Genome-wide screening of genes required for swarming motility in Escherichia coli K-12

Escherichia coli K-12 has the ability to migrate on semisolid media by means of swarming motility. A systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection) was used to identify the genes involved in the swarming motility of this bacterium. Of the 3,985 nonessential gene mutants, 294 were found to exhibit a strongly repressed-swarming phenotype. Further, 216 of the 294 mutants displayed no significant defects in swimming motility; therefore, the 216 genes were considered to be specifically associated with the swarming phenotype. The swarming-associated genes were classified into various functional categories, indicating that swarming is a specialized form of motility that requires a wide variety of cellular activities. These genes include genes for tricarboxylic acid cycle and glucose metabolism, iron acquisition, chaperones and protein-folding catalysts, signal transduction, and biosynthesis of cell surface components, such as lipopolysaccharide, the enterobacterial common antigen, and type 1 fimbriae. Lipopolysaccharide and the enterobacterial common antigen may be important surface-acting components that contribute to the reduction of surface tension, thereby facilitating the swarm migration in the E. coli K-12 strain.     

Genome-wide screening of genes affecting glycogen metabolism in Escherichia coli K-12

A systematic and comprehensive gene-disrupted mutant collection of E. coli K-12 was used to identify genes whose deletions affect glycogen accumulation. Of the 3985 non-essential gene mutants of the collection, 35 displayed a glycogen-excess phenotype, whereas 30 displayed either glycogen-less or glycogen-deficient phenotypes. The genes whose deletions affect glycogen accumulation were classified into various functional categories, including energy production, envelope composition and integrity, protein translation and stability, transport of inorganic ions and nucleotides, and metabolism of carbohydrates and amino acids. The overall data indicate that glycogen metabolism is highly interconnected with a wide variety of cellular processes in E. coli.     

Azaserine resistance in Escherichia coli: chromosomal location of multiple genes.

Resistance to azaserine in Escherichia coli is the result of mutations in at least three different loci. All spontaneously arising azaserine-resistant mutants harbor a lesion in the aroP gene. However, a lesion in this gene is not solely responsible for resistance. All spontaneously arising intermediate-level azaserine-resistant mutants also harbor a lesion in a gene designated azaA, which lies near min 43 on the chromosome. High-level resistant mutants harbor lesions in the aroP and azaA genes and in a third gene designated azaB, which lies near min 69 on the chromosome. Transport studies demonstrate that mutants harboring lesions in the azaA gene are not defective in the transport of the aromatic amino acids, but that mutants which harbor lesions in the azaB gene are defective in phenylalanine transport but not in tyrosine or tryptophan transport.     

Development and validation of an oligonucleotide microarray for detection of multiple virulence and antimicrobial resistance genes in Escherichia coli

An oligonucleotide microarray detecting 189 Escherichia coli virulence genes or markers and 30 antimicrobial resistance genes was designed and validated using DNA from known reference strains. This microarray was confirmed to be a powerful diagnostic tool for monitoring emerging E. coli pathotypes and antimicrobial resistance, as well as for environmental, epidemiological, and phylogenetic studies including the evaluation of genome plasticity.     

Identifying Escherichia coli genes involved in intrinsic multidrug resistance

Multidrug resistance is a major cause of clinical failure in treating bacterial infections. Increasing evidence suggests that bacteria can resist multiple antibiotics through intrinsic mechanisms that rely on gene products such as efflux pumps that expel antibiotics and special membrane proteins that block the penetration of drug molecules. In this study, Escherichia coli was used as a model system to explore the genetic basis of intrinsic multidrug resistance. A random mutant library was constructed in E. coli EC100 using transposon mutagenesis. The library was screened by growth measurement to identify the mutants with enhanced or reduced resistance to chloramphenicol (Cm). Out of the 4,000 mutants screened, six mutants were found to be more sensitive to Cm and seven were more resistant compared to the wild-type EC100. Mutations in 12 out of the 13 mutants were identified by inverse polymerase chain reaction. Mutants of the genes rob, garP, bipA, insK, and yhhX were more sensitive to Cm compared to the wild-type EC100, while the mutation of rhaB, yejM, dsdX, nagA, yccE, atpF, or htrB led to higher resistance. Overexpression of rob was found to increase the resistance of E. coli biofilms to tobramycin (Tob) by 2.7-fold, while overexpression of nagA, rhaB, and yccE significantly enhanced the susceptibility of biofilms by 2.2-, 2.5-, and 2.1-fold respectively.     

