Expression and phase variation of gonococcal P.II genes in Escherichia coli involves ribosomal frameshifting and slipped-strand mispairing

Abstract

Expression and phase variation of Neisseria gonorrhoeae P.II genes in Escherichia coli were studied using TnphoA fusions. Fusions were created in the P.IIc gene of N. gonorrhoeae JS3 using lambda TnphoA-1 and were characterized by restriction digestion and dideoxy sequencing. Three fusions were chosen for further study; Tnp7 (fusion junction at mature amino acid 7), Tnp57 (amino acid 57), Tnp66 (amino acid 66). All fusions were in frame with the P.IIc coding sequence but were out of frame with the purported initiation codon. All fusion constructions were shown to phase vary in E. coli in an analogous fashion to that seen in N. gonorrhoeae, i.e. phase changes (in a recA background) at a frequency of c. 10(-3) accompanied by an alteration at the DNA level of the number of coding repeats (CRs). In vitro mutagenesis of the fusion constructions indicated that expression of out of frame P.II genes in E. coli was probably the result of ribosomal frameshifting within the run of 'A' residues immediately preceding the CR region and not due to low-level false initiation at codons other than the ATG initiation codon (as had previously been suggested). The mechanism for P.IIc::phoA phase variation appears to be related to the 'slipped-strand mispairing' mechanism responsible for frameshift mutations in a number of other bacterial genes containing short, direct, tandem repeats.