Vitreoscilla hemoglobin promotes biofilm expansion and mitigates sporulation in Bacillus subtilis DK1042

Biofilm formation is considered as a stress combating strategy adopted by bacteria in response to variety of cellular and environmental signals. Impaired respiration due to low oxygen concentrations is one such signal that triggers wrinkling and robust biofilm formation in Bacillus subtilis. Vitreoscilla hemoglobin (VHb) improves microaerobic growth and bioproduct synthesis in a variety of bacteria by supplying oxygen to the respiratory chain. Present study was carried out to determine the effect of VHb on multicellularity of B. subtilis. Thus, B. subtilis DK1042 (WT) was genetically modified to express vgb and gfp genes under the control of P43 promoter at amyE locus by double cross over events. Biofilm formation by the integrant NRM1113 and WT was monitored on Lysogeny broth (LB) and LB containing glycerol and manganese (LBGM) medium. The WT produced more wrinkled colonies than NRM1113 on LB and LBGM medium. Concomitantly, biofilm-associated sporulation and production of pulcherriminic acid was decreased in NRM1113 as compared to WT on LB as well as LBGM. Expression studies of genes encoding structural components of biofilms revealed similar to 70% down-regulation of bslA gene in NRM1113 on both LB and LBGM which is correlated with reduced wrinkling in NRM1113. Moreover, NRM1113 showed increased colony expansion compared to WT in LB, LBGM and high osmolarity conditions. VHb expression alters various processes in different host cells, our study represents that VHb modulates biofilm formation, sporulation and pulcherriminic acid formation in B. subtilis DK1042.