Interaction and signalling networks: a report from the fourth 'Young Microbiologists Symposium on Microbe Signalling, Organisation and Pathogenesis'.

At the end of June, over 120 microbiologists from 18 countries gathered in Dundee, Scotland for the fourth edition of the Young Microbiologists Symposium on 'Microbe Signalling, Organisation and Pathogenesis'. The aim of the symposium was to give early career microbiologists the opportunity to present their work in a convivial environment and to interact with senior world-renowned scientists in exciting fields of microbiology research. The meeting was supported by the Microbiology Society, the Society of Applied Microbiology and the American Society for Microbiology with further sponsorship from the European Molecular Biology Organisation and the Royal Society of Edinburgh. In this report, we highlight some themes that emerged from the many interesting talks and poster presentations, as well as some of the other activities that were on offer at this energetic meeting.

Genome-wide DNA methylation analysis of human pancreatic islets from type 2 diabetic and non-diabetic donors identifies candidate genes that influence insulin secretion.

Impaired insulin secretion is a hallmark of type 2 diabetes (T2D). Epigenetics may affect disease susceptibility. To describe the human methylome in pancreatic islets and determine the epigenetic basis of T2D, we analyzed DNA methylation of 479,927 CpG sites and the transcriptome in pancreatic islets from T2D and non-diabetic donors. We provide a detailed map of the global DNA methylation pattern in human islets, ß- and α-cells. Genomic regions close to the transcription start site showed low degrees of methylation and regions further away from the transcription start site such as the gene body, 3'UTR and intergenic regions showed a higher degree of methylation. While CpG islands were hypomethylated, the surrounding 2 kb shores showed an intermediate degree of methylation, whereas regions further away (shelves and open sea) were hypermethylated in human islets, ß- and α-cells. We identified 1,649 CpG sites and 853 genes, including TCF7L2, FTO and KCNQ1, with differential DNA methylation in T2D islets after correction for multiple testing. The majority of the differentially methylated CpG sites had an intermediate degree of methylation and were underrepresented in CpG islands (∼ 7%) and overrepresented in the open sea (∼ 60%). 102 of the differentially methylated genes, including CDKN1A, PDE7B, SEPT9 and EXOC3L2, were differentially expressed in T2D islets. Methylation of CDKN1A and PDE7B promoters in vitro suppressed their transcriptional activity. Functional analyses demonstrated that identified candidate genes affect pancreatic ß- and α-cells as Exoc3l silencing reduced exocytosis and overexpression of Cdkn1a, Pde7b and Sept9 perturbed insulin and glucagon secretion in clonal ß- and α-cells, respectively. Together, our data can serve as a reference methylome in human islets. We provide new target genes with altered DNA methylation and expression in human T2D islets that contribute to perturbed insulin and glucagon secretion. These results highlight the importance of epigenetics in the pathogenesis of T2D.

New layers of regulation of the general stress response sigma factor RpoS.

The general stress response (GSR) sigma factor RpoS from Escherichia coli has emerged as one of the key paradigms for study of how numerous signal inputs are accepted at multiple levels into a single pathway for regulation of gene expression output. While many studies have elucidated the key pathways controlling the production and activity of this sigma factor, recent discoveries have uncovered still more regulatory mechanisms which feed into the network. Moreover, while the regulon of this sigma factor comprises a large proportion of the E. coli genome, the downstream expression levels of all the RpoS target genes are not identically affected by RpoS upregulation but respond heterogeneously, both within and between cells. This minireview highlights the most recent developments in our understanding of RpoS regulation and expression, in particular those which influence the regulatory network at different levels from previously well-studied pathways.

The uncharacterized PA3040-3042 operon is part of the cell envelope stress response and a tobramycin resistance determinant in a clinical isolate of Pseudomonas aeruginosa.

