Expression Analysis of the Pseudomonas aeruginosa AlgZR Two-Component Regulatory System

Abstract

cPseudomonas aeruginosa virulence components are subject to complex regulatory control primarily through two-component regulatory systems that allow for sensing and responding to environmental stimuli. In this study, the expression and regulation of the P. aeruginosa AlgZR two-component regulatory system were examined. Primer extension and S1 nuclease protection assays were used to identify two transcriptional initiation sites for algR within the algZ coding region, and two additional start sites were identified upstream of the algZ coding region. The two algR transcriptional start sites, RT1 and RT2, are directly regulated by AlgU, consistent with previous reports of increased algR expression in mucoid backgrounds, and RpoS additionally plays a role in algR transcription. The expression of the first algZ promoter, ZT1, is entirely dependent upon Vfr for expression, whereas Vfr, RpoS, or AlgU does not regulate the second algZ promoter, ZT2. Western blot, real-time quantitative PCR (RTqPCR), and transcriptional fusion analyses show that algZR expression is Vfr dependent. The algZ and algR genes also are cotranscribed in both nonmucoid and mucoid backgrounds. Furthermore, algZR was found to be cotranscribed with hemCD by RT-PCR. RT-qPCR confirmed that hemC transcription in the PAO1 ⌬algZ mutant was 40% of the level of the wild-type strain. Taken together, these results indicate that algZR transcription involves multiple factors at multiple start sites that control individual gene expression as well as coexpression of this two-component system with heme biosynthetic genes. Pseudomonas aeruginosa is a human opportunistic pathogen capable of causing fatal infections in individuals with compromised innate immunity, such as those undergoing cancer treatment or those with severe burn wounds or cystic fibrosis (CF) (1-3). P. aeruginosa is one of the most clinically relevant organisms in CF patients due to its ability to establish chronic infections, characterized by the overproduction of an exopolysaccharide called alginate. Alginate overproduction (a phenotype called mucoidy) increases P. aeruginosa resistance against antimicrobials and phagocytosis and is a hallmark of clinical decline and worsening prognosis in patients with CF.Alginate production is regulated in a complex manner by several transcriptional regulators, including the extracytoplasmic sigma factor AlgU (AlgT) (4-6). The activity of AlgU is increased in mucoid P. aeruginosa due to mutations in the mucA anti-sigma factor gene that releases AlgU from the inner membrane (6-8). The enzymes encoded by the algC gene and the algD operon are responsible for alginate biosynthesis, modification, and export. The AlgR transcriptional regulator is an essential activator for the algD operon and the algC gene; deletion of algR in mucoid P. aeruginosa backgrounds results in a nonmucoid phenotype (9-11). As part of its regulon, AlgU also activates the transcription of the algR gene to increase alginate production. An AlgU-dependent algR transcriptional start site was dete...

Regulation of ribonucleotide synthesis by the Pseudomonas aeruginosa two-component system AlgR in response to oxidative stress

“…Examining this throughout all biofilm life cycle, when planktonic cells suffer the presence of a stress condition (oxidative stress, antibiotic treatment, etc.) it activates AlgR 13 , 14 , 33 . Under these conditions, the algR gene is expressed from promoters ZT1 (further activated by Vfr) and ZT2 (constitutive) as fimS-algR 33 , a combination that favors phosphorylation.…”

The anti-sigma factor MucA is required for viability inPseudomonas aeruginosa

“…For all strains tested via our allelic exchange assay, all observed isolates had the wild-type mucA allele (p < 0.0001, Fisher's exact test), strongly suggesting that mucA is essential in a variety of wild-type P. aeruginosa strains. Paradoxically, among the P. aeruginosa isolates we tested were three laboratory strains (PAO1, PAK, and PA103) for which ∆mucA mutants have previously been published [28][29][30]. Since we were unable to delete mucA from these strains, it suggests that the published ∆mucA strains in these P. aeruginosa backgrounds contain secondary site mutations that allow for their viability, as later described.…”

“…Second, mucA mutations commonly arise in clinical CF isolates [7,[20][21][22][23], and many laboratory mucA mutants exist in the literature [24][25][26][27]. Third, there are three published strains in which the entirety of mucA is removed from the genome [28][29][30].…”

The Pseudomonas aeruginosa Two-Component Regulator AlgR Directly Activates rsmA Expression in a Phosphorylation-Independent Manner

“…The rsmATF1-lacZ fusion was analyzed in the wild-type strain P. aeruginosa PAO1, ΔalgR and algZ mutants, and in the corresponding mucA22 mutants. The algZ mutant has a mutation in the conserved histidine residue, which prevents AlgZ-mediated phosphorylation of AlgR (19,32) but does not disrupt the internal algR promoter (33). As shown in Fig.…”