Biochemical Characterization of γ-Glutamyl Transpeptidase from Bacillus altitudinis IHB B1644 and Its Application in the Synthesis of l-Theanine.

An extracellular γ-glutamyl transpeptidase (GGT) produced from Bacillus altitudinis IHB B1644 was purified to homogeneity employing ion-exchange chromatography. GGT comprised two subunits of 40 and 22 kDa determined by SDS-PAGE. The maximum enzyme activity was optimal at pH 9 and 37 °C. The purified enzyme was stable from pH 5-10 and <50 °C. Steady-state kinetic studies revealed a Km value of 0.538 mM against γ-GpNA. For substrate specificity, GGT showed highest affinity for l-methionine. The inhibitors' effect demonstrated that serine or threonine and tryptophan residues are essential for enzyme activity. l-Theanine production was optimized by employing a one-variable-at-a-time approach with 60-65% conversion rate. The final reaction consisted of 20 mM l-glutamine, 200 mM ethylamine hydrochloride, and 10 U mL-1 enzyme concentration at 37 °C in Tris-Cl (50 mM, pH 9) for 5 h. l-Theanine was purified using a Dowex 50W X 8 hydrogen form resin and confirmed by HPLC and 1H NMR spectroscopies.

Defining the Rhizobium leguminosarum Species Complex.

Bacteria currently included in Rhizobium leguminosarum are too diverse to be considered a single species, so we can refer to this as a species complex (the Rlc). We have found 429 publicly available genome sequences that fall within the Rlc and these show that the Rlc is a distinct entity, well separated from other species in the genus. Its sister taxon is R. anhuiense. We constructed a phylogeny based on concatenated sequences of 120 universal (core) genes, and calculated pairwise average nucleotide identity (ANI) between all genomes. From these analyses, we concluded that the Rlc includes 18 distinct genospecies, plus 7 unique strains that are not placed in these genospecies. Each genospecies is separated by a distinct gap in ANI values, usually at approximately 96% ANI, implying that it is a 'natural' unit. Five of the genospecies include the type strains of named species: R. laguerreae, R. sophorae, R. ruizarguesonis, "R. indicum" and R. leguminosarum itself. The 16S ribosomal RNA sequence is remarkably diverse within the Rlc, but does not distinguish the genospecies. Partial sequences of housekeeping genes, which have frequently been used to characterize isolate collections, can mostly be assigned unambiguously to a genospecies, but alleles within a genospecies do not always form a clade, so single genes are not a reliable guide to the true phylogeny of the strains. We conclude that access to a large number of genome sequences is a powerful tool for characterizing the diversity of bacteria, and that taxonomic conclusions should be based on all available genome sequences, not just those of type strains.

Statistical optimization of culture conditions of mesophillic gamma-glutamyl transpeptidase from Bacillus altitudinis IHB B1644.

Microbial gamma-glutamyl transpeptidase (GGT) is a key enzyme in production of several γ-glutamyl compounds with food and pharmaceutical applications. Bacterial GGTs are not commercially available in the market owing to their low production from various sources. Thus, the study was focused on achieving the higher GGT production from B. altitudinis IHB B1644 by optimizing the culture conditions using one-variable-at-a-time (OVAT) strategy. A mesophillic temperature of 28 °C, agitation 200 rpm and neutral pH 7 were found to be optimal for higher GGT titre. Among the medium components, the monosaccharide glucose served as the best carbon source over disaccharides, and yeast extract was the preferred organic nitrogen source over inorganic nitrogen sources. The statistical approaches (Plakett-Burman and response surface methodology) were further employed for the optimization of medium components. Medium composition: 0.1% w/v glucose, 0.3% w/v yeast extract, 0.03% w/v magnesium sulphate, 0.20% w/v potassium dihydrogen phosphate and 2.5% w/v sodium chloride with inoculum size (1% v/v) was suitable for higher GGT titres (449 U ml-1). Time kinetics showed the stability of enzyme up to 96 h of incubation suggesting its application in the industrial use. The proposed strategy resulted in 2.6-fold increase in the GGT production compared to that obtained in the unoptimized medium. The results demonstrated that RSM was fitting to identify the optimum production conditions and this finding should be of great importance for commercial GGT production.

