title: Enhancement of in vitro transcription by addition of cloned, overexpressed major sigma factor of Chlamydia psittaci 6BC.
abstract: 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 omega(66) had no effect on transcription by E. coli core polymerase. The addition of recombinant omega(66) to the in vitro system should be useful for distinguishing omega(66)-dependent transcription of developmentally regulated chlamydial genes from omega(66)-independent transcription.
authors.affiliation: Department of Microbiology and Immunology, University of Tennessee, Memphis 38163.
authors.forename: A L
authors.initials: AL
authors.lastname: Douglas
authors.forename: N K
authors.initials: NK
authors.lastname: Saxena
authors.forename: T P
authors.initials: TP
authors.lastname: Hatch
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D001483:Base Sequence; D002474:Cell-Free System; D002692:Chlamydia trachomatis / Q000201:enzymology; D002691:Chlamydophila psittaci / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D012808:Sigma Factor / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases