An oxalate decarboxylase-like cupin domain containing protein is involved in imparting acid stress tolerance in Bacillus amyloliquefaciens MBNC.

We report here the structural and functional properties of an oxalate decarboxylase (OxDC)-like cupin domain-containing protein of Bacillus amyloliquefaciens MBNC and its role in imparting tolerance to acid stress conditions. Quantitative real-time PCR (qPCR) analysis revealed 32-fold and 20-fold upregulation of the target gene [(OxDC')cupin] under acetic acid stress and hydrochloric acid stress, respectively, indicating its association with the acid stress response. Bacterial cells with targeted inactivation of the (OxDC')cupin gene using the pMUTIN4 vector system showed decreased growth and survival rate in acidic pH, with drastically reduced exopolysaccharide production. In Silico protein-protein interaction studies revealed seven genes (viz. glmS, nagA, nagB, tuaF, tuaF, gcvT, and ykgA) related to cell wall biosynthesis and biofilm production to interact with OxDC-like cupin domain containing protein. While all these seven genes were upregulated in B. amyloliquefaciens MBNC after 6 h of exposure to pH 4.5, the mutant cells containing the inactivated (OxDC')cupin gene displayed significantly lower expression (RQ: 0.001-0.02) (compared to the wild-type cells) in both neutral and acidic pH. Our results indicate that the OxDC-like cupin domain containing protein is necessary for cell wall biosynthesis and biofilm production in Bacillus amyloliquefaciens MBNC for survival in acid-stress conditions.

Genomic insights into Bacillus subtilis MBB3B9 mediated aluminium stress mitigation for enhanced rice growth.

Aluminium (Al) toxicity in acid soil ecosystems is a major impediment to crop production as it drastically affects plant root growth, thereby acquisition of nutrients from the soil. Plant growth-promoting bacteria offers an interesting avenue for promoting plant growth under an Al-phytotoxic environment. Here, we report the plant growth-promoting activities of an acid-tolerant isolate of Bacillus subtilis that could ameliorate acid-induced Al-stress in rice (Oryza sativa L.). The whole genome sequence data identified the major genes and genetic pathways in B. subtilis MBB3B9, which contribute to the plant growth promotion in acidic pH. Genetic pathways for organic acid production, denitrification, urea metabolism, indole-3-acetic acid (IAA) production, and cytokinin biosynthesis were identified as major genetic machinery for plant growth promotion and mitigation of Al-stress in plants. The in-vitro analyses revealed the production of siderophores and organic acid production as primary mechanisms for mitigation of Al-toxicity. Other plant growth-promoting properties such as phosphate solubilization, zinc solubilization, and IAA production were also detected in significant levels. Pot experiments involving rice under acidic pH and elevated concentrations of aluminium chloride (AlCl3) suggested that soil treatment with bacterial isolate MBB3B9 could enhance plant growth and productivity compared to untreated plants. A significant increase in plant growth and productivity was recorded in terms of plant height, chlorophyll content, tiller number, panicle number, grain yield, root growth, and root biomass production.

Imidacloprid degrading efficiency of Pseudomonas plecoglossicida MBSB-12 isolated from pesticide contaminated tea garden soil of Assam.

Long-term use of toxic pesticides in agricultural grounds has led to adverse effects on the environment and human health. Microbe-mediated biodegradation of pollutants is considered an effective strategy for the removal of contaminants in agricultural and environmental sustainability. Imidacloprid, a neonicotinoid class of pesticides, was widely applied insecticide in the control of pests in agricultural fields including the tea gardens of Assam. Here, native bacteria from imidacloprid contaminating tea garden soils were isolated and screened for imidacloprid degradation efficiency under laboratory conditions. Out of the 30 bacterial isolates, 4 were found to tolerate high concentrations of imidacloprid (25,000 ppm), one of which isolate MBSB-12 showed the highest efficiency for imidacloprid tolerance and utilization as the sole carbon source. Morphological, biochemical, and 16 S ribosomal RNA gene sequencing-based characterization revealed the isolate as Pseudomonas plecoglossicida MBSB-12. The isolate reduced 87% of extractable imidacloprid from the treated soil in 90 days compared to the control soil (without bacterial treatment). High-Resolution Mass Spectrometry (HRMS) analysis indicated imidacloprid breakdown to comparatively less harmful products viz., imidacloprid guanidine olefin [m/z = 209.0510 (M + H)+], imidacloprid urea [m/z = 212.0502 (M + H)+] and a dechlorinated degraded product of imidacloprid with m/z value 175.0900 (M + H)+. Further investigation on the molecular machinery of P. plecoglossicida MBSB-12 involved in the degradation of imidacloprid is expected to provide a better understanding of the degradation pathway.

