Showing papers in "Journal of Bacteriology in 2017"

Polysaccharide Utilization Loci: Fueling Microbial Communities.

Abstract: The complex carbohydrates of terrestrial and marine biomass represent a rich nutrient source for free-living and mutualistic microbes alike. The enzymatic saccharification of these diverse substrates is of critical importance for fueling a variety of complex microbial communities, including marine, soil, ruminant, and monogastric microbiota. Consequently, highly specific carbohydrate-active enzymes, recognition proteins, and transporters are enriched in the genomes of certain species and are of critical importance in competitive environments. In Bacteroidetes bacteria, these systems are organized as polysaccharide utilization loci (PULs), which are strictly regulated, colocalized gene clusters that encode enzyme and protein ensembles required for the saccharification of complex carbohydrates. This review provides historical perspectives and summarizes key findings in the study of these systems, highlighting a critical shift from sequence-based PUL discovery to systems-based analyses combining reverse genetics, biochemistry, enzymology, and structural biology to precisely illuminate the molecular mechanisms underpinning PUL function. The ecological implications of dynamic PUL deployment by key species in the human gastrointestinal tract are explored, as well as the wider distribution of these systems in other gut, terrestrial, and marine environments.

Impact of Membrane Phospholipid Alterations in Escherichia coli on Cellular Function and Bacterial Stress Adaptation.

Abstract: Bacteria have evolved multiple strategies to sense and rapidly adapt to challenging and ever-changing environmental conditions. The ability to alter membrane lipid composition, a key component of the cellular envelope, is crucial for bacterial survival and adaptation in response to environmental stress. However, the precise roles played by membrane phospholipids in bacterial physiology and stress adaptation are not fully elucidated. The goal of this study was to define the role of membrane phospholipids in adaptation to stress and maintenance of bacterial cell fitness. By using genetically modified strains in which the membrane phospholipid composition can be systematically manipulated, we show that alterations in major Escherichia coli phospholipids transform these cells globally. We found that alterations in phospholipids impair the cellular envelope structure and function, the ability to form biofilms, and bacterial fitness and cause phospholipid-dependent susceptibility to environmental stresses. This study provides an unprecedented view of the structural, signaling, and metabolic pathways in which bacterial phospholipids participate, allowing the design of new approaches in the investigation of lipid-dependent processes involved in bacterial physiology and adaptation.IMPORTANCE In order to cope with and adapt to a wide range of environmental conditions, bacteria have to sense and quickly respond to fluctuating conditions. In this study, we investigated the effects of systematic and controlled alterations in bacterial phospholipids on cell shape, physiology, and stress adaptation. We provide new evidence that alterations of specific phospholipids in Escherichia coli have detrimental effects on cellular shape, envelope integrity, and cell physiology that impair biofilm formation, cellular envelope remodeling, and adaptability to environmental stresses. These findings hold promise for future antibacterial therapies that target bacterial lipid biosynthesis.

Defining Electron Bifurcation in the Electron-Transferring Flavoprotein Family

Abstract: Electron bifurcation is the coupling of exergonic and endergonic redox reactions to simultaneously generate (or utilize) low- and high-potential electrons. It is the third recognized form of energy conservation in biology and was recently described for select electron-transferring flavoproteins (Etfs). Etfs are flavin-containing heterodimers best known for donating electrons derived from fatty acid and amino acid oxidation to an electron transfer respiratory chain via Etf-quinone oxidoreductase. Canonical examples contain a flavin adenine dinucleotide (FAD) that is involved in electron transfer, as well as a non-redox-active AMP. However, Etfs demonstrated to bifurcate electrons contain a second FAD in place of the AMP. To expand our understanding of the functional variety and metabolic significance of Etfs and to identify amino acid sequence motifs that potentially enable electron bifurcation, we compiled 1,314 Etf protein sequences from genome sequence databases and subjected them to informatic and structural analyses. Etfs were identified in diverse archaea and bacteria, and they clustered into five distinct well-supported groups, based on their amino acid sequences. Gene neighborhood analyses indicated that these Etf group designations largely correspond to putative differences in functionality. Etfs with the demonstrated ability to bifurcate were found to form one group, suggesting that distinct conserved amino acid sequence motifs enable this capability. Indeed, structural modeling and sequence alignments revealed that identifying residues occur in the NADH- and FAD-binding regions of bifurcating Etfs. Collectively, a new classification scheme for Etf proteins that delineates putative bifurcating versus nonbifurcating members is presented and suggests that Etf-mediated bifurcation is associated with surprisingly diverse enzymes.IMPORTANCE Electron bifurcation has recently been recognized as an electron transfer mechanism used by microorganisms to maximize energy conservation. Bifurcating enzymes couple thermodynamically unfavorable reactions with thermodynamically favorable reactions in an overall spontaneous process. Here we show that the electron-transferring flavoprotein (Etf) enzyme family exhibits far greater diversity than previously recognized, and we provide a phylogenetic analysis that clearly delineates bifurcating versus nonbifurcating members of this family. Structural modeling of proteins within these groups reveals key differences between the bifurcating and nonbifurcating Etfs.

