266 research outputs found

    An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants

    Get PDF
    BACKGROUND: Traditional gene replacement procedures are still time-consuming. They usually necessitate cloning of the gene to be mutated, insertional inactivation of the gene with an antibiotic resistance cassette and exchange of the plasmid-borne mutant allele with the bacterial chromosome. PCR and recombinational technologies can be exploited to substantially accelerate virtually all steps involved in the gene replacement process. RESULTS: We describe a method for rapid generation of unmarked P. aeruginosa deletion mutants. Three partially overlapping DNA fragments are amplified and then spliced together in vitro by overlap extension PCR. The resulting DNA fragment is cloned in vitro into the Gateway vector pDONR221 and then recombined into the Gateway-compatible gene replacement vector pEX18ApGW. The plasmid-borne deletions are next transferred to the P. aeruginosa chromosome by homologous recombination. Unmarked deletion mutants are finally obtained by Flp-mediated excision of the antibiotic resistance marker. The method was applied to deletion of 25 P. aeruginosa genes encoding transcriptional regulators of the GntR family. CONCLUSION: While maintaining the key features of traditional gene replacement procedures, for example, suicide delivery vectors, antibiotic resistance selection and sucrose counterselection, the method described here is considerably faster due to streamlining of some of the key steps involved in the process, especially plasmid-borne mutant allele construction and its transfer into the target host. With appropriate modifications, the method should be applicable to other bacteria

    Neue Soziologie der Kindheit – ‚veränderte Kindheit‘ in einer ‚alternden Gesellschaft‘

    Get PDF
    Der Autor geht davon aus, dass mit den gesellschaftlichen und insbesondere mit den demographischen Veränderungen auch eine grundlegende Veränderung dessen, was wir ‚Kindheit‘ nennen, verbunden ist. Im Beitrag werden die gesellschaftlichen Voraussetzungen und wissenssoziologischen Horizonte einer neuen Kindheitssoziologie erläutert. Vor dem Hintergrund einer Konzeption der ‚gesellschaftlichen Konstruktion der kindlichen Wirklichkeit‘ im Berger/Luckmannschen Sinne wird die Kindheit als Kette oder Textur von Übergängen beschrieben. Schließlich wird die These von einer ‚veränderten Kindheit‘ noch einmal auf die Situation von Kindern und Heranwachsenden in der ‚alternden Gesellschaft‘ hin zugespitzt

    Evidence of MexT-Independent Overexpression of MexEF-OprN Multidrug Efflux Pump of Pseudomonas aeruginosa in Presence of Metabolic Stress

    Get PDF
    The Pseudomonas aeruginosa MexEF-OprN efflux pump confers resistance to clinically significant antibiotics. Regulation of mexEF-oprN operon expression is multifaceted with the MexT activator being one of the most prominent regulatory proteins.We have exploited the impaired metabolic fitness of a P. aeruginosa mutant strain lacking several efflux pump of the resistance nodulation cell division superfamily and the TolC homolog OpmH, and isolated derivatives (large colony variants) that regained fitness by incubation on nutrient-rich medium in the absence of antibiotics. Although the mexEF-oprN operon is uninducible in this mutant due to a 8-bp mexT insertion present in some P. aeruginosa PAO1 strains, the large colony variants expressed high levels of MexEF-OprN. Unlike large colony variants obtained after plating on antibiotic containing medium which expressed mexEF-oprN in a MexT-dependent fashion as evidenced by clean excision of the 8-bp insertion from mexT, mexEF-oprN expression was MexT-independent in the large colony variants obtained by plating on LB alone since the mexT gene remained inactivated. A search for possible regulators of mexEF-oprN expression using transposon mutagenesis and genomic library expression approaches yielded several candidates but proved inconclusive.Our results show that antibiotic and metabolic stress lead to up-regulation of MexEF-OprN expression via different mechanisms and that MexEF-OprN does not only extrude antimicrobials but rather serves other important metabolic functions

    Identification and DNA sequence of tdcR , a positive regulatory gene of the tdc operon of Escherichia coli

