidic pI were expressed in the presence of simple sugars (glucose, arabinose, and xylose), whereas xylanase of both acidic as well as basic pI (EX(1,) EX(2,) EX(3), and EX(5)) were expressed in the presence of fructose, xylan, and combination of xylan and alcohol. The combination of fructose and xylan also led to expression of an additional xylanase (EX(4)). The positional isomer (iso-X4) was found to be the key transglycosylation product when cultures were grown in the presence of fructose and xylan. In the presence of alcohols, the higher expression of xylanase was ascribed to the synergistic effect of alkyl glycoside and other transglycosylation products present in the culture extracts.

Production of feed enzymes (phytase and plant cell wall hydrolyzing enzymes) by Mucor indicus MTCC 6333: purification and characterization of phytase.

The production of phytase and associated feed enzymes (phosphatase, xylanase, CMCase, alpha-amylase and beta-glucosidase) was determined in a thermotolerant fungus Mucor indicus MTCC 6333, isolated from composting soil. Solid-substrate culturing on wheat bran and optimizing other culture conditions (C and N sources, level of N, temperature, pH, culture age, inoculum level), increased the yield of phytase from 266 +/- 0.2 to 513 +/- 0.4 nkat/g substrate dry mass. The culture extract also contained 112, 194, 171, 396, and 333 nkat/g substrate of phosphatase, xylanase, CMCase, beta-glucosidase and alpha-amylase activities, respectively. Simple 2-step purification employing anion exchange and gel filtration chromatography resulted in 21.9-fold purified phytase. The optimum pH and temperature were pH 6.0 and 70 degrees C, respectively. The phytase was thermostable under acidic conditions, showing 82% residual activity after exposure to 60 degrees C at pH 3.0 and 5.0 for 2 h, and displayed broad substrate specificity. The Km was 200 nmol/L and v(lim) of 113 nmol/s per mg protein with dodecasodium phytate as substrate. In vitro feed trial with feed enzyme resulted in the release of 1.68 g inorganic P/kg of feed after 6 h of incubation at 37 degrees C.

Production and characterization of thermostable alkaline phytase from Bacillus laevolacticus isolated from rhizosphere soil.

A novel phytase producing thermophilic strain of Bacillus laevolacticus insensitive to inorganic phosphate was isolated from the rhizosphere soil of leguminous plant methi (Medicago falacata). The culture conditions for production of phytase by B. laevolacticus under shake flask culture were optimized to obtain high levels of phytase (2.957 +/- 0.002 U/ml). The partially purified phytase from B. laevolacticus strain was optimally active at 70 degrees C and between pH 7.0 and pH 8.0. The enzyme exhibited thermostability with approximately 80% activity at 70 degrees C and pH 8.0 for up to 3 h in the presence/absence of 5 mM CaCl(2). The phytase from B. laevolacticus showed high specificity for phytate salts of Ca(+) > Na(+). The enzyme showed an apparent K (m) 0.526 mM and V (max) 12.3 mumole/min/mg of activity against sodium phytate.

Production of multiple xylanolytic and cellulolytic enzymes by thermophilic fungus Myceliophthora sp. IMI 387099.

This study reports the production of xylanolytic and cellulolytic enzymes by a thermophilic fungal isolate Myceliophthora sp. using a cheap medium containing rice straw and chemically defined basal medium under solid-state culture. A combination of one factor at a time approach followed by response surface methodology using Box-Behnken design of experiments resulted in 2.5, 1.25, 1.28 and 4.23 fold increase in xylanase, endoglucanase, beta-glucosidase and FPase activity, respectively. The zymograms developed against IEF gels showed that multiple isoforms of xylanase (5), endoglucanase (4) and beta-glucosidase (2) were produced under optimized culture conditions. Moreover, thiol containing serine proteases produced during the growth of the culture had no role in the post-translational modification of these xylanases.

Agrococcus lahaulensis sp. nov., isolated from a cold desert of the Indian Himalayas.

The taxonomic position of a lemon-yellow-pigmented actinobacterium, strain K22-21(T), isolated from a soil sample from Lahaul-Spiti Valley in the Indian Himalayas, was determined using a polyphasic approach. The strain had phenotypic and chemical properties that were consistent with its classification in the genus Agrococcus. Alignment of the 16S rRNA gene sequence of strain K22-21(T) with sequences from Agrococcus jenensis DSM 9580(T), Agrococcus baldri DSM 14215(T) and Agrococcus citreus DSM 12453(T) revealed similarities of 98.5, 96.8 and 96.6 %, respectively. However, the level of DNA-DNA relatedness between strain K22-21(T) and A. jenensis was 55.1 %. The novel strain could be distinguished from type strains of the three species of the genus Agrococcus using DNA-DNA relatedness and phenotypic data. Based on these differences, strain K22-21(T) (=MTCC 7154(T)=DSM 17612(T)) should be classified as the type strain of a novel species of Agrococcus, for which the name Agrococcus lahaulensis sp. nov. is proposed.