Common scab disease-induced changes in geocaulosphere microbiome assemblages and functional processes in landrace potato (Solanum tuberosum var. Rongpuria) of Assam, India.

Common scab (CS) caused by pathogenic Streptomyces spp. plays a decisive role in the qualitative and quantitative production of potatoes worldwide. Although the CS pathogen is present in Assam's soil, disease signs and symptoms are less obvious in the landrace Rongpuria potatoes that indicate an interesting interaction between the plant and the geocaulosphere microbial population. Toward this, a comparative metagenomics study was performed to elucidate the geocaulosphere microbiome assemblages and functions of low CS-severe (LSG) and moderately severe (MSG) potato plants. Alpha diversity indices showed that CS occurrence modulated microbiome composition and decreased overall microbial abundances. Functional analysis involving cluster of orthologous groups (COG) too confirmed reduced microbial metabolism under disease incidence. The top-three most dominant genera were Pseudomonas (relative abundance: 2.79% in LSG; 12.31% in MSG), Streptomyces (2.55% in LSG; 5.28% in MSG), and Pantoea (2.30% in LSG; 3.51% in MSG). As shown by the high Pielou's J evenness index, the potato geocaulosphere core microbiome was adaptive and resilient to CS infection. The plant growth-promoting traits and potential antagonistic activity of major taxa (Pseudomonads, non-pathogenic Streptomyces spp., and others) against the CS pathogen, i.e., Streptomyces scabiei, point toward selective microbial recruitment and colonization strategy by the plants to its own advantage. KEGG Orthology analysis showed that the CS infection resulted in high abundances of ATP-binding cassette transporters and a two-component system, ubiquitous to the transportation and regulation of metabolites. As compared to the LSG metagenome, the MSG counterpart had a higher representation of important PGPTs related to 1-aminocyclopropane-1-carboxylate deaminase, IAA production, betaine utilization, and siderophore production.

Agricultural Land Use Influences Bacteriophage Community Diversity, Richness, and Heterogeneity.

The last two decades have witnessed a large-scale conversion of crop cultivation areas into small and mid-sized tea plantations in Assam, India. Agricultural land-use pattern positively or negatively influences native hydrology and above- and belowground biodiversity. Very little is known about the effect of agricultural land-use patterns on the soil virus (especially, bacteriophage) community structure and function. This metagenomic-based study evaluated the rhizosphere viral community structure of three interlinked cultivation areas, viz., mixed cropping area (coded as CP1), tea-seed orchard (CP2), and monocropping tea cultivation (CP3). The bacteriophages belonged to four major classes with the dominance of Malgrandaviricetes (CP1: 79.37%; CP2: 64.62%; CP3: 4.85%) followed by Caudoviricetes (CP1: 20.49%; CP2: 35.22%; CP3: 90.29%), Faserviricetes (CP1: 0.03%; CP2: 0.08%; CP3: 3.88%), and Tectiliviricetes (CP1: 0.12%; CP2: 0.07%; CP3: 0.97%). Microviruses dominated the phage population in both CP1 and CP2, representing 79.35% and 64.59% of total bacteriophage abundance. Both CP1 and CP2 had higher bacteriophage richness (species richness, R in CP1: 65; R in CP2: 66) and lower evenness (Pielou's evenness index, J in CP1: 0.531; J in CP2: 0.579) compared to the CP3 (R: 30; J: 0.902). Principal component analysis of edaphic soil factors and bacteriophage community structure showed a reverse-proportional correlation between the levels of Al saturation, and exchangeable Al3+ ions with that of soil pH, and bacteriophage abundance. Our study indicates that monocropping tea cultivation soil bears less viral richness, abundance, and heterogeneity.

Proline confers acid stress tolerance to Bacillus megaterium G18.

