Search

The identification of most or all of the genetic functions that are required for a particular biological process could be achieved through phenotypic studies of high genome-coverage mutant collections. Technologies for creating such collections, in the form of mixed populations or individually arrayed sequence-defined mutants, are now available for numerous bacterial species. The analysis of mixed mutant collections using microarray-based detection procedures appears to be particularly effective in identifying functions required for complex processes such as virulence. The phenotypic analysis of sequence-defined mutant libraries provides a virtually complete identification of nonessential genes required for processes for which suitable screens can be devised. Such libraries also serve as a source of individual mutants for examining the biological relevance of gene associations revealed by transcriptional profiling or homology. [Copyright &y& Elsevier]

As a result of the explosive growth of bacterial genomic and postgenomic information, there is a pressing need for efficient, inexpensive strategies for characterizing the in vivo behavior and function of newly identified gene products. We describe here an internal tagging procedure, based on transposon technology, to facilitate the analysis of membrane-bound and secreted proteins in Gram-negative bacteria. The technique is based on a broad?host range transposon (ISphoA/hah), which may be used to generate both alkaline phosphatase (AP) gene fusions and 63-codon in-frame insertions in the genome. The 63-codon insertion encodes an influenza hemagglutinin epitope and a hexahistidine sequence, permitting sensitive detection and metal affinity purification of tagged proteins. For each gene targeted, it is thus possible to monitor the disruption of phenotype (using the transposon insertion), the gene's transcription and translation (using the AP reporter activity), and the behavior of the unfused protein (using the internal tag). Studies on a sequence-defined collection of Escherichia coli strains generated using the transposon showed that the synthesis and subcellular localization of tagged proteins could be readily monitored. The use of ISphoA/hah should provide a cost-effective approach for genome-wide in vivo studies of the behavior of exported proteins in a number of bacterial species. [ABSTRACT FROM AUTHOR]

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.

Fusions of the secreted protein alkaline phosphatase to an integral cytoplasmic membrane protein ofEscherichia coli showed different activities depending on where in the membrane protein the alkaline phosphatase was fused. Fusions to positions in or near the periplasmic domain led to high alkaline phosphatase activity, whereas those to positions in the cytoplasmic domain gave low activity. Analysis of alkaline phosphatase fusions to membrane proteins of unknown structure may thus be generally useful in deterImining their membrane topologies. [ABSTRACT FROM AUTHOR]

Focuses on experiments involving a limited set of membrane proteins that have served as models in genetic and molecular studies of membrane protein assembly. Vulnerable lipid bilayer; Membrane protein structures; Model of membrane protein assembly; Establishing membrane topology; Involvement of secretion machinery in membrane protein insertion.

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.

Mutant phenotype analysis of bacteria has been revolutionized by genome-scale screening procedures, but essential genes have been left out of such studies because mutants are missing from the libraries analyzed. Since essential genes control the most fundamental processes of bacterial life, this is a glaring deficiency. To address this limitation, we developed a procedure for transposon insertion mutant sequencing that includes essential genes. The method, called transformation transposon insertion mutant sequencing (TFNseq), employs saturation-level libraries of bacterial mutants generated by natural transformation with chromosomal DNA mutagenized heavily by in vitro transposition. The efficient mutagenesis makes it possible to detect large numbers of insertions in essential genes immediately after transformation and to follow their loss during subsequent growth. It was possible to order 45 essential processes based on how rapidly their inactivation inhibited growth. Inactivating ATP production, deoxyribonucleotide synthesis, or ribosome production blocked growth the fastest, whereas inactivating cell division or outer membrane protein synthesis blocked it the slowest. Individual mutants deleted of essential loci formed microcolonies of nongrowing cells whose sizes were generally consistent with the TFNseq ordering. The sensitivity of essential functions to genetic inactivation provides a metric for ranking their relative importance for bacterial replication and growth. Highly sensitive functions could represent attractive antibiotic targets since even partial inhibition should reduce growth. [ABSTRACT FROM AUTHOR]

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]

