Regulated Stochasticity in a Bacterial Signaling Network Permits Tolerance to a Rapid Environmental Change.

Microbial populations can maximize fitness in dynamic environments through bet hedging, a process wherein a subpopulation assumes a phenotype not optimally adapted to the present environment but well adapted to an environment likely to be encountered. Here, we show that oxygen induces fluctuating expression of the trimethylamine oxide (TMAO) respiratory system of Escherichia coli, diversifying the cell population and enabling a bet-hedging strategy that permits growth following oxygen loss. This regulation by oxygen affects the variance in gene expression but leaves the mean unchanged. We show that the oxygen-sensitive transcription factor IscR is the key regulator of variability. Oxygen causes IscR to repress expression of a TMAO-responsive signaling system, allowing stochastic effects to have a strong effect on the output of the system and resulting in heterogeneous expression of the TMAO reduction machinery. This work reveals a mechanism through which cells regulate molecular noise to enhance fitness.

A commensal-encoded genotoxin drives restriction of Vibrio cholerae colonization and host gut microbiome remodeling.

SignificanceIn a polymicrobial battlefield where different species compete for nutrients and colonization niches, antimicrobial compounds are the sword and shield of commensal microbes in competition with invading pathogens and each other. The identification of an Escherichia coli-produced genotoxin, colibactin, and its specific targeted killing of enteric pathogens and commensals, including Vibrio cholerae and Bacteroides fragilis, sheds light on our understanding of intermicrobial interactions in the mammalian gut. Our findings elucidate the mechanisms through which genotoxins shape microbial communities and provide a platform for probing the larger role of enteric multibacterial interactions regarding infection and disease outcomes.

Amyloid-DNA Composites of Bacterial Biofilms Stimulate Autoimmunity.

Research on the human microbiome has established that commensal and pathogenic bacteria can influence obesity, cancer, and autoimmunity through mechanisms mostly unknown. We found that a component of bacterial biofilms, the amyloid protein curli, irreversibly formed fibers with bacterial DNA during biofilm formation. This interaction accelerated amyloid polymerization and created potent immunogenic complexes that activated immune cells, including dendritic cells, to produce cytokines such as type I interferons, which are pathogenic in systemic lupus erythematosus (SLE). When given systemically, curli-DNA composites triggered immune activation and production of autoantibodies in lupus-prone and wild-type mice. We also found that the infection of lupus-prone mice with curli-producing bacteria triggered higher autoantibody titers compared to curli-deficient bacteria. These data provide a mechanism by which the microbiome and biofilm-producing enteric infections may contribute to the progression of SLE and point to a potential molecular target for treatment of autoimmunity.

Comparative Proteomic Analysis Revealing ActJ-Regulated Proteins in Sinorhizobium meliloti.

To adapt to different environmental conditions, Sinorhizobium meliloti relies on finely tuned regulatory networks, most of which are unexplored to date. We recently demonstrated that deletion of the two-component system ActJK renders an acid-vulnerable phenotype in S. meliloti and negatively impacts bacteroid development and nodule occupancy as well. To fully understand the role of ActJ in acid tolerance, S. meliloti wild-type and S. meliloti ΔactJ proteomes were compared in the presence or absence of acid stress by nanoflow ultrahigh-performance liquid chromatography coupled to mass spectrometry. The analysis demonstrated that proteins involved in the synthesis of exopolysaccharides (EPSs) were notably enriched in ΔactJ cells in acid pH. Total EPS quantification further revealed that although EPS production was augmented at pH 5.6 in both the ΔactJ and the parental strain, the lack of ActJ significantly enhanced this difference. Moreover, several efflux pumps were found to be downregulated in the ΔactJ strain. Promoter fusion assays suggested that ActJ positively modulated its own expression in an acid medium but not at under neutral conditions. The results presented here identify several ActJ-regulated genes in S. meliloti, highlighting key components associated with ActJK regulation that will contribute to a better understanding of rhizobia adaptation to acid stress.

Chromosome-Membrane Interactions in Bacteria.

