Transcriptional control of the hydrogen cyanide biosynthetic genes hcnABC by the anaerobic regulator ANR and the quorum-sensing regulators LasR and RhlR in Pseudomonas aeruginosa

Abstract

Virulence factors of Pseudomonas aeruginosa include hydrogen cyanide (HCN). This secondary metabolite is maximally produced at low oxygen tension and high cell densities during the transition from exponential to stationary growth phase. The hcnABC genes encoding HCN synthase were identified on a genomic fragment complementing an HCN-deficient mutant of P. aeruginosa PAO1. The hcnA promoter was found to be controlled by the FNR-like anaerobic regulator ANR and by the quorum-sensing regulators LasR and RhlR. Primer extension analysis revealed two transcription starts, T1 and T2, separated by 29 bp. Their function was confirmed by transcriptional lacZ fusions. The promoter sequence displayed an FNR/ANR box at -42.5 bp upstream of T2 and a lux box centered around -42.5 bp upstream of T1. Expression of the hcn genes was completely abolished when this lux box was deleted or inactivated by two point mutations in conserved nucleotides. The lux box was recognized by both LasR [activated by N-(oxododecanoyl)-homoserine lactone] and RhlR (activated by N-butanoyl-homoserine lactone), as shown by expression experiments performed in quorum-sensing-defective P. aeruginosa mutants and in the N-acyl-homoserine lactone-negative heterologous host P. fluorescens CHA0. A second, less conserved lux box lying 160 bp upstream of T1 seems to account for enhanced quorum-sensing-dependent expression. Without LasR and RhlR, ANR could not activate the hcn promoter. Together, these data indicate that expression of the hcn promoter from T1 can occur under quorum-sensing control alone. Enhanced expression from T2 appears to rely on a synergistic action between LasR, RhlR, and ANR.

FIG. 1

Cell density-dependent HCN production in P. aeruginosa. The wild type PAO1 (□), the anr mutant PAO6261 (○), and the rhlR mutant PDO111 (▵) were grown with oxygen limitation in 40 ml of MMC at 37°C. HCN was measured as described in Materials and Methods. Each point is the mean of three independent experiments.

FIG. 2

The hcnABC cluster, its promoter region, and lacZ constructs. (A) The hcnABC biosynthetic genes (indicated by arrows) were cloned on a 4.25-kb SalI/XbaI fragment into pBRR1MCS, giving pME3333. (B) The hcnA promoter region (indicated by ▩ in panel A) was mapped as shown in Fig. 4. Numbering is relative to the hcnA ATG start codon. The transcriptional start sites T1 and T2 are indicated by arrows. The putative translation initiation codon (deduced by comparison with the HcnA polypeptide of P. fluorescens [34]) and the potential ribosome binding site (S/D) are underlined. Double underlining indicates the −10 promoter regions. The ANR box and the palindromic lux boxes α and β (Fig. 5) are indicated in boldface. The primer (P_E) used in the primer extension experiment (Fig. 4) is indicated by an arrow above the sequence. The insertion of an SphI restriction site in plasmid pME3852 (Fig. 6) is indicated by an inverted triangle. (C) A translational hcnA′-′lacZ fusion was integrated into the chromosome of the wild type and various mutants (Fig. 3) via homologous recombination. (D) PCR-amplified fragments, containing 130 bp upstream of T1 or 159 bp upstream of T2, were fused to the promoterless lacZ gene of pME6522, resulting in two transcriptional fusions, pME3850.1 and pME3850.2, respectively. The EcoRI site used was created by PCR.

FIG. 3

Cell density-dependent expression of a translational hcnA′-′lacZ fusion in P. aeruginosa. Cultures of PAO6289 (□), the anr mutant PAO6290 (○), the rhlR mutant PAO6293 (▵), and the lasR mutant PAO6326 (■) were grown with oxygen limitation in 40 ml of MMC at 37°C. Each β-galactosidase measurement is the mean of three independent experiments.

FIG. 4

Primer extension analysis of the 5′ end of the hcnABC transcripts. The 5′-end-labeled oligonucleotide P_E (Fig. 2B) was used as described in Materials and Methods. Lanes 1 and 2 show the extension products obtained with RNA from P. aeruginosa PAO1 grown semianaerobically to OD₆₀₀ of 2.3 and 0.4, respectively, under the same conditions as for Fig. 1 and 3. Lane 3 contains the extension product of the hcn transcript from PAO1 grown to an OD₆₀₀ of 0.9 under anaerobic conditions (in 60 ml of MMC; tightly closed 125-ml bottles). Lanes 4 and 5 show transcriptional start sites obtained with RNA from an anr mutant (PAO6261) and an rhlR mutant (PDO111), respectively, grown semianaerobically to an OD₆₀₀ of 0.9. In lanes A, G, C, and T, the sequencing ladders obtained with the unlabeled oligonucleotide P_E were run in parallel. Because of the 5′ phosphate, the primer extension products move 0.5 nucleotide ahead of the corresponding band in the ladder.

FIG. 5

Alignment of the lux boxes found in the hcnABC promoter region with other lux boxes of P. aeruginosa. The sequences shown are found in the promoters of four autoinducible genes, rhlI (42), lasB (48), rhlA (43), and phzA (GenBank accession number AF005404). A similar palindromic (→ ←) consensus sequence has been proposed previously by Whiteley et al. (56). Highly conserved nucleotides (six out of seven) are boxed, and less conserved nucleotides (five out of seven) are indicated in boldface. Two nucleotides that were mutated in the hcnA′ α lux box of pME3844 are shown in ovals.

FIG. 6

Effects of deletions and site-directed mutations on hcnA promoter activity. β-Galactosidase activities were determined in P. aeruginosa wild type PAO1 and in mutants PDO111 (rhlR), PAO6330 (lasRI), and PAO6261 (anr) containing the hcnA′-′lacZ fusion plasmids shown. Cells were grown in 40 ml of MMC with mild oxygen limitation to an OD₆₀₀ of 1.0 to 1.3. Restriction sites introduced artificially are indicated by ∗. The mutated lux box α of pME3844 is indicated by two nucleotides mutated (T, A), and the hcnA gene is shown in gray. β-Galactosidase activity is provided along with the standard deviation (mean of three independent experiments).

FIG. 7

Model for the recognition of the hcn promoter P2 by the transcriptional regulators ANR, LasR, and RhlR and their interaction with RNA polymerase. αCTD (C-terminal domain of α) and αNTD (N-terminal domain of α) of RNA polymerase are shown as separate domains joined by a linker. Positions relative to the transcriptional start site T2 are indicated below the sequence. This model has been adapted from that proposed by Belyaeva et al. (4) for CRP-dependent promoter activation. During oxygen limitation, ANR blocks transcription from site T1 by binding to the corresponding −10 sequence (in brackets).