Highlights • Ser/Thr phosphorylation is involved in the regulation of cell wall synthesis and translation. • Ser/Thr kinases phosphorylate elements of two-component systems. • Ser/Thr kinases ...phosphorylate other known transcriptional regulatory mechanisms.
Peptidoglycan serves as a key structure of the bacterial cell by determining cell shape and providing resistance to internal turgor pressure. However, in addition to these essential and well-studied ...functions, bacterial signaling by peptidoglycan fragments, or muropeptides, has been demonstrated by recent work. Actively growing bacteria release muropeptides as a consequence of cell wall remodeling during elongation and division. Therefore, the presence of muropeptide synthesis is indicative of growth-promoting conditions and may serve as a broadly conserved signal for nongrowing cells to reinitiate growth. In addition, muropeptides serve as signals between bacteria and eukaryotic organisms during both pathogenic and symbiotic interactions. The increasingly appreciated role of the microbiota in metazoan organisms suggests that muropeptide signaling likely has important implications for homeostatic mammalian physiology.
As rapidly growing bacteria begin to exhaust essential nutrients, they enter a state of reduced growth, ultimately leading to stasis or quiescence. Investigation of the response to nutrient ...limitation has focused largely on the consequences of amino acid starvation, known as the "stringent response." Here, an uncharged tRNA in the A-site of the ribosome stimulates the ribosome-associated protein RelA to synthesize the hyperphosphorylated guanosine nucleotides (p)ppGpp that mediate a global slowdown of growth and biosynthesis. Investigations of the stringent response typically employ experimental methodologies that rapidly stimulate (p)ppGpp synthesis by abruptly increasing the fraction of uncharged tRNAs, either by explicit amino starvation or by inhibition of tRNA charging. Consequently, these methodologies inhibit protein translation, thereby interfering with the cellular pathways that respond to nutrient limitation. Thus, complete and/or rapid starvation is a problematic experimental paradigm for investigating bacterial responses to physiologically relevant nutrient-limited states.
...a single phylogenetically conserved domain, called Lysin (LysM), is found in specific receptors in signaling pathways responsive to one or more of these three related carbohydrates in bacteria, ...plants, and fungi and possibly mammalian systems. ...promiscuous activation could occur when a structurally similar but physiologically inappropriate ligand binds and thereby aberrantly activates an incorrect LysM domain-containing receptor. In support of this idea, Nod factors suppress an innate immune reaction in Arabidopsis thaliana, even though this species lacks a Nod receptor 14. Since this effect depends on the presence of the LYK3 LysM-containing receptor kinase that functions as an innate immune receptor, the Nod factor could therefore act as a competitive antagonist of LYK3. Specifically, peptidoglycan is released by growing bacterial cells, and the substantial levels of peptidoglycan fragments generated by the microbiota have important systemic immunological effects mediated by key innate immune proteins like Nod1 30. ...perhaps the levels of these molecules relative to their receptors is affected by the presence of chitin, which changes the normal ligand:receptor stoichiometry. ...inflammatory bowel disease is another example in which interactions between chitin- and peptidoglycan-signaling systems could be relevant.
Phenotypic heterogeneity of microbial populations can facilitate survival in dynamic environments by generating sub-populations of cells that may have differential fitness in a future environment. ...Bacillus subtilis cultures experiencing nutrient limitation contain distinct sub-populations of cells exhibiting either comparatively high or low protein synthesis activity. This heterogeneity requires the production of phosphorylated guanosine nucleotides (pp)pGpp by three synthases: SasA, SasB, and RelA. Here we show that these enzymes differentially affect this bimodality: RelA and SasB are necessary to generate the sub-population of cells exhibiting low protein synthesis whereas SasA is necessary to generate cells exhibiting comparatively higher protein synthesis. Previously, it was reported that a RelA product allosterically activates SasB and we find that a SasA product competitively inhibits this activation. Finally, we provide in vivo evidence that this antagonistic interaction mediates the observed heterogeneity in protein synthesis. This work therefore identifies the mechanism underlying phenotypic heterogeneity in protein synthesis.
