Two small c-type cytochromes affect virulence gene expression in Bacillus anthracis

Abstract

Regulated expression of the genes for anthrax toxin proteins is essential for the virulence of the pathogenic bacterium Bacillus anthracis. Induction of toxin gene expression depends on several factors, including temperature, bicarbonate levels, and metabolic state of the cell. To identify factors that regulate toxin expression, transposon mutagenesis was performed under non-inducing conditions and mutants were isolated that untimely expressed high levels of toxin. A number of these mutations clustered in the haem biosynthetic and cytochrome c maturation pathways. Genetic analysis revealed that two haem-dependent, small c-type cytochromes, CccA and CccB, located on the extracellular surface of the cytoplasmic membrane, regulate toxin gene expression by affecting the expression of the master virulence regulator AtxA. Deregulated AtxA expression in early exponential phase resulted in increased expression of toxin genes in response to loss of the CccA-CccB signalling pathway. This is the first function identified for these two small c-type cytochromes of Bacillus species. Extension of the transposon screen identified a previously uncharacterized protein, BAS3568, highly conserved across many bacterial and archeal species, as involved in cytochrome c activity and virulence regulation. These findings are significant not only to virulence regulation in B. anthracis, but also to analysis of virulence regulation in many pathogenic bacteria and to the study of cytochrome c activity in Gram-positive bacteria.

Figure 1

Transcription analysis of pagA and atxA expression in transposon mutant strains. Strains carrying a pagA-lacZ or atxA-lacZ fusion on the replicative vector pTCV-lac were grown in LB broth supplemented with kanamycin at 37°C. β-galactosidase assays were carried out on samples taken at hourly intervals as indicated. A. Cell growth of pagA-lacZ reporter strains (cell growth of atxA-lacZ reporter strains was similar) B. β-galactosidase activity of pagA-lacZ reporter strains. C. β-galactosidase activity of atxA-lacZ reporter strains. Symbols in all three panels: -◆- 34F2; -▪- 34F2tB18 (resC); -X- 34F2tB21 (resB); -●- 34F2tB23 (BAS3568); -▲- 34F2tB24 (hemL).

Figure 2

Transcription analysis of pagA and atxA expression in resB, resBC and hemL mutant strains. Strains carrying a pagA-lacZ or atxA-lacZ fusion on the replicative vector pTCV-lac were grown in LB broth supplemented with kanamycin at 37°C. β-galactosidase assays were carried out on samples taken at hourly intervals as indicated. A. Cell growth of pagA-lacZ reporter strains (cell growth of atxA-lacZ reporter strains were similar) B. β-galactosidase activity of pagA-lacZ reporter strains. C. β-galactosidase activity of atxA-lacZ reporter strains. Symbols in all three panels: -◆- 34F2; -▪- 34F2△resB; -▲- 34F2△ resBC; -●- 34F2△hemL. The inset in panel C represents the Western blot analysis of AtxA on B. anthracis cell lysates collected after 3 and 8 hr of growth in LB Broth at 37°C. The amount of sample loaded on a 10% SDS-PAGE was normalized relative to cell growth. Lane 1: Magic Mark XP (Invitrogen); Lane 2: 34F2 after 3 hr of growth, Lane 3: 34F2△resB after 3 hr of growth; Lane 4: 34F2 after 8 hr of growth; Lane 5: 34F2△resB after 8 hr of growth. A full size of this Western blot is shown in Fig. S1.

Figure 3

Analysis of cytochrome c oxidase activity in B. anthracis parental and hemL mutant strains using TMPD oxidase staining. Strains were isolated on NSMP-agar plates in the absence (−) or presence (+) of 5 mg/ml 5-ALA. Upon exposure to TMPD, the staining for oxidase activity of strain 34F2 (full activity) or 34F2△ctaC (lack of activity) was not affected by the addition of 5-ALA. The partial staining of strain 34F2△hemL in the absence of 5-ALA was increased to the level of the parental strain when 5-ALA was present in the medium.