Proline plays a multifunctional role in several organisms including bacteria in conferring protection under stress conditions. In this paper we report the role of proline in conferring acid tolerance to Bacillus megaterium G18. An acid susceptible mutant of B. megaterium G18 which required proline for its growth under acid stress condition was generated through Tn5 mutagenesis. Further, targeted inactivation of proC involved in osmo-adaptive proline synthesis in B. megaterium G18 resulted in the loss of ability of the bacterium to grow at low pH (pH 4.5). Exogenous supply of proline (1 mM) to the growth medium restored the ability of the mutant cells to grow at pH 4.5 which was not the same in case of other osmoprotectants tested. Proline was produced and secreted to extracellular medium by B. megaterium G18 when growing in low pH condition as evidenced by the use of Escherichia coli proline auxotrophs and HPLC analysis. Further, pHT01 vector based expression of full length proC gene in the ∆proC mutant cells restored the survival capacity of the mutant cells in acidic pH, suggesting that proline production is an important strategy employed by B. megaterium G18 to survive under acid stress induced osmotic stress.

Identification of Putative Vaccine and Drug Targets against the Methicillin-Resistant Staphylococcus aureus by Reverse Vaccinology and Subtractive Genomics Approaches.

Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen and responsible for causing life-threatening infections. The emergence of hypervirulent and multidrug-resistant (MDR) S. aureus strains led to challenging issues in antibiotic therapy. Consequently, the morbidity and mortality rates caused by S. aureus infections have a substantial impact on health concerns. The current worldwide prevalence of MRSA infections highlights the need for long-lasting preventive measures and strategies. Unfortunately, effective measures are limited. In this study, we focus on the identification of vaccine candidates and drug target proteins against the 16 strains of MRSA using reverse vaccinology and subtractive genomics approaches. Using the reverse vaccinology approach, 4 putative antigenic proteins were identified; among these, PrsA and EssA proteins were found to be more promising vaccine candidates. We applied a molecular docking approach of selected 8 drug target proteins with the drug-like molecules, revealing that the ZINC4235426 as potential drug molecule with favorable interactions with the target active site residues of 5 drug target proteins viz., biotin protein ligase, HPr kinase/phosphorylase, thymidylate kinase, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-L-lysine ligase, and pantothenate synthetase. Thus, the identified proteins can be used for further rational drug or vaccine design to identify novel therapeutic agents for the treatment of multidrug-resistant staphylococcal infection.

Niche differentiation of belowground microorganisms and their functional signatures in Assam type tea (Camellia sinensis var. assamica).

We employed an Illumina-based high-throughput metagenomics sequencing approach to unveil the rhizosphere and root endosphere microbial community associated with an organically grown Camellia population located at the Experimental Garden for Plantation Crops, Assam (India). The de novo assembled tea root endosphere metagenome contained 24,231 contigs (total 7,771,089 base pairs with an average length of 321 bps), while tea rhizosphere soil metagenome contained 261,965 sequences (total 230,537,174 base pairs, average length 846). The most prominent rhizobacteria belonged to the genera, viz., Bacillus (10.35%), Candidatus Solibacter (6.36%), Burkholderia (5.19%), Pseudomonas (3.9%), Streptomyces (3.52%), and Bradyrhizobium (2.77%), while the root endosphere was dominated by bacterial genera, viz., Serratia (46.64%), Methylobacterium (8.02%), Yersinia (5.97%), Burkholderia (2.05%), etc. The presence of few agronomically important bacterial genera, Bradyrhizobium, Rhizobium (each 0.93%), Sinorhizobium (0.34%), Azorhizobium, and Flavobacterium (0.17% each), was also detected in the root endosphere. KEGG pathway mapping indicated the presence of microbial metabolic pathway genes related to tyrosine metabolism, tryptophan metabolism, glyoxylate, and dicarboxylate metabolism which play important roles in endosphere activities, including survival, growth promotion, and host adaptation. The root endosphere microbiome also contained few important plant growth promoting traits related to phytohormone production, abiotic stress alleviation, mineral solubilization, and plant disease suppression.

Comparative assessment of multi-trait plant growth-promoting endophytes associated with cultivated and wild Oryza germplasm of Assam, India.

This paper presents a comparative study of endophytic bacteria from cultivated (Oryza sativa) and wild rice (Oryza rufipogon) plants and their functional traits related to plant growth promotion. A total of 70 bacterial isolates were characterized by both biochemical and molecular identification methods. Taxonomic classification showed dominance of three major phyla, viz, Firmicutes (57.1%), Actinobacteria (20.0%) and Proteobacteria (22.8%). Screening for in vitro plant growth-promoting activities revealed a hitherto unreported endophytic bacterium from wild rice germplasm, Microbacterium laevaniformans RS0111 with highest indole acetic acid (28.39 ± 1.39 µg/ml) and gibberellic acid (67.23 ± 1.83 µg/ml) producing efficiency. Few other endophytic isolates from cultivated rice germplasm such as Bacillus tequilensis RHS01 showed highest phosphate solubilizing activity (81.70 ± 1.98 µg/ml), while Microbacterium testaceum MKLS01 and Microbacterium enclense MI03 L05 showed highest potassium (53.42 ± 0.75 µg/ml) and zinc solubilizing activity (157.50%). Fictibacillus aquaticus LP20 05 produced highest siderophore (64.8%). In vivo evaluation of plant growth-promoting efficiencies of the isolates showed that Microbacterium laevaniformans RS0111, Microbacterium testaceum MKLS01 and Bacillus tequilensis RHS 01 could increase rice grain yield by 3.4-fold when compared to the control group. This study indicates the potentiality of rice endophytes isolates as an effective bioinoculants.

