Showing papers in "Molecular Microbiology in 2014"

Biofilm formation by Bacillus subtilis: new insights into regulatory strategies and assembly mechanisms

Abstract: Biofilm formation is a social behaviour that generates favourable conditions for sustained survival in the natural environment. For the Gram-positive bacterium Bacillus subtilis the process involves the differentiation of cell fate within an isogenic population and the production of communal goods that form the biofilm matrix. Here we review recent progress in understanding the regulatory pathways that control biofilm formation and highlight developments in understanding the composition, function and structure of the biofilm matrix.

Phenazine redox cycling enhances anaerobic survival in Pseudomonas aeruginosa by facilitating generation of ATP and a proton-motive force

Abstract: While many studies have explored the growth of Pseudomonas aeruginosa, comparatively few have focused on its survival. Previously, we reported that endogenous phenazines support the anaerobic survival of P. aeruginosa, yet the physiological mechanism underpinning survival was unknown. Here, we demonstrate that phenazine redox cycling enables P. aeruginosa to oxidize glucose and pyruvate into acetate, which promotes survival by coupling acetate and ATP synthesis through the activity of acetate kinase. By measuring intracellular NAD(H) and ATP concentrations, we show that survival is correlated with ATP synthesis, which is tightly coupled to redox homeostasis during pyruvate fermentation but not during arginine fermentation. We also show that ATP hydrolysis is required to generate a proton-motive force using the ATP synthase complex during fermentation. Together, our results suggest that phenazines enable maintenance of the proton-motive force by promoting redox homeostasis and ATP synthesis. This work demonstrates the more general principle that extracellular redox-active molecules, such as phenazines, can broaden the metabolic versatility of microorganisms by facilitating energy generation.

Genetically distinct pathways guide effector export through the type VI secretion system

Abstract: Bacterial secretion systems often employ molecular chaperones to recognize and facilitate export of their substrates. Recent work demonstrated that a secreted component of the type VI secretion system (T6SS), hemolysin co-regulated protein (Hcp), binds directly to effectors, enhancing their stability in the bacterial cytoplasm. Herein, we describe a quantitative cellular proteomics screen for T6S substrates that exploits this chaperone-like quality of Hcp. Application of this approach to the Hcp secretion island I-encoded T6SS (H1-T6SS) of Pseudomonas aeruginosa led to the identification of a novel effector protein, termed Tse4 (type VI secretion exported 4), subsequently shown to act as a potent intra-specific H1-T6SS-delivered antibacterial toxin. Interestingly, our screen failed to identify two predicted H1-T6SS effectors, Tse5 and Tse6, which differ from Hcp-stabilized substrates by the presence of toxin-associated PAAR-repeat motifs and genetic linkage to members of the valine-glycine repeat protein G (vgrG) genes. Genetic studies further distinguished these two groups of effectors: Hcp-stabilized effectors were found to display redundancy in interbacterial competition with respect to the requirement for the two H1-T6SS-exported VgrG proteins, whereas Tse5 and Tse6 delivery strictly required a cognate VgrG. Together, we propose that interaction with either VgrG or Hcp defines distinct pathways for T6S effector export.

Different walls for rods and balls: the diversity of peptidoglycan

Abstract: Peptidoglycan performs the essential role of resisting turgor in the cell walls of most bacteria It determines cell shape, and its biosynthesis is the target for many important antibiotics The fundamental chemical building blocks of peptidoglycan are conserved: repeating disaccharides cross-linked by peptides However, these blocks come in many varieties and can be assembled in different ways So beyond the fundamental similarity, prodigious chemical, organizational and architectural diversity is revealed Here, we track the evolution of our current understanding of peptidoglycan and underpinning technical and methodological developments The origin and function of chemical diversity is discussed with respect to some well-studied example species We then explore how this chemistry is manifested in elegant and complex peptidoglycan organization and how this is interpreted in different and sometimes controversial architectural models We contend that emerging technology brings about the possibility of achieving a complete understanding of peptidoglycan chemistry, through architecture, to the way in which diverse species and populations of cells meet the challenges of maintaining viability and growth within their environmental niches, by exploiting the bioengineering versatility of peptidoglycan

Slow growth of Mycobacterium tuberculosis at acidic pH is regulated by phoPR and host‐associated carbon sources