质粒介导的大肠埃希菌对喹诺酮类抗菌药物耐药机制的研究

目的了解临床分离的耐环丙沙星的大肠埃希菌质粒介导的喹诺酮类耐药基因的携带情况,并进行相关耐药机制的分析。方法采用VITEK-2全自动微生物检测系统鉴定细菌,用K-B法检测细菌对16种常用抗生素的敏感性,采用聚合酶链反应检测喹诺酮类耐药基因qnrS、qnrA、qnrB、qepA和aac（6′）-Ib,并对阳性的aac（6′）-Ib结果进行测序分析。结果 30株耐环丙沙星的大肠埃希菌中,2株（6.67%）检出qepA基因,8株（26.67%）检出aac（6′）-Ib基因,经测序证实其中6株为aac（6）′-Ib-cr（20.0%）。未检出qnrS、qnrA和qnrB基因。结论对环丙沙星耐药的大肠埃希菌携带aac（6′）-Ib-cr和qepA基因,引起质粒介导的对喹诺酮类抗菌药物的低水平耐药。     

Genome-wide association mapping for resistance to leaf rust,stripe rust and tan spot in wheat reveals potential candidate genes

Key message

Genome-wide association mapping in conjunction with population sequencing map and Ensembl plants was used to identify markers/candidate genes linked to leaf rust, stripe rust and tan spot resistance in wheat.

Abstract

Leaf rust (LR), stripe rust (YR) and tan spot (TS) are some of the important foliar diseases in wheat (Triticum aestivum L.). To identify candidate resistance genes for these diseases in CIMMYT’s (International Maize and Wheat Improvement Center) International bread wheat screening nurseries, we used genome-wide association studies (GWAS) in conjunction with information from the population sequencing map and Ensembl plants. Wheat entries were genotyped using genotyping-by-sequencing and phenotyped in replicated trials. Using a mixed linear model, we observed that seedling resistance to LR was associated with 12 markers on chromosomes 1DS, 2AS, 2BL, 3B, 4AL, 6AS and 6AL, and seedling resistance to TS was associated with 14 markers on chromosomes 1AS, 2AL, 2BL, 3AS, 3AL, 3B, 6AS and 6AL. Seedling and adult plant resistance (APR) to YR were associated with several markers at the distal end of chromosome 2AS. In addition, YR APR was also associated with markers on chromosomes 2DL, 3B and 7DS. The potential candidate genes for these diseases included several resistance genes, receptor-like serine/threonine-protein kinases and defense-related enzymes. However, extensive LD in wheat that decays at about 5?×?10⁷ bps, poses a huge challenge for delineating candidate gene intervals and candidates should be further mapped, functionally characterized and validated. We also explored a segment on chromosome 2AS associated with multiple disease resistance and identified seventeen disease resistance linked genes. We conclude that identifying candidate genes linked to significant markers in GWAS is feasible in wheat, thus creating opportunities for accelerating molecular breeding.

     

Construction and screening of metagenomic libraries derived from enrichment cultures: generation of a gene bank for genes conferring alcohol oxidoreductase activity on Escherichia coli

