ANALYSIS OF THE CPXA LOCUS OF ESCHERICHIA COLI K-12 (F PLASMID, RECOMBINANT DNA, CONJUGATION)
ALBIN, RANDI L.
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Mutations at the Escherichia coli K-12 locus cpxA pleiotropically affect the expression of conjugal DNA donor activity and surface exclusion in F' or Hfr strains, isoleucine and valine biosynthesis, and envelope protein composition. These effects are made more severe in a strain that also carries a mutation at the cpxB locus. However, a cpxB mutation by itself is cryptic. To resolve how a cpxA mutation is able to affect such diverse cellular functions it is first necessary to understand the cpxA locus with respect to the number of genes it contains and the proteins which it encodes.;The cpxA locus was mapped by genetic and physical methods to the 87-88 min interval of the E. coli K-12 chromosome. A cloned restriction fragment from that interval fully complemented a chromosomal cpxA mutation. However, expression of cpxA activity from the cloned fragment required exogenous transcription and translation initiation sequences. Tn5 mutagenesis, analysis of plasmid-encoded CpxA fusion proteins, and DNA sequence analysis established the physical limits and protein coding capacity of the cpxA locus on the cloned fragment. The sizes of fusion transcripts, determined by RNA filter ("Northern") blot hybridization were consistent with the molecular weights of the fusion proteins. It was concluded from these analyses that the cpxA locus consists of a single gene and that the cpxA gene product itself is sufficient and necessary to fully complement all of the phenotypic defects associated with a chromosomal cpxA mutation.;To study the CpxA protein a large lacZ'('-)'cpxA fusion gene was constructed. The large ((TURN)140 kDa) hybrid protein was purified and used to produce a polyclonal anti-CpxA protein antiserum. "Western" blot and immunoprecipitation analyses established that the antiserum contained anti-CpxA protein antibodies. Immunological analysis of cell-free extracts identified the cpxA gene product as a 52 kDa polypeptide. The size of this protein corresponded to that identified in immune precipitates of proteins synthesized by an in vitro translation system directed by either of two plasmids that contain the intact cpxA coding sequence. Moreover, the expected molecular weight of the CpxA protein, predicted by its DNA nucleotide sequence, agrees very well with the observed value of 52,000.