Abstract: During pathogenesis, Mycobacterium tuberculosis (Mtb) colonizes environments, such as the macrophage or necrotic granuloma, that are acidic and rich in cholesterol and fatty acids. The goal of this study was to examine how acidic pH and available carbon sources interact to regulate Mtb physiology. Here we report that Mtb growth at acidic pH requires host-associated carbon sources that function at the intersection of glycolysis and the TCA cycle, such as pyruvate, acetate, oxaloacetate and cholesterol. In contrast, in other tested carbon sources, Mtb fully arrests its growth at acidic pH and establishes a state of non-replicating persistence. Growth-arrested Mtb is resuscitated by the addition of pyruvate suggesting that growth arrest is due to a pH-dependent checkpoint on metabolism. Additionally, we demonstrate that the phoPR two-component regulatory system is required to slow Mtb growth at acidic pH and functions to maintain redox homeostasis. Transcriptional profiling and functional metabolic studies demonstrate that signals from acidic pH and carbon source are integrated to remodel pathways associated with anaplerotic central metabolism, lipid anabolism and the regeneration of oxidized cofactors. Because phoPR is required for Mtb virulence in animals, we suggest that pH-driven adaptation may be critical to Mtb pathogenesis.

Histone H3K9 and H3K27 methylation regulates fungal alkaloid biosynthesis in a fungal endophyte–plant symbiosis

Abstract: Summary Epichloё festucae is a filamentous fungus that forms a mutually beneficial symbiotic association with Lolium perenne. This endophyte synthesizes bioprotective lolitrems (ltm) and ergot alkaloids (eas) in planta but the mechanisms regulating expression of the corresponding subtelomeric gene clusters are not known. We show here that the status of histone H3 lysine 9 and lysine 27 trimethylation (H3K9me3/H3K27me3) at these alkaloid gene loci are critical determinants of transcriptional activity. Using ChIP-qPCR we found that levels of H3K9me3 and H3K27me3 were reduced at these loci in plant infected tissue compared to axenic culture. Deletion of E. festucae genes encoding the H3K9- (ClrD) or H3K27- (EzhB) methyltransferases led to derepression of ltm and eas gene expression under non-symbiotic culture conditions and a further enhancement of expression in the double deletion mutant. These changes in gene expression were matched by corresponding reductions in H3K9me3 and H3K27me3 marks. Both methyltransferases are also important for the symbiotic interaction between E. festucae and L. perenne. Our results show that the state of H3K9 and H3K27 trimethylation of E. festucae chromatin is an important regulatory layer controlling symbiosis-specific expression of alkaloid bioprotective metabolites and the ability of this symbiont to form a mutualistic interaction with its host.

GIL, a new c-di-GMP-binding protein domain involved in regulation of cellulose synthesis in enterobacteria.

Abstract: Summary In contrast to numerous enzymes involved in c-di-GMP synthesis and degradation in enterobacteria, only a handful of c-di-GMP receptors/effectors have been identified. In search of new c-di-GMP receptors, we screened the Escherichia coli ASKA overexpression gene library using the Differential Radial Capillary Action of Ligand Assay (DRaCALA) with fluorescently and radioisotope-labelled c-di-GMP. We uncovered three new candidate c-di-GMP receptors in E. coli and characterized one of them, BcsE. The bcsE gene is encoded in cellulose synthase operons in representatives of Gammaproteobacteria and Betaproteobacteria. The purified BcsE proteins from E. coli, Salmonella enterica and Klebsiella pneumoniae bind c-di-GMP via the domain of unknown function, DUF2819, which is hereby designated GIL, GGDEF I-site like domain. The RxGD motif of the GIL domain is required for c-di-GMP binding, similar to the c-di-GMP-binding I-site of the diguanylate cyclase GGDEF domain. Thus, GIL is the second protein domain, after PilZ, dedicated to c-di-GMP-binding. We show that in S. enterica, BcsE is not essential for cellulose synthesis but is required for maximal cellulose production, and that c-di-GMP binding is critical for BcsE function. It appears that cellulose production in enterobacteria is controlled by a two-tiered c-di-GMP-dependent system involving BcsE and the PilZ domain containing glycosyltransferase BcsA.

The ins and outs of bacterial iron metabolism.

Abstract: Iron is a critical nutrient for the growth and survival of most bacterial species. Accordingly, much attention has been paid to the mechanisms by which host organisms sequester iron from invading bacteria and how bacteria acquire iron from their environment. However, under oxidative stress conditions such as those encountered within phagocytic cells during the host immune response, iron is released from proteins and can act as a catalyst for Fenton chemistry to produce cytotoxic reactive oxygen species. The transitory efflux of free intracellular iron may be beneficial to bacteria under such conditions. The recent discovery of putative iron efflux transporters in Salmonella enterica serovar Typhimurium is discussed in the context of cellular iron homeostasis.

