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The Lrp/AsnC-Type Regulator PA2577 Controls the EamA-like Transporter Gene PA2576 in Pseudomonas aeruginosa.

ABSTRACT: The regulatory network of gene expression in Pseudomonas aeruginosa, an opportunistic human pathogen, is very complex. In the PAO1 reference strain, about 10% of genes encode transcriptional regulators, many of which have undefined regulons and unknown functions. The aim of this study is the characterization of PA2577 protein, a representative of the Lrp/AsnC family of transcriptional regulators. This family encompasses proteins involved in the amino acid metabolism, regulation of transport processes or cell morphogenesis. The transcriptome profiling of P. aeruginosa cells with mild PA2577 overproduction revealed a decreased expression of the PA2576 gene oriented divergently to PA2577 and encoding an EamA-like transporter. A gene expression analysis showed a higher mRNA level of PA2576 in P. aeruginosa ΔPA2577, indicating that PA2577 acts as a repressor. Concomitantly, ChIP-seq and EMSA assays confirmed strong interactions of PA2577 with the PA2577/PA2576 intergenic region. Additionally, phenotype microarray analyses indicated an impaired metabolism of ΔPA2576 and ΔPA2577 mutants in the presence of polymyxin B, which suggests disturbances of membrane functions in these mutants. We show that PA2576 interacts with two proteins, PA5006 and PA3694, with a predicted role in lipopolysaccharide (LPS) and membrane biogenesis. Overall, our results indicate that PA2577 acts as a repressor of the PA2576 gene coding for the EamA-like transporter and may play a role in the modulation of the cellular response to stress conditions, including antimicrobial peptides, e.g., polymyxin B.

SUBMITTER: Modrzejewska M

PROVIDER: S-EPMC8707732 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

Project description:UnlabelledIn the presence of alanine, AldR, which belongs to the Lrp/AsnC family of transcriptional regulators and regulates ald encoding alanine dehydrogenase in Mycobacterium smegmatis, changes its quaternary structure from a homodimer to an octamer with an open-ring conformation. Four AldR-binding sites (O2, O1, O4, and O3) with a consensus sequence of GA/T-N2-NWW/WWN-N2-A/TC were identified upstream of the M. smegmatis ald gene by means of DNase I footprinting analysis. O2, O1, and O4 are required for the induction of ald expression by alanine, while O3 is directly involved in the repression of ald expression. In addition to O3, both O1 and O4 are also necessary for full repression of ald expression in the absence of alanine, due to cooperative binding of AldR dimers to O1, O4, and O3. Binding of a molecule of the AldR octamer to the ald control region was demonstrated to require two AldR-binding sites separated by three helical turns between their centers and one additional binding site that is in phase with the two AldR-binding sites. The cooperative binding of AldR dimers to DNA requires three AldR-binding sites that are aligned with a periodicity of three helical turns. The aldR gene is negatively autoregulated independently of alanine. Comparative analysis of ald expression of M. smegmatis and Mycobacterium tuberculosis in conjunction with sequence analysis of both ald control regions led us to suggest that the expression of the ald genes in both mycobacterial species is regulated by the same mechanism.ImportanceIn mycobacteria, alanine dehydrogenase (Ald) is the enzyme required both to utilize alanine as a nitrogen source and to grow under hypoxic conditions by maintaining the redox state of the NADH/NAD(+) pool. Expression of the ald gene was reported to be regulated by the AldR regulator that belongs to the Lrp/AsnC (feast/famine) family, but the underlying mechanism was unknown. This study revealed the regulation mechanism of ald in Mycobacterium smegmatis and Mycobacterium tuberculosis. Furthermore, a generalized arrangement pattern of cis-acting regulatory sites for Lrp/AsnC (feast/famine) family regulators is suggested in this study.

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The Lrp/AsnC-Type Regulator PA2577 Controls the EamA-like Transporter Gene PA2576 in Pseudomonas aeruginosa.

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