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20202
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Author: Chowdhury, Naimisha × Start date: 2020 ×
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    ThesisItemOpen Access
    Study on Acid Stress Response in Bacillus amyloliquefaciens
    (AAU, Jorhat, 2020-01) Chowdhury, Naimisha; Barooah, Madhumita
    Soil bacteria have evolved various mechanisms to adapt to stress environmental conditions such as temperature, salinity, drought and low pH condition of soil. Among the several environmental stress conditions, soil acidity an important factor influencing physicochemical and biological properties of soil along with microbial diversity and crop production is an emerging issue of immense concern due to its wide spread distribution across the globe. Although low soil pH restricts the number and diversity of bacteria, it is known that some soil bacteria are able to thrive in such conditions having evolved various mechanisms. The isolate Bacillus amyloliquefaciens MBNC can survive in low pH condition (upto pH 4.0). Moreover it can retain its antagonistic and plant growth promotion attributes under acidic condition. Early log phase cells of B.amyloliquefaciens MBNC were more susceptible to extreme pH than the late log phase cells. In addition, pre-exposed B.amyloliquefaciens MBNC cells to moderate acid condition had higher survival rate in extreme acid. In-vitro production of indole-3-acetic acid and gibberellic acid by B.amyloliquefaciens MBNC at neutral condition was higher (12.82±0.12 μg/ml and 35.15±0.005 μg/ml respectively) than at acidic condition (07.27±0.06 μg/ml and 26.66±0.003 μg/ml). Maximum antagonistic effect against fungal pathogens were shown 3 days post inoculation under acidic condition. High Resolution Mass Spectroscopy results shows the presence of surfactin C13 (exact mol.mass 1007.651836 g/mol) and surfactin C14 (exact mol.mass 1021.6677486 g/mol) in chloroform-methanol extract of both pH 7.0 and pH 4.5. Surfactin (exact mol.mass 1035.683136 g/mol) and iturinD was detected only in the extract of pH 4.5 and pH 7.0 respectively. For differential expression analysis a total of 22 genes were selected. Genes relating to maintenance of cell integrity (membrane integrity) showed increase expression under acid stress with an exception of spoA, flotilin and pdaA. Acid stress induce the environmental and energy stress regulon SigB, general stress protein gspA and the major molecular chaperones DnaK and GroES. pH-responsive gene, atpB (coding for the beta sub-unit of the F1F0-ATPase enzyme),cytochrome bd oxidase (cydA) and gene for nitrate reductase was upregulated. Cadmium, cobalt, and zinc/H/K antiporter (czcD), Iron-uptake system responsive genes (feuABC) and osmotic stress related gene pyrroline-5-carboxylate reductase (proC) was upregulated. Acidic pH upregulated acetoin production (alsDS), a pathway known to shunt fermentation into neutral products, minimizing acid production. Acid also upregulated a large number of NAD(P)-dependent dehydrogenases: alcohol dehydrogenase (eutG), formate dehydrogenases (fdhA), malate dehydrogenase (mdh). These enzymes are capable of removing acidity through NAD(P)H which transfers electrons to the electron transport system (ETS) and pumps protons out of the cell. The cupin gene (cupin superfamily of proteins) involved in the modification of cell wall carbohydrates showed manifold upregulation under acid stress. To further validate this, targeted mutagenesis of cupin in B.amyloliquefaciens MBNC was performed using pMUTIN4 integration vector. The transformation efficiency was very low with 32 transformants/μg pmutcupin DNA. The integration of pmutcupin into the chromosome of B.amyloliquefaciens MBNC was confirmed by PCR amplification of ermAM and bla gene using the genomic DNA of the transformed B.amyloliquefaciens MBNC. The mutant isolate, Δcupin failed to grow in nutrient broth at pH 4.5 which confirms the role of cupin gene in acid stress. Inability to form biofilm and scanning electron microscopy (SEM) of B.amyloliquefaciens MBNC wild type and its cupin mutant (Δcupin) at neutral pH confirms the targeted mutagenesis of cupin gene.