Extracellular vesicles produced by the Gram‐positive bacterium Bacillus subtilis are disrupted by the lipopeptide surfactin

Abstract: Previously, extracellular vesicle production in Gram-positive bacteria was dismissed due to the absence of an outer membrane, where Gram-negative vesicles originate, and the difficulty in envisioning how such a process could occur through the cell wall. However, recent work has shown that Gram-positive bacteria produce extracellular vesicles and that the vesicles are biologically active. In this study, we show that Bacillus subtilis produces extracellular vesicles similar in size and morphology to other bacteria, characterized vesicles using a variety of techniques, provide evidence that these vesicles are actively produced by cells, show differences in vesicle production between strains, and identified a mechanism for such differences based on vesicle disruption. We found that in wild strains of B. subtilis, surfactin disrupted vesicles while in laboratory strains harbouring a mutation in the gene sfp, vesicles accumulated in the culture supernatant. Surfactin not only lysed B. subtilis vesicles, but also vesicles from Bacillus anthracis, indicating a mechanism that crossed species boundaries. To our knowledge, this is the first time a gene and a mechanism has been identified in the active disruption of extracellular vesicles and subsequent release of vesicular cargo in Gram-positive bacteria. We also identify a new mechanism of action for surfactin.

The Yersinia pestis siderophore, yersiniabactin, and the ZnuABC system both contribute to zinc acquisition and the development of lethal septicaemic plague in mice

Abstract: Bacterial pathogens must overcome host sequestration of zinc (Zn(2+) ), an essential micronutrient, during the infectious disease process. While the mechanisms to acquire chelated Zn(2+) by bacteria are largely undefined, many pathogens rely upon the ZnuABC family of ABC transporters. Here we show that in Yersinia pestis, irp2, a gene encoding the synthetase (HMWP2) for the siderophore yersiniabactin (Ybt) is required for growth under Zn(2+) -deficient conditions in a strain lacking ZnuABC. Moreover, growth stimulation with exogenous, purified apo-Ybt provides evidence that Ybt may serve as a zincophore for Zn(2+) acquisition. Studies with the Zn(2+) -dependent transcriptional reporter znuA::lacZ indicate that the ability to synthesize Ybt affects the levels of intracellular Zn(2+) . However, the outer membrane receptor Psn and TonB as well as the inner membrane (IM) ABC transporter YbtPQ, which are required for Fe(3+) acquisition by Ybt, are not needed for Ybt-dependent Zn(2+) uptake. In contrast, the predicted IM protein YbtX, a member of the Major Facilitator Superfamily, was essential for Ybt-dependent Zn(2+) uptake. Finally, we show that the ZnuABC system and the Ybt synthetase HMWP2, presumably by Ybt synthesis, both contribute to the development of a lethal infection in a septicaemic plague mouse model.

The binding force of the staphylococcal adhesin SdrG is remarkably strong

Abstract: SdrG is a cell surface adhesin from Staphylococcus epidermidis which binds to the blood plasma protein fibrinogen (Fg). Ligand binding follows a 'dock, lock and latch' model involving dynamic conformational changes of the adhesin that result in a greatly stabilized adhesin-ligand complex. To date, the force and dynamics of this multistep interaction are poorly understood. Here we use atomic force microscopy (AFM) to unravel the binding strength and cell surface localization of SdrG at molecular resolution. Single-cell force spectroscopy shows that SdrG mediates time-dependent attachment to Fg-coated surfaces. Single-molecule force spectroscopy with Fg-coated AFM tips demonstrates that the adhesin forms nanoscale domains on the cell surface, which we believe contribute to strengthen cell adhesion. Notably, we find that the rupture force of single SdrG-Fg bonds is very large, ∼2nN, equivalent to the strength of a covalent bond, and shows a low dissociation rate, suggesting that the bond is very stable. The strong binding force, slow dissociation and clustering of SdrG provide a molecular foundation for the ability of S. epidermidis to colonize implanted biomaterials and to withstand physiological shear forces. © 2014 John Wiley & Sons Ltd.

Transcriptome-wide analysis of trypanosome mRNA decay reveals complex degradation kinetics and suggests a role for co-transcriptional degradation in determining mRNA levels.

Abstract: African trypanosomes are an excellent system for quantitative modelling of post-transcriptional mRNA control Transcription is constitutive and polycistronic; individual mRNAs are excised by trans splicing and polyadenylation We here measure mRNA decay kinetics in two life cycle stages, bloodstream and procyclic forms, by transcription inhibition and RNASeq Messenger RNAs with short half-lives tend to show initial fast degradation, followed by a slower phase; they are often stabilized by depletion of the 5-3 exoribonuclease XRNA Many longer-lived mRNAs show initial slow degradation followed by rapid destruction: we suggest that the slow phase reflects gradual deadenylation Developmentally regulated mRNAs often show regulated decay, and switch their decay pattern Rates of mRNA decay are good predictors of steady state levels for short mRNAs, but mRNAs longer than 3kb show unexpectedly low abundances Modelling shows that variations in splicing and polyadenylation rates can contribute to steady-state mRNA levels, but this is completely dependent on competition between processing and co-transcriptional mRNA precursor destruction

