We describe swarming in Pseudomonas aeruginosa as a third mode of surface translocation in addition to the previously described swimming and twitching motilities. Swarming in P. aeruginosa is induced on semisolid surfaces (0.5 to 0.7% agar) under conditions of nitrogen limitation and in response to certain amino acids. Glutamate, aspartate, histidine, or proline, when provided as the sole source of nitrogen, induced swarming, while arginine, asparagine, and glutamine, among other amino acids, did not sustain swarming. Cells from the edge of the swarm were about twice as long as cells from the swarm center. In both instances, bacteria possessing two polar flagella were observed by light and electron microscopy. While a fliC mutant of P. aeruginosa displayed slightly diminished swarming, a pilR and a pilA mutant, both deficient in type IV pili, were unable to swarm. Furthermore, cells with mutations in the las cell-to-cell signaling system showed diminished swarming behavior, while rhl mutants were completely unable to swarm. Evidence is presented for rhamnolipids being the actual surfactant involved in swarming motility, which explains the involvement of the cell-to-cell signaling circuitry of P. aeruginosa in this type of surface motility.Pseudomonas aeruginosa is a gram-negative bacterium living in soil and aqueous environments, where it survives due to its extraordinary metabolic abilities. P. aeruginosa is also a typical opportunistic pathogen which colonizes the lungs of cystic fibrosis patients and causes severe infections in immunocompromised hosts. Due to its notorious elevated intrinsic resistance to antimicrobial agents and its ability to attach to and to form biofilms on medical devices (9), P. aeruginosa is difficult to eradicate in the hospital environment.P. aeruginosa has a single polar flagellum which enables the cell to swim in aqueous environments and in low-agar (Ͻ0.4%) medium. The flagellum and the chemotaxis system, consisting of chemoreceptors (11, 49) and a signal relay system similar to that of Escherichia coli (25,31), allow the bacterium to respond to attractants and repellents. In addition, P. aeruginosa is able to propagate at surface interfaces by twitching motility, which is mediated by type IV pili (5,12,53). Twitching motility is believed to result from the extension and retraction of the pilus filament, which propels the cells across a surface. Pilus synthesis and assembly require at least 40 genes which are located in several unlinked regions on the chromosome (22). The nature of the environmental signal that triggers the expression of pili is not known. Pili are important for attachment to epithelial cells (8, 17) and contribute to the virulence of P. aeruginosa in animal models (19,50,51). Furthermore, twitching motility and, hence, type IV pili are required for the formation of biofilms on abiotic surfaces (38).Besides swimming and twitching, several gram-negative bacteria are able to propagate on semisolid surfaces (i.e., 0.4 to 1.0% agar) in a coordinated manner by swarming ...

Antibiotic-resistant mutants of Pseudomonas aeruginosa were generated using chloramphenicol and ciprofloxacin as selective agents. These mutants displayed a multidrug phenotype and overexpressed an outer membrane protein of 50 kDa, which was shown by Western blot analysis to correspond to OprN. A cosmid clone harbouring the oprN gene was isolated by partial complementation of a mutant deficient in OprM, the outer membrane component of the mexAB-oprM efflux operon. Antibiotic-accumulation studies indicated that OprN was part of an energy-dependent antibiotic-efflux system. Sequencing of a 6180bp fragment from the complementing cosmid revealed the presence of three open reading frames (ORFs), which exhibited amino acid similarity to the components of the mexAB-oprM and mexCD-oprJ efflux operons of P. aeruginosa. The ORFs were designated MexE, MexF and OprN. Mutation of the mexE gene eliminated the multidrug-resistance phenotype in an OprN-overexpressing strain, but did not affect the susceptibility profile of the wild-type strain. Expression of the mexEF-oprN operon was shown to be positively regulated by a protein encoded on a 1.5 kb DNA fragment located upstream of mexE and belonging to the LysR family of transcriptional activators. The presence of a plasmid containing this DNA fragment was sufficient to confer a multidrug phenotype onto the wild-type strain but not onto the mexE mutant. Evidence is provided to show that the mexEF-oprN operon may be involved in the excretion of intermediates for the biosynthesis of pyocyanin, a typical secondary metabolite of P. aeruginosa.