Bacteriophages (hereafter "phages") are ubiquitous predators of bacteria in the natural world, but interest is growing in their development into antibacterial therapy as complement or replacement for antibiotics. However, bacteria have evolved a huge variety of antiphage defense systems allowing them to resist phage lysis to a greater or lesser extent. In addition to dedicated phage defense systems, some aspects of the general stress response also impact phage susceptibility, but the details of this are not well known. In order to elucidate these factors in the opportunistic pathogen Pseudomonas aeruginosa, we used the laboratory-conditioned strain PAO1 as host for phage infection experiments as it is naturally poor in dedicated phage defense systems. Screening by transposon insertion sequencing indicated that the uncharacterized operon PA3040-PA3042 was potentially associated with resistance to lytic phages. However, we found that its primary role appeared to be in regulating biofilm formation, particularly in a clinical isolate of P. aeruginosa in which it also altered tobramycin resistance. Its expression was highly growth-phase dependent and responsive to phage infection and cell envelope stress. Our results suggest that this operon may be a cryptic but important locus for P. aeruginosa stress tolerance. IMPORTANCE: An important category of bacterial stress response systems is bacteriophage defense, where systems are triggered by bacteriophage infection and activate a response which may either destroy the phage genome or destroy the infected cell so that the rest of the population survives. In some bacteria, the cell envelope stress response is activated by bacteriophage infection, but it is unknown whether this contributes to the survival of the infection. We have found that a conserved uncharacterized operon (PA3040-PA3042) of the cell envelope stress regulon in Pseudomonas aeruginosa, which has very few dedicated phage defense systems, responds to phage infection and stationary phase as well as envelope stress and is important for growth and biofilm formation in a clinical isolate of P. aeruginosa, even in the absence of phages. As homologs of these genes are found in other bacteria, they may be a novel component of the general stress response.

Reflections on a sticky situation: how surface contact pulls the trigger for bacterial adhesion.

Adhesion of bacterial cells to surfaces can be mediated by a wide variety of extracellular structures, which can either recognize specific molecular motifs or adhere in non-specific ways to multiple types of surfaces. The attachment is thought to be highly regulated, but the underlying sensory mechanism(s) are poorly understood. In the α-proteobacterium Caulobacter crescentus, the formation of adhesive organelles is 'hardwired' into the cell cycle regulatory circuitry. In this issue of Molecular Microbiology, Li et al. (2011) employed this model organism to examine the adhesion process and the transition from temporary to permanent attachment using total internal reflection fluorescence (TIRF) microscopy. Surprisingly, they observed that adhesin production was not only under developmental control, but was also stimulated by surface contact. Initial reversible contact of the pili with the surface was followed by flagellum rotation arrest and subsequent induction of the holdfast to allow irreversible surface adhesion. These findings demonstrate that Caulobacter produces its holdfast only at the appropriate time for surface attachment, preventing premature export of the adhesin, which could then be inactivated by 'curing' or be masked by occluding particles. Importantly, their results support the notion that the flagellum serves as a mechanosensor for adhesion.

DNA methylation of the glucagon-like peptide 1 receptor (GLP1R) in human pancreatic islets.

BACKGROUND: Insulin secretion is enhanced upon the binding of Glucagon-like peptide-1 (GLP-1) to its receptor (GLP1R) in pancreatic ß cells. Although a reduced expression of GLP1R in pancreatic islets from type 2 diabetic patients and hyperglycaemic rats has been established, it is still unknown if this is caused by differential DNA methylation of GLP1R in pancreatic islets of type 2 diabetic patients. METHODS: In this study, DNA methylation levels of 12 CpG sites close to the transcription start site of GLP1R were analysed in pancreatic islets from 55 non-diabetic and 10 type 2 diabetic human donors as well as in ß and α cells isolated from human pancreatic islets. DNA methylation of GLP1R was related to GLP1R expression, HbA1c levels and BMI. Moreover, mRNA expression of MECP2, DNMT1, DNMT3A and DNMT3B was analysed in pancreatic islets of the non-diabetic and type 2 diabetic donors. RESULTS: One CpG unit, at position +199 and +205 bp from the transcription start site, showed a small increase in DNA methylation in islets from donors with type 2 diabetes compared to non-diabetic donors (0.53%, p=0.02). Furthermore, DNA methylation levels of one CpG site located 376 bp upstream of the transcription start site of GLP1R correlated negatively with GLP1R expression (rho=-0.34, p=0.008) but positively with BMI and HbA1c (rho=0.30, p=0.02 and rho=0.30, p=0.03, respectively). This specific CpG site is located in an area with known SP1 and SP3 transcription factor binding sites. Moreover, when we compared the DNA methylation of the GLP1R promoter in isolated human ß and α cells, we found that it was higher in α- compared with ß-cells (p=0.009). Finally, there was a trend towards decreased DNMT3A expression (p=0.056) in type 2 diabetic compared with non-diabetic islets. CONCLUSIONS: Together, our study shows that while BMI and HbA1c are positively associated with DNA methylation levels of GLP1R, its expression is negatively associated with DNA methylation of GLP1R in human pancreatic islets.

