Microcin H47, a gene-encoded peptide antibiotic produced by a natural Escherichia coli strain, was shown to be secreted by a three-component ATP-binding cassette exporter which was revealed to be strongly related to that of colicin V. The results of sequence and gene fusion analyses, as well as heterologous complementation assays, are presented.For gram-negative bacteria, several three-component ATPbinding cassette (ABC) exporters dedicated to protein secretion have been described. They are composed of an ABC transporter protein, a second component of the membrane fusion protein (MFP) family, and an outer membrane protein (3,4,21). There is a single peptide described to be secreted by a three-component ABC apparatus: Escherichia coli colicin V (ColV), an antibiotic of the microcin family. Its exporter comprises the ABC protein CvaB, the MFP CvaA, and the outer membrane protein TolC (6). The ColV ABC transporter contains a proteolytic domain, and consistent with this, the ColV precursor bears a double glycine leader peptide which is processed during export (8, 9).In this work, results are presented on the mode of secretion of microcin H47 (MccH47), an E. coli antibiotic peptide. Genes for its synthesis, immunity, and secretion are clustered in a 10-kb DNA segment (Fig. 1A) (5,11,16,17). The secretion function was assigned to the products of two genes, mchE and mchF. In addition, tolC mutants were shown to produce reduced amounts of microcin. It has been proposed that MccH47 is secreted by an ABC exporter, constituted by MchF, MchE, and TolC (5). A DNA segment containing the mchE and mchF genes was sequenced, partly in our laboratory (18) and partly in the DNA Sequencing Core Laboratory Service of the University of Florida. Two open reading frames were found in the positions expected for these genes (Fig. 1B)
RecA-independent recombination events between short direct repeats, leading to deletion of the intervening sequences, were found to occur in two genetic models in the Escherichia coli K12 background. The first model was a small E. coli genomic island which had been shown to be mobile in its strain of origin and, when cloned, in the E. coli K12 context too. However, it did not encode a site-specific recombinase as mobile genomic island usually do. Then, it was deduced that the host cells should provide the recombination function. This latter was searched for by means of a PCR approach to detect the island excision in E. coli K12 mutants affected in a number of recombination functions, including the 16 E. coli K12 site-specific recombinases, the RecET system, and multiple proteins that participate in the RecA-dependent pathways of homologous recombination. None of these appeared to be involved in the island excision. The second model, analyzed in a RecA deficient context, was a plasmid construction containing a short direct repeat proceeding from Saccharomyces cerevisiae, which flanked the cat gene. The excision of this gene by recombination of the DNA repeats was confirmed by PCR and through the detection, recovery and characterization of the plasmid deleted form. In sum, we present new evidence on the occurrence of RecA-independent recombination events in E. coli K12. Although the mechanism underlying these processes is still unknown, their existence suggests that RecA-independent recombination may confer mobility to other genetic elements, thus contributing to genome plasticity.
RecA-independent recombination events between short direct repeats, leading to deletion of the intervening sequences, were found to occur in two genetic models in the Escherichia coli K12 background. The first model was a small E. coli genomic island which had been shown to be mobile in its strain of origin and, when cloned, also in the E. coli K12 context. However, it did not encode a site-specific recombinase as mobile genomic islands usually do. It was then deduced that the host cells should provide the recombination function. This latter was searched for by means of a PCR approach to detect the island excision in E. coli K12 mutants affected in a number of recombination functions, including the 16 E. coli K12 site-specific recombinases, the RecET system, and multiple proteins that participate in the RecA-dependent pathways of homologous recombination. None of these appeared to be involved in the island excision. The second model, analyzed in a RecA deficient context, was a plasmid construction containing a short direct repeat proceeding from Saccharomyces cerevisiae, which flanked the cat gene. The excision of this gene by recombination of the DNA repeats was confirmed by PCR and through the detection, recovery and characterization of the plasmid deleted form. In sum, we present new evidence on the occurrence of RecA-independent recombination events in E. coli K12. Although the mechanism underlying these processes is still unknown, their existence suggests that RecA-independent recombination may confer mobility to other genetic elements, thus contributing to genome plasticity.
RecA-independent recombination events between short direct repeats, leading to deletion of the intervening sequences, were found to occur in two genetic models in the Escherichia coli K12 background. The first model was a small E. coli genomic island which had been shown to be mobile in its strain of origin and, when cloned, in the E. coli K12 context too. However, it did not encode a site-specific recombinase as mobile genomic island usually do. Then, it was deduced that the host cells should provide the recombination function. This latter was searched for by means of a PCR approach to detect the island excision in E. coli K12 mutants affected in a number of recombination functions, including the 16 E. coli K12 site-specific recombinases, the RecET system, and multiple proteins that participate in the RecA-dependent pathways of homologous recombination. None of these appeared to be involved in the island excision. The second model, analyzed in a RecA deficient context, was a plasmid construction containing a short direct repeat proceeding from Saccharomyces cerevisiae, which flanked the cat gene. The excision of this gene by recombination of the DNA repeats was confirmed by PCR and through the detection, recovery and characterization of the plasmid deleted form. In sum, we present new evidence on the occurrence of RecA-independent recombination events in E. coli K12. Although the mechanism underlying these processes is still unknown, their existence suggests that RecA-independent recombination may confer mobility to other genetic elements, thus contributing to genome plasticity.
Within the framework of the β-hemolytic streptococci surveillance carried out by the National Reference Laboratory from Uruguay, three putative Streptococcus equi subsp. zooepidemicus (SEZ) were received from different health centers. Being these the first reports associated with human infections in Uruguay, the objective of this work was to confirm their identification, to determine their genetic relationship and to study their antibiotic susceptibility. Using four different methods, they were identified as SEZ, a subspecies which has been described as the etiologic agent of rare and severe zoonosis in a few cases in other countries. The three isolates presented different pulsotypes by PFGE; however, two of them appeared to be related and were confirmed as ST431 by MLST, while the remaining isolate displayed ST72. Their resistance profile exhibited an unexpected feature: despite all of them were susceptible to macrolides, they showed different levels of resistance to clindamycin, i.e. they had the so-called “L phenotype”. This rare trait is known to be due to a nucleotidyl-transferase, encoded by genes of the lnu family. Although this phenotype was previously described in a few SEZ isolates, its genetic basis has not been studied yet. This was now analyzed by PCR in the three isolates and they were found to contain a lnuB gene. The lnuB sequence was identical among the three isolates and with many lnuB sequences deposited in data banks. In conclusion, for the first time in Uruguay, three SEZ isolates recovered from non-epidemiologically related cases of human invasive infection were identified. Moreover, this is the first report about the presence of a lnu gene in the S. equi species, revealing the active lateral spread of the lnuB in a new streptococcal host.
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