Mechanisms of gene regulation in Chlamydia: The role of sigma-28 RNA polymerase

The main objective of this work is focused on understanding the role of sigma28 RNA polymerase in regulating gene expression in the pathogenic bacterium, Chlamydia. sigma28 RNA polymerase is one of two alternative forms of RNA polymerase predicted to be present in this organism, but the function of these regulatory enzymes have not been studied. Regulation of gene expression is believed to be critical to the ability of chlamydiae to survive and replicate in mammalian host cells. In the absence of a chlamydial genetic system, we have pursued an in vitro approach. In this study, we report the development of a completely chlamydial in vitro transcription system for sigma 28 RNA polymerase. Accomplishing this goal required active sigma 28 RNA polymerase, a chlamydial sigma28-dependent promoter, and appropriate transcription conditions. We were able to reconstitute active chlamydial sigma28 RNA polymerase from recombinant chlamydial sigma28 protein and native core enzyme that was partially purified from chlamydiae grown in tissue culture. We identified a candidate sigma28-dependent promoter upstream of hctB because of sequence similarity to sigma28 promoters in other bacteria and showed that this promoter was transcriptionally active. Transcription of the hctB promoter was highly salt-tolerant, which is a feature of sigma28-regulated promoters in other bacteria. This work identified a target gene of sigma28 in Chlamydia for the first time, and since hctB encodes a histone-like protein that is only expressed late in the developmental cycle, we hypothesize that sigma28 RNA polymerase has a role in late gene expression in Chlamydia..;Our in vitro assay provided a powerful tool for studying transcription by sigma28 RNA polymerase. In the first systematic analysis of a bacterial sigma28 promoter, we defined the sequences recognized by chlamydial sigma28 RNA polymerase through a comprehensive mutational analysis of the hctB promoter. The optimal -35 promoter element that we determined closely resembled the consensus sigma28 promoter of E. coli and Salmonella, and provided support for an extended sigma28 promoter that has been proposed, but not validated. In the -10 promoter element, we identified a distinct, but previously unrecognized, CGA motif that contributes significantly to promoter activity. In a parallel analysis, we found that the sequences recognized by sigma28 RNA polymerase from Chlamydia and E. coli were well conserved, and we infer from these results that sigma28 promoter recognition is likely to be conserved among bacteria. We have now utilized the results from this analysis of the sigma28 promoter to identify other sigma 28-regulated genes. Towards this end, we developed a computer algorithm to search for sigma28-like promoter sequences based on a model generated from our functional data. We tested selected top-ranking candidate promoters with our sigma28 in vitro transcription assay and identified five promoters that are specifically transcribed by sigma 28 RNA polymerase.;With the identification of additional target genes of sigma 28 RNA polymerase, we can begin to examine the role of this alternative RNA polymerase in the regulation of gene expression in Chlamydia. Among our total of six sigma28-regulated genes are three late genes and one heat shock-responsive gene, dnaK. The identification of additional late genes as target genes of sigma 28 provides further evidence that sigma28 has a role in late temporal gene regulation. The finding that dnaK is regulated by a sigma28-dependent promoter was unexpected, however, but it might be an important clue in understanding the regulation of late genes by sigma28 RNA polymerase. One possible model is that expression of late genes by sigma28 RNA polymerase is part of a general stress response that occurs late in the chlamydial developmental cycle, perhaps due to changes such as nutrient depletion or altered intrachlamydial conditions. This is the first functional identification of a temporal regulator in Chlamydia, and the strong inference that sigma 28 RNA polymerase may also regulate gene expression in response to environmental stimuli, such as heat shock, provides valuable insights into the transcriptional mechanisms vital to chlamydial survival and pathogenesis.