    Full text link
    Efficient in vivo expression of the biodegradative threonine dehydratase ( tdc ) operon of Escherichia coli is dependent on a regulatory gene, tdcR . The tdcR gene is located 198 base pairs upstream of the tdc operon and is transcribed divergently from this operon. The nucleotide sequence of tdcR and two unrelated reading frames has been determined. The deduced amino acid sequence of TdcR indicates that is is a polypeptide of M r 12000 with 99 amino acid residues and contains a potential helix-turnhelix DNA binding motif. Deletion analysis and minicell expression of the tdcR gene suggest that TdcR may serve as a trans -acting positive activator for the tdc operon.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47570/1/438_2004_Article_BF00332418.pd

    A simple method for construction of pir+ Enterobacterial hosts for maintenance of R6K replicon plasmids

    Get PDF
    <p>Abstract</p> <p>Background</p> <p>The R6K replicon is one of the best studied bacterial plasmid replicons. Replication of the R6K plasmid and derivatives harboring its γ origin of replication (<it>ori</it><sub>R6Kγ</sub>) is dependent on the <it>pir </it>gene-encoded π protein. Originally encoded by R6K, this protein is usually provided <it>in trans </it>in hosts engineered to support replication of plasmids harboring <it>ori</it><sub>R6Kγ</sub>. In <it>Escherichia coli </it>this is commonly achieved by chromosomal integration of <it>pir </it>either via lysogenization with a λ<it>pir </it>phage or homologous recombination at a pre-determined locus.</p> <p>Findings</p> <p>Current methods for construction of host strains for <it>ori</it><sub>R6Kγ</sub>-containing plasmids involve procedures that do not allow selection for presence of the <it>pir </it>gene and require cumbersome and time-consuming screening steps. In this study, we established a mini-Tn<it>7</it>-based method for rapid and reliable construction of <it>pir</it><sup>+ </sup>host strains. Using a curable mini-Tn<it>7 </it>delivery plasmid, <it>pir </it>expressing derivatives of several commonly used <it>E. coli </it>cloning and mobilizer strains were isolated using both the wild-type <it>pir<sup>+ </sup></it>gene as well as the copy-up <it>pir-116 </it>allele. In addition, we isolated <it>pir</it><sup>+ </sup>and <it>pir-116 </it>expressing derivatives of a clinical isolate of <it>Salmonella enterica </it>serovar Typhimurium. In both <it>E. coli </it>and <it>S. enterica </it>serovar Typhimurium, the presence of the <it>pir<sup>+ </sup></it>wild-type or <it>pir-116 </it>alleles allowed the replication of <it>ori</it><sub>R6Kγ</sub>-containing plasmids.</p> <p>Conclusions</p> <p>A mini-Tn<it>7 </it>system was employed for rapid and reliable engineering of <it>E. coli </it>and <it>S. enterica </it>serovar Typhimurium host strains for plasmids containing <it>ori</it><sub>R6Kγ</sub>. Since mini-Tn7 elements transpose in most, if not all, Gram negative bacteria, we anticipate that with relatively minor modifications this newly established method will for the first time allow engineering of other bacterial species to enable replication of plasmids with <it>ori</it><sub>R6Kγ</sub>.</p

    Does microbicide use in consumer products promote antimicrobial resistance? A critical review and recommendations for a cohesive approach to risk assessment

    Get PDF
    The increasing use of microbicides in consumer products is raising concerns related to enhanced microbicide resistance in bacteria and potential cross resistance to antibiotics. The recently published documents on this topic from the European Commission have spawned much interest to better understand the true extent of the putative links for the benefit of the manufacturers, regulators, and consumers alike. This white paper is based on a 2-day workshop (SEAC-Unilever, Bedford, United Kingdom; June 2012) in the fields of microbicide usage and resistance. It identifies gaps in our knowledge and also makes specific recommendations for harmonization of key terms and refinement/standardization of methods for testing microbicide resistance to better assess the impact and possible links with cross resistance to antibiotics. It also calls for a better cohesion in research in this field. Such information is crucial to developing any risk assessment framework on microbicide use notably in consumer products. The article also identifies key research questions where there are inadequate data, which, if addressed, could promote improved knowledge and understanding to assess any related risks for consumer and environmental safety