Quorum Sensing Gene Regulation by LuxR/HapR Master Regulators in Vibrios.

Abstract: The coordination of group behaviors in bacteria is accomplished via the cell-cell signaling process called quorum sensing. Vibrios have historically been models for studying bacterial communication due to the diverse and remarkable behaviors controlled by quorum sensing in these bacteria, including bioluminescence, type III and type VI secretion, biofilm formation, and motility. Here, we discuss the Vibrio LuxR/HapR family of proteins, the master global transcription factors that direct downstream gene expression in response to changes in cell density. These proteins are structurally similar to TetR transcription factors but exhibit distinct biochemical and genetic features from TetR that determine their regulatory influence on the quorum sensing gene network. We review here the gene groups regulated by LuxR/HapR and quorum sensing and explore the targets that are common and unique among Vibrio species.

Regulation, Function, and Detection of Protein Acetylation in Bacteria.

Abstract: Ne-Lysine acetylation is now recognized as an abundant posttranslational modification (PTM) that influences many essential biological pathways. Advancements in mass spectrometry-based proteomics have led to the discovery that bacteria contain hundreds of acetylated proteins, contrary to the prior notion of acetylation events being rare in bacteria. Although the mechanisms that regulate protein acetylation are still not fully defined, it is understood that this modification is finely tuned via both enzymatic and nonenzymatic mechanisms. The opposing actions of Gcn5-related N-acetyltransferases (GNATs) and deacetylases, including sirtuins, provide the enzymatic control of lysine acetylation. A nonenzymatic mechanism of acetylation has also been demonstrated and proven to be prominent in bacteria, as well as in mitochondria. The functional consequences of the vast majority of the identified acetylation sites remain unknown. From studies in mammalian systems, acetylation of critical lysine residues was shown to impact protein function by altering its structure, subcellular localization, and interactions. It is becoming apparent that the same diversity of functions can be found in bacteria. Here, we review current knowledge of the mechanisms and the functional consequences of acetylation in bacteria. Additionally, we discuss the methods available for detecting acetylation sites, including quantitative mass spectrometry-based methods, which promise to promote this field of research. We conclude with possible future directions and broader implications of the study of protein acetylation in bacteria.

An ompR-envZ Two-Component System Ortholog Regulates Phase Variation, Osmotic Tolerance, Motility, and Virulence in Acinetobacter baumannii Strain AB5075.

Abstract: Recently, a novel phase-variable colony opacity phenotype was discovered in Acinetobacter baumannii strain AB5075, where colonies interconvert between opaque and translucent variants. Opaque colonies become mottled or sectored after 24 h of growth due to translucent variants arising within the colony. This easily distinguishable opaque-colony phenotype was used to screen for random transposon insertions that increased the frequency of sectoring at a time point when wild-type colonies were uniformly opaque. A colony was identified that contained multiple papillae of translucent variants, and the insertion in this mutant mapped to an ortholog of the two-component system response regulator ompR Subsequent investigation of in-frame deletions of ompR and the sensor kinase envZ (located adjacent to ompR) showed that the switching frequency from opaque to translucent was increased 401- and 281-fold, respectively. The ompR mutant also exhibited sensitivity to sodium chloride in growth medium, whereas the envZ mutation did not elicit sensitivity to sodium chloride. Mutation of either gene reduced motility in A. baumannii strain AB5075, but a mutation in both ompR and envZ produced a more profound effect. The ompR and envZ genes were cotranscribed but were not subject to autoregulation by OmpR. Both ompR and envZ mutant opaque variants were attenuated in virulence in the Galleria mellonella infection model, whereas mutation of ompR had no effect on the virulence of the translucent variant. IMPORTANCEAcinetobacter baumannii is a well-known antibiotic-resistant pathogen; many clinical isolates can only be treated by a very small number of antibiotics (including colistin), while some exhibit panresistance. The current antimicrobial arsenal is nearing futility in the treatment of Acinetobacter infections, and new avenues of treatment are profoundly needed. Since phase variation controls the transition between opaque (virulent) and translucent (avirulent) states in A. baumannii, this may represent an "Achilles' heel" that can be targeted via the development of small molecules that lock cells in the translucent state and allow the host immune system to clear the infection. A better understanding of how phase variation is regulated may allow for the development of methods to target this process. The ompR-envZ two-component system ortholog negatively regulates phase variation in A. baumannii, and perturbation of this system leads to the attenuation of virulence in an invertebrate infection model.

