Effects of microcin J25 on the gene expression profile in Escherichia coli

SOCIAS, SERGIO BENJAMIN, VALLEJOS ALICIA, DAVIES JULIAN, SALOMON RAúL A.

San Miguel de Tucumán

Congreso; Reunión Anual de la Sociedad Argentina de Microbiología; 2011

Based on the idea that the primary action of most organic compounds of low molecular weight secreted by microbes in their natural habitat, including antibiotics, is to serve as signal molecules that regulate gene expression in microbial populations we investigated the effect of the peptide antibiotic microcin J25 (MccJ25) on gene regulation in E. coli. In a preliminary experiment, we examined the effect of microcins J25, B17, C7 and H47 on the expression of plasmid-borne lux fusions to the phoB, fadB, yrbC, tsr, ilvlG and lasI promoters. We observed that the expression of the lasI promoter is induced in the presence of subinhibitory concentrations of microcins B17 and J25, the latter showing the highest inducing effect. These experiments were done with supernatants from microcin-producer strains. Subsequently, in order to analyze the effect of MccJ25 on the gene expression profile of E. coli, we tested a genomic library of E. coli promoters which employs the gene encoding the Green Fluorescent Protein (GFP) as a reporter. First, we determined that the minimum inhibitory concentration (MIC) of MccJ25 for the host strain MG1655 was 0.46 ìM. Then, in preliminary experiments, we examined the effect of MccJ25 on gene expression, using the antibiotic at a concentration ten times lower than the MIC. Under these conditions, it did not significantly alter gene expression of E. coli. When the experiment was repeated using a MccJ25 concentration four times lower than the MIC we were able to identify E. coli promoters whose expression was selectively affected by MccJ25. Some promoters were induced while others were repressed by the antibiotic. In order to determine whether the effect of MccJ25 on the expression of the target promoters depends on the bacterial growth phase we performed growth curves in the presence and absence of the antibiotic. This showed that the effect of MccJ25 intensifies during the stationary phase of growth. In sum, in the present study we have identified 14 genes that respond specifically to the antibiotic MccJ25. These results could provide clues on the natural function of MccJ25. Particularly interesting is the effect of the antibiotic on the expression of the genes tig and mraZ, both involved in cell division. It has been previously reported that alterations in the expression levels of these genes lead to cell filamentation, which is one of the phenotypes observed when bacterial cells are exposed to MccJ25, and whose mechanism has not yet been clarified.E. coli. In a preliminary experiment, we examined the effect of microcins J25, B17, C7 and H47 on the expression of plasmid-borne lux fusions to the phoB, fadB, yrbC, tsr, ilvlG and lasI promoters. We observed that the expression of the lasI promoter is induced in the presence of subinhibitory concentrations of microcins B17 and J25, the latter showing the highest inducing effect. These experiments were done with supernatants from microcin-producer strains. Subsequently, in order to analyze the effect of MccJ25 on the gene expression profile of E. coli, we tested a genomic library of E. coli promoters which employs the gene encoding the Green Fluorescent Protein (GFP) as a reporter. First, we determined that the minimum inhibitory concentration (MIC) of MccJ25 for the host strain MG1655 was 0.46 ìM. Then, in preliminary experiments, we examined the effect of MccJ25 on gene expression, using the antibiotic at a concentration ten times lower than the MIC. Under these conditions, it did not significantly alter gene expression of E. coli. When the experiment was repeated using a MccJ25 concentration four times lower than the MIC we were able to identify E. coli promoters whose expression was selectively affected by MccJ25. Some promoters were induced while others were repressed by the antibiotic. In order to determine whether the effect of MccJ25 on the expression of the target promoters depends on the bacterial growth phase we performed growth curves in the presence and absence of the antibiotic. This showed that the effect of MccJ25 intensifies during the stationary phase of growth. In sum, in the present study we have identified 14 genes that respond specifically to the antibiotic MccJ25. These results could provide clues on the natural function of MccJ25. Particularly interesting is the effect of the antibiotic on the expression of the genes tig and mraZ, both involved in cell division. It has been previously reported that alterations in the expression levels of these genes lead to cell filamentation, which is one of the phenotypes observed when bacterial cells are exposed to MccJ25, and whose mechanism has not yet been clarified.