Local and Global Regulators Linking Anaerobiosis tocupAFimbrial Gene Expression inPseudomonas aeruginosa

Abstract

The cupA gene cluster of Pseudomonas aeruginosa encodes components and assembly factors of a putative fimbrial structure that enable this opportunistic pathogen to form biofilms on abiotic surfaces. In P. aeruginosa the control of cupA gene expression is complex, with the H-NS-like MvaT protein functioning to repress phase-variable (on/off) expression of the operon. Here we identify four positive regulators of cupA gene expression, including three unusual regulators encoded by the cgrABC genes and Anr, a global regulator of anaerobic gene expression. We show that the cupA genes are expressed in a phase-variable manner under anaerobic conditions and that the cgr genes are essential for this expression. We show further that cgr gene expression is negatively controlled by MvaT and positively controlled by Anr and anaerobiosis. Expression of the cupA genes therefore appears to involve a regulatory cascade in which anaerobiosis, signaled through Anr, stimulates expression of the cgr genes, resulting in a concomitant increase in cupA gene expression. Our findings thus provide mechanistic insight into the regulation of cupA gene expression and identify anaerobiosis as an inducer of phase-variable cupA gene expression, raising the possibility that phase-variable expression of fimbrial genes important for biofilm formation may occur in P. aeruginosa persisting in the largely anaerobic environment of the cystic fibrosis host lung.The gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen of humans that is notorious for being the principal cause of morbidity and mortality in cystic fibrosis (CF) patients; chronic colonization of the CF lung by P. aeruginosa typically leads to progressive lung damage and eventually respiratory failure and death (13). In the CF lung the organism is thought to persist as a biofilm, forming clusters of cells encased in a polymeric matrix (32). In this biofilm mode of growth P. aeruginosa exhibits increased resistance to antibiotics and is better able to evade the host immune response (6). Recent evidence suggests that the microbial environment in the CF lung is largely anaerobic (43) and that cells of P. aeruginosa persist in the CF lung in anaerobic biofilms (47). Indeed, the biofilm formed by cells of P. aeruginosa under anaerobic conditions is especially robust (47).The cupA gene cluster of P. aeruginosa encodes components of a putative fimbrial structure that enables this organism to form biofilms on abiotic surfaces (36). Under standard laboratory growth conditions, expression of the cupA gene cluster is tightly repressed by MvaT (37), a putative transcription regulator that is thought to functionally resemble members of the H-NS family of nucleoid-associated proteins (34). MvaT from P. aeruginosa was originally identified as a global regulator of virulence gene expression (7), and recent microarray analyses have revealed that MvaT controls the expression of at least 150 or so genes in P. aeruginosa, with the cupA genes being the most tightly repressed (37). Several o...

Anr and Its Activation by PlcH Activity in Pseudomonas aeruginosa Host Colonization and Virulence

“…As PAO1 strains can vary between labs, the ⌬anr mutant biofilm defect was confirmed in two distinct PAO1 WT and ⌬anr mutant pairs (see Fig. S2) (17,37) and was also observed in genetically distinct P. aeruginosa strain PA14 (see Fig. S3) (48), demonstrating that Anr was important for P. aeruginosa biofilm formation in different strain backgrounds.…”

“…The n-fold differences between the WT and the ⌬plcHR mutant for Anr-regulated genes ranged from 5-to 36-fold (see Table S1 in the supplemental material), and differentially regulated transcripts include those involved in energy generation under low-oxygen conditions, such as those that encode high-affinity cytochrome oxidases and fermentation and denitrification enzymes. The Anrregulated operon cgrA-cgrC, which encodes transcriptional regulators of CupA fimbriae (37,46), was also differentially expressed. Transcripts that were more highly expressed in the ⌬plcHR mutant but not known to be controlled by Anr are shown in Table S2 in the supplemental material; the majority of these transcripts encode proteins that have not yet been characterized.…”

“…To monitor the induction of promoters under the control of Anr, we used a construct with the nirS or cgrA promoter region fused to lacZ (36,37). Cells were grown overnight at 37°C in LB and then diluted into 5 ml of fresh MOPS medium supplemented as indicated in culture tubes at a starting optical density at 600 nm (OD 600 ) of 0.05.…”

Revamping the role of biofilm regulating operons in device-associated Staphylococci and Pseudomonas aeruginosa

“…[55] Besides, upregulation of cupA operon in a phase variable manner is carried out by cupA gene regulator (cgr) comprising four genes cgrA, cgrB, cgrC and a global regulator of anaerobic gene expression (Anr) which is present upstream to cupA operon. [56] The presence of anaerobic environment in P. aeruginosa triggers higher expression of biofilm aided by Anr gene regulation. [56] These data suggest that the expression of cupA operon requires multiple factors and have a tremendous impact on biofilm expression when compared to other cup operons.…”

“…[56] The presence of anaerobic environment in P. aeruginosa triggers higher expression of biofilm aided by Anr gene regulation. [56] These data suggest that the expression of cupA operon requires multiple factors and have a tremendous impact on biofilm expression when compared to other cup operons.…”