We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene. About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in E. coli, we estimate that the actual number of essential genes is 300 -400. Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies. Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities. The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering.hole-genome sequences provide the foundation for the creation of relatively complete collections of strains carrying defined mutations in individual genes. Such libraries should facilitate the comprehensive identification of genes required for a wide range of biological processes. A nearly complete library of single-gene deletions of Saccharomyces cerevisiae has been assembled by an international consortium using a PCR-based mutagenesis approach (1). Other projects, also following a strategy of gene-by-gene disruption, are underway for Escherichia coli (E. coli genome project, www. genome.wisc.edu͞functional͞tnmutagenesis.htm), and have recently been completed for Bacillus subtilis (2).An alternative strategy for generating mutant libraries consists of ''random'' whole-genome transposon-insertion mutagenesis followed by sequence-based identification of insertion sites. The approach is cost-effective and applicable to a wide variety of microbes (3, 4). Studies with yeast, in which a collection of mutants corresponding to about one-third of the genes were represented, have illustrated that the generation of large, arrayed collections of insertion mutants is feasible (5). Other studies with bacteria have analyzed large numbers of transposon insertion mutants to identify genes essential for growth, although the mutants were analyzed within populations rather than being archived in a format allowing additional phenotypes to be examined (6)(7)(8). In this report, we describe the generation and initial phenotypic analysis of a near-saturation library of transposon insertion mutants of the opportunistic pathogen Pseudomonas aeruginos...

We constructed a derivative of transposon

A set of 30 mutants exhibiting reduced production of the phenazine poison pyocyanin were isolated following transposon mutagenesis of Pseudomonas aeruginosa PAO1. The mutants could be subdivided into those with defects in the primary phenazine biosynthetic pathway and those with more pleiotropic defects. The largest set of pleiotropic mutations blocked the production of the extracellular Pseudomonas quinolone signal (PQS), a molecule required for the synthesis of secondary metabolites and extracellular enzymes. Most of these pqs mutations affected genes which appear to encode PQS biosynthetic functions, although a transcriptional regulator and an apparent response effector were also represented. Two of the genes required for PQS synthesis (phnA and phnB) had previously been assumed to encode phenazine biosynthetic functions. The transcription of one of the genes required for PQS synthesis (PA2587/pqsH) was regulated by the LasI/R quorum-sensing system, thereby linking quorum sensing and PQS regulation. Others of the pleiotropic phenazine-minus mutations appear to inactivate novel components of the quorum-sensing regulatory network, including one regulator (np20) previously shown to be required for virulence in neutropenic mice.A complex network of regulatory factors governs the production of secondary metabolites and other virulence factors in the opportunistic pathogen Pseudomonas aeruginosa. This network regulates gene expression in response to stimuli such as growth phase, culture density, and oxygen and iron availability (12,26,28,37). Central components of the network are the las and rhl quorum-sensing systems, which activate gene expression in response to culture density (13). Each system is made up of two genes, one encoding an enzyme which produces a specific acylated homoserine lactone autoinducer (lasI/rhlI), and a second encoding a transcriptional activator that binds the corresponding autoinducer (lasR/rhlR). The las system directs expression of virulence factors such as elastases A and B and alkaline protease (16,25). The rhl system directs expression of rhamnolipid biosynthesis enzymes, pyocyanin biosynthesis enzymes, and hydrogen cyanide synthase (3,24,28). In addition, LasI/R regulates expression of both itself and rhlI (1, 26). The las and rhl systems together have been shown to influence the expression of over two hundred genes (36).Recently, a third signaling system based on 2-heptyl-3-hydroxy-4-quinolone, designated the Pseudomonas quinolone signal (PQS), has been shown to be a part of the quorumsensing regulatory network in P. aeruginosa (27). The production of PQS depends on lasR (27), and exogenous PQS strongly induces expression of elastase B and rhlI in a lasR mutant background (22). These results place PQS between the las and rhl quorum-sensing systems in the quorum-sensing regulatory network (22).We have described a process ("paralytic killing") in which P. aeruginosa PAO1 rapidly kills the nematode Caenorhabditis elegans by cyanide poisoning (8, 14). Previous studies of a different P....

Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats, ranging from soil to immunocompromised people. The formation of surface-associated communities called biofilms is one factor thought to enhance colonization and persistence in these diverse environments. Another factor is the ability of P. aeruginosa to diversify genetically, generating phenotypically distinct subpopulations. One manifestation of diversification is the appearance of colony morphology variants on solid medium. Both laboratory biofilm growth and chronic cystic fibrosis (CF) airway infections produce rugose small-colony variants (RSCVs) characterized by wrinkled, small colonies and an elevated capacity to form biofilms. Previous reports vary on the characteristics attributable to RSCVs. Here we report a detailed comparison of clonally related wild-type and RSCV strains isolated from both CF sputum and laboratory biofilm cultures. The clinical RSCV had many characteristics in common with biofilm RSCVs. Transcriptional profiling and Biolog phenotypic analysis revealed that RSCVs display increased expression of the pel and psl polysaccharide gene clusters, decreased expression of motility functions, and a defect in growth on some amino acid and tricarboxylic acid cycle intermediates as sole carbon sources. RSCVs also elicited a reduced chemokine response from polarized airway epithelium cells compared to wild-type strains. A common feature of all RSCVs analyzed in this study is increased levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP). To assess the global transcriptional effects of elevated c-di-GMP levels, we engineered an RSCV strain that had elevated c-di-GMP levels but did not autoaggregate. Our results showed that about 50 genes are differentially expressed in response to elevated intracellular c-di-GMP levels. Among these genes are the pel and psl genes, which are upregulated, and flagellum and pilus genes, which are downregulated. RSCV traits such as increased exopolysaccharide production leading to antibiotic tolerance, altered metabolism, and reduced immunogenicity may contribute to increased persistence in biofilms and in the airways of CF lungs.Pseudomonas aeruginosa is responsible for chronic infections in the airways of cystic fibrosis (CF) patients (13). During the course of chronic infection, P. aeruginosa forms biofilms, which are thought to promote persistence by protecting the bacterium from antibiotics and host clearance. P. aeruginosa also undergoes phenotypic and genotypic diversification. A manifestation of this diversification is the appearance of colony morphology variants among CF sputum sample isolates. One clear example of this phenomenon, which has been termed "dissociative" behavior (42), is the appearance of mucoid colonies. Mucoidy is characterized by overproduction of the exopolysaccharide (EPS) alginate, a polymer of 1,4-␤-linked mannuronic acid and its epimer, guluronic acid (13). The appearance of mucoid colonies is thought to correlate with a downturn in the p...

The opportunistic pathogenic bacterium Pseudomonas aeruginosa uses quorum-sensing signaling systems as global regulators of virulence genes. There are two quorum-sensing signal receptor and signal generator pairs, LasR-LasI and RhlR-RhlI. The recently completed P. aeruginosa genome-sequencing project revealed a gene coding for a homolog of the signal receptors, LasR and RhlR. Here we describe a role for this gene, which we call qscR. The qscR gene product governs the timing of quorum-sensing-controlled gene expression and it dampens virulence in an insect model. We present evidence that suggests the primary role of QscR is repression of lasI. A qscR mutant produces the LasI-generated signal prematurely, and this results in premature transcription of a number of quorum-sensing-regulated genes. When fed to Drosophila melanogaster, the qscR mutant kills the animals more rapidly than the parental P. aeruginosa. The repression of lasI by QscR could serve to ensure that quorum-sensing-controlled genes are not activated in environments where they are not useful.