Hydration solids.

Hygroscopic biological matter in plants, fungi and bacteria make up a large fraction of Earth's biomass1. Although metabolically inert, these water-responsive materials exchange water with the environment and actuate movement2-5 and have inspired technological uses6,7. Despite the variety in chemical composition, hygroscopic biological materials across multiple kingdoms of life exhibit similar mechanical behaviours including changes in size and stiffness with relative humidity8-13. Here we report atomic force microscopy measurements on the hygroscopic spores14,15 of a common soil bacterium and develop a theory that captures the observed equilibrium, non-equilibrium and water-responsive mechanical behaviours, finding that these are controlled by the hydration force16-18. Our theory based on the hydration force explains an extreme slowdown of water transport and successfully predicts a strong nonlinear elasticity and a transition in mechanical properties that differs from glassy and poroelastic behaviours. These results indicate that water not only endows biological matter with fluidity but also can-through the hydration force-control macroscopic properties and give rise to a 'hydration solid' with unusual properties. A large fraction of biological matter could belong to this distinct class of solid matter.

Metabolic Reprogramming and Longevity in Quiescence.

Since Jacques Monod's foundational work in the 1940s, investigators studying bacterial physiology have largely (but not exclusively) focused on the exponential phase of bacterial cultures, which is characterized by rapid growth and high biosynthesis activity in the presence of excess nutrients. However, this is not the predominant state of bacterial life. In nature, most bacteria experience nutrient limitation most of the time. In fact, investigators even prior to Monod had identified other aspects of bacterial growth, including what is now known as the stationary phase, when nutrients become limiting. This review will discuss how bacteria transition to growth arrest in response to nutrient limitation through changes in transcription, translation, and metabolism. We will then examine how these changes facilitate survival during potentially extended periods of nutrient limitation, with particular attention to the metabolic strategies that underpin bacterial longevity in this state.

A Wolf in Sheep's Clothing: Chromosomal Pathogenicity Islands Co-opt Phage Capsids to Facilitate Horizontal Spread.

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.

Habits of Highly Effective Biofilms: Ion Signaling.

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.

Crosstalk between guanosine nucleotides regulates cellular heterogeneity in protein synthesis during nutrient limitation.

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.

Elongation Factor P Is Important for Sporulation Initiation.

The universally conserved protein elongation factor P (EF-P) facilitates translation at amino acids that form peptide bonds with low efficiency, particularly polyproline tracts. Despite its wide conservation, it is not essential in most bacteria and its physiological role remains unclear. Here, we show that EF-P affects the process of sporulation initiation in the bacterium Bacillus subtilis. We observe that the lack of EF-P delays expression of sporulation-specific genes. Using ribosome profiling, we observe that expression of spo0A, encoding a transcription factor that functions as the master regulator of sporulation, is lower in a Δefp strain than the wild type. Ectopic expression of Spo0A rescues the sporulation initiation phenotype, indicating that reduced spo0A expression explains the sporulation defect in Δefp cells. Since Spo0A is the earliest sporulation transcription factor, these data suggest that sporulation initiation can be delayed when protein synthesis is impaired. IMPORTANCE 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. Here, we show that the absence of EF-P affects the ability of B. subtilis to initiate sporulation by preventing normal expression of Spo0A, the key transcriptional regulator of this process. These data illustrate a mechanism that accounts for the sporulation delay and further suggest that cells are capable of sensing translation stress before committing to sporulation.

A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments.

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.

The alarmones (p)ppGpp directly regulate translation initiation during entry into quiescence.

Many bacteria exist in a state of metabolic quiescence where energy consumption must be minimized so as to maximize available resources over a potentially extended period of time. As protein synthesis is the most energy intensive metabolic process in a bacterial cell, it would be an appropriate target for down-regulation during the transition from growth to quiescence. We observe that when Bacillus subtilis exits rapid growth, a subpopulation of cells emerges with very low protein synthetic activity. This phenotypic heterogeneity requires the production of the nucleotides (p)ppGpp, which we show are sufficient to inhibit protein synthesis in vivo. We then show that one of these molecules, ppGpp, inhibits protein synthesis by preventing the allosteric activation of the essential GTPase Initiation Factor 2 (IF2) during translation initiation. Finally, we demonstrate that the observed attenuation of protein synthesis during the entry into quiescence is a consequence of the direct interaction of (p)ppGpp and IF2.

Transcription regulates ribosome hibernation.

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.

Ribosome Dimerization Protects the Small Subunit.

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.

LysMD3 is a type II membrane protein without an in vivo role in the response to a range of pathogens.

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.

Device infections in implantable cardioverter defibrillators versus permanent pacemakers: A systematic review and meta-analysis.