Complete Genome Sequence of the Lysogenic Pseudomonas Bacteriophage Fyn8.

A temperate bacteriophage infecting Pseudomonas aeruginosa PAO1 was isolated from river water. Nanopore sequencing revealed that it has a circular double-stranded DNA genome of 45,617 bp, containing typical phage structural proteins and lambda-like lysogeny regulators. Putative O-antigen serotype conversion and anti-cyclic oligonucleotide-based antiphage signaling system (CBASS) defense system proteins were also identified.

Activation of the unfolded protein response pathway causes ceramide accumulation in yeast and INS-1E insulinoma cells.

Sphingolipids are not only important components of membranes but also have functions in protein trafficking and intracellular signaling. The LCB1 gene encodes a subunit of the serine palmitoyltransferase, which is responsible for the first step of sphingolipid synthesis. Here, we show that activation of the unfolded protein response (UPR) can restore normal ceramide levels and viability in yeast cells with a conditional defect in LCB1. Dependence on UPR was demonstrated by showing the HAC1-dependence of the suppression. A similar induction of ceramides by UPR seems to take place in mammalian cells. In rat pancreatic INS-1E cells, UPR activation induces the transcription of the CerS6 gene, which encodes a ceramide synthase. This correlates with the specific accumulation of ceramide with a C16 fatty acyl chain upon UPR activation. Therefore, our study reveals a novel connection between UPR induction and ceramide synthesis that seems to be conserved between yeast and mammalian cells.

Hepatic nuclear factor 1alpha (HNF1alpha) dysfunction down-regulates X-box-binding protein 1 (XBP1) and sensitizes beta-cells to endoplasmic reticulum stress.

Correct endoplasmic reticulum (ER) function is critical for the health of secretory cells, such as the pancreatic ß-cell, and ER stress is often a contributory factor to ß-cell death in type 2 diabetes. We have used an insulin-secreting cell line with inducible expression of dominant negative (DN) HNF1α, a transcription factor vital for correct ß-cell development and function, to show that HNF1α is required for Xbp1 transcription and maintenance of the normal ER stress response. DN HNF1α expression sensitizes the ß-cell to ER stress by directly down-regulating Xbp1 transcription, whereas Atf6 is unaffected. Furthermore, DN HNF1α alters calcium homeostasis, resulting in elevated cytoplasmic calcium and increased store-operated calcium entry, whereas mitochondrial calcium uptake is normal. Loss of function of XBP1 is toxic to the ß-cell and decreases production of the ER chaperone BiP, even in the absence of ER stress. DN HNF1α-induced sensitivity to cyclopiazonic acid can be partially rescued with the chemical chaperone tauroursodeoxycholate. Rat insulin 2 promoter-DN HNF1α mouse islets express lower levels of BiP mRNA, synthesize less insulin, and are sensitized to ER stress relative to matched control mouse islets, suggesting that this mechanism is also operating in vivo.

Cell dispersal in biofilms: an extracellular DNA masks nature's strongest glue.

