RESUMO
Although Yersinia enterocolitica genomes are highly heterogeneous, they contain a conserved N-acylhomoserine lactone-dependent (AHL) quorum sensing (QS) system consisting of the luxR and luxI orthologs yenR and yenI respectively. Certain hypervirulent strains also contain a putative orphan luxR gene, ycoR, that is not linked to an AHL synthase. To explore the contribution of yenR/yenI/ycoR to QS-dependent phenotypes in Yersinia enterocolitica strain 8081, single and multiple mutants were constructed. AHL profiling identified N-(3-oxohexanoyl) homoserine lactone, N-hexanoylhomoserine lactone, and N-(3-oxoseptanoyl) homoserine lactone as the most abundant. The AHL profiles of the yenR, ycoR and yenR/ycoR mutants were similar to the parent suggesting that the two LuxR homologues do not regulate AHL production while the yenI mutants were AHL-negative. A role for QS in swimming motility and cell attachment was demonstrated. Down-regulation of the virulence plasmid partition gene, spyA, in yenI and yenI/yenR/ycoR mutants is consistent with the greater loss of the Y. enterocolitica pYVe virulence plasmid in the yenI mutant during serial passage at 37 °C but not at 22 °C. A role for QS-regulated spyA in virulence plasmid maintenance is suggested.
RESUMO
Comparative analysis of the Clostridium difficile BI/NAP1/027 strain R20291 and ClosTron-derived ermB mutants in the hamster infection model are compromised by the clindamycin susceptibility of the parent. Mutants can appear more virulent. We have rectified this anomaly by genome engineering. The variant created (CRG20291) represents an ideal control strain for virulence assays of ClosTron mutants.
Assuntos
Clostridioides difficile/genética , Cricetulus/genética , Modelos Animais de Doenças , Enterocolite Pseudomembranosa/microbiologia , Genes Sintéticos , Genoma Bacteriano , Metiltransferases/genética , Animais , Antibacterianos/farmacologia , Clindamicina/farmacologia , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/patogenicidade , Cricetulus/microbiologia , Enterocolite Pseudomembranosa/tratamento farmacológico , Enterocolite Pseudomembranosa/mortalidade , Enterocolite Pseudomembranosa/patologia , Expressão Gênica , Engenharia Genética , Humanos , Testes de Sensibilidade Microbiana , Mutação , Análise de Sobrevida , VirulênciaRESUMO
Sophisticated genetic tools to modify essential biological processes at the molecular level are pivotal in elucidating the molecular pathogenesis of Clostridium difficile, a major cause of healthcare associated disease. Here we have developed an efficient procedure for making precise alterations to the C. difficile genome by pyrE-based allelic exchange. The robustness and reliability of the method was demonstrated through the creation of in-frame deletions in three genes (spo0A, cwp84, and mtlD) in the non-epidemic strain 630Δerm and two genes (spo0A and cwp84) in the epidemic PCR Ribotype 027 strain, R20291. The system is reliant on the initial creation of a pyrE deletion mutant, using Allele Coupled Exchange (ACE), that is auxotrophic for uracil and resistant to fluoroorotic acid (FOA). This enables the subsequent modification of target genes by allelic exchange using a heterologous pyrE allele from Clostridium sporogenes as a counter-/negative-selection marker in the presence of FOA. Following modification of the target gene, the strain created is rapidly returned to uracil prototrophy using ACE, allowing mutant phenotypes to be characterised in a PyrE proficient background. Crucially, wild-type copies of the inactivated gene may be introduced into the genome using ACE concomitant with correction of the pyrE allele. This allows complementation studies to be undertaken at an appropriate gene dosage, as opposed to the use of multicopy autonomous plasmids. The rapidity of the 'correction' method (5-7 days) makes pyrE(-) strains attractive hosts for mutagenesis studies.
Assuntos
Proteínas de Bactérias/genética , Clostridioides difficile/genética , Vetores Genéticos/genética , Genoma Bacteriano/genética , Alelos , Clostridioides difficile/crescimento & desenvolvimento , DNA Bacteriano/genética , Teste de Complementação Genética , Fenótipo , Deleção de SequênciaRESUMO
Most bacteria can only be transformed with circular plasmids, so robust DNA integration methods for these rely upon selection of single-crossover clones followed by counter-selection of double-crossover clones. To overcome the limited availability of heterologous counter-selection markers, here we explore novel DNA integration strategies that do not employ them, and instead exploit (i) activation or inactivation of genes leading to a selectable phenotype, and (ii) asymmetrical regions of homology to control the order of recombination events. We focus here on the industrial biofuel-producing bacterium Clostridium acetobutylicum, which previously lacked robust integration tools, but the approach we have developed is broadly applicable. Large sequences can be delivered in a series of steps, as we demonstrate by inserting the chromosome of phage lambda (minus a region apparently unstable in Escherichia coli in our cloning context) into the chromosome of C. acetobutylicum in three steps. This work should open the way to reliable integration of DNA including large synthetic constructs in diverse microorganisms.