Involvement of Two-Component Signaling on Bacterial Motility and Biofilm Development.

Abstract: Two-component signaling is a specialized mechanism that bacteria use to respond to changes in their environment Nonpathogenic strains of Escherichia coli K-12 harbor 30 histidine kinases and 32 response regulators, which form a network of regulation that integrates many other global regulators that do not follow the two-component signaling mechanism, as well as signals from central metabolism The output of this network is a multitude of phenotypic changes in response to changes in the environment Among these phenotypic changes, many two-component systems control motility and/or the formation of biofilm, sessile communities of bacteria that form on surfaces Motility is the first reversible attachment phase of biofilm development, followed by a so-called swim or stick switch toward surface organelles that aid in the subsequent phases In the mature biofilm, motility heterogeneity is generated by a combination of evolutionary and gene regulatory events

Maintenance of Microbial Cooperation Mediated by Public Goods in Single- and Multiple-Trait Scenarios

Abstract: Microbes often form densely populated communities, which favor competitive and cooperative interactions. Cooperation among bacteria often occurs through the production of metabolically costly molecules produced by certain individuals that become available to other neighboring individuals; such molecules are called public goods. This type of cooperation is susceptible to exploitation, since nonproducers of a public good can benefit from it while saving the cost of its production (cheating), gaining a fitness advantage over producers (cooperators). Thus, in mixed cultures, cheaters can increase in frequency in the population, relative to cooperators. Sometimes, and as predicted by simple game-theoretic arguments, such increases in the frequency of cheaters cause loss of the cooperative traits by exhaustion of the public goods, eventually leading to a collapse of the entire population. In other cases, however, both cooperators and cheaters remain in coexistence. This raises the question of how cooperation is maintained in microbial populations. Several strategies to prevent cheating have been studied in the context of a single trait and a unique environmental constraint. In this review, we describe current knowledge on the evolutionary stability of microbial cooperation and discuss recent discoveries describing the mechanisms operating in multiple-trait and multiple-constraint settings. We conclude with a consideration of the consequences of these complex interactions, and we briefly discuss the potential role of social interactions involving multiple traits and multiple environmental constraints in the evolution of specialization and division of labor in microbes.

Genome-Wide Transcriptional Response to Varying RpoS Levels in Escherichia coli K-12.

Abstract: The alternative sigma factor RpoS is a central regulator of many stress responses in Escherichia coli. The level of functional RpoS differs depending on the stress. The effect of these differing concentrations of RpoS on global transcriptional responses remains unclear. We investigated the effect of RpoS concentration on the transcriptome during stationary phase in rich media. We found that 23% of genes in the E. coli genome are regulated by RpoS, and we identified many RpoS-transcribed genes and promoters. We observed three distinct classes of response to RpoS by genes in the regulon: genes whose expression changes linearly with increasing RpoS level, genes whose expression changes dramatically with the production of only a little RpoS (“sensitive” genes), and genes whose expression changes very little with the production of a little RpoS (“insensitive”). We show that sequences outside the core promoter region determine whether an RpoS-regulated gene is sensitive or insensitive. Moreover, we show that sensitive and insensitive genes are enriched for specific functional classes and that the sensitivity of a gene to RpoS corresponds to the timing of induction as cells enter stationary phase. Thus, promoter sensitivity to RpoS is a mechanism to coordinate specific cellular processes with growth phase and may also contribute to the diversity of stress responses directed by RpoS.

