Cloning and characterization of RNA polymerase core subunits of Chlamydia trachomatis by using the polymerae chain reaction

Abstract

Taking advantage of sequence conservation of portions of the α, β, and β' subunits of RNA polymerase of bacteria and plant chloroplasts, we have designed degenerate oligonucleotides corresponding to these domains and used these synthetic DNA sequences as primers in a polymerase chain reaction to amplify DNA sequences from the chlamydial genome. The polymerase chain reaction products were used as a probe to recover the genomic fragments encoding the β subunit and the 5' portion of the β' subunit from a library of cloned murine Chlamydia trachomatis DNA. Similar attempts to recover the α subunit were unsuccessful. Sequence analysis demonstrated that the β subunit of RNA polymerase was located between genes encoding the L7/L12 ribosomal protein and the β' subunit of RNA polymerase; this organization is reminiscent of the rpoBC operon of Escherichia coli. The C. trachomatis β subunit overproduced in E. coli was used as an antigen in rabbits to make a polyclonal antibody to this subunit. Although this polyclonal antibody specifically immunoprecipitated the β subunit from Chlamydia-infected cells, it did not immunoprecipitate core or holoenzyme. Immunoblots with this antibody demonstrated that the β subunit appeared early in infection.