The metabolically versatile Gram-negative bacterium Pseudomonas aeruginosa inhabits terrestrial, aquatic, animal-, human-, and plant-host-associated environments and is an important causative agent of nosocomial infections, particularly in intensive-care units. The population genetics of P. aeruginosa was investigated by an approach that is generally applicable to the rapid, robust, and informative genotyping of bacteria. DNA, amplified from the bacterial colony by circles of multiplex primer extension, is hybridized onto a microarray to yield an electronically portable binary multimarker genotype that represents the core genome by single nucleotide polymorphisms and the accessory genome by markers of genomic islets and islands. The 240 typed P. aeruginosa strains of diverse habitats and geographic origin segregated into two large nonoverlapping clusters and 45 isolated clonal complexes with few or no partners. The majority of strains belonged to few dominant clones widespread in disease and environmental habitats. The most frequent genotype was represented by the sequenced strain PA14. Core and accessory genome were found to be nonrandomly assembled in P. aeruginosa. Individual clones preferred a specific repertoire of accessory segments. Even the most promiscuous genomic island, pKLC102, had integrated preferentially into a subset of clones. Moreover, some physically distant loci of the core genome, including oriC, showed nonrandom associations of genotypes, whereas other segments in between were freely recombining. Thus, the P. aeruginosa genome is made up of clone-typical segments in core and accessory genome and of blocks in the core with unrestricted gene flow in the population. bacterial evolution ͉ chip technology ͉ population genetics P seudomonas aeruginosa is a metabolically versatile Gramnegative bacterium, which inhabits terrestrial, aquatic, animal-, human-, and plant-host-associated environments (1). This opportunistic pathogen is the most dominant bacterium causing chronic infections in the cystic fibrosis (CF) lung (2) and has emerged as an important causative agent of nosocomial infections, particularly in intensive-care units (3).The P. aeruginosa genome is a mosaic of a conserved core and variable accessory segments (4). The core genome is characterized by a conserved synteny of genes, a low average nucleotide divergence of 0.5%, and multiple alleles at a few loci that are subject to diversifying selection (4-6). The accessory genome consists of a variable set of genomic islets and genomic islands, most of which belong to an ancient tRNA-integrated island type (4, 7-11). Genome size ranges from 5.2 to 7 Mbp in the P. aeruginosa population (4).Typing informative traits allows identification of bacterial isolates to the strain level and provides basic information about the evolutionary biology, population biology, taxonomy, ecology, and genetics of bacteria. Typically, strains of bacteria, including P. aeruginosa, have been differentiated on the basis of specific phenotypic traits or anonymous ...

Pseudomonas aeruginosa is an environmental bacterium involved in mineralization of organic matter. It is also an opportunistic pathogen able to cause serious infections in immunocompromised hosts. As such, it is exposed to xenobiotics including solvents, heavy metals, and antimicrobials. We studied the response of P. aeruginosa upon exposure to heavy metals or antibiotics to investigate whether common regulatory mechanisms govern resistance to both types of compounds. We showed that sublethal zinc concentrations induced resistance to zinc, cadmium, and cobalt, while lethal zinc concentrations selected mutants constitutively resistant to these heavy metals. Both zinc-induced and stable zinc-resistant strains were also resistant to the carbapenem antibiotic imipenem. On the other hand, only 20% of clones selected on imipenem were also resistant to zinc. Heavy metal resistance in the mutants could be correlated by quantitative real time PCR with increased expression of the heavy metal efflux pump CzcCBA and its cognate two-component regulator genes czcR-czcS. Western blot analysis revealed reduced expression of the basic amino acid and carbapenem-specific OprD porin in all imipenem-resistant mutants. Sequencing of the czcR-czcS DNA region in eight independent zincand imipenem-resistant mutants revealed the presence of the same V194L mutation in the CzcS sensor protein. Overexpression in a susceptible wild type strain of the mutated CzsS protein, but not of the wild type form, resulted in decreased oprD and increased