TRANSCRIPTIONAL ACTIVATOR, FLEQ, REGULATES MUCIN ADHESION AND FLAGELLAR GENE-EXPRESSION IN PSEUDOMONAS-AERUGINOSA IN A CASCADE MANNER

Previous work has demonstrated that fleR, the gene for a transcription al activator belonging to the NtrC subfamily of response regulators, i s involved in the regulation of mucin adhesion and flagellar expressio n by Pseudomonas aeruginosa. This report describes the identification and characterization of fleQ, the gene for another transcriptional reg ulator which also regulates mucin adhesion and motility in this organi sm. The complete nucleotide sequence of the fleQ gene was determined o n both DNA strands, and an open reading frame (ORF) consisting of 1,49 3 nucleotides was identified. This ORF coded for a gene product of pre dicted molecular weight, as confirmed by the overexpression of the fle Q gene as a fusion protein under an inducible promoter. The fleQ gene is flanked by a flagellar operon, fliDSorf126, at the 5' end and the f leSR operon on the 3' end; FleQ also had striking homology to a number of proteins belonging to the NtrC subfamily of response regulators, w hich work in concert with the alternate sigma factor RpoN (sigma(54)) to activate transcription. However, FleQ lacks the residues correspond ing to Asp-54 and Lys-104 of the NtrC protein which are conserved in m ost of the members belonging to this subfamily of regulators. In addit ion, unlike some of the other transcriptional activators of this group , FleQ does not appear to have a cognate sensor kinase. A chromosomal insertional mutation in the fLeQ gene abolished mucin adhesion and mot ility of P. aeruginosa PAK and PAK-NP, Both of these functions were re gained by providing the complete fleQ gene on a multicopy plasmid. The location of fleQ immediately upstream of the fleSR operon, which is a lso necessary for the same process, suggested that these regulators ma y interact in some way. We therefore examined the regulation of the Fl eSR operon by fleQ and vice versa. Promoter fusion experiments showed that the fleSR operon was regulated by RpoN and FleQ. On the other han d, the fleQ promoter was independent of RpoN and FleR. FleQ, thus, add s another level of regulation to motility and adhesion in P. aeruginos a, above that of fleSR. We therefore propose the existence of a regula tory cascade which consists of at least two transcriptional regulators , FleQ and FleR, in the control of motility and adhesion in P. aerugin osa.