Structural basis of peptidoglycan synthesis by E. coli RodA-PBP2 complex.

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.

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.

Understanding the Stringent Response: Experimental Context Matters.

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.

Serine-threonine phosphoregulation by PknB and Stp contributes to quiescence and antibiotic tolerance in Staphylococcus aureus.

Staphylococcus aureus can cause infections that are often chronic and difficult to treat, even when the bacteria are not antibiotic resistant because most antibiotics act only on metabolically active cells. Subpopulations of persister cells are metabolically quiescent, a state associated with delayed growth, reduced protein synthesis, and increased tolerance to antibiotics. Serine-threonine kinases and phosphatases similar to those found in eukaryotes can fine-tune essential bacterial cellular processes, such as metabolism and stress signaling. We found that acid stress-mimicking conditions that S. aureus experiences in host tissues delayed growth, globally altered the serine and threonine phosphoproteome, and increased threonine phosphorylation of the activation loop of the serine-threonine protein kinase B (PknB). The deletion of stp, which encodes the only annotated functional serine-threonine phosphatase in S. aureus, increased the growth delay and phenotypic heterogeneity under different stress challenges, including growth in acidic conditions, the intracellular milieu of human cells, and abscesses in mice. This growth delay was associated with reduced protein translation and intracellular ATP concentrations and increased antibiotic tolerance. Using phosphopeptide enrichment and mass spectrometry-based proteomics, we identified targets of serine-threonine phosphorylation that may regulate bacterial growth and metabolism. Together, our findings highlight the importance of phosphoregulation in mediating bacterial quiescence and antibiotic tolerance and suggest that targeting PknB or Stp might offer a future therapeutic strategy to prevent persister formation during S. aureus infections.

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.

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.

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.

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.

Bioluminescence dynamics in single germinating bacterial spores reveal metabolic heterogeneity.

Spore-forming bacteria modulate their metabolic rate by over five orders of magnitude as they transition between dormant spores and vegetative cells and thus represent an extreme case of phenotypic variation. During environmental changes in nutrient availability, clonal populations of spore-forming bacteria exhibit individual differences in cell fate, the timing of phenotypic transitions and gene expression. One potential source of this variability is metabolic heterogeneity, but this has not yet been measured, as existing single-cell methods are not easily applicable to spores due to their small size and strong autofluorescence. Here, we use the bacterial bioluminescence system and a highly sensitive microscope to measure metabolic dynamics in thousands of B. subtilis spores as they germinate. We observe and quantitate large variations in the bioluminescence dynamics across individual spores that can be decomposed into contributions from variability in germination timing, the amount of endogenously produced luminescence substrate and the intracellular reducing power. This work shows that quantitative measurement of spore metabolism is possible and thus it opens avenues for future study of the thermodynamic nature of dormant states.

Escherichia coli YegI is a novel Ser/Thr kinase lacking conserved motifs that localizes to the inner membrane.

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 in the Escherichia coli genome, 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 motifs including residues 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.

Epidemiology of infective endocarditis in transcatheter aortic valve replacement: systemic review and meta-analysis.

INTRODUCTION: Infective endocarditis following transcatheter aortic valve replacement (TAVR) is an emerging problem, with a high rate of morbidity and mortality. However, little is known about the burden of disease, and data on infective endocarditis incidence are scarce. This study aimed to evaluate the incidence of infective endocarditis in TAVR by performing a systematic review and meta-analysis of the literature. METHODS: We comprehensively searched the databases of MEDLINE and EMBASE from inception to October 2019. Included studies were prospective or retrospective cohort studies that reported the event rate of infective endocarditis in patients who underwent TAVR. Data from each study were combined using the random-effects method to calculate pooled incidence with 95% confidence intervals (CIs). RESULTS: A total of 30 studies consisting of 73 780 patients undergoing TAVR were included in this meta-analysis. Overall, the pooled estimated incidence of infective endocarditis following TAVR was 7 in 1000 patients (95% CI: 0.5-1%). For early infective endocarditis, the pooled estimated incidence was 8 per 1000 patients (95% CI: 0.5-1.1%). For late infective endocarditis, the pooled estimated incidence was 2 in 1000 patients (95% CI: 0.1-0.4%). Significantly, the overall pooled infective endocarditis mortality rate was 39% (95% CI: 28.7-49.4%). CONCLUSION: The current study demonstrates the incidence of overall, early, and late infective endocarditis following TAVR, ranging from 2 to 8 per 1000 patients. Although it remains a rare event, infective endocarditis following TAVR is associated with high mortality.

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.

Anaerobiosis: A Surfactant Helps Bacteria Breathe a Sigh of Relief.

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.

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.

Clickable methionine as a universal probe for labelling intracellular bacteria.

Despite their clinical and biological importance, the cell biology of obligate intracellular bacteria is less well understood than that of many free-living model organisms. One reason for this is that they are mostly genetically intractable. As a consequence, it is not possible to engineer strains expressing fluorescent proteins and therefore fluorescence light microscopy - a key tool in host-pathogen cell biology studies - is difficult. Strain diversity also limits the universality of antibody-based immunofluorescence approaches. Here, we have developed a universal labelling protocol for intracellular bacteria based on a clickable methionine analog. Whilst we have applied this to obligate intracellular bacteria, we expect it to be useful for labelling free living bacteria as well as other intracellular pathogens.

Multisite phosphorylation drives phenotypic variation in (p)ppGpp synthetase-dependent antibiotic tolerance.

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.

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.

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.

Tricuspid Valve Infective Endocarditis Due to Klebsiella pneumoniae in Intravenous Drug User.

Infective endocarditis is a high morbidity-mortality condition despite advancements in supportive care and medical therapy. One of the strongest risk factors is intravenous drug use, which has high prevalence in the Hawai'i population. Klebsiella pneumoniae is a rare but aggressive pathogen causing infective endocarditis. There is no strong evidence to guide management. We present a rare case of isolated tricuspid valve infective endocarditis due to Klebsiella pneumoniae in an intravenous drug user causing septic pulmonary emboli and multiple abscesses. The patient was managed with combined 6-week ceftriaxone and 2-week gentamicin together with early tricuspid valve repair.