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Characterization of the Pseudomonas aeruginosa metalloendopeptidase, Mep72, a member of the Vfr regulon.

Balyimez A, Colmer-Hamood JA, San Francisco M, Hamood AN - BMC Microbiol. (2013)

Bottom Line: A mutation in vfr significantly reduced the expression of both genes.Vfr directly regulates the expression of the PA2782-mep72 operon by binding to its upstream region.However, unlike other Vfr-targeted genes, Vfr binding does not require an intact Vfr consensus binding sequence.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology and Molecular Microbiology, Texas Tech University Health Sciences Center, Lubbock, TX, USA. abdul.hamood@ttuhsc.edu.

ABSTRACT

Background: Pseudomonas aeruginosa Vfr (the virulence factor regulator) enhances P. aeruginosa virulence by positively regulating the expression of numerous virulence genes. A previous microarray analysis identified numerous genes positively regulated by Vfr in strain PAK, including the yet uncharacterized PA2782 and PA2783.

Results: In this study, we report the detailed characterization of PA2783 in the P. aeruginosa strain PAO1. RT-PCR analysis confirmed that PA2782-PA2783 constitute an operon. A mutation in vfr significantly reduced the expression of both genes. The predicted protein encoded by PA2783 contains a typical leader peptide at its amino terminus end as well as metalloendopeptidase and carbohydrate binding motifs at its amino terminus and carboxy terminus regions, respectively. An in-frame PA2783::phoA fusion encoded a hybrid protein that was exported to the periplasmic space of Escherichia coli and P. aeruginosa. In PAO1, the proteolytic activity of the PA2783-encoded protein was masked by other P. aeruginosa extracellular proteases but an E. coli strain carrying a PA2783 recombinant plasmid produced considerable proteolytic activity. The outer membrane fraction of an E. coli strain in which PA2783 was overexpressed contained specific endopeptidase activity. In the presence of cAMP, purified recombinant Vfr (rVfr) bound to a 98-bp fragment within the PA2782-PA2783 upstream region that carries a putative Vfr consensus sequence. Through a series of electrophoretic mobility shift assays, we localized rVfr binding to a 33-bp fragment that contains part of the Vfr consensus sequence and a 5-bp imperfect (3/5) inverted repeat at its 3' and 5' ends (TGGCG-N22-CGCTG). Deletion of either repeat eliminated Vfr binding.

Conclusions: PA2782 and PA2783 constitute an operon whose transcription is positively regulated by Vfr. The expression of PA2783 throughout the growth cycle of P. aeruginosa follows a unique pattern. PA2783 codes for a secreted metalloendopeptidase, which we named Mep72. Mep72, which has metalloendopeptidase and carbohydrate-binding domains, produced proteolytic and endopeptidase activities in E. coli. Vfr directly regulates the expression of the PA2782-mep72 operon by binding to its upstream region. However, unlike other Vfr-targeted genes, Vfr binding does not require an intact Vfr consensus binding sequence.

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Vfr regulates the transcription of PA2782 and PA2783 at early and late stages of growth of PAO1. PAO1 and PAOΔvfr strains were grown in LB broth overnight and subcultured into fresh LB broth to a starting OD600 of 0.02. Cells were harvested at 4 h and 6 h, OD600 of 0.37 and 0.79 for PAO1 and 0.41 and 0.89 for PAOΔvfr, respectively, and total RNA was extracted. Levels of PA2782 and PA2783 mRNA in each sample were determined by qRT-PCR using specifically-designed primers. Values represent the means of three independent experiments ± SEM. ***P <0.001.
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Figure 2: Vfr regulates the transcription of PA2782 and PA2783 at early and late stages of growth of PAO1. PAO1 and PAOΔvfr strains were grown in LB broth overnight and subcultured into fresh LB broth to a starting OD600 of 0.02. Cells were harvested at 4 h and 6 h, OD600 of 0.37 and 0.79 for PAO1 and 0.41 and 0.89 for PAOΔvfr, respectively, and total RNA was extracted. Levels of PA2782 and PA2783 mRNA in each sample were determined by qRT-PCR using specifically-designed primers. Values represent the means of three independent experiments ± SEM. ***P <0.001.

Mentions: A previous microarray analysis revealed that Vfr regulates the expression of the P. aeruginosa genes PA2782 and PA2783[19]. PA2783 expression was significantly reduced in the vfr deletion mutant PAK∆vfr compared with its parent strain PAK [19]. While PAK has been extensively studied in lung and corneal infections [21-23], its effects in wound infections, a major emphasis in our laboratory, is less characterized. P. aeruginosa strain PAO1 is highly virulent in wound infections, including burn wounds, and has been well-studied in connection with infections in those with cystic fibrosis [24-27]. Therefore, using qRT-PCR, we determined whether Vfr regulates the expression of PA2782 and PA2783 in PAO1. We compared the expression of both genes in PAO1 and its vfr isogenic mutant PAO∆vfr at early (OD600 of 0.37 and 0.41) and mid (OD600 of 0.79 and 0.89) logarithmic phases of growth. As shown in Figure 2, at both time points and compared with PAO1, the expression of PA2782 and PA2783 was significantly reduced in PAO∆vfr.