Assessment of microbial quality and health risks associated with traditional rice wine starter Xaj-pitha of Assam, India: a step towards defined and controlled fermentation.

This study reports the microbial quality of ethnic starter culture Xaj-pitha used for rice wine fermentation in Assam. Here, we collected 60 Xaj-pitha samples belonging to Ahom community of the state and enumerated the microorganisms using spread plate technique. Illumina-based whole genome shotgun sequencing detected the presence of microbial contaminants like Acidovorax, Herbaspirillum, Methylobacterium, Pantoea, Pseudomonas, Stenotrophomonas, Staphylococcus, Micrococcus, Acinetobacter, etc. Presence of major health hazards associated with spontaneous rice wine fermentation necessitated method optimization through the development of a defined mixed starter culture. For this, functionally important α-amylase producers viz., Penicillium sp. ABTSJ23, Rhizopus oryzae ABTSJ63, Mucor guilliermondii ABTSJ72 and Amylomyces rouxii ABTSJ82 and eight yeasts viz., Saccharomyces cerevisiae ABTY1J, ABTY1S, ADJ5 & ADJ1, Wickerhamomyces anomalus ADJ2, Saccharomycopsis malanga ADJ3, Saccharomycopsis fibuligera ADJ4 and Saccharomycopsis malanga ADJ6 were retrieved using appropriate media. All the mould cultures tested negative for aflotoxins production. Among the yeasts, Saccharomyces cerevisiae ABTY1S and ADJ1 decarboxylated lysine HCl and tyramine HCl, respectively, indicating their biogenic amine production ability. For defined mixed starter culture, Amylomyces rouxii ABT82 with α-amylase (5.92 U/ml) and glucoamylase (7.50 U/ml) activities was selected as fungal partner; while Saccharomycopsis fibuligera ADJ4 and Saccharomyces cerevisiae ABT-Y1J with high ethanol production (up to 10.11% and 9.88% v/v, respectively) were selected as yeast partners. The mixed culture was able to produce high amount of glucose, ethanol and liquid (glucose 10.91% w/v; ethanol 7.5% w/v; liquid 51.0% w/v). Therefore, this study demonstrated the efficiency of mixed starter cultures for safe and controlled rice wine production.

Diversity and functional properties of acid-tolerant bacteria isolated from tea plantation soil of Assam.

In this study, we report on the bacterial diversity and their functional properties prevalent in tea garden soils of Assam that have low pH (3.8-5.5). Culture-dependent studies and phospholipid fatty acid analysis revealed a high abundance of Gram-positive bacteria. Further, 70 acid-tolerant bacterial isolates characterized using a polyphasic taxonomy approach could be grouped to the genus Bacillus, Lysinibacillus, Staphylococcus, Brevundimonas, Alcaligenes, Enterobacter, Klebsiella, Escherichia, and Aeromonas. Among the 70 isolates, 47 most promising isolates were tested for their plant growth promoting activity based on the production of Indole Acetic Acid (IAA), siderophore, and HCN as well as solubilization of phosphate, zinc, and potassium. Out of the 47 isolates, 10 isolates tested positive for the entire aforesaid plant growth promoting tests and further tested for quantitative analyses for production of IAA, siderophore, and phosphate solubilization at the acidic and neutral condition. Results indicated that IAA and siderophore production, as well as phosphate solubilization efficiency of the isolates decreased significantly (P ≤ 0.05) in the acidic environment. This study revealed that low soil pH influences bacterial community structure and their functional properties.

A metagenomic approach to decipher the indigenous microbial communities of arsenic contaminated groundwater of Assam.