Enrichment of microorganisms with special traits and the construction of metagenomic libraries by direct cloning of environmental DNA have great potential for identifying genes and gene products for biotechnological purposes. We have combined these techniques to isolate novel genes conferring oxidation of short-chain (C(2) to C(4)) polyols or reduction of the corresponding carbonyls. In order to favor the growth of microorganisms containing the targeted genes, samples collected from four different environments were incubated in the presence of glycerol and 1,2-propanediol. Subsequently, the DNA was extracted from the four samples and used to construct complex plasmid libraries. Approximately 100,000 Escherichia coli strains of each library per test substrate were screened for the production of carbonyls from polyols on indicator agar. Twenty-four positive E. coli clones were obtained during the initial screen. Sixteen of them contained a plasmid (pAK101 to pAK116) which conferred a stable carbonyl-forming phenotype. Eight of the positive clones exhibited NAD(H)-dependent alcohol oxidoreductase activity with polyols or carbonyls as the substrates in crude extracts. Sequencing revealed that the inserts of pAK101 to pAK116 encoded 36 complete and 17 incomplete presumptive protein-encoding genes. Fifty of these genes showed similarity to sequenced genes from a broad collection of different microorganisms. The genes responsible for the carbonyl formation of E. coli were identified for nine of the plasmids (pAK101, pAK102, pAK105, pAK107 to pAK110, pAK115, and pAK116). Analyses of the amino acid sequences deduced from these genes revealed that three (orf12, orf14, and orf22) encoded novel alcohol dehydrogenases of different types, four (orf5, sucB, fdhD, and yabF) encoded novel putative oxidoreductases belonging to groups distinct from alcohol dehydrogenases, one (glpK) encoded a putative glycerol kinase, and one (orf1) encoded a protein which showed no similarity to any other known gene product.     

Farm disinfectants select for cyclohexane resistance, a marker of multiple antibiotic resistance, in Escherichia coli

AIMS: The aim of this study was to determine if three classes of farm disinfectants were able to select for ciprofloxacin or cyclohexane tolerant [indicative of a multiple antibiotic resistance (MAR) phenotype] Escherichia coli and if cyclohexane-tolerant E. coli could be isolated from farms. METHODS AND RESULTS: Chicken slurry containing ca 1 : 99 ratio ciprofloxacin resistant : susceptible E. coli (10 different resistant strains examined) was treated for 24 h with each of the disinfectants and examined for survival of resistant : susceptible strains. Ciprofloxacin-sensitive (n=5) and -resistant (n=5) E. coli were grown with sublethal concentrations of the disinfectants and then plated to agar containing ciprofloxacin or overlaid with cyclohexane. Escherichia coli (n=389) isolated from farms were tested for cyclohexane tolerance. Minimum inhibitory concentrations (MIC) were determined against representative isolates and mutants. The disinfectants did not select for the ciprofloxacin-resistant E. coli in poultry slurry but following growth with each of the three disinfectants, higher numbers (P < or = 0.023) of cyclohexane-tolerant E. coli were isolated and these had a MAR phenotype. Of the 389 farm E. coli tested, only one was cyclohexane tolerant. CONCLUSIONS: It is possible that in a farm environment, E. coli could be exposed to similar concentrations of the disinfectants that are selected for MAR type organisms under these laboratory conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Data from this study suggest that cyclohexane-resistant E. coli are not common on farms, but in view of the ease of isolating them in the laboratory with farm disinfectants, further investigations on farms are warranted.     

Genome-wide screening for genes associated with FK506 sensitivity in fission yeast

We have been studying calcineurin signal transduction pathway in fission yeast Schizosaccharomyces pombe (S. pombe) by developing a genetic screen for mutants that show hypersensitivity to the immunosuppressive calcineurin inhibitor FK506 (tacrolimus). In the present study, to identify nonessential genes that are functionally related to the calcineurin signaling pathway, we performed a genome-wide screen of 3004 haploid deletion strains and confirmed 72 deletion strains to be FK506 sensitive. These 72 genes are classified into nine functional groups to include membrane trafficking (16 genes), signal transduction (10 genes), ubiquitination (8 genes), chromatin remodeling (6 genes), cytokinesis (4 genes), ribosomal protein (3 genes), RNA binding protein (3 genes), and a variety of other known functions (17 genes) or still unknown functions (5 genes) in the biological system. In our previous screening of FK506-sensitive mutants we isolated several membrane-trafficking mutants showing defective cell wall integrity. Here, we further examined the vacuolar fusion, the v-SNARE synaptobrevin Syb1 localization, and the sensitivity to the β-glucan synthase inhibitor micafungin in these 72 FK506-sensitive strains. Results showed that 25 deletion strains exhibited abnormal vacuole fusion, 19 deletion strains exhibited Syb1 mislocalization, and 14 deletion strains exhibited both abnormal vacuole fusion and Syb1 mislocalization, while 42 deletion strains showed both normal vacuole fusion and Syb1 localization. Likewise, 16 deletion strains showed sensitivity to micafungin. Altogether, our present study indicates that calcineurin mediates a plethora of physiological processes in fission yeast, and that calcineurin is extensively involved in cross-talk between signaling pathways.     

