Elongation factor P (EF-P) is a universally conserved translation factor that prevents ribosome stalling at amino acids that form peptide bonds with low efficiency, particularly polyproline tracts. Phenotypes associated with EF-P deletion are pleiotropic, and the mechanistic basis underlying many of these phenotypes is unclear.

Peptidoglycan (PG) is an essential structural component of the bacterial cell wall that is synthetized during cell division and elongation. PG forms an extracellular polymer crucial for cellular viability, the synthesis of which is the target of many antibiotics. PG assembly requires a glycosyltransferase (GT) to generate a glycan polymer using a Lipid II substrate, which is then crosslinked to the existing PG via a transpeptidase (TP) reaction. A Shape, Elongation, Division and Sporulation (SEDS) GT enzyme and a Class B Penicillin Binding Protein (PBP) form the core of the multi-protein complex required for PG assembly. Here we used single particle cryo-electron microscopy to determine the structure of a cell elongation-specific E. coli RodA-PBP2 complex. We combine this information with biochemical, genetic, spectroscopic, and computational analyses to identify the Lipid II binding sites and propose a mechanism for Lipid II polymerization. Our data suggest a hypothesis for the movement of the glycan strand from the Lipid II polymerization site of RodA towards the TP site of PBP2, functionally linking these two central enzymatic activities required for cell wall peptidoglycan biosynthesis.

In bacteria, signaling phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys in phylogenetically diverse bacteria over the past decade have identified numerous proteins that are phosphorylated on Ser and/or Thr residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes such as E. coli , which encodes at least three Ser/Thr kinases. Here we identify a previously uncharacterized ORF, yegI , and demonstrate that it encodes a novel Ser/Thr kinase. YegI lacks several conserved residues including those important for Mg2+ binding seen in other bacterial Ser/Thr kinases, suggesting that the consensus may be too stringent. We further find that YegI is a two-pass membrane protein with both N- and C-termini located intracellularly.

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 (Δhpf) 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.IMPORTANCE 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 Bacillus subtilis 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.

Oxygen is essential for many organisms who have therefore evolved mechanisms to enable survival during hypoxia. A new study describes how a well-known bacterial surfactant, called surfactin, facilitates bacterial viability when oxygen becomes limiting by reducing oxygen consumption.

Volume 200, no. 18, 2018, [https://doi.org/10.1128/JB.00225-18][1]. Page 1: This article was published on 24 August 2018 with a byline that lacked Elizabeth Nagle. The byline was updated in the version posted on 22 October 2019. Page 12, Acknowledgments, line 1: The second sentence should be

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.

Fillol-Salom et al. describe a mechanism by which an Escherichia coli pathogenicity island is preferentially packaged into a phage particle, thus promoting the spread of pathogenic traits among Gram-negative bacteria while protecting them from lytic infection.

The formation of ribosome dimers during periods of quiescence is widespread among bacteria and some higher eukaryotes. However, the mechanistic importance of dimerization is not well understood. In bacteria ribosome dimerization is mediated by the Hibernation Promoting Factor (HPF). Here, we report that HPF from the Gram-positive bacterium Bacillus subtilis preserves active ribosomes by preventing the loss of essential ribosomal proteins. Ribosomes isolated from strains either lacking HPF (Δ hpf ) 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 at the ribosome dimer interface. We used single particle cryo-EM to further characterize ribosomes isolated from a Δ hpf mutant strain and observed that many 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. Significance Statement When nutrients become scarce, many bacterial species enter an extended state of quiescence. A major challenge of this state is how to attenuate protein synthesis, the most energy consuming cellular process, while preserving ribosomes for the return to favorable conditions. Here, we show that the ribosome-binding protein HPF which dimerizes ribosomes functions to protect essential ribosomal proteins at the dimer interface. HPF is almost universally conserved in bacteria and HPF deletions in diverse species exhibit decreased viability under nutrient limitation. Our data provide mechanistic insight into this phenotype and establish a role for HPF in maintaining translationally competent ribosomes during quiescence.

