Highlights d Discovery of prokaryotic ribosome-associated quality control (RQC) with origin in LUCA d Bacterial RQC is partially redundant with ssrA/tmRNA d Bacterial Rqc2 homolog extends substrates with C-terminal Ala tails that act as degrons d Bacterial RQC protects cells against translation inhibition and environmental stresses

SummaryExpression of genes for Bacillus anthracis toxin and capsule virulence factors are dependent upon the AtxA transcription factor. The mechanism by which AtxA regulates the transcription of its target genes is unknown. Here we report that bioinformatic analyses suggested the presence in AtxA of two PTS (phosphenolpyruvate : sugar phosphotransferase system) regulation domains (PRD) generally regulated by phosphorylation/dephosphorylation at conserved histidine residues. By means of amino acid substitutions that mimic the phosphorylated (H to D) or the unphosphorylated (H to A) state of the protein, we showed that phosphorylation of H199 of PRD1 is likely to be necessary for AtxA activation while phosphorylation of H379 in PRD2 is inhibitory to toxin gene transcription. In vivo labelling experiments with radioactive phosphate allowed us to propose that H199 and H379 are AtxA residues subject to regulated phosphorylation. In support to these notions, we also show that deletion of ptsHI, encoding the HPr intermediate and the EI enzymes of PTS, or growth in the presence of glucose affect positively and negatively, respectively, the activity of AtxA. Our results link virulence factor production in B. anthracis to carbohydrate metabolism and, for the first time, provide a mechanistic explanation for AtxA transcriptional activity.

The initiation of sporulation in Bacillus species is regulated by the phosphorelay signal transduction pathway, which is activated by several histidine sensor kinases in response to cellular and metabolic signals.Comparison of the protein components of the phosphorelay between Bacillus subtilis and Bacillus anthracis revealed high homology in the phosphorelay orthologs of Spo0F, Spo0B, and Spo0A. The sensor domains of sensor histidine kinases are poorly conserved between species, making ortholog recognition tenuous. Putative sporulation sensor histidine kinases of B. anthracis were identified by homology to the HisKA domain of B. subtilis sporulation sensor histidine kinases, which interacts with Spo0F. Nine possible kinases were uncovered, and their genes were assayed for complementation of kinase mutants of B. subtilis, for ability to drive lacZ expression in B. subtilis and B. anthracis, and for the effect of deletion of each on the sporulation of B. anthracis. Five of the nine sensor histidine kinases were inferred to be capable of inducing sporulation in B. anthracis. Four of the sensor kinases could not be shown to induce sporulation; however, the genes for two of these were frameshifted in all B. anthracis strains and one of these was also frameshifted in the pathogenic pXO1-bearing Bacillus cereus strain G9241. It is proposed that acquisition of plasmid pXO1 and pathogenicity may require a dampening of sporulation regulation by mutational selection of sporulation sensor histidine kinase defects. The sporulation of B. anthracis ex vivo appears to result from any one or a combination of the sporulation sensor histidine kinases remaining.

SummaryThe initiation of sporulation in aerobic Bacillus species is regulated by the phosphorelay consisting of several sensor histidine kinases, the Spo0F response regulator, the Spo0B phosphotransferase and the Spo0A transcription factor that upon phosphorylation represses genes for growth and activates the developmental process. Clostridium species lack both Spo0F and Spo0B and the identities of the sensor histidine kinases are unknown. The amino acid sequence of Spo0A is highly conserved in Clostridium botulinum relative to Bacillus subtilis but the cloned C. botulinum Spo0A was unable to complement a spo0A mutant of B. subtilis for sporulation. However, it was able to repress the abrB gene of B. subtilis . Active site mutations in Spo0A still repressed, indicating this activity was independent of phosphorylation. An orphan sensor histidine kinase of C. botulinum appeared to normally phosphorylate C. botulinum Spo0A and expression of this kinase in combination with C. botulinum Spo0A in B. subtilis was lethal, suggesting phosphorylation of C. botulinum Spo0A repressed essential growth genes as a prerequisite to sporulation but could not compensate for this effect by inducing sporulation. A chimera Spo0A consisting of a B. subtilis Spo0A response regulator domain fused to a C. botulinum DNA-binding domain was capable of restoring sporulation to a spo0A mutant of B. subtilis albeit at less than wild-type levels. The data suggest that induction of sporulation requires interactions of both domains of Spo0A with other conserved proteins and despite the high conservation of the amino acid sequence of C. botulinum Spo0A, some of these interactions have been lost.