The History of Bordetella pertussis Genome Evolution Includes Structural Rearrangement.

Abstract: Despite high pertussis vaccine coverage, reported cases of whooping cough (pertussis) have increased over the last decade in the United States and other developed countries. Although Bordetella pertussis is well known for its limited gene sequence variation, recent advances in long-read sequencing technology have begun to reveal genomic structural heterogeneity among otherwise indistinguishable isolates, even within geographically or temporally defined epidemics. We have compared rearrangements among complete genome assemblies from 257 B. pertussis isolates to examine the potential evolution of the chromosomal structure in a pathogen with minimal gene nucleotide sequence diversity. Discrete changes in gene order were identified that differentiated genomes from vaccine reference strains and clinical isolates of various genotypes, frequently along phylogenetic boundaries defined by single nucleotide polymorphisms. The observed rearrangements were primarily large inversions centered on the replication origin or terminus and flanked by IS481, a mobile genetic element with >240 copies per genome and previously suspected to mediate rearrangements and deletions by homologous recombination. These data illustrate that structural genome evolution in B. pertussis is not limited to reduction but also includes rearrangement. Therefore, although genomes of clinical isolates are structurally diverse, specific changes in gene order are conserved, perhaps due to positive selection, providing novel information for investigating disease resurgence and molecular epidemiology.IMPORTANCE Whooping cough, primarily caused by Bordetella pertussis, has resurged in the United States even though the coverage with pertussis-containing vaccines remains high. The rise in reported cases has included increased disease rates among all vaccinated age groups, provoking questions about the pathogen's evolution. The chromosome of B. pertussis includes a large number of repetitive mobile genetic elements that obstruct genome analysis. However, these mobile elements facilitate large rearrangements that alter the order and orientation of essential protein-encoding genes, which otherwise exhibit little nucleotide sequence diversity. By comparing the complete genome assemblies from 257 isolates, we show that specific rearrangements have been conserved throughout recent evolutionary history, perhaps by eliciting changes in gene expression, which may also provide useful information for molecular epidemiology.

Probing for Binding Regions of the FtsZ Protein Surface through Site-Directed Insertions: Discovery of Fully Functional FtsZ-Fluorescent Proteins.

Abstract: FtsZ, a bacterial tubulin homologue, is a cytoskeleton protein that assembles into protofilaments, one subunit thick. These protofilaments assemble further to form a “Z ring” at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane, and also serves as a scaffold to recruit cell-wall remodeling proteins for complete cell division in vivo. One model of the Z ring proposes that protofilaments associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of Escherichia coli FtsZ by inserting either small peptides or whole fluorescent proteins (FP). Of the four lateral surfaces on FtsZ protofilaments, we obtained inserts on the front and back surfaces that were functional for cell division. We conclude that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174 located on the left and right surfaces, completely blocked function, and are identified as possible sites for essential lateral interactions. However, the insert at R174 did not interfere with association of protofilaments into sheets and bundles in vitro. Another goal was to find a location within FtsZ that supported insertion of FP reporter proteins, while allowing the FtsZ-FP to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by super-resolution techniques. IMPORTANCE One model for the Z-ring structure proposes that protofilaments are assembled into ribbons by lateral bonds between FtsZ subunits. Our study excluded the front and back faces of the protofilament as involved in essential interactions in vivo, but pointed to two potential lateral bond sites on the right and left sides. We also identified an FtsZ loop where various fluorescent proteins could be inserted without blocking function; these FtsZ-FPs functioned as the sole source of FtsZ. This advance provides improved tools for all fluorescent imaging of the Z ring, and may be especially important for super-resolution imaging.

