Structural and functional characterization of Rv0792c from Mycobacterium tuberculosis: identifying small molecule inhibitors against GntR protein

ABSTRACTIn order to adapt in host tissues, microbial pathogens regulate their gene expression through an array of transcription factors. Here, we have functionally characterized Rv0792c, a GntR homolog from M. tuberculosis. In comparison to the parental strain, ΔRv0792c mutant strain of M. tuberculosis was compromised for survival upon exposure to oxidative stress, cell wall agents and infection in guinea pigs. RNA-seq analysis revealed that Rv0792c regulates the expression of genes that are involved in stress adaptation and virulence of M. tuberculosis. Solution small angle X-ray scattering (SAXS) data steered model building confirmed that the C-terminal region plays a pivotal role in dimer formation. Systematic evolution of ligands by exponential enrichment resulted in identification of ssDNA aptamers that can be used as a tool to identify small molecule inhibitors targeting Rv0792c. Using SELEX and SAXS data based modelling, we identified residues essential for the DNA binding activity of Rv0792c and I-OMe-Tyrphostin as an inhibitor of Rv0792c aptamer binding activity. Taken together, we provide a detailed shape-function characterization of GntR family of transcription factors from M. tuberculosis. To the best of our knowledge, this is the first study that has resulted in the identification of small molecule inhibitors against GntR family of transcription factors from bacterial pathogens.

Regulation of alginate catabolism involves a GntR family repressor in the marine flavobacterium Zobellia galactanivorans DsijT

Marine flavobacteria possess dedicated Polysaccharide Utilization Loci (PULs) enabling efficient degradation of a variety of algal polysaccharides. The expression of these PULs is tightly controlled by the presence of the substrate, yet details on the regulatory mechanisms are still lacking. The marine flavobacterium Zobellia galactanivorans DsijT digests many algal polysaccharides, including alginate from brown algae. Its complex Alginate Utilization System (AUS) comprises a PUL and several other loci. Here, we showed that the expression of the AUS is strongly and rapidly (<30 min) induced upon addition of alginate, leading to biphasic substrate utilization. Polymeric alginate is first degraded into smaller oligosaccharides that accumulate in the extracellular medium before being assimilated. We found that AusR, a GntR family protein encoded within the PUL, regulates alginate catabolism by repressing the transcription of most AUS genes. Based on our genetic, genomic, transcriptomic and biochemical results, we propose the first model of regulation for a PUL in marine bacteria. AusR binds to promoters of AUS genes via single, double or triple copies of operator. Upon addition of alginate, secreted enzymes expressed at a basal level catalyze the initial breakdown of the polymer. Metabolic intermediates produced during degradation act as effectors of AusR and inhibit the formation of AusR/DNA complexes, thus lifting transcriptional repression.

Regulation of Glutarate Catabolism by GntR Family Regulator CsiR and LysR Family Regulator GcdR in Pseudomonas putida KT2440

ABSTRACT Glutarate, a metabolic intermediate in the catabolism of several amino acids and aromatic compounds, can be catabolized through both the glutarate hydroxylation pathway and the glutaryl-coenzyme A (glutaryl-CoA) dehydrogenation pathway in Pseudomonas putida KT2440. The elucidation of the regulatory mechanism could greatly aid in the design of biotechnological alternatives for glutarate production. In this study, it was found that a GntR family protein, CsiR, and a LysR family protein, GcdR, regulate the catabolism of glutarate by repressing the transcription of csiD and lhgO, two key genes in the glutarate hydroxylation pathway, and by activating the transcription of gcdH and gcoT, two key genes in the glutaryl-CoA dehydrogenation pathway, respectively. Our data suggest that CsiR and GcdR are independent and that there is no cross-regulation between the two pathways. l-2-Hydroxyglutarate (l-2-HG), a metabolic intermediate in the glutarate catabolism with various physiological functions, has never been elucidated in terms of its metabolic regulation. Here, we reveal that two molecules, glutarate and l-2-HG, act as effectors of CsiR and that P. putida KT2440 uses CsiR to sense glutarate and l-2-HG and to utilize them effectively. This report broadens our understanding of the bacterial regulatory mechanisms of glutarate and l-2-HG catabolism and may help to identify regulators of l-2-HG catabolism in other species. IMPORTANCE Glutarate is an attractive dicarboxylate with various applications. Clarification of the regulatory mechanism of glutarate catabolism could help to block the glutarate catabolic pathways, thereby improving glutarate production through biotechnological routes. Glutarate is a toxic metabolite in humans, and its accumulation leads to a hereditary metabolic disorder, glutaric aciduria type I. The elucidation of the functions of CsiR and GcdR as regulators that respond to glutarate could help in the design of glutarate biosensors for the rapid detection of glutarate in patients with glutaric aciduria type I. In addition, CsiR was identified as a regulator that also regulates l-2-HG metabolism. The identification of CsiR as a regulator that responds to l-2-HG could help in the discovery and investigation of other regulatory proteins involved in l-2-HG catabolism.

Molecular and Functional Insights into the Regulation of D-Galactonate Metabolism by the Transcriptional Regulator DgoR inEscherichia coli

ABSTRACTd-Galactonate, an aldonic sugar acid, is used as a carbon source byEscherichia coli, and the structuraldgogenes involved in its metabolism have previously been investigated. Here, using genetic, biochemical and bioinformatics approaches, we present the first detailed molecular and functional insights into the regulation ofd-galactonate metabolism inE. coliK-12 by the transcriptional regulator DgoR. We found thatdgoRdeletion accelerates the growth ofE. coliind-galactonate concomitant with the strong constitutive expression ofdgogenes. In thedgolocus, sequence upstream ofdgoRalone harbors thed-galactonate-inducible promoter that likely drives the expression of alldgogenes. DgoR exerts repression on thedgooperon by binding two inverted repeats overlapping thedgopromoter. Binding ofd-galactonate induces a conformational change in DgoR to derepress thedgooperon. The findings from our work firmly place DgoR in the GntR family of transcriptional regulators: DgoR binds an operator sequence [5′-TTGTA(G/C)TACA(A/T)-3′] matching the signature of GntR family members that recognize inverted repeats [5′-(N)yGT(N)xAC(N)y-3′, wherexandyindicate the number of nucleotides, which varies], and it shares critical protein-DNA contacts. We also identified features in DgoR that are otherwise less conserved in the GntR family. Recently, missense mutations indgoRwere recovered in a naturalE. coliisolate adapted to the mammalian gut. Our results show these mutants to be DNA binding defective, emphasizing that mutations in thedgo-regulatory elements are selected in the host to allow simultaneous induction ofdgogenes. The present study sets the basis to explore the regulation ofdgogenes in additional enterobacterial strains where they have been implicated in host-bacterium interactions.IMPORTANCEd-Galactonate is a widely prevalent aldonic sugar acid. Despite the proposed significance of thed-galactonate metabolic pathway in the interaction of enteric bacteria with their hosts, there are no details on its regulation even inEscherichia coli, which has been known to utilized-galactonate since the 1970s. Here, using multiple methodologies, we identified the promoter, operator, and effector of DgoR, the transcriptional repressor ofd-galactonate metabolism inE. coli. We establish DgoR as a GntR family transcriptional regulator. Recently, a human urinary tract isolate ofE. coliintroduced in the mouse gut was found to accumulate missense mutations indgoR. Our results show these mutants to be DNA binding defective, hence emphasizing the role of thed-galactonate metabolic pathway in bacterial colonization of the mammalian gut.