The native Pseudomonas stutzeri strain Q chromosomal integron can capture and express cassette-associated genes.
The integron-gene cassette system contributes to multiple antibiotic resistance in bacteria and is likely to be of broader evolutionary significance. However, the majority of integron diversity consists of chromosomal integrons (CIs), with mostly unknown phenotypes, which are poorly characterized. A pUC-based reporter plasmid (pUS23) was developed containing a recombination site [aadB 59 base element (59-be)] upstream of promoterless aadB [gentamicin (Gm) resistance] and gfp (green fluorescence) genes, and this construct was used to investigate the recombination and expression activities of the CI in Pseudomonas stutzeri strain Q. Electroporation of pUS23 into P. stutzeri Q gave ampicillin-resistant transformants, which yielded Gm(R) green fluorescent recombinants after plating on Gm medium. Site-specific integration of pUS23 at attI was detected by PCR in 8 % of Gm(R) colonies and the frequency of attI integration was estimated as 2.0 x 10(-8) per P. stutzeri Q(pUS23) cell. RT-PCR confirmed integron-mediated expression of aadB in one recombinant strain (Q23-17) and a promoter (P(c)) was localized to the 5' end of the intI gene. The integrated pUS23 and flanking integron DNA were cloned from genomic DNA of strain Q23-17 and sequenced, confirming that site-specific integration of the entire reporter plasmid had occurred at the attI site. An insertion sequence (ISPst5; IS5 family) was discovered in the vector backbone of the reporter plasmid integrated at attI and also in a pUS23 derivative recovered as a plasmid in Escherichia coli JM109. This is the first demonstration that wild-type CIs can capture gene cassettes and express cassette-associated genes.
Coleman
Nicholas V
NV
School of Molecular and Microbial Biosciences, Building G08, University of Sydney, NSW, 2006, Australia.
Holmes
Andrew J
AJ
School of Molecular and Microbial Biosciences, Building G08, University of Sydney, NSW, 2006, Australia.
D000668:Ampicillin Resistance; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D015870:Gene Expression; D017930:Genes, Reporter; D005839:Gentamicins / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D032023:Integrons / Q000502:physiology*; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D044223:Pseudomonas stutzeri / Q000235:genetics*; D011995:Recombination, Genetic; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA
D000900:Anti-Bacterial Agents; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D005839:Gentamicins; D049452:Green Fluorescent Proteins; D009713:Nucleotidyltransferases; C020565:gentamicin 2''-nucleotidyltransferase