Global effects of virulence gene regulators in a Bacillus anthracis strain with both virulence plasmids

Abstract

Control of anthrax toxin and capsule synthesis, the two major virulence factors of Bacillus anthracis, has been associated with two regulatory genes, atxA and acpA, located on virulence plasmids pXO1 and pXO2, respectively. We used transcriptional profiling to determine whether atxA and/or acpA control genes other than those already described and to investigate functional similarities of the regulators. Transcription was assessed in a pXO1(+) pXO2(+) parent strain and in isogenic mutants in which one or both regulatory genes were deleted. We determined that in addition to the toxin and capsule genes, atxA controls expression of numerous other genes on both plasmids and the chromosome. Generally, plasmid-encoded genes were more highly regulated than chromosomal genes, and both positive and negative effects were observed. Certain atxA-regulated genes were affected synergistically in an atxA acpA mutant. Yet overall, acpA appears to be a minor regulator with fewer targets than atxA. In contrast to previous reports of acpA function in attenuated strains, acpA had a minimal influence on capsule gene transcription and capsule synthesis in a genetically complete strain. Surprisingly, acpA expression was positively affected by atxA, although atxA-activated capsule gene transcription is not acpA dependent. The newly discovered atxA-regulated targets include genes predicted to encode secreted proteins and proteins with roles in transcriptional regulation and signaling. Regulation of chromosomal genes by atxA is particularly intriguing, given that many of the target genes have homologues in other Bacillus species that lack atxA homologues. Given the global effect of atxA on gene expression in B. anthracis, previous assumptions regarding reduced virulence of strains harboring single plasmids must be reassessed and the potential roles of newly identified atxA-regulated genes should be investigated.

FIG. 1.

Plasmid gene expression profile. Only genes printed on the DNA array are represented. Cells were grown to mid-exponential phase in CACO₃ with shaking at 37°C in a 5% CO₂ atmosphere. Genes positively regulated by atxA are shown in green. The negatively regulated gene is shown in red. Nonregulated genes are shown in blue. Genes shown in white were not expressed. The pathogenicity island of pXO1 is indicated by a grey line. pXO1 is 181.6 kb (accession no. AF065404), and pXO2 is 96.2 kb (accession no. AF188935).

FIG. 2.

atxA and acpA synergy. Data points shown represent genes regulated (P < 0.05) in all UT500-UT501 and UT500-UT503 experiments. Numbers shown represent the plasmid ORF names. Both axes represent fold regulation (log scale). ◊ = chromosomal genes; ▴ = pXO1 genes; • = pXO2 genes.

FIG. 3.

Capsule production by the B. anthracis parent strain (UT500) and isogenic mutants. Cells were grown in CACO₃ with shaking at 37°C in a 5% CO₂ atmosphere. At the growth phases indicated, cells were removed and capsule was visualized with India ink. UT500, parent; UT501, atxA; UT502, acpA; UT503, atxA acpA. Bar = 5 μm.

FIG. 4.

Toxin synthesis by the B. anthracis parent strain (UT500) and isogenic mutants. Cells were grown as described in the legend for Fig. 1. Filtered culture supernates from the growth phases indicated were subjected to Western hybridization using antitoxin antiserum as indicated. PA, protective antigen; LF, lethal factor; EF, edema factor. UT500, parent; UT501, atxA; UT502, acpA; UT503, atxA acpA.