Functional characterization of Tn1331 gene cassettes.
OBJECTIVE
The transposon Tn1331 possesses a region including three antibiotic resistance genes with the structure aac(6')-Ib-attC-aadA1-attI1*-bla(OXA-9)-attC, which potentially includes four gene cassettes. Experimental data on the mobility of fusion cassettes as well as those on mobility of cassettes in a genetic environment such as Tn1331, which lacks an integrase gene, are limited. Therefore, experiments using pJHCMW1, a plasmid harbouring this transposon, in the presence of IntI1 supplied in trans were carried out to define which cassettes are mobile in vivo.


METHODS
In vivo excision of resistance genes was investigated in Escherichia coli cells harbouring pJHCMW1 and in a recombinant clone that included the intI1 gene under the control of the P(tac) promoter. Plasmid DNA was purified and subjected to PCR analysis, and DNA sequencing of PCR products was performed to determine whether excision had occurred.


CONCLUSIONS
In vivo recombination experiments showed that the fused aadA1-attI1*-bla(OXA-9)-attC gene cassette was excised in the presence of IntI1. The excision of a DNA fragment including aadA1-attI1* was also detected but at a lower frequency. The analysis of the latter recombination reaction showed that, although attI1* includes only a small fraction of the complete attI1 sequence, it is still used as a substrate by IntI1, albeit in a very inefficient manner.
Ramirez
Maria S
MS
Center for Applied Biotechnology Studies, Department of Biological Science, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA 92834-6850, USA.
Parenteau
T Richard
TR
Centron
Daniela
D
Tolmasky
Marcelo E
ME
D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000378:metabolism; D005798:Genes, Bacterial; D019426:Integrases / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D019426:Integrases; C115006:integron integrase IntI1