Journal of Bacteriology, December 2000, p. 6940-6949, Vol. 182, No. 24
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Laboratoire de Biologie Microbienne, Université de Lausanne, CH-1015 Lausanne, Switzerland
Received 8 June 2000/Accepted 30 August 2000
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.
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