Deletion of the cyclic di‐AMP phosphodiesterase gene (cnpB) in Mycobacterium tuberculosis leads to reduced virulence in a mouse model of infection

Abstract: Summary Tuberculosis (TB) remains a major cause of morbidity and mortality worldwide. The pathogenesis by the causative agent, Mycobacterium tuberculosis, is still not fully understood. We have previously reported that M. tuberculosis Rv3586 (disA) encodes a diadenylate cyclase, which converts ATP to cyclic di-AMP (c-di-AMP). In this study, we demonstrated that a protein encoded by Rv2837c (cnpB) possesses c-di-AMP phosphodiesterase activity and cleaves c-di-AMP exclusively to AMP. Our results showed that in M. tuberculosis, deletion of disA abolished bacterial c-di-AMP production, whereas deletion of cnpB significantly enhanced the bacterial c-di-AMP accumulation and secretion. The c-di-AMP levels in both mutants could be corrected by expressing the respective gene. We also found that macrophages infected with ΔcnpB secreted much higher levels of IFN-β than those infected with the wild type (WT) or the complemented mutant. Interestingly, mice infected with M. tuberculosis ΔcnpB displayed significantly reduced inflammation, less bacterial burden in the lungs and spleens, and extended survival compared with those infected with the WT or the complemented mutant. These results indicate that deletion of cnpB results in attenuated virulence, which is correlated with elevated c-di-AMP levels.

Differential affinities of MinD and MinE to anionic phospholipid influence Min patterning dynamics in vitro

Abstract: Summary The E. coli Min system forms a cell-pole-to-cell-pole oscillator that positions the divisome at mid-cell. The MinD ATPase binds the membrane and recruits the cell division inhibitor MinC. MinE interacts with and releases MinD (and MinC) from the membrane. The chase of MinD by MinE creates the in vivo oscillator that maintains a low level of the division inhibitor at mid-cell. In vitro reconstitution and visualization of Min proteins on a supported lipid bilayer has provided significant advances in understanding Min patterns in vivo. Here we studied the effects of flow, lipid composition, and salt concentration on Min patterning. Flow and no-flow conditions both supported Min protein patterns with somewhat different characteristics. Without flow, MinD and MinE formed spiraling waves. MinD and, to a greater extent MinE, have stronger affinities for anionic phospholipid. MinD-independent binding of MinE to anionic lipid resulted in slower and narrower waves. MinE binding to the bilayer was also more susceptible to changes in ionic strength than MinD. We find that modulating protein diffusion with flow, or membrane binding affinities with changes in lipid composition or salt concentration, can differentially affect the retention time of MinD and MinE, leading to spatiotemporal changes in Min patterning.

Crystal structure of an HD-GYP domain cyclic-di-GMP phosphodiesterase reveals an enzyme with a novel trinuclear catalytic iron centre.

Abstract: Bis-(3',5') cyclic di-guanylate (c-di-GMP) is a key bacterial second messenger that is implicated in the regulation of many crucial processes that include biofilm formation, motility and virulence. Cellular levels of c-di-GMP are controlled through synthesis by GGDEF domain diguanylate cyclases and degradation by two classes of phosphodiesterase with EAL or HD-GYP domains. Here, we have determined the structure of an enzymatically active HD-GYP domain protein from Persephonella marina (PmGH) alone, in complex with substrate (c-di-GMP) and final reaction product (GMP). The structures reveal a novel trinuclear iron binding site, which is implicated in catalysis and identify residues involved in recognition of c-di-GMP. This structure completes the picture of all domains involved in c-di-GMP metabolism and reveals that the HD-GYP family splits into two distinct subgroups containing bi- and trinuclear metal centres.

The γ-aminobutyric acid shunt contributes to closing the tricarboxylic acid cycle in Synechocystis sp. PCC 6803.

