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.

Cinnabarinic acid from Trametes coccinea fruiting bodies exhibits antibacterial activity through inhibiting the biofilm formation.

Wild mushrooms are rich sources of natural compounds with potent bioactive properties. Several important metabolites have been reported from mushrooms, which possess clinically important bioactive properties like antibacterial, anticancer, antidiabetic, and neuroprotective activity. In this study, we have evaluated the antimicrobial activity of Trametes coccinea fruiting body extracts against different bacterial isolates, viz., Bacillus subtilis, Bacillus cereus, and Escherichia coli. Fruiting bodies of three T. coccinea samples, of which two were collected from Santipur, Arunachal Pradesh and one collected from Jorhat, Assam, were used for extraction using methanol. The extracts showed significant antimicrobial activity against all the test bacteria. Minimum Inhibitory Concentration (MIC) of the extracts against Bacillus subtilis, Bacillus cereus, and Escherichia coli was recorded as 400 µg/ml, 400 µg/ml, and 300 µg/ml, respectively. Furthermore, the bioactive compounds of the extract were separated and detected using Thin Layer Chromatography (TLC). Presence of cinnabarinic acid (CBA)-a potent antimicrobial compound- was detected in TLC, which was further confirmed through High Performance Liquid Chromatography (HPLC) and Electrospray Ionization-Mass Spectrometry (ESI-MS). Cinnabarinic acid was able to inhibit the formation of biofilms in Bacillus subtilis and B. cereus, suggesting that the compound can be beneficial in the management of biofilm-based antimicrobial resistance.

Acid tolerant bacterium Bacillus amyloliquefaciens MBNC retains biocontrol efficiency against fungal phytopathogens in low pH.

Soil pH conditions have important consequences for microbial community structure, their dynamics, ecosystem processes, and interactions with plants. Low soil pH affects the growth and functional activity of bacterial biocontrol agents which may experience a paradigm shift in their ability to act antagonistically against fungal phytopathogens. In this study, the antifungal activity of an acid-tolerant soil bacterium Bacillus amyloliquefaciens MBNC was evaluated under low pH and compared to its activity in neutral pH conditions. Bacterial supernatant from 3-day-old culture (approximately 11.2 × 108 cells/mL) grown in low pH conditions was found more effective against fungal pathogens. B. amyloliquefaciens MBNC harboured genes involved in the synthesis of secondary metabolites of which surfactin homologues, with varying chain length (C11-C15), were identified through High-Resolution Mass Spectroscopy. The pH of the medium influenced the production of these metabolites. Surfactin C15 was exclusive to the extract of pH 4.5; production of iturinA and surfactin C11 was detected only in pH 7.0, while surfactin C12, C13 and C14 were detected in extracts of both the pH conditions. The secretion of phytohormones viz. indole acetic acid and gibberellic acid by B. amyloliquefaciens MBNC was detected in higher amounts in neutral condition compared to acidic condition. Although, secretion of metabolites and phytohormones in B. amyloliquefaciens MBNC was influenced by the pH condition of the medium, the isolate retained its antagonistic efficiency against several fungal phyto-pathogens under acidic condition.

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.

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.

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.

Lipopeptide mediated biocontrol activity of endophytic Bacillus subtilis against fungal phytopathogens.

BACKGROUND: The use of chemical fungicides against fungal pathogens adversely affects soil and plant health thereby resulting in overall environmental hazards. Therefore, biological source for obtaining antifungal agents is considered as an environment-friendly alternative for controlling fungal pathogens. RESULTS: In this study, seven endophytic bacteria were isolated from sugarcane leaves and screened for its antifungal activity against 10 fungal isolates belonging to the genera Alternaria, Cochliobolus, Curvularia, Fusarium, Neodeightonia, Phomopsis and Saccharicola isolated from diseased leaves of sugarcane. Among the seven bacterial isolates, SCB-1 showed potent antagonistic activity against the tested fungi. Based on the phenotypic data, Fatty Acid Methyl Esters (FAME) and 16S rRNA gene sequence analysis, the isolate SCB-1 was identified as Bacillus subtilis. The bacterial isolate was screened negative for chitinase production; however, chloroform and methanol extracts of the bacterial culture caused significant inhibition in the growth of the fungal isolates on semisolid media. Volatile component assay showed highest inhibitory activity against Saccharicola bicolor (SC1.4). A PCR based study detected the presence of the genes involved in biosynthesis of surfactin, bacillaene, difficidin, macrolactins and fengycin. Mass spectrometric analysis of the bacterial extract detected the presence of antifungal lipopeptide surfactin, but other metabolites were not detected. The biocontrol activity of the bacterial isolate was established when bacterial pretreated mung bean seeds were able to resist Fusarium infection, however, the untreated seeds failed to germinate. CONCLUSION: The antifungal potential of isolate Bacillus subtilis SCB-1 was established against taxonomically diverse fungal pathogens including the genera Saccharicola, Cochliobolus, Alternaria and Fusarium. The potent antifungal compound surfactin as well as volatiles produced by the bacterial isolate could be responsible for its bio-control activity against fungal infections.

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.

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.

Fungal interactions induce changes in hyphal morphology and enzyme production.

In nature, species interacts/competes with one other within their surrounding for food and space and the type of interactions are unique to each species. The interacting partners secrete different metabolites, which may have high importance in human welfare. Fungal-fungal interactions are complex mechanisms that need better understanding. Here, 14 fungal isolates were facilitated in 105 possible combinations to interact on potato dextrose agar. Morphologically, no changes were observed when the same fungal isolates were allowed to interact within them. However, 10 interactions between different fungal isolates showed mutual replacement with each fungus; capturing territory from the other. Contrastingly, 35 interactions resulted into complete replacement as one of the fungi was inhibited by rapid growth of the other fungus. In 46 interactions, formation of barrage was observed leading to deadlock type of interaction wherein both fungi have restricted growth. To study in details about the barrage formation, two fungal interactions were taken (i) T. coccinea vs. L. lactinea and (ii) T. coccinea vs. T. versicolor. Microscopic changes in the hyphal growth during interaction were observed. There was significant increase in the enzymatic activities including cellulase, xylanase and chitinase during in-vitro fungal-fungal interaction, suggesting the importance of such interactions for commercial enzyme production.

Melanin production and laccase mediated oxidative stress alleviation during fungal-fungal interaction among basidiomycete fungi.

Fungal-fungal interaction often leads to the change in metabolite profile of both the interacting fungus which may have potential implication in industry or agriculture. In the present study, we performed two sets of fungal-fungal interaction-Trametes coccinea (F3) with Leiotrametes lactinea (F9) and T. coccinea (F3) with T. versicolor (F1) to understand the changes in the metabolite profile during the interaction process and how this process impacts the hyphal/mycelial morphology of the participating fungi. The metabolites produced during interaction of T. coccinea (F3) with L. lactinea (F9) and T. coccinea (F3) with T. versicolor (F1) was analysed through liquid chromatography coupled to mass spectroscopy (LC-MS). Most of the metabolites secreted or produced during interaction are associated with defensive response. Further, visualization with scanning electron microscopy revealed that interaction between the tested fungi led to the changes in the hyphal morphology. The bipartite fungal interaction resulted in the production of a dark brown colour pigment-melanin as confirmed by the LC-MS, FTIR and NMR analysis. Moreover, the fungal-fungal interaction also led to increase in the production of laccase, a group of multicopper oxidases involved in detoxification of toxic compounds. Further, increased activity of superoxide dismutase, an enzyme that catalyzes the dismutation of the superoxide anion to hydrogen peroxide was also recorded during fungal-fungal interaction. Quantitative real-time PCR revealed upregulation of lcc1 (encoding a laccase enzyme) and few other stress related genes of T. versicolor during its hyphal interaction with T. coccinea, suggesting a direct correlation between laccase production and melanin production.

Mechanism of interaction of an endofungal bacterium Serratia marcescens D1 with its host and non-host fungi.

Association of bacteria with fungi is a major area of research in infection biology, however, very few strains of bacteria have been reported that can invade and reside within fungal hyphae. Here, we report the characterization of an endofungal bacterium Serratia marcescens D1 from Mucor irregularis SS7 hyphae. Upon re-inoculation, colonization of the endobacterium S. marcescens D1 in the hyphae of Mucor irregularis SS7 was demonstrated using stereo microscopy. However, S. marcescens D1 failed to invade into the hyphae of the tested Ascomycetes (except Fusarium oxysporum) and Basidiomycetes. Remarkably, Serratia marcescens D1 could invade and spread over the culture of F. oxysporum that resulted in mycelial death. Prodigiosin, the red pigment produced by the Serratia marcescens D1, helps the bacterium to invade fungal hyphae as revealed by the increasing permeability in fungal cell membrane. On the other hand, genes encoding the type VI secretion system (T6SS) assembly protein TssJ and an outer membrane associated murein lipoprotein also showed significant up-regulation during the interaction process, suggesting the involvement of T6SS in the invasion process.