Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa

Abstract

Bacteria communicate with each other to coordinate expression of specific genes in a cell density-dependent fashion, a phenomenon called quorum sensing and response. Although we know that quorum sensing via acyl-homoserine lactone (HSL) signals controls expression of several virulence genes in the human pathogen Pseudomonas aeruginosa, the number and types of genes controlled by quorum sensing have not been studied systematically. We have constructed a library of random insertions in the chromosome of a P. aeruginosa acyl-HSL synthesis mutant by using a transposon containing a promoterless lacZ. This library was screened for acyl-HSL induction of lacZ. Thirty-nine quorum sensing-regulated genes were identified. The genes were organized into classes depending on the pattern of regulation. About half of the genes appear to be in seven operons, some seem organized in large patches on the genome. Many of the quorum sensing-regulated genes code for putative virulence factors or production of secondary metabolites. Many of the genes identified showed a high level of induction by acyl-HSL signaling.

Figure 1

Patterns of β-galactosidase expression in representative P. aeruginosa MW1 qsc mutants and in a strain with a lasB∷lacZ chromosomal fusion generated by site-specific mutation. Units of β-galactosidase are given as a function of culture density for cells grown without added signal molecules (○), with added 3OC₁₂-HSL (●), with added C₄-HSL (■), or with both signals added (□). The background levels of β-galactosidase without added signals were 4–8 units for qsc103, 6–11 units for qsc109, 1–2 units for qsc124, 190 units for qsc133A, and 90 units for lasB∷lacZ. Stationary phase begins at a culture density of about 1.5.

Figure 2

Diagrams of seven putative qsc operons. ORFs are indicated by the arrows. ORFs discovered in the qsc screen are indicated by their qsc numbers.

Figure 3

Map of the qsc insertions on the P. aeruginosa chromosome. Arrowheads indicate the direction of lacZ transcription. Class I insertions are black, class II red, class III blue, and class IV green. In addition to the qsc mutants we identified, we have mapped lasR, lasI, and rhlR (gold) and lasB (green). The locations of las-box-like elements are shown as black dots between the two DNA strands. The numbers indicate distance in Mb on the approximately 6.3-Mb chromosome. Numbering starts at oriC.

Figure 4

las-Box-like sequences in upstream DNA regions of qsc mutants. Bases highlighted in black are conserved in all sequences and those in boxes are identical in eight of the 10 sequences.