Abstract: A traditional 2-oxoglutarate dehydrogenase complex is missing in the cyanobacterial tricarboxylic acid cycle. To determine pathways that convert 2-oxoglutarate into succinate in the cyanobacterium Synechocystis sp. PCC 6803, a series of mutant strains, Δsll1981, Δslr0370, Δslr1022 and combinations thereof, deficient in 2-oxoglutarate decarboxylase (Sll1981), succinate semialdehyde dehydrogenase (Slr0370), and/or in γ-aminobutyrate metabolism (Slr1022) were constructed. Like in Pseudomonas aeruginosa, N-acetylornithine aminotransferase, encoded by slr1022, was shown to also function as γ-aminobutyrate aminotransferase, catalysing γ-aminobutyrate conversion to succinic semialdehyde. As succinic semialdehyde dehydrogenase converts succinic semialdehyde to succinate, an intact γ-aminobutyrate shunt is present in Synechocystis. The Δsll1981 strain, lacking 2-oxoglutarate decarboxylase, exhibited a succinate level that was 60% of that in wild type. However, the succinate level in the Δslr1022 and Δslr0370 strains and the Δsll1981/Δslr1022 and Δsll1981/Δslr0370 double mutants was reduced to 20-40% of that in wild type, suggesting that the γ-aminobutyrate shunt has a larger impact on metabolite flux to succinate than the pathway via 2-oxoglutarate decarboxylase. (13) C-stable isotope analysis indicated that the γ-aminobutyrate shunt catalysed conversion of glutamate to succinate. Independent of the 2-oxoglutarate decarboxylase bypass, the γ-aminobutyrate shunt is a major contributor to flux from 2-oxoglutarate and glutamate to succinate in Synechocystis sp. PCC 6803.

AdcA and AdcAII employ distinct zinc acquisition mechanisms and contribute additively to zinc homeostasis in Streptococcus pneumoniae.

Abstract: Streptococcus pneumoniae is a globally significant human pathogen responsible for nearly 1 million deaths annually. Central to the ability of S. pneumoniae to colonize and mediate disease in humans is the acquisition of zinc from the host environment. Zinc uptake in S. pneumoniae occurs via the ATP-binding cassette transporter AdcCB, and, unusually, two zinc-binding proteins, AdcA and AdcAII. Studies have suggested that these two proteins are functionally redundant, although AdcA has remained uncharacterized by biochemical methods. Here we show that AdcA is a zinc-specific substrate-binding protein (SBP). By contrast with other zinc-binding SBPs, AdcA has two zinc-binding domains: a canonical amino-terminal cluster A-I zinc-binding domain and a carboxy-terminal zinc-binding domain, which has homology to the zinc-chaperone ZinT from Gram-negative organisms. Intriguingly, this latter feature is absent from AdcAII and suggests that the two zinc-binding SBPs of S. pneumoniae employ different modalities in zinc recruitment. We further show that AdcAII is reliant upon the polyhistidine triad proteins for zinc in vitro and in vivo. Collectively, our studies suggest that, despite the overlapping roles of the two SBPs in zinc acquisition, they may have unique mechanisms in zinc homeostasis and act in a complementary manner during host colonization.

Structure of the bacterial type II NADH dehydrogenase: a monotopic membrane protein with an essential role in energy generation

Abstract: Summary Non-proton pumping type II NADH dehydrogenase (NDH-2) plays a central role in the respiratory metabolism of bacteria, and in the mitochondria of fungi, plants and protists. The lack of NDH-2 in mammalian mitochondria and its essentiality in important bacterial pathogens suggests these enzymes may represent a potential new drug target to combat microbial pathogens. Here, we report the first crystal structure of a bacterial NDH-2 enzyme at 2.5 A resolution from Caldalkalibacillus thermarum. The NDH-2 structure reveals a homodimeric organization that has a unique dimer interface. NDH-2 is localized to the cytoplasmic membrane by two separated C-terminal membrane-anchoring regions that are essential for membrane localization and FAD binding, but not NDH-2 dimerization. Comparison of bacterial NDH-2 with the yeast NADH dehydrogenase (Ndi1) structure revealed non-overlapping binding sites for quinone and NADH in the bacterial enzyme. The bacterial NDH-2 structure establishes a framework for the structure-based design of small-molecule inhibitors.

Adapting to new threats: the generation of memory by CRISPR-Cas immune systems

Abstract: Clustered, regularly interspaced, short palindromic repeats (CRISPR) loci and their associated genes (cas) confer bacteria and archaea with adaptive immunity against phages and other invading genetic elements. A fundamental requirement of any immune system is the ability to build a memory of past infections in order to deal more efficiently with recurrent infections. The adaptive feature of CRISPR-Cas immune systems relies on their ability to memorize DNA sequences of invading molecules and integrate them in between the repetitive sequences of the CRISPR array in the form of 'spacers'. The transcription of a spacer generates a small antisense RNA that is used by RNA-guided Cas nucleases to cleave the invading nucleic acid in order to protect the cell from infection. The acquisition of new spacers allows the CRISPR-Cas immune system to rapidly adapt against new threats and is therefore termed 'adaptation'. Recent studies have begun to elucidate the genetic requirements for adaptation and have demonstrated that rather than being a stochastic process, the selection of new spacers is influenced by several factors. We review here our current knowledge of the CRISPR adaptation mechanism.

