Virulence gene expression is independent of ResDE-regulated respiration control in Bacillus anthracis

Abstract

The ResDE two-component system regulates the synthesis of several components of the aerobic and anaerobic respiratory pathways in bacilli. The ResD response regulator transcription factor has been implicated in the regulation of virulence factors in a number of gram-positive species, including Bacillus anthracis. The precise deletions of resD and resE in B. anthracis that retained the classical respiratory phenotypes did not affect the expression of the gene for the protective antigen of the anthrax toxin, pagA, or that of the toxin regulator, atxA. The results indicate that the loss of ResDE-controlled respiratory capacity does not affect the synthesis of anthrax toxin.

FIG. 1.

Cell growth and virulence factor gene expression in B. anthracis 34F2 parental and resD deletion strains. Cells carrying a pagA-lacZ or atxA-lacZ fusion were grown in LB medium supplemented with kanamycin. β-Galactosidase assays were carried out on samples taken at hourly intervals as indicated. (A) Cell growth of pagA-lacZ strain (growth was similar for atxA-lacZ strains); (B) pagA-lacZ expression; (C) atxA-lacZ expression. Symbols in all three panels: ♦, parental strain; ×, resD mutant; ▪, resD_cmp1 mutant; ▴, resD_cmp2 mutant.

FIG. 2.

Cell growth and virulence factor expression in B. anthracis 34F2 parental and resD deletion strains. Cells carrying a pagA-lacZ or atxA-lacZ fusion were grown in R medium (containing 0.25% glucose) supplemented with kanamycin in a 5% CO₂ atmosphere. β-Galactosidase assays were carried out on samples taken at hourly intervals as indicated. (A) Cell growth of pagA-lacZ fusion-carrying strains (growth was similar for atxA-lacZ strains); (B) pagA-lacZ expression; (C) atxA-lacZ expression. Symbols in all three panels: ♦, parental strain; ×, resD mutant; ▪, resD_cmp1 mutant; ▴, resD_cmp2 mutant.

FIG. 3.

Toxin and AtxA protein levels in B. anthracis 34F2 parental strain and isogenic resD deletion mutants. (A) Western blot analysis carried out on B. anthracis culture supernatants collected from two independent cultures grown in R medium in a 5% CO₂ atmosphere and detected with polyclonal anti-PA antibody. Lanes: 1, MagicMarker XP (Invitrogen); 2, parental strain 1; 3, parental strain 2; 4, resD 1 mutant; 5, resD 2 mutant. (B) Western blot analysis carried out on B. anthracis cell lysates collected from the same two independent cultures used for panel A and detected with polyclonal anti-AtxA antibody. Lanes: 1, purified AtxA; 2, MagicMarker XP; 3, parental strain 1; 4, parental strain 2; 5, resD culture 1; 6, resD culture 2. Sample preparation, antibody production, and Western blot analyses were carried out as described by Tsvetanova et al. (23). Samples were collected after 9 h of growth and normalized to an optical density at 600 nm of 1.5.