Most bacteria are quiescent, typically as a result of nutrient limitation. In order to minimize energy consumption during this potentially prolonged state, quiescent bacteria substantially attenuate ...protein synthesis, the most energetically costly cellular process. Ribosomes in quiescent bacteria are present as dimers of two 70S ribosomes. Dimerization is dependent on a single protein, hibernation promoting factor (HPF), that binds the ribosome in the mRNA channel. This interaction indicates that dimers are inactive, suggesting that HPF inhibits translation. However, we observe that HPF does not significantly affect protein synthesis in vivo suggesting that dimerization is a consequence of inactivity, not the cause. The HPF–dimer interaction further implies that re‐initiation of translation when the bacteria exit quiescence requires dimer resolution. We show that ribosome dimers quickly resolve in the presence of nutrients, and this resolution is dependent on transcription, indicating that mRNA synthesis is required for dimer resolution. Finally, we observe that ectopic HPF expression in growing cells where mRNA is abundant does not significantly affect protein synthesis despite stimulating dimer formation, suggesting that dimerization is dynamic. Thus, the extensive transcription that occurs in response to nutrient availability rapidly re‐activates the translational apparatus of a quiescent cell and induces dimer resolution.
Ribosomes in quiescent bacteria form inactive 100S dimers that must be resolved to reinitiate growth. Here, we demonstrate that this process is under direct control of mRNA synthesis, thus coupling the re‐activation of ribosomes with production of their substrate. Furthermore, our data suggest that ribosome dimerization is not required for attenuation of protein synthesis during stationary phase in Bacillus subtilis and that dimerization is dynamic and results from reduced transcription and translation.
Isogenic populations of cells exhibit phenotypic variability that has specific physiological consequences. Individual bacteria within a population can differ in antibiotic tolerance, but whether this ...variability can be regulated or is generally an unavoidable consequence of stochastic fluctuations is unclear. Here we report that a gene encoding a bacterial (p)ppGpp synthetase in Bacillus subtilis, sasA, exhibits high levels of extrinsic noise in expression. We find that sasA is regulated by multisite phosphorylation of the transcription factor WalR, mediated by a Ser/Thr kinase-phosphatase pair PrkC/PrpC, and a Histidine kinase WalK of a two-component system. This regulatory intersection is crucial for controlling the appearance of outliers; rare cells with unusually high levels of sasA expression, having increased antibiotic tolerance. We create a predictive model demonstrating that the probability of a given cell surviving antibiotic treatment increases with sasA expression. Therefore, multisite phosphorylation can be used to strongly regulate variability in antibiotic tolerance.
When nutrients become scarce, bacteria can enter an extended state of quiescence. A major challenge of this state is how to preserve ribosomes for the return to favorable conditions. Here, we show ...that the ribosome dimerization protein hibernation-promoting factor (HPF) functions to protect essential ribosomal proteins. Ribosomes isolated from strains lacking HPF (Δ
) or encoding a mutant allele of HPF that binds the ribosome but does not mediate dimerization were substantially depleted of the small subunit proteins S2 and S3. Strikingly, these proteins are located directly at the ribosome dimer interface. We used single-particle cryo-electron microscopy (cryo-EM) to further characterize these ribosomes and observed that a high percentage of ribosomes were missing S2, S3, or both. These data support a model in which the ribosome dimerization activity of HPF evolved to protect labile proteins that are essential for ribosome function. HPF is almost universally conserved in bacteria, and HPF deletions in diverse species exhibit decreased viability during starvation. Our data provide mechanistic insight into this phenotype and establish a mechanism for how HPF protects ribosomes during quiescence.
The formation of ribosome dimers during periods of dormancy is widespread among bacteria. Dimerization is typically mediated by a single protein, hibernation-promoting factor (HPF). Bacteria lacking HPF exhibit strong defects in viability and pathogenesis and, in some species, extreme loss of rRNA. The mechanistic basis of these phenotypes has not been determined. Here, we report that HPF from the Gram-positive bacterium
preserves ribosomes by preventing the loss of essential ribosomal proteins at the dimer interface. This protection may explain phenotypes associated with the loss of HPF, since ribosome protection would aid survival during nutrient limitation and impart a strong selective advantage when the bacterial cell rapidly reinitiates growth in the presence of sufficient nutrients.
Bacteria can respond to adverse environmental conditions by drastically reducing or even ceasing metabolic activity. They must then determine that conditions have improved before exiting dormancy, ...and one indication of such a change is the growth of other bacteria in the local environment. Growing bacteria release muropeptide fragments of the cell wall into the extracellular milieu, and we report here that these muropeptides are potent germinants of dormant
Bacillus subtilis spores. The ability of a muropeptide to act as a germinant is determined by the identity of a single amino acid. A well-conserved, eukaryotic-like Ser/Thr membrane kinase containing an extracellular domain capable of binding peptidoglycan is necessary for this response, and a small molecule that stimulates related eukaryotic kinases is sufficient to induce germination. Another small molecule, staurosporine, that inhibits related eukaryotic kinases blocks muropeptide-dependent germination. Thus, in contrast to traditional antimicrobials that inhibit metabolically active cells, staurosporine acts by blocking germination of dormant spores.
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