Transcriptional analysis of the Bacillus anthracis capsule regulators

Abstract

The poly-d-glutamic acid capsule of Bacillus anthracis is essential for virulence. Control of capsule synthesis occurs at the level of transcription and involves positive regulation of the capsule biosynthetic operon capBCAD by a CO2/bicarbonate signal and three plasmid-borne regulators: atxA, acpA, and acpB. Although the molecular mechanism for control of cap transcription is unknown, atxA affects cap expression via positive control of acpA and acpB, two genes with partial functional similarity. Transcriptional analyses of a genetically complete strain indicate that capB expression is several hundred-fold higher during growth in 5% CO2 compared to growth in air. atxA was expressed appreciably during growth in air and induced only 2.5-fold by CO2. In contrast, expression of acpA and acpB was induced up to 23-fold and 59-fold, respectively, by CO2. The 5'-end mapping of gene transcripts revealed atxA-regulated and atxA-independent apparent transcription start sites for capB, acpA, and acpB. Transcripts mapping to all atxA-regulated start sites were increased during growth in elevated CO2. The acpA gene has one atxA-regulated and one atxA-independent start site. acpB lies downstream of capBCAD. A single atxA-independent start site maps immediately upstream of acpB. atxA-mediated control of acpB appears to occur via transcriptional read-through from atxA-dependent start sites 5' of capB. One atxA-independent and two atxA-regulated start sites map upstream of capB. Transcription from the atxA-regulated start sites of capBCAD was reduced significantly in an acpA acpB double mutant but unaffected in mutants with deletion of only acpA or acpB, in agreement with the current model for epistatic relationships between the regulators.

FIG. 1.

Real-time transcript levels detected during growth in elevated CO₂ and during growth in air for (A) capB and (B) atxA, acpA, and acpB. The transcript levels shown represent four different data sets that were normalized to gyrB transcript levels. A representative growth curve is shown for each experiment. The inset in panel A shows capB transcript levels using a different scale.

FIG. 2.

(A) Model showing transcriptional start sites for the capsule biosynthetic gene operon (capBCAD) and the cap gene regulators, acpA and acpB. (B) DNA sequence of the region upstream of capB. The predicted translational start site is underlined and capitalized. Nucleotides corresponding to the transcriptional start sites are in bold and capitalized. * denotes atxA/CO₂-regulated start site. P1 and P2 upstream of capB were reported previously by Uchida and coworkers (17).

FIG. 2.

(A) Model showing transcriptional start sites for the capsule biosynthetic gene operon (capBCAD) and the cap gene regulators, acpA and acpB. (B) DNA sequence of the region upstream of capB. The predicted translational start site is underlined and capitalized. Nucleotides corresponding to the transcriptional start sites are in bold and capitalized. * denotes atxA/CO₂-regulated start site. P1 and P2 upstream of capB were reported previously by Uchida and coworkers (17).

FIG. 3.

Primer extension analysis of capB transcripts. PE2 primer was employed (22). (A) RNA was extracted from cells grown in 5% CO₂. Lane 1, UT500 (Parent); lane 2, UT501 (atxA); lane 3, UT502 (acpA); lane 4, UT525 (acpB); lane 5, UT526 (acpA acpB). (B) RNA was extracted from cells as shown. Lane 1, UT500 (Parent) grown in 5% CO₂; lane 2, UT500 grown in air.

FIG. 4.

Primer extension analysis of acpB transcripts. MD65 primer was employed (See Materials and Methods). RNA was extracted from cells grown in 5% CO₂. Lane 1, UT500; lane 2, UT501 (atxA).

FIG. 5.

RT-PCR of the capD-acpB region. (A) capD-acpB transcript was detected. Lane M, DNA markers with sizes as indicated; lane 1, cDNA template plus reverse transcriptase; lane 2, cDNA template minus reverse transcriptase; lane 3, DNA template, PCR control. (B) Illustration showing location of primers MD62 and MD129 employed in the PCRs used in panel A. The open arrow indicates the position of pXO2-54.

FIG. 6.

Primer extension analysis of acpA transcripts. MD28 primer was employed. (A) RNA was extracted from cells grown in 5% CO₂. Lane 1, UT500 (Parent); lane 2, UT501 (atxA); lane 3, UT502 (acpA); lane 4, UT525 (acpB); lane 5, UT526 (acpA acpB). (B) RNA was extracted from cells as shown. Lane 1, UT500 (Parent) grown in 5% CO₂; lane 2, UT500 grown in air.