Growth in biofilms provides bacterial species with many advantages over growth in suspension, e.g. colonization of nutrient-rich areas. In the α-proteobacterium Caulobacter crescentus biofilm formation is facilitated through its asymmetric cell division, where one daughter cell becomes a motile flagellated swarmer cell able to colonize new surfaces while the other remains as a stalked cell attached to the substrate through the adhesive holdfast. The Caulobacter biofilm consists of stalked cells arranged either in a monolayer or in a multicellular 'mushroom' structure. In this issue of Molecular Microbiology, Berne et al. demonstrate that extracellular DNA (eDNA) from lysed cells prevents biofilm maturation. eDNA masks the adhesive properties of newly synthesized holdfast to enable the escape of swarmer cells from the biofilm. By contrast, holdfasts on previously attached stalked cells remain unaffected by eDNA. Surprisingly, the inhibitory effect was genus-specific, as only DNA from Caulobacter, but not from other genera, could interfere with biofilm maturation. This study reveals a new role for DNA in biofilms, as a regulatory rather than a structural component, and a novel mechanism to facilitate the escape of cells from biofilms. A compelling case is made for the existence of a new type of genus-specific 'macromolecular language'.

Mitochondrial dysfunction contributes to impaired insulin secretion in INS-1 cells with dominant-negative mutations of HNF-1alpha and in HNF-1alpha-deficient islets.

Maturity Onset Diabetes of the Young-type 3 (MODY-3) has been linked to mutations in the transcription factor hepatic nuclear factor (HNF)-1alpha, resulting in deficiency in glucose-stimulated insulin secretion. In INS-1 cells overexpressing doxycycline-inducible HNF-1alpha dominant-negative (DN-) gene mutations, and islets from Hnf-1alpha knock-out mice, insulin secretion was impaired in response to glucose (15 mm) and other nutrient secretagogues. Decreased rates of insulin secretion in response to glutamine plus leucine and to methyl pyruvate, but not potassium depolarization, indicate defects specific to mitochondrial metabolism. To identify the biochemical mechanisms responsible for impaired insulin secretion, we used (31)P NMR measured mitochondrial ATP synthesis (distinct from glycolytic ATP synthesis) together with oxygen consumption measurements to determine the efficiency of mitochondrial oxidative phosphorylation. Mitochondrial uncoupling was significantly higher in DN-HNF-1alpha cells, such that rates of ATP synthesis were decreased by approximately one-half in response to the secretagogues glucose, glutamine plus leucine, or pyruvate. In addition to closure of the ATP-sensitive K(+) channels with mitochondrial ATP synthesis, mitochondrial production of second messengers through increased anaplerotic flux has been shown to be critical for coupling metabolism to insulin secretion. (13)C-Isotopomer analysis and tandem mass spectrometry measurement of Krebs cycle intermediates revealed a negative impact of DN-HNF-1alpha and Hnf-1alpha knock-out on mitochondrial second messenger production with glucose but not amino acids. Taken together, these results indicate that, in addition to reduced glycolytic flux, uncoupling of mitochondrial oxidative phosphorylation contributes to impaired nutrient-stimulated insulin secretion with either mutations or loss of HNF-1alpha.

There is no hiding if you Seq: recent breakthroughs in Pseudomonas aeruginosa research revealed by genomic and transcriptomic next-generation sequencing.

The advent of next-generation sequencing technology has revolutionized the field of prokaryotic genetics and genomics by allowing interrogation of entire genomes, transcriptomes and global transcription factor binding profiles. As more studies employing these techniques have been performed, the state of the art regarding prokaryotic gene regulation has developed from the level of individual genes to genetic regulatory networks and systems biology. When applied to bacterial pathogens, particularly valuable insights have been gained into the regulation of virulence-associated genes, their relative importance to bacterial survival in planktonic, biofilm or host infection scenarios, antimicrobial resistance and the molecular details of host-pathogen interactions. This review outlines some of the latest developments and applications of next-generation sequencing techniques that have used primarily Pseudomonas aeruginosa as a model system. We focus particularly on insights into Pseudomonas virulence and infection that have been gained from these approaches and the future directions in which this field could develop.

