Fusion of the N-terminal 119 amino acids of RelA with the CTD domain render growth inhibitory effects of the latter, (p)ppGpp-dependent.

The guanosine nucleotide derivatives ppGpp and pppGpp are central to the remarkable capacity of bacteria to adapt to fluctuating environments and metabolic perturbations. They are synthesized by two proteins, RelA and SpoT in E. coli and the activities of each of the two enzymes are highly regulated for homeostatic control of intracellular (p)ppGpp levels. Characterization of the mutant studied here indicates that moderate level expression of RelA appreciably reduces growth of cells wherein the basal levels of (p)ppGpp are higher than in the wild type without elevating the levels further. Consistent with this result, a large part of the growth inhibition effect is reproduced by overexpression of RelA NTD-CTD fusion lacking the (p)ppGpp synthesis function. A null mutation in relA abolishes this growth inhibitory effect suggesting its requirement for basal level synthesis of (p)ppGpp. Accordingly, increase in the (p)ppGpp levels in the relA1 mutant by spoT202 mutation largely restored the growth inhibitory effects of overexpression of RelA NTD-CTD fusion. Expression of this construct consisting of 119 amino acids of the N-terminal hydrolytic domain (HD) fused in-frame with the CTD domain (±TGS domain) renders the growth inhibitory effects (p)ppGpp-responsive-inhibited growth only of spoT1 and spoT202 relA1 mutants. This finding uncovered an hitherto unrealized (p)ppGpp-dependent regulation of RelA-CTD function, unraveling the importance of RelA NTD-HD domain for its regulatory role. An incremental rise in the (p)ppGpp levels is proposed to progressively modulate the interaction of RelA-CTD with the ribosomes with possible implications in the feedback regulation of the (p)ppGpp synthesis function, a proposal that accounts for the nonlinear kinetics of (p)ppGpp synthesis and increased ratio of RelA:ribosomes, both in vitro as well as in vivo.

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 ~ 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.

Chromomycin, an antibiotic produced by Streptomyces flaviscleroticus might play a role in the resistance to oxidative stress and is essential for viability in stationary phase.

The well-known role of antibiotics in killing sensitive organisms has been challenged by the effects they exert at subinhibitory concentrations. Unfortunately, there are very few published reports on the advantages these molecules may confer to their producers. This study describes the construction of a genetically verified deletion mutant of Streptomyces flaviscleroticus unable to synthesize chromomycin. This mutant was characterized by a rapid loss of viability in stationary phase that was correlated with high oxidative stress and altered antioxidant defences. Altered levels of key metabolites in the mutant signalled a redistribution of the glycolytic flux toward the PPP to generate NADPH to fight oxidative stress as well as reduction of ATP-phosphofructokinase and Krebs cycle enzymes activities. These changes were correlated with a shift in the preference for carbon utilization from glucose to amino acids. Remarkably, chromomycin at subinhibitory concentration increased longevity of the non-producer and restored most of the phenotypic features' characteristic of the wild type strain. Altogether these observations suggest that chromomycin may have antioxidant properties that would explain, at least in part, some of the phenotypes of the mutant. Our observations warrant reconsideration of the secondary metabolite definition and raise the possibility of crucial roles for their producers.