Regulons and protein–protein interactions of PRD‐containing Bacillus anthracis virulence regulators reveal overlapping but distinct functions

Abstract

Bacillus anthracis produces three regulators, AtxA, AcpA and AcpB, which control virulence gene transcription and belong to an emerging class of regulators termed 'PCVRs' (Phosphoenolpyruvate-dependent phosphotransferase regulation Domain-Containing Virulence Regulators). AtxA, named for its control of toxin gene expression, is the master virulence regulator and archetype PCVR. AcpA and AcpB are less well studied. Reports of PCVR activity suggest overlapping function. AcpA and AcpB independently positively control transcription of the capsule biosynthetic operon capBCADE, and culture conditions that enhance AtxA level or activity result in capBCADE transcription in strains lacking acpA and acpB. We used RNA-Seq to assess the regulons of the paralogous regulators in strains constructed to express individual PCVRs at native levels. Plasmid and chromosome-borne genes were PCVR controlled, with AtxA, AcpA and AcpB having a ≥ 4-fold effect on transcript levels of 145, 130 and 49 genes respectively. Several genes were coregulated by two or three PCVRs. We determined that AcpA and AcpB form homomultimers, as shown previously for AtxA, and we detected AtxA-AcpA heteromultimers. In co-expression experiments, AcpA activity was reduced by increased levels of AtxA. Our data show that the PCVRs have specific and overlapping activity and that PCVR stoichiometry and potential heteromultimerization can influence target gene expression.

AtxA-Controlled Small RNAs of Bacillus anthracis Virulence Plasmid pXO1 Regulate Gene Expression in trans

“…Read-through of a weak terminator hairpin at the end of the capsule biosynthesis operon transcript leads to transcription of the downstream acpB gene, which in turn positively regulates the operon in a feed-forward loop ( Drysdale et al, 2005 ). The AtxA protein and its paralogs are members of an emerging class of virulence regulators designated P RD- c ontaining v irulence r egulators (PCVRs), so termed due to the presence of phosphoenolpyruvate p hosphotransferase system - r egulated d omains (PRDs) in their protein structures ( Hondorp et al, 2013 ; Raynor et al, 2018 ).…”

Rap Protein Paralogs of Bacillus thuringiensis: a Multifunctional and Redundant Regulatory Repertoire for the Control of Collective Functions

“…Few studies have addressed multicellular behaviors such as differentiation, cell specialization, collective functions, and the resulting ecological interactions in species from the B. cereus group (11,58). Similarly, molecular mechanisms for the control of differentiation processes in the B. cereus group bacteria remain understudied (48,49,51,58,59). In this work, we uncovered the functions of Rap protein paralogs from Bt8741.…”

Influence of the phosphoenolpyruvate:carbohydrate phosphotransferase system on toxin gene expression and virulence in Bacillus anthracis

“…Markerless deletions in B. anthracis were constructed using the temperature‐sensitive integration vector pHY304 as described previously (Raynor et al , ). The ptsHI operon was removed from the ANR‐1 chromosome via homologous recombination.…”

“…The promoter of GBAA_2500 was chosen to control atxA transcription because GBAA_2500 transcript levels are comparable to atxA transcript levels in cultures grown in toxin‐inducing conditions (Raynor et al , ). The atxA ‐His6 construct was fused to the promoter region and cloned into the plcR locus, a nonfunctional gene in B. anthracis that has been used to express cloned genes (Raynor et al , ). To confirm the promoter was not controlled by the PTS, expression of the promoter‐ atxA fusion in a parent strain and a ptsHI ‐null mutant was compared using western blotting (data now shown).…”

“…As might be expected for such a pathogen, control of classic virulence factor synthesis in B. anthracis is intertwined with regulation of key physiological processes. AtxA, a well‐established transcription regulator of B. anthracis , positively controls the structural genes for the anthrax toxin proteins and the capsule biosynthesis operon, but also affects expression of genes associated with sporulation, branched chain amino acid biosynthesis and transport and other aspects of metabolism (Raynor et al ., ). Moreover, while carbohydrate utilization has not been directly associated with AtxA, transcription of the atxA gene is influenced by glucose availability via carbon catabolite repression (Chiang et al ., ), and toxin synthesis is high when the bacterium is cultured in defined medium containing glucose (Ristroph and Ivins, ).…”