LasR, a transcriptional activator of Pseudomonas aeruginosa virulence genes, functions as a multimer

Abstract

The Pseudomonas aeruginosa LasR protein functions in concert with N-3-oxo-dodecanoyl-L-homoserine lactone (3O-C(12)-HSL) to coordinate the expression of target genes, including many genes that encode virulence factors, with cell density. We used a LexA-based protein interaction assay to demonstrate that LasR forms multimers only when 3O-C(12)-HSL is present. A series of LasR molecules containing internal deletions or substitutions in single, conserved amino acid residues indicated that the N-terminal portion of LasR is required for multimerization. Studies performed with these mutant versions of LasR demonstrated that the ability of LasR to multimerize correlates with its ability to function as a transcriptional activator of lasI, a gene known to be tightly regulated by the LasR-3O-C(12)-HSL regulatory system. A LasR molecule that carries a C-terminal deletion can function as a dominant-negative mutant in P. aeruginosa, as shown by its ability to decrease expression of lasB, another LasR-3O-C(12)-HSL target gene. Taken together, our data strongly support the hypothesis that LasR functions as a multimer in vivo.

FIG. 1.

LasR forms multimers in the presence of 3O-C₁₂-HSL. The multimerization of LasR was determined by the ability of LexA-LasR protein fusions to inhibit lacZ expression in the reporter strain E. coli SU101. β-Gal activity from the strains was normalized to the activity of E. coli SU101 containing the vector control (pSR658). A strain carrying the LexA DBD-NeuD protein encoded on pSR658-neuD was used as a positive control. When required, 100 nM 3O-C₁₂-HSL or 500 nM C₄-HSL was added to cultures. The results are averages for three independent assays. pSR658, LexA DBD; pSR658-neuD, LexA DBD-NeuD; pLXR, LexA DBD-LasR.

FIG. 2.

Multimerization of LasR deletion constructs. (A) Plasmids pLXR to pLXR6 encoding the lasR deletion constructs. (B) Abilities of the LexA DBD-LasR constructs to express protein capable of forming multimers in the presence or absence of 100 nM 3O-C₁₂-HSL. The results are the averages for three independent determinations.

FIG. 3.

Abilities of LasR deletions to function as activators of lasI. The truncated LasR mutants (pLXR2 to pLXR6), as well as full-length LasR (pLXR), were examined for the ability to activate lasI expression from λI₁4 in E. coli strain MG4 carrying a lasI::lacZ fusion. Expression of lasI was determined by assaying β-Gal activity in the presence or absence of 100 nM 3O-C₁₂-HSL, and the data are expressed relative to the value for the positive control (pLXR), which was normalized to 100%. The results are averages for three independent assays.

FIG. 4.

LasR carrying a C-terminal deletion can function as a dominant-negative mutant to inhibit multimerization of LasR. The ability of a LasR mutant (LasR_Δ176-237) to interfere with the formation of functional LasR multimers was determined by measuring the expression of lasB::lacZ in P. aeruginosa PAO230. Expression of lasB was determined by measuring β-Gal activity in strains harboring pEX1.8 (vector control), pEXRΔC (ptac-lasR_Δ176-237), or pEXR1 (plasR-lasR ptac-lasR_Δ176-237). 3O-C₁₂-HSL was not added or was added to a final concentration of 1 or 10 μM as indicated. The results are averages for three independent assays.

FIG. 5.

LasR_Δ176-237 inhibits proteolysis in P. aeruginosa PAO230. P. aeruginosa PAO230 carrying either pEX1.8 (vector control) or pEXRΔC (ptac-lasR_Δ176-237) was assayed for proteolytic activity by plating on skim milk agar in the presence or absence of 1 mM IPTG, which was used to induce expression of LasR_Δ176-237. Proteolysis was visualized as a clear zone surrounding the bacterial streak. P. aeruginosa PAO231(pEX1.8), a lasI null mutant, was utilized as a negative control.