  • Thumbnail Image
    ThesisItemOpen Access
    Study on Acid Stress Response in Bacillus amyloliquefaciens
    (AAU, Jorhat, 2020-01) Chowdhury, Naimisha; Barooah, Madhumita
    Soil bacteria have evolved various mechanisms to adapt to stress environmental conditions such as temperature, salinity, drought and low pH condition of soil. Among the several environmental stress conditions, soil acidity an important factor influencing physicochemical and biological properties of soil along with microbial diversity and crop production is an emerging issue of immense concern due to its wide spread distribution across the globe. Although low soil pH restricts the number and diversity of bacteria, it is known that some soil bacteria are able to thrive in such conditions having evolved various mechanisms. The isolate Bacillus amyloliquefaciens MBNC can survive in low pH condition (upto pH 4.0). Moreover it can retain its antagonistic and plant growth promotion attributes under acidic condition. Early log phase cells of B.amyloliquefaciens MBNC were more susceptible to extreme pH than the late log phase cells. In addition, pre-exposed B.amyloliquefaciens MBNC cells to moderate acid condition had higher survival rate in extreme acid. In-vitro production of indole-3-acetic acid and gibberellic acid by B.amyloliquefaciens MBNC at neutral condition was higher (12.82±0.12 μg/ml and 35.15±0.005 μg/ml respectively) than at acidic condition (07.27±0.06 μg/ml and 26.66±0.003 μg/ml). Maximum antagonistic effect against fungal pathogens were shown 3 days post inoculation under acidic condition. High Resolution Mass Spectroscopy results shows the presence of surfactin C13 (exact mol.mass 1007.651836 g/mol) and surfactin C14 (exact mol.mass 1021.6677486 g/mol) in chloroform-methanol extract of both pH 7.0 and pH 4.5. Surfactin (exact mol.mass 1035.683136 g/mol) and iturinD was detected only in the extract of pH 4.5 and pH 7.0 respectively. For differential expression analysis a total of 22 genes were selected. Genes relating to maintenance of cell integrity (membrane integrity) showed increase expression under acid stress with an exception of spoA, flotilin and pdaA. Acid stress induce the environmental and energy stress regulon SigB, general stress protein gspA and the major molecular chaperones DnaK and GroES. pH-responsive gene, atpB (coding for the beta sub-unit of the F1F0-ATPase enzyme),cytochrome bd oxidase (cydA) and gene for nitrate reductase was upregulated. Cadmium, cobalt, and zinc/H/K antiporter (czcD), Iron-uptake system responsive genes (feuABC) and osmotic stress related gene pyrroline-5-carboxylate reductase (proC) was upregulated. Acidic pH upregulated acetoin production (alsDS), a pathway known to shunt fermentation into neutral products, minimizing acid production. Acid also upregulated a large number of NAD(P)-dependent dehydrogenases: alcohol dehydrogenase (eutG), formate dehydrogenases (fdhA), malate dehydrogenase (mdh). These enzymes are capable of removing acidity through NAD(P)H which transfers electrons to the electron transport system (ETS) and pumps protons out of the cell. The cupin gene (cupin superfamily of proteins) involved in the modification of cell wall carbohydrates showed manifold upregulation under acid stress. To further validate this, targeted mutagenesis of cupin in B.amyloliquefaciens MBNC was performed using pMUTIN4 integration vector. The transformation efficiency was very low with 32 transformants/μg pmutcupin DNA. The integration of pmutcupin into the chromosome of B.amyloliquefaciens MBNC was confirmed by PCR amplification of ermAM and bla gene using the genomic DNA of the transformed B.amyloliquefaciens MBNC. The mutant isolate, Δcupin failed to grow in nutrient broth at pH 4.5 which confirms the role of cupin gene in acid stress. Inability to form biofilm and scanning electron microscopy (SEM) of B.amyloliquefaciens MBNC wild type and its cupin mutant (Δcupin) at neutral pH confirms the targeted mutagenesis of cupin gene.