Isolation and analysis of a circular form of the IncJ conjugative transposon-like elements, R391 and R997: implications for IncJ incompatibility.
The incompatibility between the chromosomally integrating, conjugative transposon-like, IncJ elements R997 (ampicillin resistant) and R391 (kanamycin resistant) was examined by constructing strains harbouring both elements. Unusually, recA(+) strains harbouring the resistance determinants of both elements could be isolated but all strains lacked detectable extrachromosomal DNA. The phenotypic characteristics and transfer patterns observed suggested the formation of recombinant hybrids rather than strains harbouring both elements independently. Formation of strains harbouring two IncJ elements in a recA background was thus examined and resulted in the visualisation of extrachromosomal DNA. When R391 was transferred to a recA strain containing integrated R997, both elements co-existed stably and resulted in the isolation of a plasmid of 93.9 kb. When R997 was transferred to a recA strain harbouring an integrated R391, a plasmid of 85 kb was isolated. Comparison of restriction patterns for both elements revealed many common and several distinct fragments indicating a close physical relationship. These data suggest that although IncJ elements normally integrate at a unique site in the Escherichia coli chromosome, they possess the ability for autonomous replication which becomes manifest in a recA background when this site is occupied. This observation has implications for the nature of the incompatibility associated with IncJ elements and also provides a reliable method for isolating IncJ elements for molecular characterisation.
Pembroke
J T
JT
Molecular Biochemistry Group, University of Limerick, Limerick, Ireland. tony.pembroke@ul.ie
Murphy
D B
DB
D000668:Ampicillin Resistance / Q000235:genetics; D003227:Conjugation, Genetic; D004251:DNA Transposable Elements; D004270:DNA, Circular / Q000235:genetics* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D007613:Kanamycin Resistance / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D015183:Restriction Mapping
D004251:DNA Transposable Elements; D004270:DNA, Circular