The Bacillus anthracis toxin genes, cya, lef, and pag, can be viewed as a regulon, in which transcription of all three genes is activated in trans by the same regulatory gene, atxA, in response to the same signal, CO2. In atxA+ strains, toxin gene expression is increased 5 to 20-fold in cells grown in 5% CO2, relative to cells grown in air. CO2-enhanced toxin gene transcription is not observed in atxA-null mutants. I hypothesized that atxA regulates genes in addition to those that encode the toxin proteins. I determined that several phenotypes are associated with the atxA gene. In addition to being toxin-deficient (Tox-), an atxA-null mutant grows poorly on minimal media and sporulates early compared to the parent strain. Furthermore, an atxA-null mutant has an altered 2-D gel protein profile compared to the parent. Thus, atxA appears to affect non-toxin gene expression. I used a genetic approach to find new atxA-regulated genes. Random transcriptional lacZ fusions were generated in B. anthracis using transposon Tn917-LTV3. Transposon-insertion libraries were screened for mutants expressing increased beta-galactosidase activity in 5% CO2. Introduction of an atxA-null mutation in these mutants revealed that 79% of the CO2-regulated fusions were also atxA-dependent. DNA sequence analysis of transposon insertion sites in seventeen mutants carrying CO2-enhanced atxA-dependent fusions revealed ten mutants carrying independent insertions that did not map to the toxin genes. These regulated fusions were designated as tcr-lacZ (tcr for “toxin-coregulated”). Nine of twelve tcr-lacZ fusions are located within the pXO1 pathogenicity island. These results indicate a clear association of atxA with CO2-enhanced gene expression in B. anthracis and provide evidence that atxA regulates genes other than the structural genes for the anthrax toxin proteins. Sequence analysis of DNA at one tcr locus led to the identification of a 300-bp gene located downstream of pag. The gene is co-transcribed with pag and represses expression of the pag operon, lef, and cya. I have designated this gene pagR (for protective antigen repressor). Two pag mRNA transcripts were detected in cells producing PA: a short 2.7-kb transcript corresponding to the pag gene, and a longer 4.2-kb transcript representing a bicistronic message derived from pag and pagR. The 3′ end of the short transcript mapped adjacent to an inverted repeat sequence, indicating that the sequence can act as a transcription terminator. Attenuation of termination at this site results in transcription of pagR. I propose renaming the pag gene pagA, such that the pag operon is composed of pagA and pagR. Repression of toxin gene expression by pagR may be mediated by atxA. The steady state level of atxA mRNA was also increased in the pagR mutant. Recombinant PagR protein was purified from Escherichia coli and used for DNA-binding studies. In gel retardation assays, PagR did not specifically bind the promoter regions of pagA or atxA. An unidentified factor in B. anthracis crude extracts, however, was able to cause a specific shift of the atxA promoter. The binding was not due to PagR or AtxA since crude extracts of UT119 (pagR) and UT53 (atxA) also shifted the atxA promoter probe. Further investigation into how B. anthracis regulates virulence gene expression in response to environmental stimuli will lead to a better understanding of how B. anthracis causes disease.

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atxA-Dependent Gene Expression in Bacillus anthracis