Prokaryotes, by definition, do not segregate their genetic material from the cytoplasm. Thus, there is no barrier preventing direct interactions between chromosomal DNA and the plasma membrane. The possibility of such interactions in bacteria was proposed long ago and supported by early electron microscopy and cell fractionation studies. However, the identification and characterization of chromosome-membrane interactions have been slow in coming. Recently, this subject has seen more progress, driven by advances in imaging techniques and in the exploration of diverse cellular processes. A number of loci have been identified in specific bacteria that depend on interactions with the membrane for their function. In addition, there is growing support for a general mechanism of DNA-membrane contacts based on transertion-concurrent transcription, translation, and insertion of membrane proteins. This review summarizes the history and recent results of chromosome-membrane associations and discusses the known and theorized consequences of these interactions in the bacterial cell.

Rewiring the specificity of two-component signal transduction systems.

Two-component signal transduction systems are the predominant means by which bacteria sense and respond to environmental stimuli. Bacteria often employ tens or hundreds of these paralogous signaling systems, comprised of histidine kinases (HKs) and their cognate response regulators (RRs). Faithful transmission of information through these signaling pathways and avoidance of detrimental crosstalk demand exquisite specificity of HK-RR interactions. To identify the determinants of two-component signaling specificity, we examined patterns of amino acid coevolution in large, multiple sequence alignments of cognate kinase-regulator pairs. Guided by these results, we demonstrate that a subset of the coevolving residues is sufficient, when mutated, to completely switch the substrate specificity of the kinase EnvZ. Our results shed light on the basis of molecular discrimination in two-component signaling pathways, provide a general approach for the rational rewiring of these pathways, and suggest that analyses of coevolution may facilitate the reprogramming of other signaling systems and protein-protein interactions.

The phosphohistidine phosphatase SixA dephosphorylates the phosphocarrier NPr.

Histidine phosphorylation is a posttranslational modification that alters protein function and also serves as an intermediate of phosphoryl transfer. Although phosphohistidine is relatively unstable, enzymatic dephosphorylation of this residue is apparently needed in some contexts, since both prokaryotic and eukaryotic phosphohistidine phosphatases have been reported. Here we identify the mechanism by which a bacterial phosphohistidine phosphatase dephosphorylates the nitrogen-related phosphotransferase system, a broadly conserved bacterial pathway that controls diverse metabolic processes. We show that the phosphatase SixA dephosphorylates the phosphocarrier protein NPr and that the reaction proceeds through phosphoryl transfer from a histidine on NPr to a histidine on SixA. In addition, we show that Escherichia coli lacking SixA are outcompeted by wild-type E. coli in the context of commensal colonization of the mouse intestine. Notably, this colonization defect requires NPr and is distinct from a previously identified in vitro growth defect associated with dysregulation of the nitrogen-related phosphotransferase system. The widespread conservation of SixA, and its coincidence with the phosphotransferase system studied here, suggests that this dephosphorylation mechanism may be conserved in other bacteria.

Zwitterionic surface chemistry enhances detachment of bacteria under shear.

The ubiquitous nature of microorganisms, especially of biofilm-forming bacteria, makes biofouling a prevalent challenge in many settings, including medical and industrial environments immersed in liquid and subjected to shear forces. Recent studies have shown that zwitterionic groups are effective in suppressing bacteria and protein adhesion as well as biofilm growth. However, the effect of zwitterionic groups on the removal of surface-bound bacteria has not been extensively studied. Here we present a microfluidic approach to evaluate the effectiveness in facilitating bacteria detachment by shear of an antifouling surface treatment using (3-(dimethyl;(3-trimethoxysilyl)propyl)ammonia propane-1-sulfonate), a sulfobetaine silane (SBS). Control studies show that SBS-functionalized surfaces greatly increase protein (bovine serum albumin) removal upon rinsing. On the same surfaces, enhanced bacteria (Pseudomonas aeruginosa) removal is observed under shear. To quantify this enhancement a microfluidic shear device is employed to investigate how SBS-functionalized surfaces promote bacteria detachment under shear. By using a microfluidic channel with five shear zones, we compare the removal of bacteria from zwitterionic and glass surfaces under different shear rates. At times of 15 min, 30 min, and 60 min, bacteria adhesion on SBS-functionalized surfaces is reduced relative to the control surface (glass) under quiescent conditions. However, surface-associated bacteria on the SBS-functionalized glass and control show similar percentages of live cells, suggesting minimal intrinsic biocidal effect from the SBS-functionalized surface. Notably, when exposed to shear rates ranging from 104 to 105 s-1, significantly fewer bacteria remain on the SBS-functionalized surfaces. These results demonstrate the potential of zwitterionic sulfobetaine as effective antifouling coatings that facilitate the removal of bacteria under shear.

