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.

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.

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.

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.

A pH-Dependent Gene Expression Enables Bacillus amyloliquefaciens MBNC to Adapt to Acid Stress.

Acid tolerance response (ATR), a process by which bacteria optimize their growth conditions for cellular functions, is a well-characterized bacterial stress response. A bacterial isolate identified, as Bacillus amyloliquefaciens MBNC, was isolated from acidic soil and studied for its acid tolerance response under several range of acidic stress conditions imposed through inorganic acid, organic acid, acetate buffer, and soil extract. The ability of the B. amyloliquefaciens MBNC to tolerate extreme acidic conditions (pH 4.5) increased when exposed to moderate-acidic pH (pH 5.5). Along with ATR, the bacterial cell density was also critical to its ability to tolerate low pH as the cells of late log phase were more tolerant to low pH stress compared to the early log phase cells. A comparative expression study of 28 genes of B. amyloliquefaciens MBNC was assessed in cells grown in neutral (pH 7.0) and acidic condition (pH 4.5) through qRT-PCR. Among the 28 genes analyzed, 24 genes showed increased expression whereas the expression of 4 genes was downregulated under acid stress indicating to the involvement of the genes in acid stress response.

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.

Exemplifying endophytes of banana (Musa paradisiaca) for their potential role in growth stimulation and management of Fusarium oxysporum f. sp cubense causing panama wilt.

In the present study, potentiality of endophytic microorganisms such as Rigidiporus vinctus AAU EF, Trichoderma reesei UH EF, and Sphingobacterium tabacisoli UH EB in the management of panama wilt and growth promotion of banana was assessed through artificial inoculation. During the study, a total of 220 bacterial and 110 fungal endophytes were isolated from root, pseudostem, and leaf samples of banana, and they were evaluated against Fusarium oxysporum f. sp cubense causing panama wilt. Out of total 330 bacterial and fungal endophytes, only five endophytes exhibited antagonism against Fusarium oxysporum f. sp cubense, out of which only three isolates, namely Trichoderma reesei UH EF, Rigidiporus vinctus AAU EF, and Sphingobacterium tabacisoli UH EB, produced indole acetic acid, siderophore, and hydrogen cyanide, except one bacterial strain Sphingobacterium tabacisoli UH EB which does not produce hydrogen cyanide. Furthermore, these three endophytes were identified through cultural and morphological characteristics as well as by the sequencing internal transcribed spacer (ITS) and 16S rRNA gene sequences analysis for bacteria, respectively. The response of host plant to endophyte inoculation was assessed by measuring the change in four growth parameters; plant height, pseudo stem girth (diameter), number of roots, and total number of leaves. The application of endophytes, irrespective of isolate and treatment type promoted the overall growth of the plant growth when compared with diseased plants with significant higher values recorded for all parameters assessed. The endophytes reported as growth promoters were found to have significant inhibition effect on Foc which can evidenced with lowest AUDPC values and epidemic rate at 99.09 units2 and 0.02 unit/day, respectively.

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.

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.

Bacterial exopolysaccharide promotes acid tolerance in Bacillus amyloliquefaciens and improves soil aggregation.

In this paper we report the isolation and taxonomic characterization of exopolysaccharide (EPS) producing bacteria followed by the role of EPS in conferring acid tolerance to the soil bacteria Bacillus amyloliquefaciens p16. The role of EPS in promoting soil aggregation is also presented. A total of 75 isolates were tested for acid tolerance and biofilm production under acid stress of which, 54 isolates were further tested for EPS production. Out of the 54 isolates, 28 isolates produced EPS in the range of (67.88 and 219.96 µg/ml) with B. amyloliquefaciens p16 showing the highest production. The 28 isolates characterized for phenotypic and molecular traits mostly belonged to the members of the genera Bacillus, Brevibacillus, Brevibacterium, Paenibacillus, Serretia, Pseudomonas, Arthrobacter and Lysinibacillus. The monosaccharide components of the EPS produced by B. amyloliquefaciens p16 shifted from galactose to arabinose under acid stress as revealed through HPLC analysis. Inactivation of the epsB gene encoding putative bacterial protein tyrosine kinase (BY-kinases) in B. amyloliquefaciens p16 resulted in significantly less EPS (33.23 µg/ml) production compared to wild-type (WT) (223.87 µg/ml). The mutant (B. amyloliquefaciens 6A5) was barely able to survive in pH 4.5 unlike that of the WT. Further, inoculation of the WT and mutant B. amyloliquefaciens 6A5 in the soil resulted in formation of small sized soil aggregates (42.41 mm) with less water holding capacity (27.67%) as compared to the soil treated with WT that produced larger soil aggregates of size 80.59 mm with higher 53.90% water holding capacity. This study indicates that EPS produced by acid-tolerant B. amyloliquefaciens p16 can not only impart acid tolerance to the bacteria but also aids in promoting soil aggregation when applied to the soil.

Bacillus megaterium adapts to acid stress condition through a network of genes: Insight from a genome-wide transcriptome analysis.

RNA-seq analysis of B. megaterium exposed to pH 7.0 and pH 4.5 showed differential expression of 207 genes related to several processes. Among the 207 genes, 11 genes displayed increased transcription exclusively in pH 4.5. Exposure to pH 4.5 induced the expression of genes related to maintenance of cell integrity, pH homeostasis, alternative energy generation and modification of metabolic processes. Metabolic processes like pentose phosphate pathway, fatty acid biosynthesis, cysteine and methionine metabolism and synthesis of arginine and proline were remodeled during acid stress. Genes associated with oxidative stress and osmotic stress were up-regulated at pH 4.5 indicating a link between acid stress and other stresses. Acid stress also induced expression of genes that encoded general stress-responsive proteins as well as several hypothetical proteins. Our study indicates that a network of genes aid B. megaterium G18 to adapt and survive in acid stress condition.

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.