Characterization of the Pseudomonas aeruginosa metalloendopeptidase, Mep72, a member of the Vfr regulon.

Balyimez A, Colmer-Hamood JA, San Francisco M, Hamood AN - BMC Microbiol. (2013)

Vfr regulates the transcription of PA2782 and PA2783 at early and late stages of growth of PAO1. PAO1 and PAOΔvfr strains were grown in LB broth overnight and subcultured into fresh LB broth to a starting OD600 of 0.02. Cells were harvested at 4 h and 6 h, OD600 of 0.37 and 0.79 for PAO1 and 0.41 and 0.89 for PAOΔvfr, respectively, and total RNA was extracted. Levels of PA2782 and PA2783 mRNA in each sample were determined by qRT-PCR using specifically-designed primers. Values represent the means of three independent experiments ± SEM. ***P <0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4222646&req=5

Figure 2: Vfr regulates the transcription of PA2782 and PA2783 at early and late stages of growth of PAO1. PAO1 and PAOΔvfr strains were grown in LB broth overnight and subcultured into fresh LB broth to a starting OD600 of 0.02. Cells were harvested at 4 h and 6 h, OD600 of 0.37 and 0.79 for PAO1 and 0.41 and 0.89 for PAOΔvfr, respectively, and total RNA was extracted. Levels of PA2782 and PA2783 mRNA in each sample were determined by qRT-PCR using specifically-designed primers. Values represent the means of three independent experiments ± SEM. ***P <0.001.
Mentions: A previous microarray analysis revealed that Vfr regulates the expression of the P. aeruginosa genes PA2782 and PA2783[19]. PA2783 expression was significantly reduced in the vfr deletion mutant PAK∆vfr compared with its parent strain PAK [19]. While PAK has been extensively studied in lung and corneal infections [21-23], its effects in wound infections, a major emphasis in our laboratory, is less characterized. P. aeruginosa strain PAO1 is highly virulent in wound infections, including burn wounds, and has been well-studied in connection with infections in those with cystic fibrosis [24-27]. Therefore, using qRT-PCR, we determined whether Vfr regulates the expression of PA2782 and PA2783 in PAO1. We compared the expression of both genes in PAO1 and its vfr isogenic mutant PAO∆vfr at early (OD600 of 0.37 and 0.41) and mid (OD600 of 0.79 and 0.89) logarithmic phases of growth. As shown in Figure 2, at both time points and compared with PAO1, the expression of PA2782 and PA2783 was significantly reduced in PAO∆vfr.

Bottom Line: A mutation in vfr significantly reduced the expression of both genes.Vfr directly regulates the expression of the PA2782-mep72 operon by binding to its upstream region.However, unlike other Vfr-targeted genes, Vfr binding does not require an intact Vfr consensus binding sequence.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology and Molecular Microbiology, Texas Tech University Health Sciences Center, Lubbock, TX, USA. abdul.hamood@ttuhsc.edu.

ABSTRACT

Background: Pseudomonas aeruginosa Vfr (the virulence factor regulator) enhances P. aeruginosa virulence by positively regulating the expression of numerous virulence genes. A previous microarray analysis identified numerous genes positively regulated by Vfr in strain PAK, including the yet uncharacterized PA2782 and PA2783.

Results: In this study, we report the detailed characterization of PA2783 in the P. aeruginosa strain PAO1. RT-PCR analysis confirmed that PA2782-PA2783 constitute an operon. A mutation in vfr significantly reduced the expression of both genes. The predicted protein encoded by PA2783 contains a typical leader peptide at its amino terminus end as well as metalloendopeptidase and carbohydrate binding motifs at its amino terminus and carboxy terminus regions, respectively. An in-frame PA2783::phoA fusion encoded a hybrid protein that was exported to the periplasmic space of Escherichia coli and P. aeruginosa. In PAO1, the proteolytic activity of the PA2783-encoded protein was masked by other P. aeruginosa extracellular proteases but an E. coli strain carrying a PA2783 recombinant plasmid produced considerable proteolytic activity. The outer membrane fraction of an E. coli strain in which PA2783 was overexpressed contained specific endopeptidase activity. In the presence of cAMP, purified recombinant Vfr (rVfr) bound to a 98-bp fragment within the PA2782-PA2783 upstream region that carries a putative Vfr consensus sequence. Through a series of electrophoretic mobility shift assays, we localized rVfr binding to a 33-bp fragment that contains part of the Vfr consensus sequence and a 5-bp imperfect (3/5) inverted repeat at its 3' and 5' ends (TGGCG-N22-CGCTG). Deletion of either repeat eliminated Vfr binding.

Conclusions: PA2782 and PA2783 constitute an operon whose transcription is positively regulated by Vfr. The expression of PA2783 throughout the growth cycle of P. aeruginosa follows a unique pattern. PA2783 codes for a secreted metalloendopeptidase, which we named Mep72. Mep72, which has metalloendopeptidase and carbohydrate-binding domains, produced proteolytic and endopeptidase activities in E. coli. Vfr directly regulates the expression of the PA2782-mep72 operon by binding to its upstream region. However, unlike other Vfr-targeted genes, Vfr binding does not require an intact Vfr consensus binding sequence.

Show MeSH
Related in: MedlinePlus