Bioprospecting Psychrotrophic Bacteria for Serine-Type Proteases from the Cold Areas of Western Himalayas.

The aim of present study was to analyze the prevalence of protease diversity among psychrotrophic bacteria in Lahaul and Spiti of the Western Himalayas. A total of 459 bacteria were screened and protease activity was observed in 150 isolates at 5 °C. Furthermore, 55 isolates showed protease activity up to pH 10 at 5 °C. Based on the hydrolytic zone, 22 isolates were selected for protease quantification. The protease activity varied from 58-377 U mL-1 at 10 °C, 49-396 U mL-1 at 28 °C and 31-407 U mL-1 at 37 °C. Similarly, protease activity ranged from 36-353 U mL-1 at pH 7, 40-306 U mL-1 at pH 9 and 33-304 U mL-1 at pH 10. The isolates were identified based on 16S rRNA gene sequencing and showed phylogenetic relationship to Arthrobacter belonging to the class Actinobacteria, Bacillus, Exiguobacterium, Paenibacillus, and Planomicrobium to Bacilli, and Pseudomonas, Serratia, and Stenotrophomonas to Gammaproteobacteria. Zymogram analysis revealed variations in protease isoforms ranging from 20 to 250 kDa which were strongly inhibited in the presence of phenylmethylsulfonyl fluoride, thus indicated serine-type nature. The extensive number of serine proteases among these bacteria was confirmed by annotating genomes of the reported genera for prevalence of protease isoforms. The properties of proteases including low-temperature activity with alkaline stability and detergent compatibility suggested their suitability as bio-additives in laundry.

Psychromicrobium lacuslunae sp. nov., isolated from a high altitude lake.

The novel strain IHBB 11108T was a psychrotolerant and alkaliphilic bacterium isolated from the subsurface water of Chandra Tal Lake in the Lahaul-Spiti valley located in the Indian trans-Himalayas. Cells were Gram-stain-positive, aerobic, non-motile, catalase-positive and oxidase-negative. The strain grew at 5-37 °C (optimum 28 °C), pH 5.0-12.0 (optimum pH 7.0) and with up to 8 % NaCl (optimum 1 %). The 16S rRNA gene sequence analysis revealed the highest relatedness of strain IHBB 11108T with Psychromicrobium silvestre DSM 102047T (97.5 %), Arthrobacter russicus DSM 14555T (97.4 %) and Renibacterium salmoninarum ATCC 33209T (97.4 %). The strain contained a quinone system with 57.2 % MK-9(H2), 39.1 % MK-10(H2), 3.0 % MK-8(H2) and 0.7 % MK-7(H2). The polar lipids detected were diphosphatidylglycerol, dimannosylglyceride, phosphatidylinositol, phosphatidylglycerol, monogalactosyldiacylglycerol, one unidentified glycolipid and four unidentified lipids. The cell-wall peptidoglycan structure type was A3α l-Lys-l-Thr-l-Ala with substitution of the α-carboxyl group of d-Glu by alanine amide. Anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0 were the predominant fatty acids. The genomic DNA G+C content was 59.0 mol%. The DNA-DNA relatedness of strain IHBB 11108T was 46.7±2.2, 43.1±2.5 and 19.1±2.4 % with P. silvestre DSM 102047T, A. russicus DSM 14555T and R. salmoninarum ATCC 33209T, respectively. On the basis of the results of the phenotypic, chemotaxonomic and phylogenetic analyses, IHBB 11108T is considered to represent a novel species of the genus Psychromicrobium for which the name Psychromicrobium lacuslunae sp. nov. is proposed. The type strain is IHBB 11108T (=MTCC 12460T=MCC 2780T=JCM 31143T=KACC 19070T).

Chryseobacterium glaciei sp. nov., isolated from the surface of a glacier in the Indian trans-Himalayas.