Loss of Bacterial Cell Pole Stabilization in Caulobacter crescentus Sensitizes to Outer Membrane Stress and Peptidoglycan-Directed Antibiotics.

Rod-shaped bacteria frequently localize proteins to one or both cell poles in order to regulate processes such as chromosome replication or polar organelle development. However, the roles of polar factors in responses to extracellular stimuli have been generally unexplored. We employed chemical-genetic screening to probe the interaction between one such factor from Caulobacter crescentus, TipN, and extracellular stress and found that TipN is required for normal resistance of cell envelope-directed antibiotics, including vancomycin which does not normally inhibit growth of Gram-negative bacteria. Forward genetic screening for suppressors of vancomycin sensitivity in the absence of TipN revealed the TonB-dependent receptor ChvT as the mediator of vancomycin sensitivity. Loss of ChvT improved resistance to vancomycin and cefixime in the otherwise sensitive ΔtipN strain. The activity of the two-component system regulating ChvT (ChvIG) was increased in ΔtipN cells relative to the wild type under some, but not all, cell wall stress conditions that this strain was sensitized to, in particular cefixime and detergent exposure. Together, these results indicate that TipN contributes to cell envelope stress resistance in addition to its roles in intracellular development, and its loss influences signaling through the ChvIG two-component system which has been co-opted as a sensor of cell wall stress in CaulobacterIMPORTANCE Maintenance of an intact cell envelope is essential for free-living bacteria to protect themselves against their environment. In the case of rod-shaped bacteria, the poles of the cell are potential weak points in the cell envelope due to the high curvature of the layers and the need to break and reform the cell envelope at the division plane as the cells divide. We have found that TipN, a factor required for correct division and cell pole development in Caulobacter crescentus, is also needed for maintaining normal levels of resistance to cell wall-targeting antibiotics such as vancomycin and cefixime, which interfere with peptidoglycan synthesis. Since TipN is normally located at the poles of the cell and at the division plane just before cells complete division, our results suggest that it is involved in stabilization of these weak points of the cell envelope as well as its other roles inside the cell.

Growth control switch by a DNA-damage-inducible toxin-antitoxin system in Caulobacter crescentus.

Bacterial toxin-antitoxin systems (TASs) are thought to respond to various stresses, often inducing growth-arrested (persistent) sub-populations of cells whose housekeeping functions are inhibited. Many such TASs induce this effect through the translation-dependent RNA cleavage (RNase) activity of their toxins, which are held in check by their cognate antitoxins in the absence of stress. However, it is not always clear whether specific mRNA targets of orthologous RNase toxins are responsible for their phenotypic effect, which has made it difficult to accurately place the multitude of TASs within cellular and adaptive regulatory networks. Here, we show that the TAS HigBA of Caulobacter crescentus can promote and inhibit bacterial growth dependent on the dosage of HigB, a toxin regulated by the DNA damage (SOS) repressor LexA in addition to its antitoxin HigA, and the target selectivity of HigB's mRNA cleavage activity. HigB reduced the expression of an efflux pump that is toxic to a polarity control mutant, cripples the growth of cells lacking LexA, and targets the cell cycle circuitry. Thus, TASs can have outcome switching activity in bacterial adaptive (stress) and systemic (cell cycle) networks.

Synthetic interaction between the TipN polarity factor and an AcrAB-family efflux pump implicates cell polarity in bacterial drug resistance.

Quinolone antibiotics are clinically important drugs that target bacterial DNA replication and chromosome segregation. Although the AcrAB-family efflux pumps generally protect bacteria from such drugs, the physiological role of these efflux systems and their interplay with other cellular events are poorly explored. Here, we report an intricate relationship between antibiotic resistance and cell polarity in the model bacterium Caulobacter crescentus. We show that a polarity landmark protein, TipN, identified by virtue of its ability to direct flagellum placement to the new cell pole, protects cells from toxic misregulation of an AcrAB efflux pump through a cis-encoded nalidixic acid-responsive transcriptional repressor. Alongside the importance of polarity in promoting the inheritance and activity of virulence functions including motility, we can now ascribe to it an additional role in drug resistance that is distinct from classical efflux mechanisms.

