Investigation of a cfr-like gene from Clostridium

• Autor(a) principal: Santos, Patrícia Isabel Teixeira dos
• Data de Publicação: 2014
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/14199
• topic: Biotecnologia molecular; Bactérias patogénicas; Expressão genética; Metiltransferases; Resistência a antibióticos

The aim of this project was, primarily, to clone, express and investigate the function of a cfr-like gene from Clostridium sporogenes (clcs) in E. coli and, subsequently, to investigate the function of the clcs gene in C. sporogenes, its natural host, and ascertain possible variations in function. The cfr and cfr-like genes were cloned into a plasmid, which allowed their constitutive expression in E. coli. The ClCs protein was not able to mediate changes in the antibiotic susceptibility of E. coli compared to the PhLOPSA phenotype conferred by the Cfr methyltransferase. The lack of function of the expressed protein was also investigated by combining parts of the cfr and clcs genes, but no MIC changes were observed. Attending to the Cfr methyltransferase function, the verification of the presence or absence of the RNA methylation at A2503 in 23S rRNA from E. coli was checked by primer extension, showing that the ClCs-containing strain E. coli JW2501-1 does not give rise to any stop at A2503, revealing that the Cfr-like protein from C. sporogenes does not methylate E. coli 23S RNA, which is consistent with the MIC results. Since it was not possible to conclude that ClCs does not have a Cfr-like function, further investigation was required to determine if ClCs could be able to methylate C. sporogenes 23S RNA and act as Cfr. Two C. sporogenes strains reported as cfr-like gene carriers were investigated. The attempts to amplify the cfr-like genes revealed that the assumption that both these C. sporogenes strains contained a cfr-like gene seemed to be true for only one of them. MICs showed that the cfr-like gene-containing C. sporogenes strain has lower susceptibility to all the PhLOPSA antibiotics tested than the presumably Cfr-lacking C. sporogenes strain. The uncertain function of the ClCs protein was then investigated by primer extension to look for an indication of modification at A2503 23S RNA from C. sporogenes (E. coli numbering). As a similar stop was observed for both strains, mass spectrometry was performed revealing a mono-methylation at A2503, probably caused by the housekeeping RlmN protein and not by Cfr. Another possible modification in the area around A2503 was detected and should be further analyzed.