AbstractIn bacteria, signaling phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys in phylogenetically diverse bacteria over the past decade have identified numerous proteins that are phosphorylated on Ser and/or Thr residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes such asE. coli, which encodes at least three Ser/Thr kinases. Here we identify a previously uncharacterized ORF,yegI, and demonstrate that it encodes a novel Ser/Thr kinase. YegI lacks several conserved residues including those important for Mg2+binding seen in other bacterial Ser/Thr kinases, suggesting that the consensus may be too stringent. We further find that YegI is a two-pass membrane protein with both N- and C-termini located intracellularly.

Germline-encoded receptors recognizing common pathogen-associated molecular patterns are a central element of the innate immune system and play an important role in shaping the host response to infection. Many of the innate immune molecules central to these signaling pathways are evolutionarily conserved. LysMD3 is a novel molecule containing a putative peptidoglycan-binding domain that has orthologs in humans, mice, zebrafish, flies, and worms. We found that the lysin motif (LysM) of LysMD3 is likely related to a previously described peptidoglycan-binding LysM found in bacteria. Mouse LysMD3 is a type II integral membrane protein that co-localizes with GM130+ structures, consistent with localization to the Golgi apparatus. We describe here two lines of mLysMD3-deficient mice for in vivo characterization of mLysMD3 function. We found that mLysMD3-deficient mice were born at Mendelian ratios and had no obvious pathological abnormalities. They also exhibited no obvious immune response deficiencies in a number of models of infection and inflammation. mLysMD3-deficient mice exhibited no signs of intestinal dysbiosis by 16S analysis or alterations in intestinal gene expression by RNA sequencing. We conclude that mLysMD3 contains a LysM with cytoplasmic orientation, but we were unable to define a physiological role for the molecule in vivo.

Isogenic populations of cells exhibit phenotypic variability that has specific physiological consequences. For example, individual bacteria within a population can differ in their sensitivity to an antibiotic, but whether this variability can be regulated or is generally an unavoidable consequence of stochastic fluctuations is unclear. We observed that a bacterial stress response gene, the (p)ppGpp synthetase sasA , exhibits high levels of extrinsic noise in expression, suggestive of a regulatory process. We traced this variability to the convergence of two signaling systems that together control the multisite phosphorylation of a transcription factor, an event largely unexplored in bacteria, This regulatory intersection between a Ser/Thr kinase and a prototypical two component system is crucial for controlling the appearance of outliers, rare cells with unusually high levels of sasA expression. Additionally, by examining the full distributions of gene expression we calculated the contribution of the additional Ser/Thr kinase-dependent phosphorylation in setting the relative abundance of cells with a given a level of SasA. We then created a predictive model for the probability of a given cell surviving antibiotic treatment as a function of sasA expression. Therefore, our data show that multisite phosphorylation can be used to strongly regulate variations in phenotypes across a bacterial population.

In bacteria, signaling phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys over the past decade in phylogenetically diverse bacteria have identified numerous proteins that are phosphorylated on Ser and/or Thr residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes, such as that of Escherichia coli, which encodes at least three Ser/Thr kinases. Since Ser/Thr phosphorylation is a stable modification, a dedicated phosphatase is necessary to allow reversible regulation. Ser/Thr phosphatases belonging to several conserved families are found in bacteria. One family of particular interest are Ser/Thr phosphatases, which have extensive sequence and structural homology to eukaryotic Ser/Thr protein phosphatase 2C (PP2C) phosphatases. These proteins, called eukaryote-like Ser/Thr phosphatases (eSTPs), have been identified in a number of bacteria but not in E. coli Here, we describe a previously unknown eSTP encoded by an E. coli open reading frame (ORF), yegK, and characterize its biochemical properties, including its kinetics, substrate specificity, and sensitivity to known phosphatase inhibitors. We investigate differences in the activity of this protein in closely related E. coli strains. Finally, we demonstrate that this eSTP acts to dephosphorylate a novel Ser/Thr kinase that is encoded in the same operon.IMPORTANCE Regulatory protein phosphorylation is a conserved mechanism of signaling in all biological systems. Recent phosphoproteomic analyses of phylogenetically diverse bacteria, including the model Gram-negative bacterium Escherichia coli, demonstrate that many proteins are phosphorylated on serine or threonine residues. In contrast to phosphorylation on histidine or aspartate residues, phosphorylation of serine and threonine residues is stable and requires the action of a partner Ser/Thr phosphatase to remove the modification. Although a number of Ser/Thr kinases have been reported in E. coli, no partner Ser/Thr phosphatases have been identified. Here, we biochemically characterize a novel Ser/Thr phosphatase that acts to dephosphorylate a Ser/Thr kinase that is encoded in the same operon.