SecA Cotranslationally Interacts with Nascent Substrate Proteins In Vivo

Abstract: SecA is an essential component of the Sec machinery in bacteria, which is responsible for transporting proteins across the cytoplasmic membrane. Recent work from our laboratory indicates that SecA binds to ribosomes. Here, we used two different approaches to demonstrate that SecA also interacts with nascent polypeptides in vivo and that these polypeptides are Sec substrates. First, we photo-cross-linked SecA to ribosomes in vivo and identified mRNAs that copurify with SecA. Microarray analysis of the copurifying mRNAs indicated a strong enrichment for proteins containing Sec-targeting sequences. Second, we used a 2-dimensional (2-D) gel approach to analyze radioactively labeled nascent polypeptides that copurify with SecA, including maltose binding protein, a well-characterized SecA substrate. The interaction of SecA with nascent chains was not strongly affected in cells lacking SecB or trigger factor, both of which also interact with nascent Sec substrates. Indeed, the ability of SecB to interact with nascent chains was disrupted in strains in which the interaction between SecA and the ribosome was defective. Analysis of the interaction of SecA with purified ribosomes containing arrested nascent chains in vitro indicates that SecA can begin to interact with a variety of nascent chains when they reach a length of ∼110 amino acids, which is considerably shorter than the length required for interaction with SecB. Our results suggest that SecA cotranslationally recognizes nascent Sec substrates and that this recognition could be required for the efficient delivery of these proteins to the membrane-embedded Sec machinery. Importance SecA is an ATPase that provides the energy for the translocation of proteins across the cytoplasmic membrane by the Sec machinery in bacteria. The translocation of most of these proteins is uncoupled from protein synthesis and is frequently described as "posttranslational." Here, we show that SecA interacts with nascent Sec substrates. This interaction is not dependent on SecB or trigger factor, which also interact with nascent Sec substrates. Moreover, the interaction of SecB with nascent polypeptides is dependent on the interaction of SecA with the ribosome, suggesting that interaction of the nascent chain with SecA precedes interaction with SecB. Our results suggest that SecA could recognize substrate proteins cotranslationally in order to efficiently target them for uncoupled protein translocation.

Cystic Fibrosis Airway Microbiome: Overturning the Old, Opening the Way for the New.

Abstract: The genetic disease cystic fibrosis (CF) is associated with chronic airway infections that are a proximal cause of death in many patients with this affliction. Classic microbiology studies focusing on canonical pathogens resulted in the development of a common set of views regarding the nature of the airway infections associated with this disease, and these ideas have influenced everything from the way infections are treated to how clinical trials for new CF-targeted antibiotics are designed and the focus of CF-related research topics. Recent culture-independent studies have prompted us to rethink, and in some cases discard, some of these long-held views. In this piece, I argue that an updated view of the complicated chronic infections associated with CF, thanks in large part to culture-independent studies of sputum and bronchoalveolar lavage fluid samples, should be leveraged to develop new strategies to treat these recalcitrant infections.

The Small Protein SgrT Controls Transport Activity of the Glucose-Specific Phosphotransferase System.

Abstract: The bacterial small RNA (sRNA) SgrS has been a fruitful model for discovery of novel RNA-based regulatory mechanisms and new facets of bacterial physiology and metabolism. SgrS is one of only a few characterized dual-function sRNAs. SgrS can control gene expression posttranscriptionally via sRNA-mRNA base-pairing interactions. Its second function is coding for the small protein SgrT. Previous work demonstrated that both functions contribute to relief of growth inhibition caused by glucose-phosphate stress, a condition characterized by disrupted glycolytic flux and accumulation of sugar phosphates. The base-pairing activity of SgrS has been the subject of numerous studies, but the activity of SgrT is less well characterized. Here, we provide evidence that SgrT acts to specifically inhibit the transport activity of the major glucose permease PtsG. Superresolution microscopy demonstrated that SgrT localizes to the cell membrane in a PtsG-dependent manner. Mutational analysis determined that residues in the N-terminal domain of PtsG are important for conferring sensitivity to SgrT-mediated inhibition of transport activity. Growth assays support a model in which SgrT-mediated inhibition of PtsG transport activity reduces accumulation of nonmetabolizable sugar phosphates and promotes utilization of alternative carbon sources by modulating carbon catabolite repression. The results of this study expand our understanding of a basic and well-studied biological problem, namely, how cells coordinate carbohydrate transport and metabolism. Further, this work highlights the complex activities that can be carried out by sRNAs and small proteins in bacteria.IMPORTANCE Sequencing, annotation and investigation of hundreds of bacterial genomes have identified vast numbers of small RNAs and small proteins, the majority of which have no known function. In this study, we explore the function of a small protein that acts in tandem with a well-characterized small RNA during metabolic stress to help bacterial cells maintain balanced metabolism and continue growing. Our results indicate that this protein acts on the glucose transport system, inhibiting its activity under stress conditions in order to allow cells to utilize alternative carbon sources. This work sheds new light on a key biological problem: how cells coordinate carbohydrate transport and metabolism. The study also expands our understanding of the functional capacities of small proteins.