Bioactive cell-like hybrids from dendrimersomes with a human cell membrane and its components.

Cell-like hybrids from natural and synthetic amphiphiles provide a platform to engineer functions of synthetic cells and protocells. Cell membranes and vesicles prepared from human cell membranes are relatively unstable in vitro and therefore are difficult to study. The thicknesses of biological membranes and vesicles self-assembled from amphiphilic Janus dendrimers, known as dendrimersomes, are comparable. This feature facilitated the coassembly of functional cell-like hybrid vesicles from giant dendrimersomes and bacterial membrane vesicles generated from the very stable bacterial Escherichia coli cell after enzymatic degradation of its outer membrane. Human cells are fragile and require only mild centrifugation to be dismantled and subsequently reconstituted into vesicles. Here we report the coassembly of human membrane vesicles with dendrimersomes. The resulting giant hybrid vesicles containing human cell membranes, their components, and Janus dendrimers are stable for at least 1 y. To demonstrate the utility of cell-like hybrid vesicles, hybrids from dendrimersomes and bacterial membrane vesicles containing YadA, a bacterial adhesin protein, were prepared. The latter cell-like hybrids were recognized by human cells, allowing for adhesion and entry of the hybrid bacterial vesicles into human cells in vitro.

Encoding biological recognition in a bicomponent cell-membrane mimic.

Self-assembling dendrimers have facilitated the discovery of periodic and quasiperiodic arrays of supramolecular architectures and the diverse functions derived from them. Examples are liquid quasicrystals and their approximants plus helical columns and spheres, including some that disregard chirality. The same periodic and quasiperiodic arrays were subsequently found in block copolymers, surfactants, lipids, glycolipids, and other complex molecules. Here we report the discovery of lamellar and hexagonal periodic arrays on the surface of vesicles generated from sequence-defined bicomponent monodisperse oligomers containing lipid and glycolipid mimics. These vesicles, known as glycodendrimersomes, act as cell-membrane mimics with hierarchical morphologies resembling bicomponent rafts. These nanosegregated morphologies diminish sugar-sugar interactions enabling stronger binding to sugar-binding proteins than densely packed arrangements of sugars. Importantly, this provides a mechanism to encode the reactivity of sugars via their interaction with sugar-binding proteins. The observed sugar phase-separated hierarchical arrays with lamellar and hexagonal morphologies that encode biological recognition are among the most complex architectures yet discovered in soft matter. The enhanced reactivity of the sugar displays likely has applications in material science and nanomedicine, with potential to evolve into related technologies.

Effects of Regulatory Network Organization and Environment on PmrD Connector Activity and Polymyxin Resistance in Klebsiella pneumoniae and Escherichia coli.

