Structure and function of conjugative pili: inducible synthesis of functional F pili by Escherichia coli K-12 containing a lac-tra operon fusion.
In vivo and in vitro recombination methods were used to construct the recombinant plasmid pTG801, in which the F-plasmid DNA transfer (tra) genes required for the formation of functional F pili were placed under the lac transcriptional control sequences of pUC19. The 20 kilobases of cloned F DNA includes genes traA through the 5'-terminal part of traG; the plasmid lacks the positive regulatory gene traJ and all of the known tra genes required for the DNA transfer stage of conjugation. pTG801 transformants were sensitive to the donor-specific bacteriophages Q beta and f1, as measured by the formation of infectious centers. They were relatively insensitive to bacteriophage R17, as expected from the absence of traD. In the presence of a lacIq allele, sensitivity of pTG801 transformants to f1 and Q beta depended on the concentration of inducer (isopropyl-beta-D-thiogalactopyranoside [IPTG]). Viewed by electron microscopy, pTG801 transformants elaborated 7- to 10-nm-diameter filaments that could be laterally decorated with RNA bacteriophage particles, consistent with the formation of F pili. In stationary-phase cultures, these filaments formed massive aggregates and could be seen to adhere lengthwise to the cell surface; few pili accumulated in the medium as single filaments.
Grossman
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Department of Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10462.
Silverman
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PM
D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000235:genetics* / Q000648:ultrastructure; D005144:F Factor; D010861:Fimbriae, Bacterial / Q000502:physiology* / Q000648:ultrastructure; D005798:Genes, Bacterial; D008854:Microscopy, Electron; D009876:Operon; D015183:Restriction Mapping