Plasmid-encoded blaPER-2 could be regulated by two putative promoters

DI CONZA JOSÉ; POWER PABLO; GUTKIND GABRIEL; AYALA JUAN

Chicago, EEUU

Congreso; 47th annual Interscience Conference on Antimicrobial Agents and Chemotherapy - ICAAC; 2007

ASM

Background PER-2 was the first detected and the second most prevalent ESBL in clinical pathogens isolated in Argentina; its presences has been only reported in other South-American countries. The presence of similar structures upstream of blaPER- genes suggests common elements involved in blaPER-2 expression. Methods Citrobacter freundii 33587 total RNA was extracted using an RNeasy Midi kit (Qiagen, USA). 5´-rapid amplification of cDNA ends (5´-RACE system kit, Invitrogen, USA) was performed to detect the transcription initiation sites. Conjugation assay was performed by solid-medium mating using E. coli CAG12177 as recipient. Results blaPER-2 and its associated IS are located in a conjugative plasmid. We were able to detect two +1 transcription starting sites. One of them was located 227 bp upstream from the blaPER-2 start codon embedded within the ISPa12/IS1387a. We deduced a -35 consensus sequence (TTCAAA) separated 17 bp from a -10 box (TAATTT), constituting the blaPER-2 PCf1 promoter. These elements are homologous to that described for blaPER-1 in different P. aeruginosa isolates, differing only in the +1 starting site location and the -10 consensus sequence. On the other hand, the +1 transcription starting site of the second putative promoter, named PCf2, was located 63 bp upstream from the blaPER-2 start codon. Only a putative -10 box could be deduced (TATGAA), whereas a clear -35 consensus sequence could not be inferred. This promoter does not belong to the ISPa12/IS1387a backbone, but to an additional 127-bp sequence which is absent in P. aeruginosa blaPER-1 upstream region. Conclusions In all the previously reported cases, transcription of blaPER- genes seems to be directed by an only promoter embedded in the ISPa12/IS1387a upstream element. We describe a putative second transcription site origin and the corresponding putative promoter located outside the IS backbone, which could probably modify the expression of blaPER-2.