A Second Operator Is Involved in Pseudomonas aeruginosa Elastase (lasB) Activation

Pseudomonas aeruginosa LasB elastase is a well-characterized exoenzyme that contributes to pathogenesis of the organism in animal models (28). Suggestive of its pathogenic potential is its broad substrate range. LasB elastase acts alone or together with other P. aeruginosa proteases to degrade or inactivate several biologically important substrates, including connective tissues and immune system components (29). Detection of lasB transcript in the sputa of cystic fibrosis patients (44) and of antibodies to elastase in cystic fibrosis patients (1, 23) demonstrates that LasB elastase is produced in the human host. LasB elastase gene (lasB) transcription depends on a cell density-dependent (quorum-sensing) mechanism of gene activation (35). Bacteria with quorum-sensing systems respond to the attainment of a critical culture density by activating transcription of target genes. Quorum sensing as a regulatory mechanism was first described for the marine bacteria Vibrio harveyi and V. fischeri, in which the attainment of a critical mass of bacteria in the symbiotic host’s light organ results in expression of the bioluminescence (lux) operon (for a review, see reference 11). Quorum-sensing gene activator complexes are composed of two components: a regulatory protein and an N-acylhomoserine lactone (“autoinducer”) molecule. A basal level of autoinducer is constitutively produced and, in the example of V. fischeri, diffuses freely through the cell envelope (22). The intracellular concentration of V. fischeri autoinducer (VAI) is thus dependent upon bacterial cell density. Inside the cell, the autoinducer and regulatory protein form an active complex. The complex recognizes a target gene operator and activates gene transcription (for a review, see reference 11). The first gene of the V. fischeri lux operon codes for autoinducer synthase (luxI). This induction results in a rapid increase in autoinducer concentration that further serves to induce the lux operon, resulting in light emission. P. aeruginosa quorum-sensing regulatory components are the regulatory protein, LasR, and the autoinducer, 3OC12-HSL (formerly known as PAI-1; N-3-[oxododecanoyl]homoserine lactone) (13, 35, 37). The LasR protein is 30 and 53% identical to V. fischeri regulatory protein (LuxR) at the LuxR autoinducer-binding and DNA-binding domains, respectively (13). The 3OC12-HSL N-acyl side chain is six carbons longer than VAI. Despite structural similarities, the interchangeability of system components in target gene activation is extremely limited (15). In addition, organizational and mechanistic features of the two systems are not identical. In contrast to LuxR and VAI activation of the lux operon, the LasR and 3OC12-HSL complex (LasR/3OC12-HSL) regulated genes are widely separated on the P. aeruginosa chromosome. Two protease genes in addition to lasB are dependent on the presence of LasR/3OC12-HSL: aprA (coding for alkaline protease) and lasA (coding for LasA elastase) (14, 45). Each of these must be independently activated. An additional contrasting feature of the P. aeruginosa system is the active efflux of 3OC12-HSL from the cell, recently reported by Pearson et al. (36), which would limit the intracellular concentration. Thus, cell density and intracellular 3OC12-HSL concentration are not directly proportional in P. aeruginosa. A sequence upstream of the lasB transcriptional start site, OP1 (−33 to −52 relative to the transcriptional start site; Fig. ​Fig.1),1), is a LasR-responsive operator (42). OP1 forms an inverted repeat sequence at 18 of 20 bp and is located immediately upstream of, and may overlap, the lasB promoter sequence. Mutation of OP1 at various positions reduced lasB activity by characteristic amounts (42). Expression of lasB in Escherichia coli was dependent on the presence of LasR and exogenously added 3OC12-HSL, and mutation of OP1 abolished this expression, indicating that OP1 is a LasR-responsive element. FIG. 1 Nucleotide sequence of the lasB promoter region (4). Numbering is relative to the lasB transcriptional start site (+1), which is indicated with an arrow. OP1 and OP2 are underlined and labeled accordingly. In addition to OP1, a sequence 42 bp upstream of OP1, named OP2 (−94 to −112 relative to the transcriptional start site; Fig. ​Fig.1),1), shares 70% identity with OP1. Mutation of OP2 at positions 3 and 5 together reduced lasB induction by 20% (42). These results indicate that all or part of the OP2 sequence is involved in lasB induction. In this report, we use targeted mutagenesis and sequence replacement to show the extent to which OP2 is involved in lasB induction. We express these mutant lasB upstream regions in the presence or absence of P. aeruginosa regulatory components to show that OP2 is a suboptimal, LasR/3OC12-HSL-responsive sequence. We also show that LasR lacking the putative autoinducer-binding domain elicits synergistic activation from OP1 and OP2, and propose alternative models for OP1- and OP2-mediated lasB activation based on these results.