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Sequence alterations affecting F plasmid transfer gene expression: a conjugation system dependent on transcription by the RNA polymerase of phage T7
Author(s) -
Maneewannakul Kesmanee,
Maneewannakul Sumit,
IppenIhler Karin
Publication year - 1992
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1992.tb01755.x
Subject(s) - biology , operon , pilus , pilin , plasmid , microbiology and biotechnology , lac operon , transcription (linguistics) , mutant , gene , promoter , rna polymerase , t7 rna polymerase , rna , genetics , gene expression , escherichia coli , bacteriophage , linguistics , philosophy
Summary We constructed derivatives of the Escherichia coli conjugative plasmid F that carry altered sequences in place of the major transfer operon promoter, P Y Replacement of P Y with a promoter‐deficient sequence resulted in a transfer‐deficient, F‐pilus‐specific phage‐resistant plasmid (pOX38‐ tra 701) that could still express TraJ and TraT; TraY, F‐pilin, TraD, and Tral were not detectable on Western blots. On a second plasmid (pOX38‐ tra 715) we replaced P Y with a phage T7 late promoter sequence. In hosts carrying a lacUV5‐promoter‐regulated T7 RNA polymerase gene, all transfer‐associated properties of pOX38‐ tra 715 could be regulated with IPTG. After induction, pOX38‐ tra715 transferred at the wild‐type frequency, expressed normal numbers of F‐pili and conferred sensitivity to pilus‐specific phages. No adverse effects on cell viability were apparent, and additional mutations could easily be crossed onto pOX38‐ tra 715. A traJ deletion (pOX36‐tra716) had no effect on the IPTG‐induced transfer phenotype. Insertion of cam into trbC , resulted in a mutant (pOX38‐tra715trbC33) which, after induction, exhibited the same phenotype associated with other trbC mutants; it could also be complemented by expression of trbC in trans. With pOX38‐ tra715 or its derivatives, we were able to label specifically the products of tra genes located throughout the long tra operon, by using rifampicin. This feature can be used to investigate transfer protein interactions and to follow changes in these proteins that are associated with conjugal mating events.