Progress in Understanding the Molecular Basis Underlying Functional Diversification of Cyclic Dinucleotide Turnover Proteins.

Abstract: Cyclic di-GMP was the first cyclic dinucleotide second messenger described, presaging the discovery of additional cyclic dinucleotide messengers in bacteria and eukaryotes. The GGDEF diguanylate cyclase (DGC) and EAL and HD-GYP phosphodiesterase (PDE) domains conduct the turnover of cyclic di-GMP. These three unrelated domains belong to superfamilies that exhibit significant variations in function, and they include both enzymatically active and inactive members, with a subset involved in synthesis and degradation of other cyclic dinucleotides. Here, we summarize current knowledge of sequence and structural variations that underpin the functional diversification of cyclic di-GMP turnover proteins. Moreover, we highlight that superfamily diversification is not restricted to cyclic di-GMP signaling domains, as particular DHH/DHHA1 domain and HD domain proteins have been shown to act as cyclic di-AMP phosphodiesterases. We conclude with a consideration of the current limitations that such diversity of action places on bioinformatic prediction of the roles of GGDEF, EAL, and HD-GYP domain proteins.

Imaging the motility and chemotaxis machineries in Helicobacter pylori by cryo-electron tomography.

Abstract: Helicobacter pylori is a bacterial pathogen that can cause many gastrointestinal diseases, including ulcers and gastric cancer. A unique chemotaxis-mediated motility is critical for H. pylori to colonize in the human stomach and to establish chronic infection, but the underlying molecular mechanisms are not well understood. Here, we employ cryo-electron tomography (cryo-ET) to reveal detailed structures of the H. pylori cell envelope, including the sheathed flagella and chemotaxis arrays. Notably, H. pylori possesses a distinctive periplasmic cage-like structure with 18-fold symmetry. We propose that this structure forms a robust platform for recruiting 18 torque generators, which likely provide the higher torque needed for swimming in high-viscosity environments. We also reveal a series of key flagellar assembly intermediates, providing structural evidence that flagellar assembly is tightly coupled with the biogenesis of the membrane sheath. Finally, we determine the structure of putative chemotaxis arrays at the flagellar pole, which have implications for how the direction of flagellar rotation is regulated. Together, our pilot cryo-ET studies provide novel structural insights into the unipolar flagella of H. pylori and lay a foundation for a better understanding of the unique motility of this organism. IMPORTANCE Helicobacter pylori is a highly motile bacterial pathogen that colonizes approximately 50% of the world's population. H. pylori can move readily within the viscous mucosal layer of the stomach. It has become increasingly clear that its unique flagella-driven motility is essential for successful gastric colonization and pathogenesis. Here, we use advanced imaging techniques to visualize novel in situ structures with unprecedented detail in intact H. pylori cells. Remarkably, H. pylori possesses multiple unipolar flagella, which are driven by one of the largest flagellar motors found in bacteria. These large motors presumably provide the higher torque needed by the bacterial pathogens to navigate in the viscous environment of the human stomach.

Cooperative Metabolism in a Three-Partner Insect-Bacterial Symbiosis Revealed by Metabolic Modeling.