A novel bacterial strain, IHBB 10212T, of the genus Chryseobacterium was isolated from a glacier near the Kunzum Pass located in the Lahaul-Spiti in the North-Western Himalayas of India. The cells were Gram-negative, aerobic, non-sporulating, single rods, lacked flagella, and formed yellow to orange pigmented colonies. The strain utilized maltose, trehalose, sucrose, gentibiose, glucose, mannose, fructose, mannitol, arabitol and salicin for growth. Flexirubin-type pigments were produced by strain IHBB 10212T. The 16S rRNA gene sequence analysis showed relatedness of strain IHBB 10212T to Chryseobacterium polytrichastri DSM 26899T (97.43 %), Chryseobacterium greenlandense CIP 110007T (97.29 %) and Chryseobacterium aquaticum KCTC 12483T (96.80 %). Iso-C15 : 0 and summed feature 3 (C16 : 1ω7c/C16 : 1ω6c) constituted the major cellular fatty acids. The polar lipids present were six unidentified aminolipids, one unidentified phospholipid and three unidentified lipids. MK-6 was identified as the major quinone. The DNA G+C content was 34.08  mol%. Digital DNA-DNA hybridization of strain IHBB 10212T with C. polytrichastri, C. greenlandense and C. aquaticum showed values far below the prescribed thresholds of 95 % for average nucleotide identity and 70 % for the Genome-to-Genome Distance Calculator for species delineation. Based on its differences from validly published Chryseobacterium species, strain IHBB 10212T is identified as a new species, for which the proposed name is Chryseobacterium glaciei sp. nov., with IHBB 10212T as the type strain (=MTCC 12457T=JCM 31156T=KACC 19170T).

Heterologous expression and structure-function relationship of low-temperature and alkaline active protease from Acinetobacter sp. IHB B 5011(MN12).

The gene encoding protease from Acinetobacter sp. IHB B 5011(MN12) was cloned and expressed in Escherichia coli BL21(DE3). The nucleotide sequence revealed 1323bp ORF encoding 441 amino acids protein with molecular weight 47.2kDa. The phylogenetic analysis showed clustering of Alp protease with subtilisin-like serine proteases of S8 family. The amino acid sequence was comprised of N-terminal signal peptide 1-21 amino acids, pre-peptide 22-143 amino acids, peptidase S8 domain 144-434 amino acids, and pro-peptide 435-441 amino acids at C-terminus. Three constructs with signal peptide pET-Alp, without signal peptide pET-Alp1 and peptidase S8 domain pET-Alp2 were prepared for expression in E. coli BL21(DE3). The recombinant proteins Alp1 and Alp2 expressed as inclusion bodies showed ∼50kDa and ∼40kDa bands, respectively. The pre-propeptide ∼11kDa removed from Alp1 resulted in mature protein of ∼35kDa with 1738Umg-1 specific activity. The recombinant protease was optimally active at 40°C and pH 9, and stable over 10-70°C and 6-12pH. The activity at low-temperature and alkaline pH was supported by high R/(R+K) ratio, more glycine, less proline, negatively charged amino acids, less salt bridges and longer loops. These properties suggested the suitability of Alp as additive in the laundry.

Paenibacillus ihbetae sp. nov., a cold-adapted antimicrobial producing bacterium isolated from high altitude Suraj Tal Lake in the Indian trans-Himalayas.