Kocuria himachalensis sp. nov., an actinobacterium isolated from the Indian Himalayas.

A reddish orange bacterium, strain K07-05(T), was isolated from soil during a study of the bacterial diversity of a cold desert of the Indian Himalayas and was studied by using a polyphasic approach. The organism had morphological and chemotaxonomic properties consistent with its classification in the genus Kocuria. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain K07-05(T) was closely related to Kocuria rosea DSM 20447(T) and Kocuria polaris MTCC 3702(T) (98.1 and 97.8 % sequence similarity, respectively), whereas the sequence similarity values with respect to the other Kocuria species with validly published names were between 96.4 and 94.2 %. However, the genomic relatedness, as shown by DNA-DNA hybridization, of strain K07-05(T) and K. polaris MTCC 3702(T) is 49.5 % and that with K. rosea MTCC 2522(T) is 24.0 %. The DNA G+C content of the strain is 75.3 mol%. The above data in combination with the phenotypic distinctiveness of K07-05(T) clearly indicate that the strain represents a novel species, for which the name Kocuria himachalensis sp. nov. is proposed. The type strain is K07-05(T) (=MTCC 7020(T)=DSM 44905(T)=JCM 13326(T)).

Ornithinimicrobium kibberense sp. nov., isolated from the Indian Himalayas.

A buff-yellow-pigmented bacterium, strain K22-20(T), which was isolated from a cold desert of the Indian Himalayas, was subjected to a polyphasic taxonomic study. Phenotypic and chemical properties of strain K22-20(T) were consistent with its classification in the genus Ornithinimicrobium. The major fatty acids of the strain were iso-C(17 : 1)omega9c (cis-15-methyl 7-hexadecenoic acid), iso-C(15 : 0) (13-methyl tetradecanoic acid), iso-C(16 : 0) (14-methyl pentadecanoic acid) and iso-C(17 : 0) (15-methyl hexadecanoic acid). The G+C content of the genomic DNA was 71 mol%. According to 16S rRNA gene sequence analysis, strain K22-20(T) was closely related to Ornithinimicrobium humiphilum HKI 0124(T) (97.7 %). However, genomic relatedness between strain K22-20(T) and O. humiphilum MTCC 6406(T), as revealed by DNA-DNA hybridization, was 64.5 %. Based on the polyphasic data, strain K22-20(T) (=MTCC 6545(T)=DSM 17687(T)=JCM 12763(T)) represents a novel species of the genus Ornithinimicrobium, for which the name Ornithinimicrobium kibberense sp. nov. is proposed.

Dietzia kunjamensis sp. nov., isolated from the Indian Himalayas.

A coral-red-pigmented actinobacterium, strain K30-10(T), was isolated from a soil sample from a cold desert of the Indian Himalayas. Chemical and phenotypic properties of strain K30-10(T) were consistent with its classification in the genus Dietzia. It showed 97.9 % 16S rRNA gene sequence similarity to Dietzia maris MTCC 7011(T); similarities to the type strains of three other species of the genus, Dietzia natronolimnaea, Dietzia psychralcaliphila and Dietzia cinnamea, were 94.4-96.0 %. The DNA-DNA relatedness between K30-10(T) and the closely related strain D. maris MTCC 7011(T) was 59.2 %. The DNA G+C content of strain K30-10(T) was 67.0 mol%. Based on physiological and biochemical tests and genotypic differences between strain K30-10(T) and its closest phylogenetic relatives, it is proposed that this strain represents a novel species, Dietzia kunjamensis sp. nov.; the type strain is K30-10(T) (=MTCC 7007(T)=DSM 44907(T)=JCM 13325(T)).

Rhodococcus kroppenstedtii sp. nov., a novel actinobacterium isolated from a cold desert of the Himalayas, India.

The taxonomic position of an actinomycete, strain K07-23T, isolated from a cold desert of the Himalayas, India, was established by a polyphasic approach. The strain exhibited phenotypic characters that were typical of the genus Rhodococcus. 16S rRNA gene sequence (1467 bases) comparisons confirmed that strain K07-23T belongs to the genus Rhodococcus. 16S rRNA sequence similarity studies showed that the isolate is very closely related to Nocardia corynebacterioides DSM 20151T (98.6 %), which has been recently reclassified as Rhodococcus corynebacterioides. It showed 94.4-96.6 % sequence similarity with other species of the genus Rhodococcus. However, genomic relatedness between strain K07-23T and R. corynebacterioides as revealed by DNA-DNA hybridization was low (62 %). Based on polyphasic analysis, strain K07-23T could be clearly distinguished from other species. It is proposed that strain K07-23T (=MTCC 6634T=DSM 44908T=JCM 13011T) represents a novel species of Rhodococcus, Rhodococcus kroppenstedtii sp. nov.