Cell polarity: ParA-logs gather around the Hub.

The chromosomal origin, chemotaxis arrays and flagellum of Vibrio cholerae congregate at the same pole of the cell. How? A recent study identifies a new pole-organizing protein, HubP, that recruits members of the ParA family of spatial regulators of subcellular structures to the pole.

Decoding Caulobacter development.

Caulobacter crescentus uses a multi-layered system of oscillating regulators to program different developmental fates into each daughter cell at division. This is achieved by superimposing gene expression, subcellular localization, phosphorylation, and regulated proteolysis to form a complex regulatory network that integrates chromosome replication, segregation, polar differentiation, and cytokinesis. In this review, we outline the current state of research in the field of Caulobacter development, emphasizing new findings that elaborate how the developmental program is modulated by factors such as the environment or the metabolic state of the cell.

Increased DNA methylation and decreased expression of PDX-1 in pancreatic islets from patients with type 2 diabetes.

Mutations in pancreatic duodenal homeobox 1 (PDX-1) can cause a monogenic form of diabetes (maturity onset diabetes of the young 4) in humans, and silencing Pdx-1 in pancreatic ß-cells of mice causes diabetes. However, it is not established whether epigenetic alterations of PDX-1 influence type 2 diabetes (T2D) in humans. Here we analyzed mRNA expression and DNA methylation of PDX-1 in human pancreatic islets from 55 nondiabetic donors and nine patients with T2D. We further studied epigenetic regulation of PDX-1 in clonal ß-cells. PDX-1 expression was decreased in pancreatic islets from patients with T2D compared with nondiabetic donors (P = 0.0002) and correlated positively with insulin expression (rho = 0.59, P = 0.000001) and glucose-stimulated insulin secretion (rho = 0.41, P = 0.005) in the human islets. Ten CpG sites in the distal PDX-1 promoter and enhancer regions exhibited significantly increased DNA methylation in islets from patients with T2D compared with nondiabetic donors. DNA methylation of PDX-1 correlated negatively with its gene expression in the human islets (rho = -0.64, P = 0.0000029). Moreover, methylation of the human PDX-1 promoter and enhancer regions suppressed reporter gene expression in clonal ß-cells (P = 0.04). Our data further indicate that hyperglycemia decreases gene expression and increases DNA methylation of PDX-1 because glycosylated hemoglobin (HbA1c) correlates negatively with mRNA expression (rho = -0.50, P = 0.0004) and positively with DNA methylation (rho = 0.54, P = 0.00024) of PDX-1 in the human islets. Furthermore, while Pdx-1 expression decreased, Pdx-1 methylation and Dnmt1 expression increased in clonal ß-cells exposed to high glucose. Overall, epigenetic modifications of PDX-1 may play a role in the development of T2D, given that pancreatic islets from patients with T2D and ß-cells exposed to hyperglycemia exhibited increased DNA methylation and decreased expression of PDX-1. The expression levels of PDX-1 were further associated with insulin secretion in the human islets.

New(s) to the (Z-)ring.

Cytokinesis in bacteria is mediated by a macromolecular machine known as the divisome, consisting of an assembly of FtsZ polymers around the cylindrical axis of the cell and the downstream regulators of division that are subsequently recruited to it. FtsZ polymerizes into filaments in a GTP-dependent manner, similarly to its eukaryotic structural homolog tubulin. The initial placement of the FtsZ polymerization site is tightly regulated by multiple mechanisms, as are the subsequent polymer reshaping and force generation that separate the two daughter cells from each other. New factors have been recently discovered that contribute to this regulation, notably affecting FtsZ polymer shaping, and modulating FtsZ polymerization in response to the metabolic or redox state of the cell.