The assessment of bacterial diversity and bioprospection of the high-altitude lake Suraj Tal microorganisms for potent antimicrobial activities revealed the presence of two Gram-stain-variable, endospore-forming, rod-shaped, aerobic bacteria, namely IHBB 9852T and IHBB 9951. Phylogenetic analysis based on 16S rRNA gene sequence showed the affiliation of strains IHBB 9852T and IHBB 9951 within the genus Paenibacillus, exhibiting the highest sequence similarity to Paenibacillus lactis DSM 15596T (97.8% and 97.7%) and less than 95.9% similarity to other species of the genus Paenibacillus. DNA-DNA relatedness among strains IHBB 9852T and IHBB 9951 was 90.2%, and with P. lactis DSM 15596T, was 52.7% and 52.4%, respectively. The novel strains contain anteiso-C15:0, iso-C15:0, C16:0 and iso-C16:0 as major fatty acids, and phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol were predominant polar lipids. The DNA G+C content for IHBB 9852T and IHBB 9951 was 52.1 and 52.2mol%. Based on the results of phenotypic and genomic characterisations, we concluded that strains IHBB 9852T and IHBB 9951 belong to a novel Paenibacillus species, for which the name Paenibacillus ihbetae sp. nov. is proposed. The type strain is IHBB 9852T (=MTCC 12459T=MCC 2795T=JCM 31131T=KACC 19072T; DPD TaxonNumber TA00046) and IHBB 9951 (=MTCC 12458=MCC 2794=JCM 31132=KACC 19073) is a reference strain.

Integrated Translatome and Proteome: Approach for Accurate Portraying of Widespread Multifunctional Aspects of Trichoderma.

Genome-wide studies of transcripts expression help in systematic monitoring of genes and allow targeting of candidate genes for future research. In contrast to relatively stable genomic data, the expression of genes is dynamic and regulated both at time and space level at different level in. The variation in the rate of translation is specific for each protein. Both the inherent nature of an mRNA molecule to be translated and the external environmental stimuli can affect the efficiency of the translation process. In biocontrol agents (BCAs), the molecular response at translational level may represents noise-like response of absolute transcript level and an adaptive response to physiological and pathological situations representing subset of mRNAs population actively translated in a cell. The molecular responses of biocontrol are complex and involve multistage regulation of number of genes. The use of high-throughput techniques has led to rapid increase in volume of transcriptomics data of Trichoderma. In general, almost half of the variations of transcriptome and protein level are due to translational control. Thus, studies are required to integrate raw information from different "omics" approaches for accurate depiction of translational response of BCAs in interaction with plants and plant pathogens. The studies on translational status of only active mRNAs bridging with proteome data will help in accurate characterization of only a subset of mRNAs actively engaged in translation. This review highlights the associated bottlenecks and use of state-of-the-art procedures in addressing the gap to accelerate future accomplishment of biocontrol mechanisms.

Genome assembly of Chryseobacterium sp. strain IHBB 10212 from glacier top-surface soil in the Indian trans-Himalayas with potential for hydrolytic enzymes.

The cold-active esterases are gaining importance due to their catalytic activities finding applications in chemical industry, food processes and detergent industry as additives, and organic synthesis of unstable compounds as catalysts. In the present study, the complete genome sequence of 4,843,645 bp with an average 34.08% G + C content and 4260 protein-coding genes are reported for the low temperature-active esterase-producing novel strain of Chrysobacterium isolated from the top-surface soil of a glacier in the cold deserts of the Indian trans-Himalayas. The genome contained two plasmids of 16,553 and 11,450 bp with 40.54 and 40.37% G + C contents, respectively. Several genes encoding the hydrolysis of ester linkages of triglycerides into fatty acids and glycerol were predicted in the genome. The annotation also predicted the genes encoding proteases, lipases, amylases, ß-glucosidases, endoglucanases and xylanases involved in biotechnological processes. The complete genome sequence of Chryseobacterium sp. strain IHBB 10212 and two plasmids have been deposited vide accession numbers CP015199, CP015200 and CP015201 at DDBJ/EMBL/GenBank.

Stress-Tolerant Viridibacillus arenosi Strain IHB B 7171 from Tea Rhizosphere as a Potential Broad-Spectrum Microbial Inoculant.

