Promoter characterization and expression of the blaKPC-2 gene in Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii.
KPC-producing pathogens exhibit variable carbapenem susceptibility levels, which is probably the result of the genetic environment of the bla KPC genes. Here we determined the transcriptional start sites (TSSs) and the expression of the bla KPC-2 gene in various genetic contexts and in different hosts ( Escherichia coli , Pseudomonas aeruginosa and Acinetobacter baumannii ).



The bla KPC-2 genes along with the upstream sequences derived from Tn 4401b (structure A), Tn 4401b interrupted by Tn 3 /IS 26 (structure B) and Tn 4401b interrupted by Tn 5563 (structure C) were cloned in two E. coli shuttle vectors (pBBR1MCS.3 for expression studies in P. aeruginosa and pIM-arr2 for expression studies in A. baumannii ). MICs were determined by Etests. 5' RACE (where RACE stands for rapid amplification of cDNA ends) and quantitative RT-PCR experiments were performed to determine TSSs and transcription levels, respectively.



Depending on the bacterial host, different promoters were used for bla KPC-2 gene expression. The highest transcriptional level was obtained in P. aeruginosa with structure C, described only in P. aeruginosa . Tn 4401b (structure A), harbouring two promoters (P1 and P2), was the most efficient in E. coli and A. baumannii . This structure was also efficient in P. aeruginosa , although the same deduced promoter was not used (P1, instead of P2 used by E. coli and A. baumannii ). Two novel TSSs and putative promoters (P2b and P3b) were identified in structure B. In this structure, P2b and P3b were preferably used in E. coli and in P. aeruginosa , respectively, whereas P1 was used in A. baumannii .



We determined the preferred TSSs of the bla KPC gene in each species and described two novel deduced promoters in structure B.
EA7361, Université Paris-Sud, Université Paris-Saclay, Associated French National Reference Center for Antibiotic Resistance 'Carbapenemase-producing Enterobacteriaceae', LabEx Lermit, Bacteriology-Hygiene Unit, APHP, Hôpital Bicêtre, Le Kremlin-Bicêtre, France.|EERA 'Evolution and Ecology of Resistance to Antibiotics' Unit, Institut Pasteur-APHP-Université Paris-Sud, Paris, France.
Delphine
D
Girlich
EA7361, Université Paris-Sud, Université Paris-Saclay, Associated French National Reference Center for Antibiotic Resistance 'Carbapenemase-producing Enterobacteriaceae', LabEx Lermit, Bacteriology-Hygiene Unit, APHP, Hôpital Bicêtre, Le Kremlin-Bicêtre, France.|EERA 'Evolution and Ecology of Resistance to Antibiotics' Unit, Institut Pasteur-APHP-Université Paris-Sud, Paris, France.
Rémy A
RA
Bonnin
EA7361, Université Paris-Sud, Université Paris-Saclay, Associated French National Reference Center for Antibiotic Resistance 'Carbapenemase-producing Enterobacteriaceae', LabEx Lermit, Bacteriology-Hygiene Unit, APHP, Hôpital Bicêtre, Le Kremlin-Bicêtre, France.|EERA 'Evolution and Ecology of Resistance to Antibiotics' Unit, Institut Pasteur-APHP-Université Paris-Sud, Paris, France.
Agnes
A
Jousset
EA7361, Université Paris-Sud, Université Paris-Saclay, Associated French National Reference Center for Antibiotic Resistance 'Carbapenemase-producing Enterobacteriaceae', LabEx Lermit, Bacteriology-Hygiene Unit, APHP, Hôpital Bicêtre, Le Kremlin-Bicêtre, France.|EERA 'Evolution and Ecology of Resistance to Antibiotics' Unit, Institut Pasteur-APHP-Université Paris-Sud, Paris, France.
Thierry
T
Naas
D040981:Acinetobacter baumannii / Q000235:genetics*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015780:Carbapenems / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D008826:Microbial Sensitivity Tests; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D024363:Transcription Initiation Site; D001618:beta-Lactamases / Q000235:genetics*
D000900:Anti-Bacterial Agents; D015780:Carbapenems; D001618:beta-Lactamases