Genome-wide expression profiling in Escherichia coli K-12.

We have established high resolution methods for global monitoring of gene expression in Escherichia coli. Hybridization of radiolabeled cDNA to spot blots on nylon membranes was compared to hybridization of fluorescently-labeled cDNA to glass microarrays for efficiency and reproducibility. A complete set of PCR primers was created for all 4290 annotated open reading frames (ORFs) from the complete genome sequence of E.coli K-12 (MG1655). Glass- and nylon-based arrays of PCR products were prepared and used to assess global changes in gene expression. Full-length coding sequences for array printing were generated by two-step PCR amplification. In this study we measured changes in RNA levels after exposure to heat shock and following treatment with isopropyl-beta-D-thiogalactopyranoside (IPTG). Both radioactive and fluorescence-based methods showed comparable results. Treatment with IPTG resulted in high level induction of the lacZYA and melAB operons. Following heat shock treatment 119 genes were shown to have significantly altered expression levels, including 35 previously uncharacterized ORFs and most genes of the heat shock stimulon. Analysis of spot intensities from hybridization to replicate arrays identified sets of genes with signals consistently above background suggesting that at least 25% of genes were expressed at detectable levels during growth in rich media.     

Virtual ligand screening against Escherichia coli dihydrofolate reductase: improving docking enrichment using physics-based methods

Motivated by their participation in the McMaster Data-Mining and Docking Competition, the authors developed 2 new computational technologies and applied them to docking against Escherichia coli dihydrofolate reductase: a receptor preparation procedure that incorporates rotamer optimization of side chains and a physics-based rescoring procedure for estimating relative binding affinities of the protein-ligand complexes. Both methods use the same energy function, consisting of the all-atom OPLS-AA force field and a generalized Born solvent model, which treats the protein receptor and small-molecule ligands in a consistent manner. Thus, the energy function is similar to that used in more sophisticated approaches, such as free-energy perturbation and the molecular mechanics Poisson-Boltzmann/surface area, but sampling during the rescoring procedure is limited to simple energy minimization of the ligand. The use of a highly efficient minimization algorithm permitted the authors to apply this rescoring procedure to hundreds of thousands of protein-ligand complexes during the competition, using a modest Linux cluster. To test these methods, they used the 12 competitive inhibitors identified in the training set, plus methotrexate, as positive controls in enrichment studies with both the training and test sets, each containing 50,000 compounds. The key conclusion is that combining the receptor preparation and rescoring methods makes it possible to identify most of the positive controls within the top few tenths of a percent of the rank-ordered training and test set libraries.     

Fusion expression of human beta-defensin-2 from multiple joined genes in Escherichia coli

Human beta-defensin-2 (hBD-2) is a cysteine-rich cationic low molecular weight antimicrobial peptide, which exhibits a broad range of antimicrobial activity without observed acquired resistance. In this work, multiple copies of the hBD-2 gene were linked in tandem and the expression of the multiple joined genes in two fusion expression system, pET28a(+) and pGEX-4T-2, was examined. Using plasmid pET28a(+) with one, two, and four copies of the hBD-2 gene, the expressed level was relatively low, whereas much higher with plasmid pGEX-4T-2, and the fusion products, most of which in insoluble form, account for approximately 26% of the total insoluble cellular proteins.     

Clinical cases,drug resistance,and virulence genes profiling in Uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) as the most important bacterial agent of urinary tract infections (UTIs) encompasses a wide treasure of virulence genes and factors. In due to this default,...