Identification of the Escherichia coli K-12 cpxA locus as a single gene: construction and analysis of biologically-active cpxA gene fusions

Summary

In the accompanying communication we showed that a 2 kb EcoRI-BamHI restriction fragment from the pfkA-rha interval of the Escherichia coli K-12 chromosome fully complemented a chromosomal cpxA mutation when the fragment was cloned in pBR325. The same fragment cloned in pBR322 lacked any complementing activity. We show here that minicells containing the pBR325 derivative (pRA310) synthesized a 33 kDa polypeptide, designated ϕ33, that was not synthesized in minicells containing the pBR322 derivative (pRA311) or either of the parent plasmids. Synthesis of the ϕ33 polypeptide did not occur in minicells containing Tn5 insertion alleles of pRA310 that inactivated its cpxA complementing activity. These insertions mapped within the vector cat (chloramphenicol acetyltransferase gene) sequence immediately adjacent to the EcoRI site of pRA310 and within the 700–800 bp of the cloned EcoRI-BamHI fragment immediately adjacent to the EcoRI site. Tn5 insertions located within the fragment but closer to the BamHI terminus affected neither the cpxA complementing activity of pRA310 nor synthesis of the ϕ33 polypeptide in minicells. Plasmid pRA311 could be converted to a plasmid with cpxA complementing activity by cloning into its EcoRI site a restriction fragment containing a hybrid trp-lacUV5 promoter, the lacZ ribosome binding site, and the first eight lacZ codons. Minicells containing the resultant plasmid (pRA312) synthesized a 30 kDa polypeptide (designated ϕ30). The same fragment but lacking the ATG initiation codon and the following lacZ codons did not confer cpxA complementing activity on pRA311, and minicells containing the resultant plasmid (pRA313) did not synthesize the ϕ30 protein. We conclude that the EcoRI terminus of the 2 kb fragment interrupts the cpxA coding sequence. pRA310 contains a ϕ(cal‘-’cpxA)hyb fusion gene that encodes a polypeptide consisting of 73 NH₂-terminal amino acids of chloramphenicol acetyltransferase fused to about 25 kDa of CpxA protein. pRA312 contains a ϕ(lacZ‘-’cpxA)hyb fusion gene that encodes a smaller protein consisting of 7 or 8 amino acids of β-galactosidase fused to the same 25 kDa of CpxA protein. Both fusion proteins retain biological activity, as judged by complementation of a chromosomal cpxA mutation. Both proteins could be detected in whole cells. Analysis of fusion gene transcripts by RNA filter (“Northern”) blot hybridization indicated that they are monocistronic.