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A novel 3-hydroxypropionic acid-inducible promoter regulated by the LysR-type transcriptional activator protein MmsR of Pseudomonas denitrificans.

ABSTRACT: MmsR (33.3?kDa) is a putative LysR-type transcriptional activator of Pseudomonas denitrificans. With the help of 3-hydroxypropionic acid (3-HP), an important platform chemical, MmsR positively regulates the expression of mmsA, which encodes methylmalonylsemialdehyde dehydrogenase, the enzyme involved in valine degradation. In the present study, the cellular function of MmsR and its binding to the regulatory DNA sequence of mmsA expression were investigated both in vivo and in vitro. Transcription of the mmsA was enhanced >140-fold in the presence of 3-HP. In the MmsR-responsive promoter region, two operators showing dyad symmetry, designated O1 and O2 and centered at the -79 and -28 positions, respectively, were present upstream of the mmsA transcription start site. An electrophoretic mobility shift assay indicated that MmsR binds to both operator sites for transcription activation, probably in cooperative manner. When either O1 or O2 or both regions were mutated, the inducibility by the MmsR-3-HP complex was significantly reduced or completely removed, indicating that both sites are required for transcription activation. A 3-HP sensor was developed by connecting the activation of MmsR to a green fluorescent readout. A more than 50-fold induction by 25?mM 3-HP was observed.

SUBMITTER: Nguyen NH

PROVIDER: S-EPMC6441110 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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A novel 3-hydroxypropionic acid-inducible promoter regulated by the LysR-type transcriptional activator protein MmsR of Pseudomonas denitrificans.

Nguyen Nam Hoai NH Ainala Satish Kumar SK Zhou Shengfang S Park Sunghoon S

Scientific reports 20190329 1

MmsR (33.3 kDa) is a putative LysR-type transcriptional activator of Pseudomonas denitrificans. With the help of 3-hydroxypropionic acid (3-HP), an important platform chemical, MmsR positively regulates the expression of mmsA, which encodes methylmalonylsemialdehyde dehydrogenase, the enzyme involved in valine degradation. In the present study, the cellular function of MmsR and its binding to the regulatory DNA sequence of mmsA expression were investigated both in vivo and in vitro. Transcriptio ...[more]

PMID: 30926872

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Project description:On the basis of the constitutive phenotypes of two catM mutants of Acinetobacter calcoaceticus, the CatM protein was proposed to repress expression of two different loci involved in catechol degradation, catA and catBCIJFD (E. Neidle, C. Hartnett, and L. N. Ornston, J. Bacteriol. 171:5410-5421, 1989). In spite of its proposed negative role as a repressor, CatM is similar in amino acid sequence to positive transcriptional activators of the LysR family. Investigating this anomaly, we found that insertional inactivation of catM did not cause the phenotype expected for the disruption of a repressor-encoding gene: in an interposon-generated catM mutant, no cat genes were expressed constitutively, but rather catA and catB were still inducible by muconate. Moreover, this catM mutant grew poorly on benzoate, a process requiring the expression of all cat genes. The inducibility of the cat genes in this catM mutant was completely eliminated by a 3.5-kbp deletion 10 kbp upstream of catM. In this double mutant, catM in trans restored muconate inducibility to both catA and catB. These results suggested the presence of an additional regulatory locus controlling cat gene expression. The ability of CatM to function as an activator was also suggested by these results. In support of this hypothesis, in vivo methylation protection assays showed that CatM protects two guanines in a dyad 65 nucleotides upstream of the catB transcriptional start site, in a location and pattern typical of LysR-type transcriptional activators. Gel mobility shift assays indicated that CatM also binds to a region upstream of catA. DNA sequence analysis revealed a nucleotide near the 3' end of catM not present in the published sequence. Translation of the corrected sequence resulted in the deduced CatM protein being 52 residues longer than previously reported. The size, amino acid sequence, and mode of action of CatM now appear similar to, and typical of, what has been found for transcriptional activators in the LysR family. Analysis of one of the constitutive alleles of catM previously thought to encode a dysfunctional repressor indicated instead that it encodes an inducer-independent transcriptional activator.

Project description:Pseudomonas aeruginosa, a facultative human pathogen causing nosocomial infections, has complex regulatory systems involving many transcriptional regulators. LTTR (LysR-Type Transcriptional Regulator) family proteins are involved in the regulation of various processes, including stress responses, motility, virulence, and amino acid metabolism. The aim of this study was to characterize the LysR-type protein BsrA (PA2121), previously described as a negative regulator of biofilm formation in P. aeruginosa. Genome wide identification of BsrA binding sites using chromatin immunoprecipitation and sequencing analysis revealed 765 BsrA-bound regions in the P. aeruginosa PAO1161 genome, including 367 sites in intergenic regions. The motif T-N11-A was identified within sequences bound by BsrA. Transcriptomic analysis showed altered expression of 157 genes in response to BsrA excess; of these, 35 had a BsrA binding site within their promoter regions, suggesting a direct influence of BsrA on the transcription of these genes. BsrA-repressed loci included genes encoding proteins engaged in key metabolic pathways such as the tricarboxylic acid cycle. The panel of loci possibly directly activated by BsrA included genes involved in pilus/fimbria assembly, as well as secretion and transport systems. In addition, DNA pull-down and regulatory analyses showed the involvement of PA2551, PA3398, and PA5189 in regulation of bsrA expression, indicating that this gene is part of an intricate regulatory network. Taken together, these findings reveal the existence of a BsrA regulon, which performs important functions in P. aeruginosa. IMPORTANCE This study shows that BsrA, a LysR-type transcriptional regulator from Pseudomonas aeruginosa, previously identified as a repressor of biofilm synthesis, is part of an intricate global regulatory network. BsrA acts directly and/or indirectly as the repressor and/or activator of genes from vital metabolic pathways (e.g., pyruvate, acetate, and tricarboxylic acid cycle) and is involved in control of transport functions and the formation of surface appendages. Expression of the bsrA gene is increased in the presence of antibiotics, which suggests its induction in response to stress, possibly reflecting the need to redirect metabolism under stressful conditions. This is particularly relevant for the treatment of infections caused by P. aeruginosa. In summary, the findings of this study demonstrate that the BsrA regulator performs important roles in carbon metabolism, biofilm formation, and antibiotic resistance in P. aeruginosa.

Dataset Information

A novel 3-hydroxypropionic acid-inducible promoter regulated by the LysR-type transcriptional activator protein MmsR of Pseudomonas denitrificans.

Publications

A novel 3-hydroxypropionic acid-inducible promoter regulated by the LysR-type transcriptional activator protein MmsR of Pseudomonas denitrificans.

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