INTRODUCTION: Recent studies suggest that implantable cardioverter defibrillators (ICDs) are associated with increased risk of cardiac implantable electronic device (CIED) infections when compared with permanent pacemakers (PPMs). However, there were controversies among studies. In this study we performed a systematic review and meta-analysis to explore the risk of device infection in ICD versus PPM. METHODS: We searched the databases of MEDLINE and EMBASE from inception to January 2019. Data from each study were combined using the random-effects, generic inverse variance method of Der Simonian and Laird to calculate odds ratios (OR) and 95% confidence intervals (CI). RESULTS: Twenty-seven studies involving 202 323 CIEDs (36 782 ICDs and 165 541 PPMs) were included. Infections occurred from 9 days to 6 years postoperatively. When compared with PPM, ICD had a significantly higher risk of device infection in overall analysis (OR = 1.62, 95% CI: 1.29-2.04). The risk was seen in subgroups such as single chamber or dual chamber device (OR = 1.57, 95% CI: 1.18-2.09), de novo implantation (OR = 1.62, 95% CI: 1.29-2.69), revision implantation (OR = 1.63, 95% CI: 1.24-2.13), and cardiac resynchronization therapy (CRT) (OR = 1.75, 95% CI: 1.18-2.60). CRT-defibrillator increased risk of infection over CRT-pacemaker in revision implantation (OR = 1.81, 95% CI: 1.20-2.74) but not in de novo implantation (OR = 1.07, 95% CI: 0.23-4.88). The increased risk of infection among defibrillator was higher in CRT compared to non-CRT but not significant (P = 0.654). CONCLUSIONS: Our meta-analysis demonstrates a statistically significant increased risk of device infection in CIED patients who received ICD when compared to PPM.

The medium is the message: interspecies and interkingdom signaling by peptidoglycan and related bacterial glycans.

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.

Identification and Biochemical Characterization of a Novel Protein Phosphatase 2C-Like Ser/Thr Phosphatase in Escherichia coli.

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.

The Eukaryotic-Like Ser/Thr Kinase PrkC Regulates the Essential WalRK Two-Component System in Bacillus subtilis.

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.

Protein synthesis during cellular quiescence is inhibited by phosphorylation of a translational elongation factor.

In nature, most organisms experience conditions that are suboptimal for growth. To survive, cells must fine-tune energy-demanding metabolic processes in response to nutrient availability. Here, we describe a novel mechanism by which protein synthesis in starved cells is down-regulated by phosphorylation of the universally conserved elongation factor Tu (EF-Tu). Phosphorylation impairs the essential GTPase activity of EF-Tu, thereby preventing its release from the ribosome. As a consequence, phosphorylated EF-Tu has a dominant-negative effect in elongation, resulting in the overall inhibition of protein synthesis. Importantly, this mechanism allows a quick and robust regulation of one of the most abundant cellular proteins. Given that the threonine that serves as the primary site of phosphorylation is conserved in all translational GTPases from bacteria to humans, this mechanism may have important implications for growth-rate control in phylogenetically diverse organisms.

In Vivo Probe of Lipid†II-Interacting Proteins.

ß-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.

Pulsed feedback defers cellular differentiation.

Environmental signals induce diverse cellular differentiation programs. In certain systems, cells defer differentiation for extended time periods after the signal appears, proliferating through multiple rounds of cell division before committing to a new fate. How can cells set a deferral time much longer than the cell cycle? Here we study Bacillus subtilis cells that respond to sudden nutrient limitation with multiple rounds of growth and division before differentiating into spores. A well-characterized genetic circuit controls the concentration and phosphorylation of the master regulator Spo0A, which rises to a critical concentration to initiate sporulation. However, it remains unclear how this circuit enables cells to defer sporulation for multiple cell cycles. Using quantitative time-lapse fluorescence microscopy of Spo0A dynamics in individual cells, we observed pulses of Spo0A phosphorylation at a characteristic cell cycle phase. Pulse amplitudes grew systematically and cell-autonomously over multiple cell cycles leading up to sporulation. This pulse growth required a key positive feedback loop involving the sporulation kinases, without which the deferral of sporulation became ultrasensitive to kinase expression. Thus, deferral is controlled by a pulsed positive feedback loop in which kinase expression is activated by pulses of Spo0A phosphorylation. This pulsed positive feedback architecture provides a more robust mechanism for setting deferral times than constitutive kinase expression. Finally, using mathematical modeling, we show how pulsing and time delays together enable "polyphasic" positive feedback, in which different parts of a feedback loop are active at different times. Polyphasic feedback can enable more accurate tuning of long deferral times. Together, these results suggest that Bacillus subtilis uses a pulsed positive feedback loop to implement a "timer" that operates over timescales much longer than a cell cycle.

Partial penetrance facilitates developmental evolution in bacteria.

Development normally occurs similarly in all individuals within an isogenic population, but mutations often affect the fates of individual organisms differently. This phenomenon, known as partial penetrance, has been observed in diverse developmental systems. However, it remains unclear how the underlying genetic network specifies the set of possible alternative fates and how the relative frequencies of these fates evolve. Here we identify a stochastic cell fate determination process that operates in Bacillus subtilis sporulation mutants and show how it allows genetic control of the penetrance of multiple fates. Mutations in an intercompartmental signalling process generate a set of discrete alternative fates not observed in wild-type cells, including rare formation of two viable 'twin' spores, rather than one within a single cell. By genetically modulating chromosome replication and septation, we can systematically tune the penetrance of each mutant fate. Furthermore, signalling and replication perturbations synergize to significantly increase the penetrance of twin sporulation. These results suggest a potential pathway for developmental evolution between monosporulation and twin sporulation through states of intermediate twin penetrance. Furthermore, time-lapse microscopy of twin sporulation in wild-type Clostridium oceanicum shows a strong resemblance to twin sporulation in these B. subtilis mutants. Together the results suggest that noise can facilitate developmental evolution by enabling the initial expression of discrete morphological traits at low penetrance, and allowing their stabilization by gradual adjustment of genetic parameters.