The virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of many virulence factors, including type IV pili, which are required for colonization of host tissues and for twitching motility. Type IV pilus function is controlled in part by the Chp chemosensory system, which includes a histidine kinase, ChpA, and two CheY-like response regulators, PilG and PilH. How the Chp components interface with the type IV pilus motor proteins PilB, PilT, and PilU is unknown. We present genetic evidence confirming the role of ChpA, PilG, and PilB in the regulation of pilus extension and the role of PilH and PilT in regulating pilus retraction. Using informative double and triple mutants, we show that (i) ChpA, PilG, and PilB function upstream of PilH, PilT, and PilU; (ii) that PilH enhances PilT function; and (iii) that PilT and PilB retain some activity in the absence of signaling input from components of the Chp system. By site-directed mutagenesis, we demonstrate that the histidine kinase domain of ChpA and the phosphoacceptor sites of both PilG and PilH are required for type IV pilus function, suggesting that they form a phosphorelay system important in the regulation of pilus extension and retraction. Finally, we present evidence suggesting that pilA transcription is regulated by intracellular PilA levels. We show that PilA is a negative regulator of pilA transcription in P. aeruginosa and that the Chp system functionally regulates pilA transcription by controlling PilA import and export.

SummaryVirulence of Pseudomonas aeruginosa involves the co-ordinate expression of a range of factors including type IV pili (tfp), the type III secretion system (TTSS) and quorum sensing. Tfp are required for twitching motility, efficient biofilm formation, and for adhesion and type III secretion (TTS)-mediated damage to mammalian cells. We describe a novel gene ( fimL ) that is required for tfp biogenesis and function, for TTS and for normal biofilm development in P. aeruginosa . The predicted product of fimL is homologous to the Nterminal domain of ChpA, except that its putative histidine and threonine phosphotransfer sites have been replaced with glutamine. fimL mutants resemble vfr mutants in many aspects including increased autolysis, reduced levels of surface-assembled tfp and diminished production of type III secreted effectors. Expression of vfr in trans can complement fimL mutants. vfr transcription and production is reduced in fimL mutants whereas cAMP levels are unaffected. Deletion and insertion mutants of fimL frequently revert to wild-type phenotypes suggesting that an extragenic suppressor mutation is able to overcome the loss of fimL . vfr transcription and production, as well as cAMP levels, are elevated in these revertants, while Pseudomonas quinolone signal (PQS) production is reduced. These results suggest that the site(s) of spontaneous mutation is in a gene(s) which lies upstream of vfr transcription, cAMP, production, and PQS synthesis. Our studies indicate that Vfr and FimL are components of intersecting pathways that control twitching motility, TTSS and autolysis in P. aeruginosa .

Three promoters have been identified as having potentially important regulatory roles in governing expression of the fla/che operon and of sigD, a gene that lies near the 3 end of the operon. Two of these promoters, fla/che P A and P D-3 , lie upstream of the >26-kb fla/che operon. The third promoter, P sigD , lies within the operon, immediately upstream of sigD. fla/che P A , transcribed by E A , lies >24 kb upstream of sigD and appears to be largely responsible for sigD expression. P D-3 , transcribed by E D , has been proposed to participate in an autoregulatory positive feedback loop. P sigD , a minor A -dependent promoter, has been implicated as essential for normal expression of the fla/che operon. We tested the proposed functions of these promoters in experiments that utilized strains that bear chromosomal deletions of fla/che P A , P D-3 , or P sigD . Our analysis of these strains indicates that fla/che P A is absolutely essential for motility, that P D-3 does not function in positive feedback regulation of sigD expression, and that P sigD is not essential for normal fla/che expression. Further, our results suggest that an additional promoter(s) contributes to sigD expression.Motility and chemotaxis functions in Bacillus subtilis are encoded within the fla/che operon. This large (Ͼ26-kb) operon includes both structural and regulatory components required for motility (6,19,32). The proximal region of the operon includes genes that encode the hook and basal body (HBB) complex, a structure that is required for tethering the flagellar filament to the cell. The distal-most region of the fla/che operon encodes the flagellum-specific sigma factor, D (19). D activity is required for transcription of the genes encoding flagellin (hag) and for the motA and motB genes, which encode the motor proteins that drive flagellar rotation (21,22 (11,15). It is believed that FlgM activity is also controlled by export through the HBB in B. subtilis (5,21,24). Expression of the fla/che operon thus controls motility in a complex manner. First, HBB components are expressed concurrently with D . Subsequent assembly of the HBB structure allows export of FlgM. This activates D to promote transcription of D -dependent motility genes. Recent studies have implicated three promoters in the expression of the fla/che operon and the sigD gene (1, 6) (see schematic, Fig. 1A). One of these promoters, P sigD , is located within the fla/che operon, immediately upstream of the sigD gene. Transcription from P sigD is dependent upon A , the major, vegetative sigma factor in B. subtilis. Previous studies indicated that P sigD contributed only weakly to overall expression of the sigD gene (1). However, genetic data suggested that this slight level of expression might be required to control temporal regulation of the entire fla/che operon (1). This requirement would presumably be indirect, since the location of P sigD precludes it from directly promoting transcription of the fla/che operon (see schematic in Fig. 1A).Two additional promoters, fla/ch...