The Serratia marcescens Group II Intron Targets Antibiotic Resistant Gene Cassettes

C. QUIROGA, P. H. ROY, D. CENTRON

Atlanta, USA

Congreso; American Society of Microbiology 105th General Meeting.; 2005

ASM

Background. Antibiotic resistant gene cassettes are mobile elements from the genome formed by a structural gene and a palindromic region of 60 to 140 bp called an attC site. Cassettes can be inserted or excised from the variable region of the integrons by a site-specific recombination mechanism mediated by the integrase, a tyrosine recombinase. A class 1 integron from Serratia marcescens possesses a group II intron inserted in an integron gene cassette at the junction of the structural gene and its attC site, at the TA/ACAA target site. Analysis of the S. marcescens GII intron (Smtr) has shown that this intron is capable of invading different antibiotic gene cassettes. Methods. Double transformation of an intron-containing plasmid (I+, donor plasmid) and 9 different target site-containing plasmids (I-, recipient plasmid) were inserted into E.coli DH5á (RecA+). The ability of the intron to recognize each target site was tested by PCR with primers in the intron and in the recipient plasmid, followed by sequence analysis. Additionally, analysis of the frequency of the event was tested by colony hybridization with probes labeled with digoxigenin following the manufacturer’s conditions. Moreover the ability of the Smtr GII intron to perform self-splicing at in vitro conditions was tested. The mRNA was synthesized with T7 RNA polymerase and exposed to Mg2+ concentrations ranging 0 to 50 mM and NH4+ concentrations ranging 0 to 500 mM. Results. Seven out of nine target sites showed insertion of the Smtr GII intron. All positives have shown insertion at the consensus region TA/ACAA, while the negative targets presented a modification in the ACAA region. Splicing in vitro of the intron was successful at 20 mM Mg2+ and 500 mM NH4+. Conclusion. The Smtr GII intron can be inserted in any antibiotic resistant gene cassettes that has the TA/ACAA target site at a low frequency. The target site corresponds to the junction point between the structural gene and its attC. The mechanism involved in the mobility of the intron into a novel target site may be mediated by the intron mRNA.