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Pseudomonas aeruginosa AlgB, a two-component response regulator of the NtrC family, is required for algD transcription.

ABSTRACT: Most strains of Pseudomonas aeruginosa isolated from the respiratory tracts of cystic fibrosis patients have a mucoid colony morphology due to the synthesis of an expolysaccharide called alginate. The algB gene product (AlgB) is necessary for the high-level production of alginate in mucoid P. aeruginosa. In this study, AlgB was shown to be involved in the transcription of algD, a gene previously demonstrated to be activated in mucoid P. aeruginosa. In vitro and in vivo expression studies reveal that algB encodes a protein with a molecular size of 49 kDa. The DNA sequence of a 2.2-kb P. aeruginosa fragment containing algB was also determined. The amino-terminal domain of AlgB was found to be conserved with the amino-terminal domains of the response regulator class of two-component regulatory proteins. The central domain of AlgB has sequences highly conserved with those in the NtrC subfamily of transcriptional activators (NtrC, NifA, HydG, DctD, FlbD, TyrR, and PgtA). The central domain of AlgB also contains a potential nucleotide binding site. AlgB is the first NtrC homolog described from P. aeruginosa. At the carboxy terminus of AlgB, a helix-turn-helix motif was observed, suggesting that AlgB is a DNA-binding protein. The strongly conserved NtrC-like central domain of AlgB is not present in AlgR, another alginate response regulator. This study therefore identifies and characterizes the second of at least two unique response regulators used by P. aeruginosa to control alginate gene expression.

SUBMITTER: Wozniak DJ

PROVIDER: S-EPMC207277 | biostudies-other | 1991 Feb

REPOSITORIES: biostudies-other

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Pseudomonas aeruginosa AlgB, a two-component response regulator of the NtrC family, is required for algD transcription.

Wozniak D J DJ Ohman D E DE

Journal of bacteriology 19910201 4

Most strains of Pseudomonas aeruginosa isolated from the respiratory tracts of cystic fibrosis patients have a mucoid colony morphology due to the synthesis of an expolysaccharide called alginate. The algB gene product (AlgB) is necessary for the high-level production of alginate in mucoid P. aeruginosa. In this study, AlgB was shown to be involved in the transcription of algD, a gene previously demonstrated to be activated in mucoid P. aeruginosa. In vitro and in vivo expression studies reveal ...[more]

PMID: 1899859

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Project description:Pseudomonas aeruginosa is an important pathogen of the immunocompromised, causing both acute and chronic infections. In cystic fibrosis (CF) patients, P. aeruginosa causes chronic disease. The impressive sensory network of P. aeruginosa allows the bacterium to sense and respond to a variety of stimuli found in diverse environments. Transcriptional regulators, including alternative sigma factors and response regulators, integrate signals changing gene expression, allowing P. aeruginosa to cause infection. The two-component transcriptional regulator AlgR is important in P. aeruginosa pathogenesis in both acute and chronic infections. In chronic infections, AlgR and the alternative sigma factor AlgU activate the genes responsible for alginate production. Previous work demonstrated that AlgU controls rsmA expression. RsmA is a posttranscriptional regulator that is antagonized by two small RNAs, RsmY and RsmZ. In this work, we demonstrate that AlgR directly activates rsmA expression from the same promoter as AlgU. In addition, phosphorylation was not necessary for AlgR activation of rsmA using algR and algZ mutant strains. AlgU and AlgR appear to affect the antagonizing small RNAs rsmY and rsmZ indirectly. RsmA was active in a mucA22 mutant strain using leader fusions of two RsmA targets, tssA1 and hcnA AlgU and AlgR were necessary for posttranscriptional regulation of tssA1 and hcnA Altogether, our work demonstrates that the alginate regulators AlgU and AlgR are important in the control of the RsmA posttranscriptional regulatory system. These findings suggest that RsmA plays an unknown role in mucoid strains due to AlgU and AlgR activities.IMPORTANCEP. aeruginosa infections are difficult to treat and frequently cause significant mortality in CF patients. Understanding the mechanisms of persistence is important. Our work has demonstrated that the alginate regulatory system also significantly impacts the posttranscriptional regulator system RsmA/Y/Z. We demonstrate that AlgR directly activates rsmA expression, and this impacts the RsmA regulon. This leads to the possibility that the RsmA/Y/Z system plays a role in helping P. aeruginosa persist during chronic infection. In addition, this furthers our understanding of the reach of the alginate regulators AlgU and AlgR.

Project description:During late stages of cystic fibrosis pulmonary infections, Pseudomonas aeruginosa often overproduces the exopolysaccharide alginate, protecting the bacterial community from host immunity and antimicrobials. The transcription of the alginate biosynthesis operon is under tight control by a number of factors, including AmrZ, the focus of this study. Interestingly, multiple transcription factors interact with the far-upstream region of this promoter (PalgD), in which one AmrZ binding site has been identified previously. The mechanisms of AmrZ binding and subsequent activation remain unclear and require more-detailed investigation. In this study, in-depth examinations elucidated four AmrZ binding sites, and their disruption eliminated AmrZ binding and promoter activation. Furthermore, our in vitro fluorescence resonance energy transfer experiments suggest that AmrZ holds together multiple binding sites in PalgD and thereafter induces the formation of higher-order DNA-AmrZ complexes. To determine the importance of interactions between those AmrZ oligomers in the cell, a DNA phasing experiment was performed. PalgD transcription was significantly impaired when the relative phase between AmrZ binding sites was reversed (5 bp), while a full-DNA-turn insertion (10 bp) restored promoter activity. Taken together, the investigations presented here provide a deeper mechanistic understanding of AmrZ-mediated binding to PalgD IMPORTANCE: Overproduction of the exopolysaccharide alginate provides protection to Pseudomonas aeruginosa against antimicrobial treatments and is associated with chronic P. aeruginosa infections in the lungs of cystic fibrosis patients. In this study, we combined a variety of microbiological, genetic, biochemical, and biophysical approaches to investigate the activation of the alginate biosynthesis operon promoter by a key transcription factor named AmrZ. This study has provided important new information on the mechanism of activation of this extremely complex promoter.

Dataset Information

Pseudomonas aeruginosa AlgB, a two-component response regulator of the NtrC family, is required for algD transcription.

Publications

Pseudomonas aeruginosa AlgB, a two-component response regulator of the NtrC family, is required for algD transcription.

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