Polymyxins are a class of cyclic peptides with antimicrobial activity against Gram-negative bacteria. In Enterobacteriaceae, the PhoQ/PhoP and PmrB/PmrA two-component systems regulate many genes that confer resistance to both polymyxins and host antimicrobial peptides. The activities of these two-component systems are modulated by additional proteins that are conserved across Enterobacteriaceae, such as MgrB, a negative regulator of PhoQ, and PmrD, a "connector" protein that activates PmrB/PmrA in response to PhoQ/PhoP stimulation. Despite the conservation of many protein components of the PhoQ/PhoP-PmrD-PmrB/PmrA network, the specific molecular interactions and regulatory mechanisms vary across different genera. Here, we explore the role of PmrD in modulating this signaling network in Klebsiella pneumoniae and Escherichia coli We show that in K. pneumoniae, PmrD is not required for polymyxin resistance arising from mutation of mgrB-the most common cause of spontaneous polymyxin resistance in this bacterium-suggesting that direct activation of polymyxin resistance genes by PhoQ/PhoP plays a critical role in this resistance pathway. However, for conditions of low pH or intermediate iron concentrations, both of which stimulate PmrB/PmrA, we find that PmrD does contribute to resistance. We further show that in E. coli, PmrD functions as a connector between PhoQ/PhoP and PmrB/PmrA, in contrast with previous reports. In this case, activity also depends on PmrB/PmrA stimulation, or on very high activation of PhoQ/PhoP. Our results indicate that the importance of the PmrD connector in modulating the polymyxin resistance network depends on both the network organization and on the environmental conditions associated with PmrB stimulation.

Non-equilibrium repressor binding kinetics link DNA damage dose to transcriptional timing within the SOS gene network.

Biochemical pathways are often genetically encoded as simple transcription regulation networks, where one transcription factor regulates the expression of multiple genes in a pathway. The relative timing of each promoter's activation and shut-off within the network can impact physiology. In the DNA damage repair pathway (known as the SOS response) of Escherichia coli, approximately 40 genes are regulated by the LexA repressor. After a DNA damaging event, LexA degradation triggers SOS gene transcription, which is temporally separated into subsets of 'early', 'middle', and 'late' genes. Although this feature plays an important role in regulating the SOS response, both the range of this separation and its underlying mechanism are not experimentally defined. Here we show that, at low doses of DNA damage, the timing of promoter activities is not separated. Instead, timing differences only emerge at higher levels of DNA damage and increase as a function of DNA damage dose. To understand mechanism, we derived a series of synthetic SOS gene promoters which vary in LexA-operator binding kinetics, but are otherwise identical, and then studied their activity over a large dose-range of DNA damage. In distinction to established models based on rapid equilibrium assumptions, the data best fit a kinetic model of repressor occupancy at promoters, where the drop in cellular LexA levels associated with higher doses of DNA damage leads to non-equilibrium binding kinetics of LexA at operators. Operators with slow LexA binding kinetics achieve their minimal occupancy state at later times than operators with fast binding kinetics, resulting in a time separation of peak promoter activity between genes. These data provide insight into this remarkable feature of the SOS pathway by demonstrating how a single transcription factor can be employed to control the relative timing of each gene's transcription as a function of stimulus dose.

Microbes vs. chemistry in the origin of the anaerobic gut lumen.

The succession from aerobic and facultative anaerobic bacteria to obligate anaerobes in the infant gut along with the differences between the compositions of the mucosally adherent vs. luminal microbiota suggests that the gut microbes consume oxygen, which diffuses into the lumen from the intestinal tissue, maintaining the lumen in a deeply anaerobic state. Remarkably, measurements of luminal oxygen levels show nearly identical pO2 (partial pressure of oxygen) profiles in conventional and germ-free mice, pointing to the existence of oxygen consumption mechanisms other than microbial respiration. In vitro experiments confirmed that the luminal contents of germ-free mice are able to chemically consume oxygen (e.g., via lipid oxidation reactions), although at rates significantly lower than those observed in the case of conventionally housed mice. For conventional mice, we also show that the taxonomic composition of the gut microbiota adherent to the gut mucosa and in the lumen throughout the length of the gut correlates with oxygen levels. At the same time, an increase in the biomass of the gut microbiota provides an explanation for the reduction of luminal oxygen in the distal vs. proximal gut. These results demonstrate how oxygen from the mammalian host is used by the gut microbiota, while both the microbes and the oxidative chemical reactions regulate luminal oxygen levels, shaping the composition of the microbial community throughout different regions of the gut.

Functional determinants of a small protein controlling a broadly conserved bacterial sensor kinase.

