Search

Examines the mechanism by which Pseudomonas aeruginosa (P. aeruginosa) rapidly paralyzes and kills the Caenorhabditis elegans (C. elegans). Influence of hydrogen cyanide produced by P. aeruginosa on the death of nematode; Investigation on the bacterial factors related to mediate killing of C. elegans by strain PAO1; Assessment on the mutations of cyanide production.

Examines a set of mutants of the Escherichia coli lac repressor. Reasons for the examination; Details on the methodology used; Results of the examination; Implications of the results for structure-function analysis.

Discusses the role of the N-terminal domain in the establishment of serine chemoreceptor membrane topology in Escherichia coli. Separation of N and C termini from a central periplasmic domain; Use of alkaline phosphate gene fusions; Effect of elimination of the positive charge of the domain; Requirements of efficient export of C-terminal domain.

The author comments on a study published within the isse which argues that the activation of CpxRA actually helps bacteria resist aminoglycoside and hydroxyurea lethality. He believes that the common pathway model for antibiotic lethality links broad areas of bacterial physiology and accounts for large sets of genome-scale data. The author finds that the model was thus a plausible working hypothesis with experimentally testable predictions.

The P. aeruginosa reference strain PAO1 has been used to delineate much of the physiology, metabolism, and fundamental biology of the species. The wild-type parent of PAO1 was lost, and PAO1 carries a regulatory mutation introduced for positive genetic selection that affects antibiotic resistance, virulence, quorum sensing, and other traits. The mutation is a loss-of-function change in an oxidoreductase gene (mexS), which constitutively activates a stress response controlled by a positive regulator (MexT). Fitness defects associated with the constitutive response have led to the inadvertent selection of mexT-minus suppressor mutations, creating genetic heterogeneity in PAO1 sublines studied in different laboratories. To help circumvent complications due to the mexS-minus phenotypes, we created a wild-type version of PAO1 (called LPAO) by "reverting" its mexS to the functional allele likely to have been in its parent. Phenotypic analysis revealed that the mexS-minus allele in PAO1 makes growth sensitive to salt (NaCl) and is lethal when combined with mutations inactivating the major sodium antiporter (ShaABCDEF). The salt sensitivity of PAO1 may underlie some complex mexS-minus phenotypes and help explain the selection of mexT-minus suppressor mutations. To facilitate genetic comparisons of PAO1, LPAO, and other P. aeruginosa strains, we developed a transformation procedure to transfer selectable alleles, such as transposon insertion alleles, between strains. Overall, the study helps explain phenotypic heterogeneity of PAO1-derived strains and provides resources to help recognize and eliminate difficulties due to it. [ABSTRACT FROM AUTHOR]

We exploited the natural histidine auxotrophy of FrawciseHa species to develop hisD (encodes histidinol dehydrogenase) as a positive selection marker. A shuttle plasmid (pBR103) carrying Escherichia coli hisD and designed for cloning of PCR fragments replicated in both attenuated and highly virulent Francisella strains. During this work, we formulated a simplified defined growth medium for Francisella novicida. [ABSTRACT FROM AUTHOR]

Rhodopseudomonas palustris is an alphaproteobacterium that has served as a model organism for studies of photophosphorylation, regulation of nitrogen fixation, production of hydrogen as a biofuel, and anaerobic degradation of aromatic compounds. This bacterium is able to transition between anaerobic photoautotrophic growth, anaerobic photoheterotrophic growth, and aerobic heterotrophic growth. As a starting point to explore the genetic basis for the metabolic versatility of R. palustris, we used transposon mutagenesis and Tn-seq to identify 552 genes as essential for viability in cells growing aerobically on semirich medium. Of these, 323 have essential gene homologs in the alphaproteobacterium Caulobacter crescentus, and 187 have essential gene homologs in Escherichia coli. There were 24 R. palustris genes that were essential for viability under aerobic growth conditions that have low sequence identity but are likely to be functionally homologous to essential E. coli genes. As expected, certain functional categories of essential genes were highly conserved among the three organisms, including translation, ribosome structure and biogenesis, secretion, and lipid metabolism. R. palustris cells divide by budding in which a sessile cell gives rise to a motile swarmer cell. Conserved cell cycle genes required for this developmental process were essential in both C. crescentus and R. palustris. Our results suggest that despite vast differences in lifestyles, members of the alphaproteobacteria have a common set of essential genes that is specific to this group and distinct from that of gammaproteobacteria like E. coli. [ABSTRACT FROM AUTHOR]

Hospital environments are excellent reservoirs for the opportunistic pathogen Acinetobacter baumannii in part because it is exceptionally tolerant to desiccation. We found that relative to other A. baumannii strains, the virulent strain AB5075 was strikingly desiccation resistant at 2% relative humidity (RH), suggesting that it is a good model for studies of the functional basis of this trait. Consistent with results from other A. baumannii strains at 40% RH, we found the global posttranscriptional regulator CsrA to be critically important for desiccation tolerance of AB5075 at 2% RH. Proteomics experiments identified proteins that were differentially present in wild-type and csrA mutant cells. Subsequent analysis of mutants in genes encoding some of these proteins revealed six genes that were required for wild-type levels of desiccation tolerance. These include genes for catalase, a universal stress protein, a hypothetical protein, and a biofilm-associated protein. Two genes of unknown function had very strong desiccation phenotypes, with one of the two genes predicting an intrinsically disordered protein (IDP) that binds to DNA. Intrinsically disordered proteins are widespread in eukaryotes but less so in prokaryotes. Our results suggest there are new mechanisms underlying desiccation tolerance in bacteria and identify several key functions involved. [ABSTRACT FROM AUTHOR]

