ENHANCEMENT OF IN-VITRO TRANSCRIPTION BY ADDITION OF CLONED, OVEREXPRESSED MAJOR SIGMA-FACTOR OF CHLAMYDIA-PSITTACI 6BC

Obligate parasitic bacteria of the genus Chlamydia possess a developmental cycle that takes place entirely within eucaryotic host cells. Because standard methods of genetic analysis are not available for chlamydiae, an in vitro transcription system has been developed to elucidate the mechanisms by which chlamydiae regulate gene expression. The in vitro system is specific for chlamydial promoters but is inefficient, presumably because the RNA polymerase is not saturated with sigma factor. Therefore, we prepared recombinant Chlamydia psittaci 6BC major sigma factor to enhance transcription in the in vitro system. The gene encoding the major sigma factor (sigA) was identified by using an rpoD box oligonucleotide and was subsequently cloned and sequenced. It was found to encode a potential 571-amino-acid protein (sigma(66)) that is greater than 90% identical to the previously identified major sigma factors from the L2 and MoPn strains of Chlamydia trachomatis. sigA was recloned into a T7 RNA polymerase expression system to produce large quantities of sigma(66) in Escherichia coli. Overexpressed sigma(66) was identified by immunoblot by using monoclonal antibodies 2G10 (reactive) and 2F8 (nonreactive) generated against E. coli sigma(70). After purification by polyacrylamide gel electrophoresis, the recombinant protein was found to stimulate, by 10-fold or more, promoter-specific in vitro transcription by C. psittaci 6BC and C. trachomatis L2 RNA polymerases. Transcription was dependent on added chlamydial sigma(66), rather than on potentially contaminating E. coli sigma(70) or other fortuitous activators, since the monoclonal antibody 2G10, and not 2F8, inhibited transcription initiation. Recombinant sigma(66) had no effect on transcription by E. coli core polymerase. The addition of recombinant sigma(66) to the in vitro system should be useful for distinguishing sigma(66)-dependent transcription of developmentally regulated chlamydial genes from sigma(66)-independent transcription.