To characterize the consequences of eliminating essential functions needed for peptidoglycan synthesis, we generated deletion mutations of Acinetobacter baylyi by natural transformation and visualized the resulting microcolonies of dead cells. We found that loss of genes required for peptidoglycan precursor synthesis or polymerization led to the formation of polymorphic giant cells with diameters that could exceed ten times normal. Treatment with antibiotics targeting early or late steps of peptidoglycan synthesis also produced giant cells. The giant cells eventually lysed, although they were partially stabilized by osmotic protection. Genome-scale transposon mutant screening (Tn-seq) identified mutations that blocked or accelerated giant cell formation. Among the mutations that blocked the process were those inactivating a function predicted to cleave murein glycan chains (the MltD murein lytic transglycosylase), suggesting that giant cell formation requires MltD hydrolysis of existing peptidoglycan. Among the mutations that accelerated giant cell formation after ß-lactam treatment were those inactivating an enzyme that produces unusual 3->3 peptide cross-links in peptidoglycan (the LdtG L,D-transpeptidase). The mutations may weaken the sacculus and make it more vulnerable to further disruption. Although the study focused on A. baylyi, we found that a pathogenic relative (A. baumannii) also produced giant cells with genetic dependencies overlapping those of A. baylyi. Overall, the analysis defines genetic pathway for giant cell formation conserved in Acinetobacter species in which independent initiating branches converge to create the unusual cells. [ABSTRACT FROM AUTHOR]

While much is known about acute infection pathogenesis, the understanding of chronic infections has lagged. Here we sought to identify the genes and functions that mediate fitness of the pathogen Pseudomonas aeruginosa in chronic wound infections, and to better understand the selective environment in wounds. We found that clinical isolates from chronic human wounds were frequently defective in virulence functions and biofilm formation, and that many virulence and biofilm formation genes were not required for bacterial fitness in experimental mouse wounds. In contrast, genes involved in anaerobic growth, some metabolic and energy pathways, and membrane integrity were critical. Consistent with these findings, the fitness characteristics of some wound impaired-mutants could be represented by anaerobic, oxidative, and membrane-stress conditions ex vivo, and more comprehensively by high-density bacterial growth conditions, in the absence of a host. These data shed light on the bacterial functions needed in chronic wound infections, the nature of stresses applied to bacteria at chronic infection sites, and suggest therapeutic targets that might compromise wound infection pathogenesis. [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]

Summary Interspecies protein-protein interactions are essential mediators of infection. While bacterial proteins required for host cell invasion and infection can be identified through bacterial mutant library screens, information about host target proteins and interspecies complex structures has been more difficult to acquire. Using an unbiased chemical crosslinking/mass spectrometry approach, we identified interspecies protein-protein interactions in human lung epithelial cells infected with Acinetobacter baumannii . These efforts resulted in identification of 3,076 crosslinked peptide pairs and 46 interspecies protein-protein interactions. Most notably, the key A. baumannii virulence factor, OmpA, was identified as crosslinked to host proteins involved in desmosomes, specialized structures that mediate host cell-to-cell adhesion. Co-immunoprecipitation and transposon mutant experiments were used to verify these interactions and demonstrate relevance for host cell invasion and acute murine lung infection. These results shed new light on A. baumannii -host protein interactions and their structural features, and the presented approach is generally applicable to other systems. [ABSTRACT FROM AUTHOR]

Summary In pathogenic Gram-negative bacteria, interactions among membrane proteins are key mediators of host cell attachment, invasion, pathogenesis, and antibiotic resistance. Membrane protein interactions are highly dependent upon local properties and environment, warranting direct measurements on native protein complex structures as they exist in cells. Here we apply in vivo chemical cross-linking mass spectrometry, to reveal the first large-scale protein interaction network in Pseudomonas aeruginosa , an opportunistic human pathogen, by covalently linking interacting protein partners, thereby fixing protein complexes in vivo. A total of 626 cross-linked peptide pairs, including previously unknown interactions of many membrane proteins, are reported. These pairs not only define the existence of these interactions in cells but also provide linkage constraints for complex structure predictions. Structures of three membrane proteins, namely, SecD-SecF, OprF, and OprI are predicted using in vivo cross-linked sites. These findings improve understanding of membrane protein interactions and structures in cells. [ABSTRACT FROM AUTHOR]