Viridibacillus arenosi strain IHB B 7171 identified based on 16S rRNA gene sequence produced colony forming units (cfu/ml) ranging from 3.3 × 104 to 1.2 × 1010 under pH 5-11, 2.2 × 102 to 1.4 × 1010 for temperature 5-40 °C, 2.4 × 102 to 1.1 × 1010 for PEG 6000 10-30%, 2.2 × 102 to 1.4 × 1010 for 2.5-10% NaCl, 3.1 × 103 to 1.7 × 109 for 2.5-7.5 mM CaCl2, 2.2 × 102 to 1.4 × 107 for 2.5-7.5 mM AlCl3, and 3.2 × 102 to 1.2 × 107 for 2.5-7.5 mM FeCl3. The activities of plant growth-promoting attributes with the increasing acidity, desiccation and salinity ranged from 408 to 101, 20 to 8, 14 to 5 µg/ml P-liberated from tri-calcium phosphate, aluminium phosphate and iron phosphate, 20-9% siderophore units, 14-4 µg/ml IAA and 190-16 α-ketobutyrate h/mg protein ACC-deaminase activity. Plant height, leaf number, and leaf weight on treatment with bacterial inoculum showed an increment of 9.5, 17.6, 54.5 and 31.0% in tea seedlings, respectively. The bacterium also enhanced plant height and yield by 10 and 13% in pea and 2.8 and 13.9% in wheat. The results exhibited stress-tolerance and plant growth-promoting activities by the strain under stressed growth-conditions with potential as a broad-spectrum plant growth-promoting rhizobacterium.

Complete genome sequence of a low-temperature active and alkaline-stable endoglucanase-producing Paenibacillus sp. strain IHB B 3084 from the Indian Trans-Himalayas.

A genome of 5.88Mb with 46.83% G+C content is reported for an endoglucanase-producing bacterium Paenibacillus sp. strain IHB B 3084 isolated from the cold environments of the Indian Trans-Himalayas. The psychrotrophic bacterium produces low-temperature active and alkaline-stable endoglucanases of industrial importance. The genomic data has provided insight into genomic basis of cellulase production and survival of the bacterium in the cold environments.

Complete Genome Sequence of the Rhizobacterium Pseudomonas trivialis Strain IHBB745 with Multiple Plant Growth-Promoting Activities and Tolerance to Desiccation and Alkalinity.

The complete genome sequence of 6.45 Mb is reported here for Pseudomonas trivialis strain IHBB745 (MTCC 5336), which is an efficient, stress-tolerant, and broad-spectrum plant growth-promoting rhizobacterium. The gene-coding clusters predicted the genes for phosphate solubilization, siderophore production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, indole-3-acetic acid (IAA) production, and stress response.

Cloning and expression of low temperature active endoglucanase EG5C from Paenibacillus sp. IHB B 3084.

The endoglucanase gene designated as EG5C encoding cold active endoglucanase produced by Paenibacillus sp. IHB B 3084 was cloned and expressed in Escherichia coli BL21(DE3). The gene consisting of 1719bp open reading frame encoded a protein of 573 amino acids with a predicted molecular weight of 63.5kDa. The presence of N-terminal catalytic domain of the glycosyl hydrolase family 5 (GH5) and C-terminal carbohydrate binding X2 domain suggested the modular nature of the enzyme. The native signal peptide of EG5C was capable of efficiently secreting the enzyme with near equal activities in the cytoplasmic and extracellular fractions. The recombinant enzyme purified 9.46 fold to homogeneity with 22.33% yield gave 7.758IU/mg specific activity. The enzyme was stable over the broad pH range of 4-12 with more than 50% residual activity. The optimal activity was at 40°C with 70% relative activity at 5°C. The low temperature activity despite the shorter linker region suggested a novel cold adaptation mechanism by the enzyme. The enzyme displayed higher activity on carboxymethylcellulose than avicel which is useful in maintaining the tensile strength of fiber. The efficient secretion and low temperature activity offer prospect for large-scale production and industrial application of the endoglucanase.

Complete Genome Sequencing of Protease-Producing Novel Arthrobacter sp. Strain IHBB 11108 Using PacBio Single-Molecule Real-Time Sequencing Technology.

