Effect of regB on expression from the P1 and P2 promoters of the Pseudomonas aeruginosa regAB operon

Storey, Douglas G.; Raivio, Tracy; Frank, Dara W.; Wick, Macdonald; Kaye, Susan A.; Iglewski, B H

doi:10.1128/jb.173.19.6088-6094.1991

Cited by 29 publications

(38 citation statements)

References 34 publications

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“…Synthesized fragments were then cloned in the previously described lacZ translational fusion vector pSW205 (14). The synthesis of the correct fragments was confirmed by nucleotide sequence analysis.…”

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“…The expression of ptxS that is carried on fragments that contain different mutations in PAO1 was determined using the previously described lacZ translational fusion vector pSW205 (14). As shown in Fig.…”

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confidence: 99%

“…(B) Localization of the ptxS promoter activity. DNA fragments that carry the indicated deletions from the 5Ј end of the ptxS upstream region were synthesized by PCR and cloned into the lacZ fusion vector pSW205 (14). Recombinant plasmids were introduced into the P. aeruginosa strains PAO1 and PAO::ptxS, and the level of ␤-galactosidase activity was determined as previously described (10).…”

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confidence: 99%

“…Despite several attempts, we were not successful in determining the ptxS transcriptional start site. Therefore, we tried to localize the ptxS promoter using the previously described lacZ translational fusion vector pSW205 (14). DNA fragments that carry different nested deletions from the 5Ј end of the 742-bp BamHI/ScaI fragment (which contains the entire ptxS upstream region) were synthesized by PCR (Fig.…”

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Autoregulation of the Pseudomonas aeruginosa Protein PtxS Occurs through a Specific Operator Site within the ptxS Upstream Region

Swanson

Hamood

2000

J Bacteriol

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We have previously shown that the Pseudomonas aeruginosa toxA regulatory protein PtxS autoregulates its own synthesis by binding to a 52-bp fragment. The 3 end of the 52-bp fragment is located 58 bp 5 of the ptxS translation start site. We have identified a 14-bp palindromic sequence (TGAAACCGGTTTCA) within the 52-bp fragment. In this study, we used site-directed mutagenesis and promoter fusion experiments to determine if PtxS binds specifically to this palindromic sequence and regulates ptxS expression. We have also tried to determine the roles of specific nucleotides within the palindromic sequence in PtxS binding and ptxS expression. Initial promoter fusion experiments confirmed that the 52-bp fragment does not overlap with the region that carries the ptxS promoter activity. PtxS binding was eliminated upon the deletion of the 14-bp palindromic sequence from the 52-bp fragment. In addition, the deletion of the 14-bp sequence caused a significant enhancement in ptxS expression in the P. aeruginosa strain PAO1 and the ptxS isogenic mutant PAO::ptxS. Mutation of specific nucleotides within the 14-bp sequence eliminated, reduced, or had no effect on PtxS binding. However, mutations of several of these nucleotides produced a significant increase in ptxS expression in both PAO1 and PAO::ptxS. These results suggest that (i) the 14-bp palindromic sequence and specific nucleotides within it play a role in PtxS binding and (ii) deletion of the palindromic sequence or changing of certain nucleotides within it interferes with another mechanism that may regulate ptxS expression.Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that causes serious infections in burned patients and immunocompromised hosts (4,22). Damage caused by P. aeruginosa is due to the production of several extracellular and cell-associated virulence factors (7, 23). Exotoxin A, which is the most toxic of the extracellular virulence factors, catalyzes the transfer of an ADP-ribosyl moiety onto elongation factor 2 in eukaryotic cells (6). This results in inhibition of protein synthesis and cell death (6, 21). It is known that exotoxin A production by P. aeruginosa is controlled by both positive and negative regulatory genes (17). We have previously described two P. aeruginosa genes, ptxR and ptxS, that regulate exotoxin A synthesis (3, 5). The ptxR gene codes for a protein that belongs to the LysR family of transcriptional activators and enhances exotoxin A synthesis at the transcriptional level (5). The ptxS gene codes for a protein that belongs to the GalRLacI family of transcriptional repressors (3). Available evidence suggests that PtxS interferes with the enhancement of exotoxin A synthesis by ptxR (3).Despite previous analyses, the mechanism(s) through which the expression of ptxS and ptxR is regulated is not known. We have recently provided evidence which suggests that ptxS negatively autoregulates its own synthesis in P. aeruginosa (15). The level of ␤-galactosidase activity produced by a ptxS-lacZ fusion plasmid in the ptxS isoge...

