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One of the characteristic features of IncHI1 plasmids is a thermosensitive process of conjugation, which is optimal between 22 6C and 30 6C but inhibited at 37 6C. R27, the prototypical IncHI1 plasmid, contains transfer genes clustered in two regions of the plasmid, Tra1 and Tra2. In the present study, transcriptional analyses of the tra genes were undertaken at both 30 6C and 37 6C. Screening of 38 tra genes showed that tra genes are transcriptionally linked in six operons, three in each Tra region. RT-PCR analysis showed that gene expression was reduced at 37 6C relative to that observed at 30 6C. The transcription start sites of the six transcripts were identified, promoters and upstream regions were cloned, and transcription was tested at both temperatures. In cells grown at 37 6C, in the presence of R27, the promoters were inhibited, except for promoters of the H operon and AN operon. Conditions that influenced DNA topology, such as osmolarity, anaerobiosis, quorum sensing and acidity, showed no significant influence on transfer frequency. These results should facilitate future understanding of the basis of temperature-sensitive transfer in this large conjugative plasmid.

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Bacterial conjugation is a horizontal gene transfer event mediated by the type IV secretion system (T4SS) encoded by bacterial plasmids. Within the T4SS, the coupling protein plays an essential role in linking the membrane-associated pore-forming proteins to the cytoplasmic, DNA-processing proteins. TraG is the coupling protein encoded by the incompatibility group HI plasmids. A hallmark feature of the IncHI plasmids is optimal conjugative transfer at 30 6C and an inability to transfer at 37 6C. Transcriptional analysis of the transfer region 1 (Tra1) of R27 has revealed that traG is transcribed in a temperature-dependent manner, with significantly reduced levels of expression at 37 6C as compared to expression at 30 6C. The R27 coupling protein contains nucleoside triphosphate (NTP)-binding domains, the Walker A and Walker B boxes, which are well conserved among this family of proteins. Site-specific mutagenesis within these motifs abrogated the conjugative transfer of R27 into recipient cells. Mutational analysis of the TraG periplasmic-spanning residues, in conjunction with bacterial two-hybrid and immunoprecipitation analysis, determined that this region is essential for a successful interaction with the T4SS protein TrhB. Further characterization of TraG by immunofluorescence studies revealed that the R27 coupling protein forms membrane-associated fluorescent foci independent of R27 conjugative proteins. These foci were found at discrete positions within the cell periphery. These results allow the definition of domains within TraG that are involved in conjugative transfer, and determination of the cellular location of the R27 coupling protein.

The present objectives of universalization of the academic and research formation for earning scientific degrees in the health care sector are added to the universalization processes of the Cuban medical education. At the same time, they respond to the need of this sector to give solution to the health problems with the help of scientific methods. The current scenarios of the primary and secondary care are real teaching and research settings that allow attaining this goal of the medical university. Universalization of PhD. formation in the health care area is encouraged by the political willingness of the Cuban state, the Commander-in-Chief´s thinking, who has stated that we are called upon to reach high performance in health services, which has been shown by health professionals in the country and in all the internationalist missions. This paper presented the Ministry of Public Health strategy with a view to expanding the formation of PhDs. and showed the results of one of the areas, that is, PhD. in health sciences as an example of the work that is being carried out throughout the country.