Metagenomic approach was used to understand the structural and functional diversity present in arsenic contaminated groundwater of the Ganges Brahmaputra Delta aquifer system. A metagene dataset (coded as TTGW1) of 89,171 sequences (totaling 125,449,864 base pairs) with an average length of 1406 bps was annotated. About 74,478 sequences containing 101,948 predicted protein coding regions passed the quality control. Taxonomical classification revealed abundance of bacteria that accounted for 98.3% of the microbial population of the metagenome. Eukaryota had an abundance of 1.1% followed by archea that showed 0.4% abundance. In phylum based classification, Proteobacteria was dominant (62.6%) followed by Bacteroidetes (11.7%), Planctomycetes (7.7%), Verrucomicrobia (5.6%), Actinobacteria (3.7%) and Firmicutes (1.9%). The Clusters of Orthologous Groups (COGs) analysis indicated that the protein regulating the metabolic functions constituted a high percentage (18,199 reads; 39.3%) of the whole metagenome followed by the proteins regulating the cellular processes (22.3%). About 0.07% sequences of the whole metagenome were related to genes coding for arsenic resistant mechanisms. Nearly 50% sequences of these coded for the arsenate reductase enzyme (EC. 1.20.4.1), the dominant enzyme of ars operon. Proteins associated with iron acquisition and metabolism were coded by 2% of the metagenome as revealed through SEED analysis. Our study reveals the microbial diversity and provides an insight into the functional aspect of the genes that might play crucial role in arsenic geocycle in contaminated ground water of Assam.

Molecular Modeling and Dynamics Simulation Analysis of KATNAL1 for Identification of Novel Inhibitor of Sperm Maturation.

BACKGROUND: Hormone based birth control often causes various side effects. A recent study revealed that temporary infertility without changing hormone levels can be attained by inhibiting Katanin p60 ATPase-containing subunit A-like 1 protein (KATNAL1) which is critical for sperm maturation in the testes. OBJECTIVE: This study aimed at attaining the most energetically stable three dimensional (3D) structure of KATNAL1 protein using comparative modeling followed by screening of a ligand library of known natural spermicidal compounds for their binding affinity with KATNAL1. This in turn may inhibit the development of mature sperm in the seminiferous epithelium. METHOD: A series of computational techniques were used for building the 3D structure of KATNAL1 which was further optimized by molecular dynamics (MD) simulation. For revealing the ATP binding mode of KATNAL1, docking study was carried out using the optimized model obtained from the MD simulation. The docking study was also employed to test the binding efficiency of the ligand library. RESULTS: Molecular docking study confirmed the ATP binding of KATNAL1 with various hydrophobic and hydrogen bond interactions. Binding efficiency of the ligand library suggested that calotropin, a cardenolide of Calotropis procera showed the highest binding efficiency against the target protein without toxicity. MD simulation of the docked complex validated the results of the docking study. CONCLUSION: This study revealed the ATP binding mode of KATNAL1 and identified calotropin as a potential lead molecule against it showing high binding efficiency with good bioavailability and no mutagenicity. Further in vitro and in vivo bioassay of calotropin could facilitate the development of novel non-hormonal male-specific contraceptive in near future.

Metagenomics analysis of microbial communities associated with a traditional rice wine starter culture (Xaj-pitha) of Assam, India.

This is the first report on the microbial diversity of xaj-pitha, a rice wine fermentation starter culture through a metagenomics approach involving Illumine-based whole genome shotgun (WGS) sequencing method. Metagenomic DNA was extracted from rice wine starter culture concocted by Ahom community of Assam and analyzed using a MiSeq® System. A total of 2,78,231 contigs, with an average read length of 640.13 bp, were obtained. Data obtained from the use of several taxonomic profiling tools were compared with previously reported microbial diversity studies through the culture-dependent and culture-independent method. The microbial community revealed the existence of amylase producers, such as Rhizopus delemar, Mucor circinelloides, and Aspergillus sp. Ethanol producers viz., Meyerozyma guilliermondii, Wickerhamomyces ciferrii, Saccharomyces cerevisiae, Candida glabrata, Debaryomyces hansenii, Ogataea parapolymorpha, and Dekkera bruxellensis, were found associated with the starter culture along with a diverse range of opportunistic contaminants. The bacterial microflora was dominated by lactic acid bacteria (LAB). The most frequent occurring LAB was Lactobacillus plantarum, Lactobacillus brevis, Leuconostoc lactis, Weissella cibaria, Lactococcus lactis, Weissella para mesenteroides, Leuconostoc pseudomesenteroides, etc. Our study provided a comprehensive picture of microbial diversity associated with rice wine fermentation starter and indicated the superiority of metagenomic sequencing over previously used techniques.