The PhoQ/PhoP two-component system plays a vital role in the regulation of Mg2+ homeostasis, resistance to acid and hyperosmotic stress, cationic antimicrobial peptides, and virulence in Escherichia coli, Salmonella and related bacteria. Previous studies have shown that MgrB, a 47 amino acid membrane protein that is part of the PhoQ/PhoP regulon, inhibits the histidine kinase PhoQ. MgrB is part of a negative feedback loop modulating this two-component system that prevents hyperactivation of PhoQ and may also provide an entry point for additional input signals for the PhoQ/PhoP pathway. To explore the mechanism of action of MgrB, we have analyzed the effects of point mutations, C-terminal truncations and transmembrane region swaps on MgrB activity. In contrast with two other known membrane protein regulators of histidine kinases in E. coli, we find that the MgrB TM region is necessary for PhoQ inhibition. Our results indicate that the TM region mediates interactions with PhoQ and that W20 is a key residue for PhoQ/MgrB complex formation. Additionally, mutations of the MgrB cytosolic region suggest that the two N-terminal lysines play an important role in regulating PhoQ activity. Alanine scanning mutagenesis of the periplasmic region of MgrB further indicates that, with the exception of a few highly conserved residues, most residues are not essential for MgrB's function as a PhoQ inhibitor. Our results indicate that the regulatory function of the small protein MgrB depends on distinct contributions from multiple residues spread across the protein. Interestingly, the TM region also appears to interact with other non-cognate histidine kinases in a bacterial two-hybrid assay, suggesting a potential route for evolving new small protein modulators of histidine kinases.

Natural variation of a sensor kinase controlling a conserved stress response pathway in Escherichia coli.

Previous studies have shown that exponentially growing Escherichia coli can detect mild acidity (~pH 5.5) and, in response, synthesize enzymes that protect against severe acid shock. This adaptation is controlled by the EvgS/EvgA phosphorelay, a signal transduction system present in virtually every E. coli isolate whose genome has been sequenced. Here we show that, despite this high level of conservation, the EvgS/EvgA system displays a surprising natural variation in pH-sensing capacity, with some strains entirely non-responsive to low pH stimulus. In most cases that we have tested, however, activation of the EvgA regulon still confers acid resistance. From analyzing selected E. coli isolates, we find that the natural variation results from polymorphisms in the sensor kinase EvgS. We further show that this variation affects the pH response of a second kinase, PhoQ, which senses pH differently from the closely related PhoQ in Salmonella enterica. The within-species diversification described here suggests EvgS likely responds to additional input signals that may be correlated with acid stress. In addition, this work highlights the fact that even for highly conserved sensor kinases, the activities identified from a subset of isolates may not necessarily generalize to other members of the same bacterial species.

Self-interrupted synthesis of sterically hindered aliphatic polyamide dendrimers.

2,2-Bis(azidomethyl)propionic acid was prepared in four steps and 85% yield from the commercially available 2,2-bis(hydroxymethyl)propionic acid and used as the starting building block for the divergent, convergent, and double-stage convergent-divergent iterative methods for the synthesis of dendrimers and dendrons containing ethylenediamine (EDA), piperazine (PPZ), and methyl 2,2-bis(aminomethyl)propionate (COOMe) cores. These cores have the same multiplicity but different conformations. A diversity of synthetic methods were used for the synthesis of dendrimers and dendrons. Regardless of the method used, a self-interruption of the synthesis was observed at generation 4 for the dendrimer with an EDA core and at generation 5 for the one with a PPZ core, whereas for the COOMe core, self-interruption was observed at generation 6 dendron, which is equivalent to generation 5 dendrimer. Molecular modeling and molecular-dynamics simulations demonstrated that the observed self-interruption is determined by the backfolding of the azide groups at the periphery of the dendrimer. The latter conformation inhibits completely the heterogeneous hydrogenation of the azide groups catalyzed by 10% Pd/carbon as well as homogeneous hydrogenation by the Staudinger method. These self-terminated polyamide dendrimers are enzymatically and hydrolytically stable and also exhibit antimicrobial activity. Thus, these nanoscale constructs open avenues for biomedical applications.

The SOS Response Mediates Sustained Colonization of the Mammalian Gut.