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Autoregulation of the Pseudomonas aeruginosa Protein PtxS Occurs through a Specific Operator Site within the ptxS Upstream Region

Swanson

Hamood

2000

J Bacteriol

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“…Ten-milliliter cultures of P. aeruginosa PAO or PAO(pPA3.5RK) were grown overnight at 37ЊC in TSBDC broth supplemented with 400 g of EDDHA per ml. These cultures were diluted 1/100 in fresh medium, and total RNA was isolated from 10 ml of cells at an optical density at 600 nm of ϳ0.5, using a hot-phenol extraction procedure (18,51). For Northern (RNA) hybridization analysis, aliquots of 12 g of total RNA were electrophoresed and transferred onto a nitrocellulose membrane, and hybridization was performed overnight at 65ЊC in 15 ml of 1% SDS-1 M NaCl-10% dextran sulfate-0.1-mg/ml salmon sperm DNA as previously described (44).…”

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Identification and characterization of the tolQRA genes of Pseudomonas aeruginosa

1996

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The tolQ, tolR, and tolA genes from Pseudomonas aeruginosa PAO were cloned using degenerate oligonucleotide PCR primers designed based on conserved transmembrane regions of Escherichia coli TolQ and TolR and E. coli and Pseudomonas putida ExbB and ExbD. The resulting PCR product was used as a probe to isolate a 6.5-kb DNA fragment containing P. aeruginosa tolQ, tolR, and tolA. The nucleotide sequence of a 2.9-kb DNA fragment containing the tolQ, tolR, and tolA genes was determined. The DNA sequence predicts TolQ to be a 25,250-Da protein exhibiting 53% identity to E. coli TolQ. TolR is predicted to be a 15,788-Da protein, sharing 38% identity with the E. coli TolR protein. The P. aeruginosa tolA sequence predicts a 37,813-Da protein with 27% identity to the E. coli TolA. The P. aeruginosa TolQRA proteins were expressed in E. coli minicells. Analysis of plasmid-encoded tolQ::lacZ and tolA::lacZ promoter fusions in E. coli indicated that these genes are expressed at different levels, suggesting transcription from different promoters. Transcriptional analysis of the tol genes in P. aeruginosa revealed that the tolQ and tolR genes are cotranscribed as an approximately 1.5-kb transcript and that tolA is transcribed from its own promoter as an approximately 1.2-kb transcript. The P. aeruginosa Tol proteins were functionally unable to complement E. coli tol mutants, although P. aeruginosa TolQ was able to complement the iron-limited growth of an E. coli exbB mutant. Introduction of the tolQRA genes in the tol-like mutant PAO 1652 restored pyocin AR41 killing, indicating that the Tol proteins are involved in the uptake of pyocin AR41 in P. aeruginosa. Attempts to inactivate the chromosomal copy of the tolA or tolQ gene in the parent strain PAO proved to be unsuccessful, and we propose that inactivation of these genes in P. aeruginosa results in a lethal phenotype.

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Regulation of Pseudomonas Aeruginosa Exotoxin a Synthesis

Hamood¹,

Colmer-Hamood²,

Carty³

2004

Pseudomonas

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Effect of regB on expression from the P1 and P2 promoters of the Pseudomonas aeruginosa regAB operon

Cited by 29 publications

References 34 publications

Autoregulation of the Pseudomonas aeruginosa Protein PtxS Occurs through a Specific Operator Site within the ptxS Upstream Region

Autoregulation of the Pseudomonas aeruginosa Protein PtxS Occurs through a Specific Operator Site within the ptxS Upstream Region

Identification and characterization of the tolQRA genes of Pseudomonas aeruginosa

Regulation of Pseudomonas Aeruginosa Exotoxin a Synthesis

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