czcC expression. We further show that zinc is released from latex urinary catheters into urine in amounts sufficient to induce carbapenem resistance in P. aeruginosa, possibly compromising treatment of urinary tract infections by this class of antibiotics.Pseudomonas aeruginosa is a Gram-negative bacterium thriving in environments polluted with organic matter. It is also an opportunistic pathogen frequently encountered in the hospital, causing morbidity and mortality in immunocompromised and cystic fibrosis patients (1). P. aeruginosa is characterized by an intrinsically high level of resistance to xenobiotics including antimicrobial agents, solvents, and heavy metals (2), which can be accounted for by a combination of its low outer membrane permeability and the presence of multiple efflux pumps (3). These pumps belong to the resistance, nodulation, cell division (RND) 1 transporter family, present in many Gramnegative bacteria (4). To extrude substrates from the cytoplasm across the two membranes, these systems are composed of a proton antiporter located in the cytoplasmic membrane, a membrane fusion protein spanning the periplasmic space, and an outer membrane protein (5). Members of the RND family, namely the Mex pumps, have recently gained increasing interest. In particular, the constitutively expressed MexAB-OprM (6, 7) and the inducible MexXY (8) efflux pumps endow the PAO1 reference strain and other clinical isolates (9) with a natural resistance to a wide range of antimicrobial agents. Proton-dr...

Intrinsic and acquired antibiotic resistance of the nosocomial pathogen Pseudomonas aeruginosa is mediated mainly by the expression of several efflux pumps of broad substrate specificity. Here we report that nfxC type mutants, overexpressing the MexEF-OprN efflux system, produce lower levels of extracellular virulence factors than the susceptible wild type. These include pyocyanin, elastase, and rhamnolipids, three factors controlled by the las and rhl quorum-sensing systems of P. aeruginosa. In agreement with these observations are the decreased transcription of the elastase gene lasB and the rhamnosyltransferase genes rhlAB measured in nfxC type mutants. Expression of the lasR and rhlR regulator genes was not affected in the nfxC type mutant. In contrast, transcription of the C4-homoserine lactone (C4-HSL) autoinducer synthase gene rhlI was reduced by 50% in the nfxC type mutant relative to that in the wild type. This correlates with a similar decrease in C4-HSL levels detected in supernatants of the nfxC type mutant. Transcription of an rhlAB-lacZ fusion could be partially restored by the addition of synthetic C4-HSL and Pseudomonas quinolone signal (PQS). It is proposed that the MexEF-OprN efflux pump affects intracellular PQS levels.Pseudomonas aeruginosa is an opportunistic pathogen which may cause pneumonia and bacteremia in immunocompromised hosts and is responsible for chronic destructive lung disease in patients suffering from cystic fibrosis. The pathogenicity of P. aeruginosa is attributable to an arsenal of virulence factors, some of which are cell associated (pili, nonpilus adhesins, lipopolysaccharide, and alginate) while others are secreted (proteases, rhamnolipids, exotoxin A, exoenzyme S, and pyocyanin). The production of many of these extracellular virulence factors is controlled by two cell-to-cell signaling systems, called las and rhl, which are both composed of a transcriptional regulator (LasR and RhlR, respectively) and an autoinducer synthase (LasI and RhlI, respectively). LasI and RhlI catalyze the last step in the synthesis of the cell-to-cell signaling molecules 3-oxo-C12-homoserine lactone (3-oxo-C12-HSL) and C4-HSL, respectively; each of these molecules binds to, and activates, its corresponding transcriptional regulator. The systems are connected via a hierarchical cascade (19) and allow coordinated production of extracellular virulence factors, which occurs only when the bacterial cell density has reached a threshold (quorum). Recently, a novel signaling molecule, called PQS, for Pseudomonas quinolone signal (39), has been identified. Furthermore, the published genome sequence of PAO1 (53) has revealed a new modulator of cellto-cell signaling, termed QscR (4). This protein is homologous to both LasR and RhlR and seems to prevent premature transcription of quorum-sensing regulated genes.Besides its pathogenic capabilities, P. aeruginosa is well known for its intrinsic resistance to a wide range of antimicrobial agents and its ability to develop multidrug resistance following antibi...