Cloning, sequence and mutagenesis of the structural gene of Pseudomonas aeruginosa CysB, which can activate algD transcription

Pseudomonas aeruginosa strains infecting patients with cystic fibrosis (CF) acquire a mucoid phenotype due to overproduction of alginate. The key enzyme in alginate synthesis is AlgD, whose promoter is transcriptionally active in mucoid strains and under the control of several trans-acting factors, including the integration host factor (IHF). The algD promoter (palgD) contains two IHF-binding sites (ihf1 and ihf2). Study of IHF binding to ihf2 of palgD, by electrophoretic mobility-shift assays, led to the discovery of a protein of 36 kDa (p36) able to bind downstream from ihf2, to the 3' region of palgD. The gene encoding p36 was isolated from the mucoid strain CHA of P. aeruginosa and sequenced. It can encode a 324-amino-acid protein, which shares a high degree of sequence identity (63%) with CysB from Escherichia coli and from Salmonella typhimurium, a transcriptional factor of the LysR superfamily. Furthermore, both p36 and S. typhimurium CysB bind the same site of palgD; p36 was therefore termed CysB and its structural gene was called cysB. Next to cysB, on the opposite DNA strand, cysH was capable of encoding a protein sharing 26% identity with CysH (PAPS reductase) of E. coli and an even greater identity (54%) with the nucleotide-deduced protein from Arabidopsis. A CysB-deficient mutant of CHA, constructed by insertional inactivation of cysB, was a cysteine auxotroph and was unable to form a specific complex with palgD in vitro. Activity of palgD in the cysB mutant, in CHA and in the non-mucoid strain PAO was assessed by the use of a transcriptional algD-xylE fusion. Cells of PAO and of the cysB mutant grown in minimal media in the presence of 0.3 M NaCl exhibited a palgD activity, which was 10% or less that of the mucoid strain CHA. Thus, P. aeruginosa CysB can act as an activator of algD expression.