    A novel phosphate-regulated expression vector in Escherichia coli

    Full text link
    The ugp promoter (pugp) responsible for expression of the binding-protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli was cloned into a small multicopy plasmid pTER5, a derivative of pBR322, between the transcription terminators rpoCt and tL1. The resulting expression vector, pPH3, permits convenient insertion of structural genes containing their own translational-initiation regions, into the multiple-cloning site derived from the pUC19 plasmid. The efficiency and regulatory properties of pugp were measured using xylE and lacZ as reporter genes, which code for the corresponding enzymes catechol-2,3-dioxygenase (C23O) and [beta]-galactosidase ([beta]Gal), respectively. Enzyme activities were virtually completely repressed in the presencee of excess inorganic phosphates (Pi) and high concentrations of glucose. Maximal induction was observed at limiting Pi (pugp -directed [beta]Gal synthesis was approx. 80% of that directed by directed by strong ptac. When the xylE gene was maximally expressed, the induced enzyme constituted approx. 50% of total cellular protein as judged by laser densitometry following sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. These results suggest the usefulness of the pugp in expression vectors for strong, but controlled, expression of cloned genes in E. coli. This Pi controlled vector can be adapted to large-scale fermentation by using Pi-limiting growth conditions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28559/1/0000361.pd

    Positive In Vivo Selection of the HIV-1 Envelope Protein gp120 Occurs at Surface-Exposed Regions

    Get PDF
    The rapid evolution of human immunodeficiency virus (HIV) envelope represents a major challenge to vaccine and drug development, particularly because the underlying mechanisms are not completely understood. To explore whether distinct patterns of positive selection within the envelope protein glycoprotein (gp) 120 exist and are associated with functionally relevant domains, we conducted a long-term survey of sequence evolution in 20 HIV-1-infected persons who interrupted antiretroviral therapy. In total, 1753 clonal sequences encompassing the C2-V3-C3 region of gp120 were derived. Strikingly, positively selected amino acids mapped almost exclusively (P=.0003) to externally accessible residues on the gp120 crystal structure. The current understanding of envelope structure and function associates the main determinants of viral entry and the targets for neutralizing antibodies with these exterior regions of gp120, strongly suggesting that the observed adaptive evolution of these sites occurs in response to respective selective force

    Molecular Investigations of PenA-mediated β-lactam Resistance in Burkholderia pseudomallei

    Get PDF
    Burkholderia pseudomallei is the etiological agent of melioidosis. Because of the bacterium’s intrinsic resistance and propensity to establish latent infections, melioidosis therapy is complicated and prolonged. Newer generation β-lactams, specifically ceftazidime, are used for acute phase therapy, but resistance to this cephalosporin has been observed. The chromosomally encoded penA gene encodes a putative twin arginine translocase (TAT)-secreted β-lactamase, and penA mutations have been implicated in ceftazidime resistance in clinical isolates. However, the role of PenA in resistance has not yet been systematically studied in isogenetic B. pseudomallei mutant backgrounds. We investigated the effects of penA deletion, point mutations, and up-regulation, as well as tat operon deletion and PenA TAT-signal sequence mutations. These experiments were made possible by employing a B. pseudomallei strain that is excluded from Select Agent regulations. Deletion of penA significantly (>4-fold) reduced the susceptibility to six of the nine β-lactams tested and ≥16-fold for ampicillin, amoxicillin, and carbenicillin. Overexpression of penA by single-copy, chromosomal expression of the gene under control of the inducible Ptac promoter, increased resistance levels for all β-lactams tested 2- to 10-fold. Recreation of the C69Y and P167S PenA amino acid substitutions previously observed in resistant clinical isolates increased resistance to ceftazidime by ≥85- and 5- to 8-fold, respectively. Similarly, a S72F substitution resulted in a 4-fold increase in resistance to amoxicillin and clavulanic acid. Susceptibility assays with PenA TAT-signal sequence and ΔtatABC mutants, as well as Western blot analysis, confirmed that PenA is a TAT secreted enzyme and not periplasmic but associated with the spheroplastic cell fraction. Lastly, we determined that two LysR-family regulators encoded by genes adjacent to penA do not play a role in transcriptional regulation of penA expression
    corecore