Characterization of divergent promoters PmaiA and Phyd from Gordonia: Co-expression and regulation by CRP.

Divergent promoters are often responsible for controlling gene expression of related genes of the same pathway or for coordinating regulation at different time points. There are relatively few reports on characterization of divergent promoters in bacteria. In the present study, microarray profiling was carried out to analyze gene expression during growth of Gordonia sp. IITR100, which led to the identification of 35 % of adjacent gene candidates that are divergently transcribed. We focus here on the in-depth characterization of one such pair of genes. Two divergent promoters, PmaiA and Phyd, drive the expression of genes encoding maleate cis-trans isomerase (maiA) and hydantoinase (hyd), respectively. Our findings reveal asymmetric promoter activity with higher activity in the reverse orientation (Phyd) as compared to the forward orientation (PmaiA). Minimal promoter region for each orientation was identified by deletion mapping. Deletion of a 5'-untranslated region of each gene resulted in an increase in promoter activity. A putative binding site for CRP (Catabolite Repressor Protein) transcription regulator was also identified in the 80 bp common regulatory region between the -35 hexamers of the two promoters. The results of this study suggest that CRP-mediated repression of PmaiA occurs only in the cells grown in glucose. Phyd, on the other hand, is not repressed by CRP. However, deletion of the CRP binding site located between -95 to -110 upstream to the transcription start site of the maiA gene resulted in increased activity of PmaiA and decreased activity of Phyd. A single CRP binding site, therefore, affects the two promoters differently.

Bending is required for activation of dsz operon by the TetR family protein (DszGR).

The dsz operon responsible for the biodesulfurization of organosulfurs is under the control of a 385 bp long promoter. Recently, a TetR family protein was identified which served as an activator of operon. Here we report that the TetR family protein (WP_058249973.1), named DszGR can specifically activate the dsz operon. Direct binding of the DszGR to DNA was observed at single molecule level by AFM. It was found that the binding of DszGR to the promoter DNA induces a bend by about âˆ¼40-50° degrees which may not be enough for the activation of the promoter. Thus, bendability in the promoter sequence was analyzed. The results show that the promoter has a curvature at around -235 and -200 bp with respect to dszA start codon. On mutating this region, a decrease in activity of the promoter was observed. Our results suggest that the DszGR protein binds to the upstream sequences and induces a bend, which is facilitated by further bending of the DNA which is required for dsz promoter activity. IHF binding site present in the promoter, and a significant reduction in desulphurization activity in the absence of either IHF subunits, suggested role of IHF in regulation of the dsz operon.

Functional characterization of the transcription regulator WhiB1 from Gordonia sp. IITR100.

WhiB is a transcription regulator which has been reported to be involved in the regulation of cell morphogenesis, cell division, antibiotic resistance, stress, etc., in several members of the family Actinomycetes. The present study describes functional characterization of a WhiB family protein, WhiB1 (protein ID: WP_065632651.1), from Gordonia sp. IITR100. We demonstrate that WhiB1 affects chromosome segregation and cell morphology in recombinant Escherichia coli, Gordonia sp. IITR100 as well as in Rhodococcus erythropolis. Multiple sequence alignment suggests that WhiB1 is a conserved protein among members of the family Actinomycetes. It has been reported that overexpression of WhiB1 leads to repression of the biodesulfurization operon in recombinant E. coli, Gordonia sp. IITR100 and R. erythropolis. A WhiB1-mut containing a point mutation Q116A in the DNA binding domain of WhiB1 led to partial alleviation of repression of the biodesulfurization operon. We show for the first time that the WhiB family protein WhiB1 is also involved in repression of the biodesulfurization operon by directly binding to the dsz promoter DNA.