Contact-dependent growth inhibition (CDI) is a mode of inter-bacterial competition mediated by the CdiB/CdiA family of two-partner secretion systems. CdiA binds to receptors on susceptible target bacteria, then delivers a toxin domain derived from its C-terminus. Studies with Escherichia coli suggest the existence of multiple CDI growth-inhibition pathways, whereby different systems exploit distinct target-cell proteins to deliver and activate toxins. Here, we explore the CDI pathway in Burkholderia using the CDIIIBp1026b system encoded on chromosome II of Burkholderia pseudomallei 1026b as a model. We took a genetic approach and selected Burkholderia thailandensis E264 mutants that are resistant to growth inhibition by CDIIIBp1026b. We identified mutations in three genes, BTH_I0359, BTH_II0599, and BTH_I0986, each of which confers resistance to CDIIIBp1026b. BTH_I0359 encodes a small peptide of unknown function, whereas BTH_II0599 encodes a predicted inner membrane transport protein of the major facilitator superfamily. The inner membrane localization of BTH_II0599 suggests that it may facilitate translocation of CdiA-CTIIBp1026b toxin from the periplasm into the cytoplasm of target cells. BTH_I0986 encodes a putative transglycosylase involved in lipopolysaccharide (LPS) synthesis. ∆BTH_I0986 mutants have altered LPS structure and do not interact with CDI+ inhibitor cells to the same extent as BTH_I0986+ cells, suggesting that LPS could function as a receptor for CdiAIIBp1026b. Although ∆BTH_I0359, ∆BTH_II0599, and ∆BTH_I0986 mutations confer resistance to CDIIIBp1026b, they provide no protection against the CDIE264 system deployed by B. thailandensis E264. Together, these findings demonstrate that CDI growth-inhibition pathways are distinct and can differ significantly even between closely related species. [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]

Teredinibacter turnerae is an intracellular bacterial symbiont in the gills of wood-eating shipworms, where it is proposed to use antibiotics to defend itself and its animal host. Several biosynthetic gene clusters are conserved in T. turnerae and their host shipworms around the world, implying that they encode defensive compounds. Here, we describe turnercyclamycins, lipopeptide antibiotics encoded in the genomes of all sequenced T. turnerae strains. Turnercyclamycins are bactericidal against challenging Gram-negative pathogens, including colistin-resistant Acinetobacter baumannii. Phenotypic screening identified the outer membrane as the likely target. Turnercyclamycins and colistin operate by similar cellular, although not necessarily molecular, mechanisms, but turnercyclamycins kill colistin-resistant A. baumannii , potentially filling an urgent clinical need. Thus, by exploring environments that select for the properties we require, we harvested the fruits of evolution to discover compounds with potential to target unmet health needs. Investigating the symbionts of shipworms is a powerful example of this principle. [Display omitted] • Microbial competition in a symbiosis leading to antibiotics • Group of lipopeptide antibiotics that targets drug-resistant Acinetobacter The symbiotic bacteria associated with marine shipworms are prolific producers of bioactive metabolites. Miller et al. use an ecology-guided approach to isolate the turnercyclamycins, proposed to be important for this association. Turnercyclamycins are described as a group of lipopeptide antibiotics with selective activity against Gram-negative pathogens, including drug-resistant Acinetobacter. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei, the etiologic agent of human melioidosis, is capable of causing severe acute infection with overwhelming septicemia leading to death. A high rate of recurrent disease occurs in adult patients, most often due to recrudescence of the initial infecting strain. Pathogen persistence and evolution during such relapsing infections are not well understood. Bacterial cells present in the primary inoculum and in late infections may differ greatly, as has been observed in chronic disease, or they may be genetically similar. To test these alternative models, we conducted wholegenome comparisons of clonal primary and relapse B. pseudomallei isolates recovered six months to six years apart from four adult Thai patients. We found differences within each of the four pairs, and some, including a 330 Kb deletion, affected substantial portions of the genome. Many of the changes were associated with increased antibiotic resistance. We also found evidence of positive selection for deleterious mutations in a TetR family transcriptional regulator from a set of 107 additional B. pseudomallei strains. As part of the study, we sequenced to base-pair accuracy the genome of B. pseudomallei strain 1026b, the model used for genetic studies of B. pseudomallei pathogenesis and antibiotic resistance. Our findings provide new insights into pathogen evolution during long-term infections and have important implications for the development of intervention strategies to combat recurrent melioidosis. [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]