Time‐dependent effects of transcription‐ and translation‐halting drugs on the spatial distributions of the Escherichia coli chromosome and ribosomes

Abstract: Summary Previously observed effects of rifampicin and chloramphenicol indicate that transcription and translation activity strongly affect the coarse spatial organization of the bacterial cytoplasm. Single-cell, time-resolved, quantitative imaging of chromosome and ribosome spatial distributions and ribosome diffusion in live Escherichia coli provides insight into the underlying mechanisms. Monte Carlo simulations of model DNA-ribosome mixtures support a novel nucleoid-ribosome mixing hypothesis. In normal conditions, 70S-polysomes and the chromosomal DNA segregate, while 30S and 50S ribosomal subunits are able to penetrate the nucleoids. Growth conditions and drug treatments determine the partitioning of ribosomes into 70S-polysomes versus free 30S and 50S subunits. Entropic and excluded volume effects then dictate the resulting chromosome and ribosome spatial distributions. Direct observation of radial contraction of the nucleoids 0–5 min after treatment with either transcription- or translation-halting drugs supports the hypothesis that simultaneous transcription, translation, and insertion of proteins into the membrane (‘transertion’) exerts an expanding force on the chromosomal DNA. Breaking of the DNA-RNA polymerase-mRNA-ribosome-membrane chain in either of two ways causes similar nucleoid contraction on a similar timescale. We suggest that chromosomal expansion due to transertion enables co-transcriptional translation throughout the nucleoids.

Pbp2x localizes separately from Pbp2b and other peptidoglycan synthesis proteins during later stages of cell division of Streptococcus pneumoniae D39

Abstract: The relative localization patterns of class B penicillin-binding proteins Pbp2x and Pbp2b were used as positional indicators of septal and peripheral (side-wall-like) peptidoglycan (PG) synthesis, respectively, in the mid-cell regions of Streptococcus pneumoniae cells at different stages of division. We confirm that Pbp2x and Pbp2b are essential in the strain D39 genetic background, which differs from that of laboratory strains. We show that Pbp2b, like Pbp2x and class A Pbp1a, follows a different localization pattern than FtsZ and remains at division septa after FtsZ reappears at the equators of daughter cells. Pulse-experiments with fluorescent D-amino acids (FDAAs) were performed in wild-type cells and in cells in which Pbp2x activity was preferentially inhibited by methicillin or Pbp2x amount was depleted. These experiments show that Pbp2x activity separates from that of other PBPs to the centres of constricting septa in mid-to-late divisional cells resolved by high-resolution 3D-SIM microscopy. Dual-protein and protein-fluorescent vancomycin 2D and 3D-SIM immunofluorescence microscopy (IFM) of cells at different division stages corroborate that Pbp2x separates to the centres of septa surrounded by an adjacent constricting ring containing Pbp2b, Pbp1a and regulators, StkP and MreC. The separate localization of Pbp2x suggests distinctive roles in completing septal PG synthesis and remodelling.

The three major types of CRISPR-Cas systems function independently in CRISPR RNA biogenesis in Streptococcus thermophilus

Abstract: CRISPR-Cas systems are small RNA-based immune systems that protect prokaryotes from invaders such as viruses and plasmids. We have investigated the features and biogenesis of the CRISPR (cr)RNAs in Streptococcus thermophilus (Sth) strain DGCC7710, which possesses four different CRISPR-Cas systems including representatives from the three major types of CRISPR-Cas systems. Our results indicate that the crRNAs from each CRISPR locus are specifically processed into divergent crRNA species by Cas proteins (and non-coding RNAs) associated with the respective locus. We find that the Csm Type III-A and Cse Type I-E crRNAs are specifically processed by Cas6 and Cse3 (Cas6e), respectively, and retain an 8-nucleotide CRISPR repeat sequence tag 5' of the invader-targeting sequence. The Cse Type I-E crRNAs also retain a 21-nucleotide 3' repeat tag. The crRNAs from the two Csn Type II-A systems in Sth consist of a 5'-truncated targeting sequence and a 3' tag; however, these are distinct in size between the two. Moreover, the Csn1 (Cas9) protein associated with one Csn locus functions specifically in the production of crRNAs from that locus. Our findings indicate that multiple CRISPR-Cas systems can function independently in crRNA biogenesis within a given organism - an important consideration in engineering coexisting CRISPR-Cas pathways.