Bacteria have a remarkable ability to survive, persist, and ultimately adapt to environmental challenges. A ubiquitous environmental hazard is DNA damage, and most bacteria have evolved a network of genes to combat genotoxic stress. This network is known as the SOS response and aids in bacterial survival by regulating genes involved in DNA repair and damage tolerance. Recently, the SOS response has been shown to play an important role in bacterial pathogenesis, and yet the role of the SOS response in nonpathogenic organisms and in physiological settings remains underexplored. Using a commensal Escherichia coli strain, MP1, we showed that the SOS response plays a vital role during colonization of the murine gut. In an unperturbed environment, the SOS-off mutant is impaired for stable colonization relative to a wild-type strain, suggesting the presence of genotoxic stress in the mouse gut. We evaluated the possible origins of genotoxic stress in the mouse gut by examining factors associated with the host versus the competing commensal organisms. In a dextran sulfate sodium (DSS) colitis model, the SOS-off colonization defect persisted but was not exacerbated. In contrast, in a germ-free model, the SOS-off mutant colonized with efficiency equal to that seen with the wild-type strain, suggesting that competing commensal organisms might be a significant source of genotoxic stress. This report extends our understanding of the importance of a functional SOS response for bacterial fitness in the context of a complex physiological environment and highlights the SOS response as a possible mechanism that contributes to ongoing genomic changes, including potential antibiotic resistance, in the microbiome of healthy hosts.

Colistin-resistance-mediated bacterial surface modification sensitizes phage infection.

Colistin is a drug of last resort for the treatment of many multidrug resistant Gram-negative bacteria, including Klebsiella pneumoniae However, bacteria readily acquire resistance to this antibiotic via lipopolysaccharide modifications caused by spontaneous mutations or from enzymes acquired by lateral gene transfer. The fitness cost associated with these modifications remains poorly understood. In this study, we show that colistin-resistant K. pneumoniae are more susceptible to killing by a newly isolated lytic phage than the colistin sensitive parent strain. We observe this behavior for colistin-resistance conferred by a horizontally transferred mcr-1 containing plasmid and also from the inactivation of the chromosomal gene mgrB By measuring zeta potentials, we found that the phage particles were negatively charged at neutral pH and that colistin-resistant bacteria had less negative zeta potentials than did wildtype. These results suggest that the decreased negative surface charge of colistin-resistant cells lowers the electrostatic repulsion between the phage and bacteria, thereby promoting phage adherence and subsequent infection. To further explore this, we tested the effect of phage treatment on K. pneumoniae growing in several different environments. We found that colistin-resistant cells were more susceptible to phage than were the wildtype cells when growing in biofilms or infected moth larvae and when colonizing the mammalian gut. A better understanding of these fitness costs may lead to new treatment approaches that minimize the emergence and spread of colistin-resistant pathogens in human and environmental reservoirs.

A bacterial signaling system regulates noise to enable bet hedging.

Phenotypic diversity helps populations persist in changing and often unpredictable environments. One diversity-generating strategy is for individuals to switch randomly between phenotypic states such that one subpopulation has high fitness in the present environment, and another subpopulation has high fitness in an environment that might be encountered in the future. This sort of biological bet hedging can be found in all domains of life. Here, we discuss a recently described example from the bacterium Escherichia coli. When exposed to both oxygen and trimethylamine oxide (TMAO), E. coli hedges its bets on the possibility of oxygen loss by generating high cell-to-cell variability in the expression of the TMAO respiratory system. If oxygen is rapidly depleted from the environment, only those cells that had been expressing the TMAO respiratory system at high levels can continue to grow. This particular bet-hedging scheme possesses some unusual characteristics, most notably the decoupling of gene expression noise from the mean expression level. This decoupling allows bacteria to sense oxygen and regulate the amount of variability in TMAO reductase expression (that is, to turn bet hedging on or off) without having to adjust the mean TMAO reductase expression level. In this review, we discuss the features of the TMAO signaling pathway that permit the decoupling of gene expression noise from the mean and the regulation of bet hedging. We also highlight some open questions regarding the TMAO respiratory system and its regulatory architecture that may be relevant to many signaling systems.