We present genetic studies that help define the functional network underlying intrinsic aminoglycoside resistance in Pseudomonas aeruginosa. Our analysis shows that proteolysis, particularly that controlled by the membrane protease FtsH, is a major determinant of resistance. First, we examined the consequences of inactivating genes controlled by AmgRS, a two-component regulator required for intrinsic tobramycin resistance. Three of the gene products account for resistance: a modulator of FtsH protease (YccA), a membrane protease (HtpX), and a membrane protein of unknown function (PA5528). Second, we screened mutations inactivating 66 predicted proteases and related functions. Insertions inactivating two FtsH protease accessory factors (HflK and HflC) and a cytoplasmic protease (HslUV) increased tobramycin sensitivity. Finally, we generated an ftsH deletion mutation. The mutation dramatically increased aminoglycoside sensitivity. Many of the functions whose inactivation increased sensitivity appeared to act independently, since multiple mutations led to additive or synergistic effects. Up to 500-fold increases in tobramycin sensitivity were observed. Most of the mutations also were highly pleiotropic, increasing sensitivity to a membrane protein hybrid, several classes of antibiotics, alkaline pH, NaCl, and other compounds. We propose that the network of proteases provides robust protection from aminoglycosides and other substances through the elimination of membrane-disruptive mistranslation products. [ABSTRACT FROM AUTHOR]

Francisella tularensis is the causative agent of tularemia and is a category A select agent. Francisella novicida, considered by some to be one of four subspecies of F. tularensis, is used as a model in pathogenesis studies because it causes a disease similar to tularemia in rodents but is not harmful to humans. F. novicida exhibits a strong restriction barrier which reduces the transformation frequency of foreign DNA up to 106-fold. To identify the genetic basis of this barrier, we carried out a mutational analysis of restriction genes identified in the F. novicida genome. Strains carrying combinations of insertion mutations in eight candidate loci were created and assayed for reduced restriction of unmodified plasmid DNA introduced by transformation. Restriction was reduced by mutations in four genes, corresponding to two type I, one type II, and one type III restriction system. Restriction was almost fully eliminated in a strain in which all four genes were inactive. The strongest contributor to the restriction barrier, the type II gene, encodes an enzyme which specifically cleaves Dam-methylated DNA. Genome comparisons show that most restriction genes in the F. tularensis subspecies are pseudogenes, explaining the unusually strong restriction barrier in F. novicida and suggesting that restriction was lost during evolution of the human pathogenic subspecies. As part of this study, procedures were developed to introduce unmodified plasmid DNA into F. novicida efficiently, to generate defined multiple mutants, and to produce chromosomal deletions of multiple adjacent genes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Mutant hunts using comprehensive sequence-defined libraries make it possible to identify virtually all of the nonessential functions required for different bacterial processes. ,However, the success of such screening depends on the accuracy of mutant identification in the mutant library used. To provide a high-quality library for Pseudomonas aeruginosa PAO1, we created a sequence-verified collection of 9,437 transposon mutants that provides genome coverage and includes two mutants for most genes. Mutants were cherry-picked from a larger library, colony-purified, and resequenced both individually using Sanger sequencing and in a pool using Tn-seq. About 8% of the insertion assignments were corrected, and in the final library nearly 93% of the transposon locations were confirmed by at least one of the resequencing procedures. The extensive sequence verification and inclusion of more than one mutant for most genes should help minimize missed or erroneous genotype-phenotype assignments in studies using the new library. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae and other carbapenem-resistant members of the family Enterobacteriaceae are a major cause of hospital-acquired infections, yet the basis of their success as nosocomial pathogens is poorly understood. To help provide a foundation for genetic analysis of K. pneumoniae, we created an arrayed, sequence-defined transposon mutant library of an isolate from the 2011 outbreak of infections at the U.S. National Institutes of Health Clinical Center. The library is made up of 12,000 individually arrayed mutants of a carbapenemase deletion parent strain and provides coverage of 85% of the predicted genes. The library includes an average of 2.5 mutants per gene, with most insertion locations identified and confirmed in two independent rounds of Sanger sequencing. On the basis of an independent transposon sequencing assay, about half of the genes lacking representatives in this "two-allele" library are essential for growth on nutrient agar. To validate the use of the library for phenotyping, we screened candidate mutants for increased antibiotic sensitivity by using custom phenotypic microarray plates. This screening identified several mutations increasing sensitivity to ß-lactams (in acrB1, mcrB, ompR, phoP1, and slt1) and found that two-component regulator cpxAR mutations increased multiple sensitivities (to an aminoglycoside, a fluoroquinolone, and several ß-lactams). Strains making up the two-allele mutant library are available through a web-based request mechanism. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

MglA is a transcriptional regulator of genes that contribute to the virulence of Francisella tularensis, a highly infectious pathogen and the causative agent of tularemia. This study used a label-free shotgun proteomics method to determine the F. tularensis subsp. novicida (F. novicida) proteins that are regulated by MglA. The differences in relative protein amounts between wild-type F. novicida and the mglA mutant were derived directly from the average peptide precursor ion intensity values measured with the mass spectrometer by using a suite of mathematical algorithms. Among the proteins whose relative amounts changed in an F. novicida mglA mutant were homologs of oxidative and general stress response proteins. The F. novicida mglA mutant exhibited decreased survival during stationary-phase growth and increased susceptibility to killing by superoxide generated by the redox-cycling agent paraquat. The F. novicida mglA mutant also showed increased survival upon exposure to hydrogen peroxide, likely due to increased amounts of the catalase KatG. Our results suggested that MglA coordinates the stress response of F. tularensis and is likely essential for bacterial survival in harsh environments. [ABSTRACT FROM AUTHOR]