Temperature‐dependent expression of conjugation pili by IncM plasmid‐harbouring bacteria: Identification of plasmid‐encoded regulatory functions

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Abstract
A 1,3 kb DNA fragment of the IncM plasmid R446, if cloned in a multicopy plasmid, inhibits in trans the expression of conjugation pili by IncM plasmid-harbouring host bacteria as indicated by their insensitivity to the IncM pilus-dependent bacteriophage ϕM (Iml, insensitivity to ϕM-mediated lysis). Determination of the nucleotide sequence of this DNA fragment, the introduction of deletions and the analysis of transposon insertions reveal two determinants, imlA and imlB, responsible for the Iml phenotype. A stretch of 80 bp of DNA containing imlA and about 450 bp of adjacent DNA comprising imlB, together, bring about inhibition of the typical expression of conjugation pili at 30 °C. The introduction of a transposable promoter probe and the construction of respective lacZ fusions indicate transcription of complementary strands in vivo overlapping in the region comprising imlA and imlB. Moreover, the expression of reporter genes discloses temperature-dependent transcription of the imlA-imlB-region in one direction. A particular subfragment of the 1.3 kb IncM plasmid-derived DNA does not inhibit conjugation pilus expression at 30 °C but stimulates in trans the formation of pili at 42 °C to give rise to untypical sensitivity to ϕM at 42 °C in addition to 30 °C. Other subfragments reveal vital interferences with IncM plasmid-harbouring host cells. The putative nature of the cloned determinants interfering with the normal expression of IncM plasmid DNA is discussed.