A previously uncharacterized species of the genus Arthrobacter, strain IHBB 11108 (MCC 2780), is a Gram-positive, strictly aerobic, nonmotile, cold-adapted, and protease-producing alkaliphilic actinobacterium, isolated from shallow undersurface water from Chandra Tal Lake, Lahaul-Spiti, India. The complete genome of the strain is 3.6 Mb in size with an average 58.97% G+C content.

Complete Genome Sequence of Paenibacillus sp. Strain IHBB 10380 Using PacBio Single-Molecule Real-Time Sequencing Technology.

The complete genome sequence of 5.77 Mb is reported for Paenibacillus sp. strain IHBB 10380, isolated from the cold desert area of the northwestern Himalayas and exhibiting amylase and cellulase activities. The gene-coding clusters predicted the presence of genes for hydrolytic enzymes in the genome.

Draft Genome Sequence of a Cellulase-Producing Psychrotrophic Paenibacillus Strain, IHB B 3415, Isolated from the Cold Environment of the Western Himalayas, India.

Paenibacillus sp. strain IHB B 3415 is a cellulase-producing psychrotrophic bacterium isolated from a soil sample from the cold deserts of Himachal Pradesh, India. Here, we report an 8.44-Mb assembly of its genome sequence with a G+C content of 50.77%. The data presented here will provide insights into the mechanisms of cellulose degradation at low temperature.

Effect of physicochemical parameters on nitrile-hydrolyzing potentials of newly isolated nitrilase of Fusarium oxysporum f. sp. lycopercisi ED-3.

In recent years, nitrilases from fungus have received increasing attention, and most of the studies are performed on nitrilases of bacterial origin. Frequently used methods are based on analytical methods such as high-performance liquid chromatography, liquid chromatography-mass spectrometry, and gas chromatography; therefore, an efficient, user friendly, and rapid method has been developed to screen nitrilase enzyme based on the principle of color change of a pH indicator. Phenol red amended with the minimal medium appears light yellow at neutral pH, which changes into pink with the formation of ammonia, indicating nitrilase activity in the reaction medium. A highly potent strain ED-3 identified as Fusarium oxysporum f. sp. lycopercisi (specific activity 17.5 µmol/Min/mg dcw) was isolated using this method. The nitrilase activity of F. oxysporum f. sp. lycopercisi ED-3 strain showed wide substrate specificity toward aliphatic nitriles, aromatic nitriles, and orthosubstituted heterocyclic nitriles. 4-Aminobenzonitrile was found to be a superior substrate among all the nitriles used in this study. This nitrilase was active within pH 5-10 and temperature ranging from 25 to 60 °C with optimal at pH 7.0 and temperature at 50 °C. The nitrilase activity was enhanced to several folds through optimization of culture and biotransformation conditions from 1,121 to 1,941 µmol/Min.

Taxonomy and compositional analysis of two new for science medicinal mushroom taxa from India.

Taxonomy and compositional evaluation of two new medicinal and edible varieties, Lentinus squarrosulus var. mangiferae and Pleurotus pulmonarius var. indicus, have been detailed in the present paper. Two new varieties were taxonomically identified by morphological, microscopical and ITS-region sequencing. Composition of proteins, carbohydrates, fat, ash content, minerals, fibers, ascorbic acid, phenolics, ß-carotene, and lycopene and alkaloids was analyzed for these varieties.

Statistical Optimization of Medium Components for Mass Production of Plant Growth-Promoting Microbial Inoculant Pseudomonas trivialis BIHB 745 (MTCC5336).

Optimizing nutritional requirements for mass production of microbial inoculants in shortened time has relevance for their economical field application. Therefore, the present study aimed at selecting suitable growth medium, optimizing its components, and up-scaling inoculum production for plant growth-promoting Pseudomonas trivialis BIHB 745. Of the different media tested, the culture exhibited maximal viable colony count in trypticase soya broth with 17.6 % increased biomass on optimizing levels of carbon source, nitrogen source, and NaCl using response surface methodology. A twofold higher biomass with 9 h shorter incubation period was obtained in optimized medium in a bioreactor in comparison to shake flasks.