Abstract: An important factor determining the impact of microbial symbionts on their animal hosts is the balance between the cost of nutrients consumed by the symbionts and the benefit of nutrients released back to the host, but the quantitative significance of nutrient exchange in symbioses involving multiple microbial partners has rarely been addressed. In this study on the association between two intracellular bacterial symbionts, "Candidatus Portiera aleyrodidarum" and "Candidatus Hamiltonella defensa," and their animal host, the whitefly Bemisia tabaci, we apply metabolic modeling to investigate host-symbiont nutrient exchange. Our in silico analysis revealed that >60% of the essential amino acids and related metabolites synthesized by "Candidatus Portiera aleyrodidarum" are utilized by the host, including a substantial contribution of nitrogen recycled from host nitrogenous waste, and that these interactions are required for host growth. In contrast, "Candidatus Hamiltonella defensa" retains most or all of the essential amino acids and B vitamins that it is capable of synthesizing. Furthermore, "Candidatus Hamiltonella defensa" suppresses host growth in silico by competition with "Candidatus Portiera aleyrodidarum" for multiple host nutrients, by suppressing "Candidatus Portiera aleyrodidarum" growth and metabolic function, and also by consumption of host nutrients that would otherwise be allocated to host growth. The interpretation from these modeling outputs that "Candidatus Hamiltonella defensa" is a nutritional parasite could not be inferred reliably from gene content alone but requires consideration of constraints imposed by the structure of the metabolic network. Furthermore, these quantitative models offer precise predictions for future experimental study and the opportunity to compare the functional organization of metabolic networks in different symbioses.IMPORTANCE The metabolic functions of unculturable intracellular bacteria with much reduced genomes are traditionally inferred from gene content without consideration of how the structure of the metabolic network may influence flux through metabolic reactions. The three-compartment model of metabolic flux between two bacterial symbionts and their insect host constructed in this study revealed that one symbiont is structured to overproduce essential amino acids for the benefit of the host, but the essential amino acid production in the second symbiont is quantitatively constrained by the structure of its network, rendering it "selfish" with respect to these nutrients. This study demonstrates the importance of quantitative flux data for elucidation of the metabolic function of symbionts. The in silico methodology can be applied to other symbioses with intracellular bacteria.

Brucella abortus Induces a Warburg Shift in Host Metabolism That Is Linked to Enhanced Intracellular Survival of the Pathogen.

Abstract: Intracellular bacterial pathogens exploit host cell resources to replicate and survive inside the host. Targeting these host systems is one promising approach to developing novel antimicrobials to treat intracellular infections. We show that human macrophage-like cells infected with Brucella abortus undergo a metabolic shift characterized by attenuated tricarboxylic acid cycle metabolism, reduced amino acid consumption, altered mitochondrial localization, and increased lactate production. This shift to an aerobic glycolytic state resembles the Warburg effect, a change in energy production that is well described in cancer cells and also occurs in activated inflammatory cells. B. abortus efficiently uses lactic acid as its sole carbon and energy source and requires the ability to metabolize lactate for normal survival in human macrophage-like cells. We demonstrate that chemical inhibitors of host glycolysis and lactate production do not affect in vitro growth of B. abortus in axenic culture but decrease its survival in the intracellular niche. Our data support a model in which infection shifts host metabolism to a Warburg-like state, and B. abortus uses this change in metabolism to promote intracellular survival. Pharmacological perturbation of these features of host cell metabolism may be a useful strategy to inhibit infection by intracellular pathogens.IMPORTANCEBrucella spp. are intracellular bacterial pathogens that cause disease in a range of mammals, including livestock. Transmission from livestock to humans is common and can lead to chronic human disease. Human macrophage-like cells infected with Brucella abortus undergo a Warburg-like metabolic shift to an aerobic glycolytic state where the host cells produce lactic acid and have reduced amino acid catabolism. We provide evidence that the pathogen can exploit this change in host metabolism to support growth and survival in the intracellular niche. Drugs that inhibit this shift in host cell metabolism inhibit intracellular replication and decrease the survival of B. abortus in an in vitro infection model; these drugs may be broadly useful therapeutics for intracellular infections.

Mechanisms of Pyocyanin Toxicity and Genetic Determinants of Resistance in Staphylococcus aureus.

