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 (...

Flagellar number in

In gram-negative bacteria, the alternate sigma factor 54 , working in concert with transcriptional activators that belong to the NtrC superfamily, activates a variety of genes that are regulated in response to external stimuli. For example, in various bacteria, 54 is required for expression of the enzymatic pathways responsible for nitrogen utilization, dicarboxylate transport, xylene degradation, and hydrogen utilization (7,16,19,21,35).54 -regulated genes may be involved in RNA modification (13), chemotaxis, development, energy transduction, fructose assimilation (21), response to heat and phage shock (33), and expression of alternate sigma factors such as H (rpoH) (23) and S (rpoS) (29). In Pseudomonas aeruginosa, 54 is also involved in the regulation of expression of virulence factors including pilin (14), flagellin (31), and alginate (34). There is no obvious theme in the repertoire of functions carried out by 54 -dependent transcripts. Flagellar biogenesis in P. aeruginosa involves more than 40 genes intertwined in a complex regulatory cascade. Its flagellar hierarchy appears to be different from the FlhDC-dependent Salmonella enterica serovar Typhimurium hierarchy (20) and resembles more closely that in Vibrio cholerae, which involves both 28 -and 54 -dependent genes (24). FleQ, a NifA/NtrCtype 54 -dependent activator, is at the highest level of the flagellar hierarchy in P. aeruginosa. Homologues of FleQ have been identified in Caulobacter crescentus (25), V. cholerae (17), and Helicobacter pylori (28) and play important roles in flagellar protein synthesis and secretion. Structurally FleQ lacks the highly conserved phospho-acceptor Asp54 and instead has a serine residue. Phosphorylation could also occur at the serine residue, but lack of evidence to that effect and absence of any cognate sensor kinase probably indicate that FleQ does not require phosphorylation for its activation (9). This is also observed in NifA of Klebsiella pneumoniae, which lacks an Nterminal phospho-acceptor domain, and FlrA of V. cholerae, which lacks the same aspartate residue.Previous studies showed that FleQ positively regulates many flagellar genes (1, 2, 10, 31). These include flhA and fliLM-NOPQ involved in flagellar export; flhF involved in the localization of the flagellar apparatus; fleSR, a two-component sensor and regulator involved in flagellin synthesis; fliEFG, encoding the flagellar basal body MS ring and motor switch complex; fliDS, encoding the flagellar cap and export proteins; and flgA, involved in P-ring formation of the flagellar basal body (N. Dasgupta, unpublished data). The mechanism by which FleQ activates these flagellar genes has not been elucidated. Also, it is unknown whether FleQ acts in the typical manner as other NtrC-like regulators by binding to consensus upstream activating enhancer elements. In this study we have randomly selected four of the above promoters and identified the binding sites of FleQ in order to derive a consensus, if evident. Four different FleQ binding sites were identified whi...

Flagellar biogenesis inPseudomonas aeruginosa is an intricate process in which various transcriptional activators (FleQ, FleR) and alternative sigma factors (RpoN/ 54 , RpoF/ 28 ) participate in the transcriptional regulation of the flagellar genes and operons (3,23,31,33). Of the two transcriptional activators identified in our laboratory, FleQ appears to be the highest-level positive regulator of flagellar biogenesis in P. aeruginosa.FleQ is homologous to the NtrC group of response regulators, which are part of a two-component signal transduction system, that works in concert with the alternative sigma factor RpoN (3). Typically, a two-component system consists of a phosphorelay between a sensor kinase and a response regulator pair (5). The sensor kinase is activated by autophosphorylation when an environmental stimulus is sensed, and the response regulator is subsequently phosphorylated at the receiver domain. The response regulators are usually DNA binding proteins, and phosphorylation activates them to promote activation or repression of the downstream genes (5). However, FleQ and several of its homologs that regulate flagellar biogenesis in other bacterial species, including FlrA of Vibrio cholerae (16), FlaK of Vibrio parahaemolyticus (15), and FlgR of Helicobacter pylori (30), do not contain the hallmark residues in their receiver domains for phosphorylation and do not have a cognate sensor kinase encoded as a part of the same operon. Therefore, phosphorelay signaling does not appear to be the modulating mechanism that regulates the activities of these response regulators. Attempts to detect phosphorylation of FleQ in vitro were not successful in our laboratory, thus raising further doubts about the existence of a putative sensor kinase for FleQ (unpublished data). One of the mechanisms employed to accomplish posttranslational modulation of FleQ activity in P. aeruginosa involves direct protein-protein interactions with FleN, an antiactivator that represses FleQ-dependent transcriptional activation (8). Whether fleQ is subject to transcriptional activation or repression in addition to posttranslational modulation has been a question. Preliminary data have demonstrated that FleQ synthesis occurs from the early log phase to the stationary phase of growth in shaken cultures of P. aeruginosa PAK (unpublished data), which suggests that constitutive expression occurs under these conditions. Whether the constitutive expression observed was driven by a constitutively expressed activator or merely by the housekeeping RNA polymerase holoenzyme is not clear.The flagellar regulon is subject to hierarchical regulation in most of the flagellated bacteria. Among the flagellar regulators of monoflagellate bacteria, flgR, the fleQ homolog of H. pylori,

The opportunistic pathogen Pseudomonas aeruginosa utilizes a type III secretion system (T3SS) to intoxicate eukaryotic host cells. Transcription of the T3SS is induced under calcium-limited growth conditions or following intimate contact of P. aeruginosa with host cells. In the present study, we demonstrate that expression of the T3SS is controlled by two distinct regulatory mechanisms and that these mechanisms are differentially activated in a host cell-dependent manner. The first mechanism is dependent upon ExsC, a regulatory protein that couples transcription of the T3SS to the activity of the type III secretion machinery. ExsC is essential for induction of the T3SS under low-calcium-growth conditions and for T3SS-dependent cytotoxicity towards social amoebae, insect cells, and erythrocytes. The second regulatory mechanism functions independently of ExsC and is sufficient to elicit T3SS-dependent cytotoxicity towards certain types of mammalian cells. Although this second pathway (ExsC independent) is sufficient, an exsC mutant demonstrates a lag in the induction of cytotoxicity towards Chinese hamster ovary cells and is attenuated for virulence in a mouse pneumonia model. We propose that the ExsC-dependent pathway is required for full cytotoxicity towards all host cell types tested whereas the ExsC-independent pathway may represent an adaptation that allows P. aeruginosa to increase expression of the T3SS in response to specific types of mammalian cells.