SummaryThe single polar flagellum of Pseudomonas aeruginosa is an important virulence and colonization factor of this opportunistic pathogen. In this study, the annotation of the genes belonging to the fla regulon was updated and their organization was analysed in strains PAK and PAO1, representative type-a and type-b strains of P. aeruginosa respectively. The flagellar genes are clustered in three non-contiguous regions of the chromosome.

Pseudomonas aeruginosa can develop resistance to polymyxin and other cationic antimicrobial peptides. Previous work has shown that mutations in the PmrAB and PhoPQ regulatory systems can confer low to moderate levels of colistin (polymyxin E) resistance in laboratory strains and clinical isolates of this organism (MICs of 8 to 64 mg/liter). To explore the role of PmrAB in high-level clinical polymyxin resistance, P. aeruginosa isolates from chronically colistin-treated cystic fibrosis patients, most with colistin MICs of >512 mg/liter, were analyzed. These cystic fibrosis isolates contained probable gain-of-function pmrB alleles that conferred polymyxin resistance to strains with a wild-type or pmrAB deletion background. Double mutant pmrB alleles that contained mutations in both the periplasmic and dimerization-phosphotransferase domains markedly augmented polymyxin resistance. Expression of mutant pmrB alleles induced transcription from the promoter of the arnB operon and stimulated addition of 4-amino-L-arabinose to lipid A, consistent with the known role of this lipid A modification in polymyxin resistance. For some highly polymyxin-resistant clinical isolates, repeated passage without antibiotic selection pressure resulted in loss of resistance, suggesting that secondary suppressors occur at a relatively high frequency and account for the instability of this phenotype. These results indicate that pmrB gain-of-function mutations can contribute to high-level polymyxin resistance in clinical strains of P. aeruginosa.

SummaryExpression of the Pseudomonas aeruginosa type III secretion system (TTSS) is coupled to the secretion status of the cells. Environmental signals such as calcium depletion activate the type III secretion channel and, as a consequence, type III gene transcription is derepressed. Two proteins, ExsA and ExsD, were shown previously to play a role in coupling transcription to secretion. ExsA is an activator of TTSS gene transcription, and ExsD is an antiactivator of ExsA. In the absence of environmental secretion cues, ExsD binds ExsA and inhibits transcription. Here, we describe the characterization of ExsC as an anti-anti-activator of TTSS expression. Transcription of the TTSS is repressed in an exsC mutant and is derepressed upon ExsC overexpression. The dependence on exsC for transcription is relieved in the absence of exsD , suggesting that ExsC and ExsD function together to regulate transcription. Consistent with this idea, ExsC interacts with ExsD in bacterial two-hybrid and co-purification assays. We propose a model in which the anti-antiactivator (ExsC) binds to and sequesters the antiactivator (ExsD) under low Ca 2+ conditions, freeing ExsA and allowing for transcription of the TTSS. The P. aeruginosa system represents the first example of an anti-activator/anti-anti-activator pair controlling transcription of a TTSS.

The single polar flagellum of Pseudomonas aeruginosa plays an important role in the pathogenesis of infection by this organism. However, regulation of the assembly of this organelle has not been delineated. In analyzing the sequence available at the Pseudomonas genome database, an open reading frame (ORF), flanked by flagellar genes flhF and fliA, that coded for a protein (280 amino acids) with an ATP-binding motif at its N terminus was found. The ORF was inactivated by inserting a gentamicin cassette in P. aeruginosa PAK and PAO1. The resulting mutants were nonmotile on motility agar plates, but under a light microscope they exhibited random movement and tumbling behavior. Electron microscopic studies of the wild-type and mutant strains revealed that the mutants were multiflagellate, with three to six polar flagella per bacterium as rather than one as in the wild type, indicating that this ORF was involved in regulating the number of flagella and chemotactic motility in P. aeruginosa. The ORF was named fleN. Flagella serve primarily as locomotory organelles in flagellated bacterial species. They have also been implicated in biofilm development in Pseudomonas aeruginosa and Escherichia coli (24,26) and in the pathogenesis of infections by P. aeruginosa, Campylobacter jejuni, Helicobacter pylori, and Vibrio cholerae (23,25). The bacterial flagellum is a complex structure requiring more than 40 genes for its assembly (18). Over the years, significant progress has been made in identifying various flagellar structural and regulatory genes, elucidating the composition of flagellar substructures, and understanding the mechanisms of its assembly in a number of bacterial species, including E. coli, Salmonella enterica serovar Typhimurium (1,18,22), and Caulobacter crescentus (28,38). Work is in progress to elucidate the pathway of flagellar assembly in the pathogens P. aeruginosa, V. cholerae, and H. pylori.Most flagellated bacterial species have extracellular flagella; exceptions are spirochetes such as pathogenic Treponema pallidum and Borrelia burgdorferi, which have flagella implanted in the periplasmic space. In some cases, extracellular flagella are covered by a sheath, as in V. cholerae and H. pylori (23). The distribution of flagella may be monotrichous polar as in P. aeruginosa (14) and V. cholerae (39) or peritrichous (5 to 10 flagella) as in E. coli and Salmonella serovar Typhimurium (18). Flagellar number, a characteristic feature of each species, is successfully maintained over the generations, but nothing is known about the genes and the mechanisms which contribute to its regulation. In a recent model proposed for Salmonella, the number of flagellar filaments has been linked to the cell cycle (2).A major goal of our laboratory is to gain an insight into the biogenesis pathway of the flagellum of P. aeruginosa. The published information from our laboratory and elsewhere (5,6,29,33) indicates that the system deviates from the well-described enteric model (18). It has certain similarities to the Caulobacter (...