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.

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.