How do neighboring bacterial biofilms sense and communicate with each other? In a recent paper, Liu et al. (2017) demonstrate how electrical signaling allows communication of metabolic states between adjacent B. subtilis biofilms, providing a possible generalizable mechanism for communication in multispecies biofilms with interdependent metabolism.

β-Lactams represent one of the most important classes of antibiotics discovered to date. These agents block Lipid II processing and cell wall biosynthesis through inactivation of penicillin-binding proteins (PBPs). PBPs enzymatically load cell wall building blocks from Lipid II carrier molecules onto the growing cell wall scaffold during growth and division. Lipid II, a bottleneck in cell wall biosynthesis, is the target of some of the most potent antibiotics in clinical use. Despite the immense therapeutic value of this biosynthetic pathway, the PBP-Lipid II association has not been established in live cells. To determine this key interaction, we designed an unnatural d-amino acid dipeptide that is metabolically incorporated into Lipid II molecules. By hijacking the peptidoglycan biosynthetic machinery, photoaffinity probes were installed in combination with click partners within Lipid II, thereby allowing, for the first time, demonstration of PBP interactions in vivo with Lipid II.

•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.

Stimuli responsive materials are in high demand for myriad applications within the fields of biomedicine, adaptive-architecture, robotics, and alternative energy. The non-pathogenic bacterial spores of Bacillus subtilis have the ability to swell as much as 12% in response to humidity gradients. Within the spore unique biomaterials drive this dynamic response, resulting in energy densities greater than 10 MJ∗m−3, more than a hundred times the potential of existing synthetics. We hypothesize that the spore's cortex, a protective layer surrounding the genetic core, is largely but not solely responsible for this peculiar water-responsive behavior. To evaluate the spore's constituent biomaterials we developed AFM techniques that assay individual spores during a thermodynamic cycle of varied applied force and relative humidity. This method resulted in energy density and strain profiles for multiple spore variants and structural mutants. Electron microscopy (FIB SEM) was then used to ascribe radial dimensions to biomaterial layers within the spore. Quantifying precise substructural contributions to the spore's hygrodynamics will allow for in vitro optimization of composite spore biomaterials and may even facilitate artificial synthesis in the future. The bacterial spore's inherent ability to form monolayers and superior performance in highly efficient energy conversion and actuation qualifies it as an intriguing material worthy of broad development.

Most bacteria contain both eukaryotic-like Ser/Thr kinases (eSTKs) and eukaryotic-like Ser/Thr phosphatases (eSTPs). Their role in bacterial physiology is not currently well understood in large part because the conditions where the eSTKs are active are generally not known. However, all sequenced Gram-positive bacteria have a highly conserved eSTK with extracellular PASTA repeats that bind cell wall derived muropeptides. Here, we report that in the Gram-positive bacterium Bacillus subtilis, the PASTA-containing eSTK PrkC and its cognate eSTP PrpC converge with the essential WalRK two-component system to regulate WalR regulon genes involved in cell wall metabolism. By continuously monitoring gene expression throughout growth, we consistently find a large PrkC-dependent effect on expression of several different WalR regulon genes in early stationary phase, including both those that are activated by WalR (yocH) as well as those that are repressed (iseA, pdaC). We demonstrate that PrkC phosphorylates WalR in vitro and in vivo on a single Thr residue located in the receiver domain. Although the phosphorylated region of the receiver domain is highly conserved among several B. subtilis response regulators, PrkC displays specificity for WalR in vitro. Consistently, strains expressing a nonphosphorylatable WalR point mutant strongly reduce both PrkC dependent activation and repression of yocH, iseA, and pdaC. This suggests a model where the eSTK PrkC regulates the essential WalRK two-component signaling system by direct phosphorylation of WalR Thr101, resulting in the regulation of WalR regulon genes involved in cell wall metabolism in stationary phase. As both the eSTK PrkC and the essential WalRK two-component system are highly conserved in Gram-positive bacteria, these results may be applicable to further understanding the role of eSTKs in Gram-positive physiology and cell wall metabolism.