Sensitivity to the two‐peptide bacteriocin lactococcin G is dependent on UppP, an enzyme involved in cell‐wall synthesis

Abstract: Most bacterially produced antimicrobial peptides (bacteriocins) are thought to kill target cells by a receptor-mediated mechanism. However, for most bacteriocins the receptor is unknown. For instance, no target receptor has been identified for the two-peptide bacteriocins (class IIb), whose activity requires the combined action of two individual peptides. To identify the receptor for the class IIb bacteriocin lactococcin G, which targets strains of Lactococcus lactis, we generated 12 lactococcin G-resistant mutants and performed whole-genome sequencing to identify mutations causing the resistant phenotype. Remarkably, all had a mutation in or near the gene uppP (bacA), encoding an undecaprenyl pyrophosphate phosphatase; a membrane protein involved in peptidoglycan synthesis. Nine mutants had stop codons or frameshifts in the uppP gene, two had point mutations in putative regulatory regions and one caused an amino acid substitution in UppP. To verify the receptor function of UppP, it was shown that growth of non-sensitive Streptococcus pneumoniae could be inhibited by lactococcin G when L. lactis uppP was expressed in this bacterium. Furthermore, we show that the related class IIb bacteriocin enterocin 1071 also uses UppP as receptor. The approach used here should be broadly applicable to identify receptors for other bacteriocins as well.

A comparative systems analysis of polysaccharide‐elicited responses in Neurospora crassa reveals carbon source‐specific cellular adaptations

Abstract: Filamentous fungi are powerful producers of hydrolytic enzymes for the deconstruction of plant cell wall polysaccharides. However, the central question of how these sugars are perceived in the context of the complex cell wall matrix remains largely elusive. To address this question in a systematic fashion we performed an extensive comparative systems analysis of how the model filamentous fungus Neurospora crassa responds to the three main cell wall polysaccharides: pectin, hemicellulose and cellulose. We found the pectic response to be largely independent of the cellulolytic one with some overlap to hemicellulose, and in its extent surprisingly high, suggesting advantages for the fungus beyond being a mere carbon source. Our approach furthermore allowed us to identify carbon source-specific adaptations, such as the induction of the unfolded protein response on cellulose, and a commonly induced set of 29 genes likely involved in carbon scouting. Moreover, by hierarchical clustering we generated a coexpression matrix useful for the discovery of new components involved in polysaccharide utilization. This is exemplified by the identification of lat-1, which we demonstrate to encode for the physiologically relevant arabinose transporter in Neurospora. The analyses presented here are an important step towards understanding fungal degradation processes of complex biomass.

The CsoR‐like sulfurtransferase repressor (CstR) is a persulfide sensor in Staphylococcus aureus

Abstract: How cells regulate the bioavailability of utilizable sulfur while mitigating the effects of hydrogen sulfide toxicity is poorly understood. CstR [Copper-sensing operon repressor (CsoR)-like sulfurtransferase repressor] represses the expression of the cst operon encoding a putative sulfide oxidation system in Staphylococcus aureus. Here, we show that the cst operon is strongly and transiently induced by cellular sulfide stress in an acute phase and specific response and that cst-encoded genes are necessary to mitigate the effects of sulfide toxicity. Growth defects are most pronounced when S. aureus is cultured in chemically defined media with thiosulfate (TS) as a sole sulfur source, but are also apparent when cystine is used or in rich media. Under TS growth conditions, cells fail to grow as a result of either unregulated expression of the cst operon in a ΔcstR strain or transformation with a non-inducible C31A/C60A CstR that blocks cst induction. This suggests that the cst operon contributes to cellular sulfide homeostasis. Tandem high-resolution mass spectrometry reveals derivatization of CstR by both inorganic tetrasulfide and an organic persulfide, glutathione persulfide, to yield a mixture of Cys31-Cys60' interprotomer cross-links, including di-, tri- and tetrasulfide bonds, which allosterically inhibit cst operator DNA binding by CstR.

Incorporation of extracellular fatty acids by a fatty acid kinase-dependent pathway in Staphylococcus aureus.

Abstract: Acyl-CoA and acyl-acyl carrier protein (ACP) synthetases activate exogenous fatty acids for incorporation into phospholipids in Gram-negative bacteria. However, Gram-positive bacteria utilize an acyltransferase pathway for the biogenesis of phosphatidic acid that begins with the acylation of sn-glycerol-3-phosphate by PlsY using an acyl-phosphate (acyl-PO4 ) intermediate. PlsX generates acyl-PO4 from the acyl-ACP end-products of fatty acid synthesis. The plsX gene of Staphylococcus aureus was inactivated and the resulting strain was both a fatty acid auxotroph and required de novo fatty acid synthesis for growth. Exogenous fatty acids were only incorporated into the 1-position and endogenous acyl groups were channeled into the 2-position of the phospholipids in strain PDJ39 (ΔplsX). Extracellular fatty acids were not elongated. Removal of the exogenous fatty acid supplement led to the rapid accumulation of intracellular acyl-ACP and the abrupt cessation of fatty acid synthesis. Extracts from the ΔplsX strain exhibited an ATP-dependent fatty acid kinase activity, and the acyl-PO4 was converted to acyl-ACP when purified PlsX is added. These data reveal the existence of a novel fatty acid kinase pathway for the incorporation of exogenous fatty acids into S. aureus phospholipids.

