Mosaic structure and regulation of conjugal transfer of the Escherichia coli plasmid pRK100

Plasmids are extrachromosomal DNA elements that can be found in prokaryotic as well as in eukaryotic cells. They can vary in size and genetic make-up. The plasmid pRK100, which is the study subject of this thesis, is a large (145 kb) natural conjugative plasmid, which was isolated from an uropathogenic E. coli strain. Genetic characterisation of pRK100 using DNA hybridisation, targeted nucleotide sequencing of hybridising as well as seemingly unique regions of pRK100, and biological assays revealed that pRK100 carries two functional replication regions, the F plasmid like RepFIB and the R1 plasmid like RepFIIA replication region, ampicillin and tetracycline resistance genes, the F plasmid like tra region needed for conjugal transfer, an pColV-K30 like aerobactin iron uptake system, an E. coli strain 536 PAI III536 like enterochelin iron uptake system, two colicins, ColV and ColIa, the insertion sequences IS1, IS2 and IS3, and a R100 like rmoA gene. Together, these data led us to suggest that pRK100 has a truly mosaic structure with genetic elements that acquired from several sources. The conjugal transfer of F and F-like plasmids has been studied already for more than 50 years. During the research of F plasmid conjugal transfer it was established that the main positive regulator of conjugation encoded by plasmid is the TraJ protein. As positive regulation of traJ promoter activity had not been described, we focussed on the activity of the promoter under various environmental conditions using a traJ-LacZ reporter system. These experiments indicated that the level of glucose in the medium affects the expression of the traJ-lacZ gene fusion and further ?-galactosidase assays with cya and crp mutant strains demonstrated that the formation of the cAMP-CRP complex is needed for a full transcription from the traJ promoter. This finding was confirmed in competitive RT-PCR assays with cya and crp mutants. EMSA showed that the CRP protein bound to the traJ promoter region. Additional DNAseI protection assays showed that the CRP binding site was centred around ?67.5 from the putative transcriptional traJ start site. Targeted mutagenesis of the identified CRP binding site influenced the activity of the traJ promoter. To corroborate the relevance of these in vitro data, mating assays were performed with crp and cya mutants and the parent strain. These experiments demonstrated a clear decrease in the mating efficiency in the mutants compared to the wild type strain. Results obtained with lrp, hns, cpx, himA, rpoS, relA spoT mutants in the traJ-lacZ reporter assays and the competitive RT-PCR indicated that both, H-NS and Lrp protein, were positive modulators of traJ transcription. EMSA showed that H-NS and Lrp protein bind to the traJ promoter, but also that this binding is less specific than the binding of the CRP protein. Mating tests with hns and lrp mutants demonstrated that H-NS and LRP were important for conjugation in vivo. Comparative analysis of the CRP, H-NS and Lrp binding sites on pRK100 with plasmid sequences in the database indicated similar sequences in the traJ promoter regions of other F-like plasmids. These data may indicate that the identified global regulators CRP, Lrp and H-NS likely act as regulators of conjugal transfer not only in pRK100, but also in other F-like plasmids

Antibiotic Resistance, Virulence Factors, Phenotyping, and Genotyping of E. coli Isolated from the Feces of Healthy Subjects

Escherichia coli may innocuously colonize the intestine of healthy subjects or may instigate infections in the gut or in other districts. This study investigated intestinal E. coli isolated from 20 healthy adults. Fifty-one strains were genotyped by molecular fingerprinting and analyzed for genetic and phenotypic traits, encompassing the profile of antibiotic resistance, biofilm production, the presence of surface structures (such as curli and cellulose), and their performance as recipients in conjugation experiments. A phylogroup classification and analysis of 34 virulence determinants, together with genes associated to the pks island (polyketide-peptide genotoxin colibactin) and conjugative elements, was performed. Most of the strains belonged to the phylogroups B1 and B2. The different phylogroups were separated in a principal coordinate space, considering both genetic and functional features, but not considering pulsed-field gel electrophoresis. Within the B2 and F strains, 12 shared the pattern of virulence genes with potential uropathogens. Forty-nine strains were sensitive to all the tested antibiotics. Strains similar to the potential pathogens innocuously inhabited the gut of healthy subjects. However, they may potentially act as etiologic agents of extra-intestinal infections and are susceptible to a wide range of antibiotics. Nevertheless, there is still the possibility to control infections with antibiotic therapy