Figure 4

Transcription analysis of pagA and atxA expression in resB and atxA-resB mutant strains. β-galactosidase assays were carried out on samples taken at hourly intervals as indicated. A. and B. Strains carrying a pagA-lacZ or atxA-lacZ fusion on the replicative vector pTCV-lac were grown in LB broth supplemented with kanamycin at 37°C. A. pagA-lacZ reporter strains. B. atxA-lacZ reporter strains. Symbols in both panels: -◆- 34F2; -▪- 34F2△resB; -▲- 34F2△atxA; -●- 34F2△atxA△resB. C. The 34F2△atxA strain carrying an isotopically integrated pagA-lacZ reporter was transformed with plasmids pTCV-lac-spac or the pTCV-spac-AtxA and β-galactosidase activity was determined. Symbols: -▪- 34F2△atxA::pagA-lacZ/pTCV-spac-AtxA; -□- 34F2△atxA::pagA-lacZ/pTCV-spac; -●- 34F2△atxA△resB::pagA-lacZ/pTCV-spac-AtxA; -□- 34F2△atxA△resB::pagA-lacZ/pTCV-spac. D. The resB deletion affects only the P1 promoter of atxA. The 34F2 and 34F2△resB strains were transformed with different atxA-lacZ fusion constructs in pTCV-lac (Bongiorni et al., 2008) and β-galactosidase activity was measured. Symbols: -▪- 34F2/pAtxA10; -□- 34F2△resB/pAtxA10; -▲- 34F2/pAtxA12; -△- 34F2△resB/pAtxA12; -◆- 34F2/pAtxA20; -◇- 34F2△resB/pAtxA20.

Figure 5

Transcription analysis of pagA and atxA expression in cytochrome c deletion strains. Strains carrying a pagA-lacZ or atxA-lacZ fusion on the replicative vector pTCV-lac were grown in LB broth supplemented with kanamycin at 37°C. β-galactosidase assays were carried out on samples taken at hourly intervals as indicated. A. pagA-lacZ reporter strains. B. atxA-lacZ reporter strains. Symbols in both panels: -◆- 34F2; -▪- 34F2△resB; -▲- 34F2△ctaC; -X- 34F2△qcrBC; -○- 34F2△cccA; -●- 34F2△cccB; -△- 34F2△cccA-B; -□- 34F2△5cyt

Figure 6

Transcription of pagA and atxA in BAS3568 and resB mutant strains. Strains carrying a pagA-lacZ or atxA-lacZ fusion on the replicative vector pTCV-lac were grown in LB broth supplemented with kanamycin at 37°C. β-galactosidase assays were carried out on samples taken at hourly intervals as indicated. A. pagA-lacZ reporter strains. B. atxA-lacZ reporter strains. Symbols in both panels: -▲- 34F2; -▪- 34F2△resB; -▲- 34F2△BAS3568; -●- 34F2△resB△BAS3568

Figure 7

Transcription analysis of pagA and atxA expression under toxin-inducing growth conditions. Strains carrying a pagA-lacZ or atxA-lacZ fusion on the replicative vector pTCV-lac were grown in R-Media with or without added NaHCO₃ supplemented with kanamycin at 37°C under 5% atmospheric CO₂. β-galactosidase assays were carried out on samples taken at hourly intervals as indicated. A. pagA-lacZ reporter strains. B. atxA-lacZ reporter strains. Symbols in both panels: -▪- 34F2 without added NaHCO₃; -□- 34F2 with 0.8% NaHCO₃; -○- 34F2△resB without added NaHCO₃; -●- 34F2△resB with 0.8% NaHCO_3.

Figure 8

Schematic representation of the small c-type cytochrome pathway regulating virulence gene expression in B. anthracis. Intracellularly synthesized heme, through a pathway that requires the Hem proteins including HemL, is transported across the membrane by the ResBC proteins (Ahuja et al., 2007) and is covalently attached to CccA and CccB with the involvement of ResA (Le Brun et al., 2000). Either CccA or CccB can then act to indirectly repress atxA transcription at the P1 promoter and their function is redundant. The BAS3568 protein, which is required for full cytochrome oxidase activity, is also involved in repression of atxA transcription in early exponential phase by an unknown mechanism (indicated by the broken arrow) likely to act on the cytochrome c maturation pathway. The BAS3568 protein does not share amino acid similarity with the Res proteins. The sidedness of the membrane is indicated by “in” and “out”.