Infection with the bacterial pathogen Francisella tularensis tularensis (F. tularensis) causes tularemia, a serious and debilitating disease. Francisella tularensis novicida strain U112 (abbreviated F. novicida), which is closely related to F. tularensis, is pathogenic for mice but not for man, making it an ideal model system for tularemia. Intracellular pathogens like Francisella inhibit the innate immune response, thereby avoiding immune recognition and death of the infected cell. Because activation of inflammatory pathways may lead to cell death, we reasoned that we could identify bacterial genes involved in inhibiting inflammation by isolating mutants that killed infected cells faster than the wild-type parent. We screened a comprehensive transposon library of F. novicida for mutant strains that increased the rate of cell death following infection in J774 macrophage-like cells, as compared to wild-type F. novicida. Mutations in 28 genes were identified as being hypercytotoxic to both J774 and primary macrophages of which 12 were less virulent in a mouse infection model. Surprisingly, we found that F. novicida with mutations in four genes (lpcC, manB, manC and kdtA) were taken up by and killed macrophages at a much higher rate than the parent strain, even upon treatment with cytochalasin D (cytD), a classic inhibitor of macrophage phagocytosis. At least 10-fold more mutant bacteria were internalized by macrophages as compared to the parent strain if the bacteria were first fixed with formaldehyde, suggesting a surface structure is required for the high phagocytosis rate. However, bacteria were required to be viable for macrophage toxicity. The four mutant strains do not make a complete LPS but instead have an exposed lipid A. Interestingly, other mutations that result in an exposed LPS core were not taken up at increased frequency nor did they kill host cells more than the parent. These results suggest an alternative, more efficient macrophage uptake mechanism for Francisella that requires exposure of a specific bacterial surface structure(s) but results in increased cell death following internalization of live bacteria. [ABSTRACT FROM AUTHOR]

This report describes the identification and analysis of a 2-component regulator of Pseudomonas aeruginosa that is a potential aminoglycoside antibiotic combination therapy target. The regulator, AmgRS, was identified in a screen of a comprehensive, defined transposon mutant library for functions whose inactivation increased tobramycin sensitivity. AmgRS mutations enhanced aminoglycoside action against bacteria grown planktonically and in antibiotic tolerant biofilms, against genetically resistant clinical isolates, and in lethal infections of mice. Drugs targeting AmgRS would thus be expected to enhance the clinical efficacy of aminoglycosides. Unexpectedly, the loss of AmgRS reduced virulence in the absence of antibiotics, indicating that its inactivation could protect against infection directly as well as by enhancing aminoglycoside action. Transcription profiling and phenotypic analysis suggested that AmgRS controls an adaptive response to membrane stress, which can be caused by aminoglycoside-induced translational misreading. These results help validate AmgRS as a potential antibiotic combination target for P. aeruginosa and indicate that fundamental stress responses may be a valuable general source of such targets. [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]

Francisella tularensis, the causative agent of tularemia, is one of the most infectious bacterial pathogens known and is a category A select agent. We created a sequence-defined, near-saturation transposon mutant library of F. tularensis novicida. a subspecies that causes a tularemia-like disease in rodents. The library consists of 16,508 unique insertions, an average of >9 insertions per gene, which is a coverage nearly twice that of the greatest previously achieved for any bacterial species. Insertions were recovered in 84% (1,490) of the predicted genes. To achieve high coverage, it was necessary to construct transposons carrying an endogenous Francisella promoter to drive expression of antibiotic resistance. An analysis of genes lacking (or with few) insertions identified nearly 400 candidate essential genes, most of which are likely to be required for growth on rich medium and which represent potential therapeutic targets. To facilitate genome-scale screening using the mutant collection, we assembled a sublibrary made up of two purified mutants per gene. The library provides a resource for virtually complete identification of genes involved in virulence and other nonessential processes. [ABSTRACT FROM AUTHOR]

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. [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]