THE PSEUDOMONAS-AERUGINOSA PILK GENE ENCODES A CHEMOTACTIC METHYLTRANSFERASE (CHER) HOMOLOG THAT IS TRANSLATIONALLY REGULATED

A new locus, designated pilK, located immediately adjacent to the prev iously described Pseudomonas aeroginosa pilG-J gene cluster, has been identified. Sequence analysis of a 1.3 kb region revealed the presence of a single open reading frame of 291 amino acid residues (M(r), 33 3 38) that contained significant homology to the chemotactic methyltrans ferase proteins of Escherichia coli, Bacillus subtilis and the gliding bacterium Myxococcus xanthus. The 60 bp pilJ-pilK intergenic region w as devoid of promoter consensus sequences, suggesting that pilJ and pi lK are contained within the same transcriptional unit. The intergenic region did contain, however, a large, highly GC-rich, inverted repeat that prevented PilK production in expression studies. To investigate t he regulatory role of these sequences, pilK-lacZ gene fusions, as well as derivatives containing sequence alterations in the potential stem- loop region, were constructed and analysed in E. coli and P. aeruginos a. Modification of the inverted repeat region in pilK-lacZ protein fus ion constructs resulted in as much as a 24-fold increase in beta-galac tosidase activity, whereas similar modifications in pilK-lacZ transcri ptional fusions had only a marginal effect on beta-galactosidase level s. These results indicated that PilK production may be largely regulat ed at the level of translation, In stark contrast to pilG-J mutants, w hich are dramatically impaired in pilus production and/or function, a PAO1 pilK deletion mutant was indistinguishable from the wild type. In addition, complementation studies suggested that the PilK and E. coli CheR proteins are not functionally interchangeable.