The bacterial septal ring protein RlpA is a lytic transglycosylase that contributes to rod shape and daughter cell separation in Pseudomonas aeruginosa

Abstract: Rare lipoprotein A (RlpA) is a widely conserved outer membrane protein of unknown function that has previously only been studied in Escherichia coli, where it localizes to the septal ring and scattered foci along the lateral wall, but mutants have no phenotypic change. Here we show rlpA mutants of Pseudomonas aeruginosa form chains of short, fat cells when grown in low osmotic strength media. These morphological defects indicate RlpA is needed for efficient separation of daughter cells and maintenance of rod shape. Analysis of peptidoglycan sacculi from an rlpA deletion mutant revealed increased tetra and hexasaccharides that lack stem peptides (hereafter called 'naked glycans'). Incubation of these sacculi with purified RlpA resulted in release of naked glycans containing 1,6-anhydro N-acetylmuramic acid ends. RlpA did not degrade sacculi from wild-type cells unless the sacculi were subjected to a limited digestion with an amidase to remove some of the stem peptides. Thus, RlpA is a lytic transglycosylase with a strong preference for naked glycan strands. We propose that RlpA activity is regulated in vivo by substrate availability, and that amidases and RlpA work in tandem to degrade peptidoglycan in the division septum and lateral wall.

Bacillithiol is a major buffer of the labile zinc pool in Bacillus subtilis.

Abstract: Summary Intracellular zinc levels are tightly regulated since zinc is an essential cofactor for numerous enzymes, yet can be toxic when present in excess. The majority of intracellular zinc is tightly associated with proteins and is incorporated during synthesis from a poorly defined pool of kinetically labile zinc. In Bacillus subtilis, this labile pool is sensed by equilibration with the metalloregulator Zur, as an indication of zinc sufficiency, and by CzrA, as an indication of zinc excess. Here, we demonstrate that the low-molecular-weight thiol bacillithiol (BSH) serves as a major buffer of the labile zinc pool. Upon shift to conditions of zinc excess, cells transiently accumulate zinc in a low-molecular-weight pool, and this accumulation is largely dependent on BSH. Cells lacking BSH are more sensitive to zinc stress, and they induce zinc efflux at lower external zinc concentrations. Thiol reactive agents such as diamide and cadmium induce zinc efflux by interfering with the Zn-buffering function of BSH. Our data provide new insights into intracellular zinc buffering and may have broad relevance given the presence of BSH in pathogens and the proposed role of zinc sequestration in innate immunity.

Iron and copper as virulence modulators in human fungal pathogens.

Abstract: Summary Fungal pathogens have evolved sophisticated machinery to precisely balance the fine line between acquiring essential metals and defending against metal toxicity. Iron and copper are essential metals for many processes in both fungal pathogens and their mammalian hosts, but reduce viability when present in excess. However, during infection, the host uses these two metals differently. Fe has a long-standing history of influencing virulence in pathogenic fungi, mostly in regards to Fe acquisition. Numerous studies demonstrate the requirement of the Fe acquisition pathway of Candida, Cryptococcus and Aspergillus for successful systemic infection. Fe is not free in the host, but is associated with Fe-binding proteins, leading fungi to develop mechanisms to interact with and to acquire Fe from these Fe-bound proteins. Cu is also essential for cell growth and development. Essential Cu-binding proteins include Fe transporters, superoxide dismutase (SOD) and cytochrome c oxidase. Although Cu acquisition plays critical roles in fungal survival in the host, recent work has revealed that Cu detoxification is extremely important. Here, we review fungal responses to altered metal conditions presented by the host, contrast the roles of Fe and Cu during infection, and outline the critical roles of fungal metal homeostasis machinery at the host–pathogen axis.

OmpA and OmpC are critical host factors for bacteriophage Sf6 entry in Shigella.

Abstract: Despite being essential for successful infection, the molecular cues involved in host recognition and genome transfer of viruses are not completely understood. Bacterial outer membrane proteins A and C co-purify in lipid vesicles with bacteriophage Sf6, implicating both outer membrane proteins as potential host receptors. We determined that outer membrane proteins A and C mediate Sf6 infection by dramatically increasing its rate and efficiency. We performed a combination of in vivo studies with three omp null mutants of Shigella flexneri, including classic phage plaque assays and time-lapse fluorescence microscopy to monitor genome ejection at the single virion level. Cryo-electron tomography of phage 'infecting' outer membrane vesicles shows the tail needle contacting and indenting the outer membrane. Lastly, in vitro ejection studies reveal that lipopolysaccharide and outer membrane proteins are both required for Sf6 genome release. We conclude that Sf6 phage entry utilizes either outer membrane proteins A or C, with outer membrane protein A being the preferred receptor.