Abstract: Pseudomonas aeruginosa and Staphylococcus aureus are commonly isolated from polymicrobial infections, such as wound infections and chronic respiratory infections of persons with cystic fibrosis. Despite their coisolation, P. aeruginosa produces substances toxic to S. aureus, including pyocyanin, a blue-pigmented molecule that functions in P. aeruginosa virulence. Pyocyanin inhibits S. aureus respiration, forcing it to derive energy from fermentation and adopt a small-colony variant (SCV) phenotype. The mechanisms by which S. aureus sustains infection in the presence of pyocyanin are not clear. We sought to clarify the mechanisms of pyocyanin toxicity in S. aureus as well as identify the staphylococcal factors involved in its resistance to pyocyanin toxicity. Nonrespiring S. aureus SCVs are inhibited by pyocyanin through pyocyanin-dependent reactive oxygen species (ROS) production, indicating that pyocyanin toxicity is mediated through respiratory inhibition and ROS generation. Selection on pyocyanin yielded a menadione auxotrophic SCV capable of growth on high concentrations of pyocyanin. Genome sequencing of this isolate identified mutations in four genes, including saeS, menD, NWMN_0006, and qsrR QsrR is a quinone-sensing repressor of quinone detoxification genes. Inactivation of qsrR resulted in significant pyocyanin resistance, and additional pyocyanin resistance was achieved through combined inactivation of qsrR and menadione biosynthesis. Pyocyanin-resistant S. aureus has an enhanced capability to inactivate pyocyanin, suggesting QsrR-regulated gene products may degrade pyocyanin to alleviate toxicity. These findings demonstrate pyocyanin-mediated ROS generation as an additional mechanism of pyocyanin toxicity and define QsrR as a key mediator of pyocyanin resistance in S. aureus IMPORTANCE Many bacterial infections occur in the presence of other microbes, where interactions between different microbes and the host impact disease. In patients with cystic fibrosis, chronic lung infection with multiple microbes results in the most severe disease manifestations. Staphylococcus aureus and Pseudomonas aeruginosa are prevalent cystic fibrosis pathogens, and infection with both is associated with worse outcomes. These organisms have evolved mechanisms of competing with one another. For example, P. aeruginosa produces pyocyanin, which inhibits S. aureus growth. Our research has identified how pyocyanin inhibits S. aureus growth and how S. aureus can adapt to survive in the presence of pyocyanin. Understanding how S. aureus sustains infection in the presence of P. aeruginosa may identify means of disrupting these microbial communities.

Bacteria, Rev Your Engines: Stator Dynamics Regulate Flagellar Motility.

Abstract: Many bacteria move through liquids and across surfaces by using flagella-filaments propelled by a membrane-embedded rotary motor. Much is known about the flagellum: its basic structure, the function of its individual motor components, and the regulation of its synthesis. However, we are only beginning to identify the dynamics of flagellar proteins and to understand how the motor structurally adapts to environmental stimuli. In this review, we discuss the external and cellular factors that influence the dynamics of stator complexes (the ion-conducting channels of the flagellar motor). We focus on recent discoveries suggesting that stator dynamics are a means for controlling flagellar function in response to different environments.

Comprehensive Spatial Analysis of the Borrelia burgdorferi Lipoproteome Reveals a Compartmentalization Bias toward the Bacterial Surface.

Abstract: The Lyme disease spirochete Borrelia burgdorferi is unique among bacteria in its large number of lipoproteins that are encoded by a small, exceptionally fragmented, and predominantly linear genome. Peripherally anchored in either the inner or outer membrane and facing either the periplasm or the external environment, these lipoproteins assume varied roles. A prominent subset of lipoproteins functioning as the apparent linchpins of the enzootic tick-vertebrate infection cycle have been explored as vaccine targets. Yet, most of the B. burgdorferi lipoproteome has remained uncharacterized. Here, we comprehensively and conclusively localize the B. burgdorferi lipoproteome by applying established protein localization assays to a newly generated epitope-tagged lipoprotein expression library and by validating the obtained individual protein localization results using a sensitive global mass spectrometry approach. The derived consensus localization data indicate that 86 of the 125 analyzed lipoproteins encoded by B. burgdorferi are secreted to the bacterial surface. Thirty-one of the remaining 39 periplasmic lipoproteins are retained in the inner membrane, with only 8 lipoproteins being anchored in the periplasmic leaflet of the outer membrane. The localization of 10 lipoproteins was further defined or revised, and 52 surface and 23 periplasmic lipoproteins were newly localized. Cross-referencing prior studies revealed that the borrelial surface lipoproteome contributing to the host-pathogen interface is encoded predominantly by plasmids. Conversely, periplasmic lipoproteins are encoded mainly by chromosomal loci. These studies close a gap in our understanding of the functional lipoproteome of an important human pathogen and set the stage for more in-depth studies of thus-far-neglected spirochetal lipoproteins.IMPORTANCE The small and exceptionally fragmented genome of the Lyme disease spirochete Borrelia burgdorferi encodes over 120 lipoproteins. Studies in the field have predominantly focused on a relatively small number of surface lipoproteins that play important roles in the transmission and pathogenesis of this global human pathogen. Yet, a comprehensive spatial assessment of the entire borrelial lipoproteome has been missing. The current study newly identifies 52 surface and 23 periplasmic lipoproteins. Overall, two-thirds of the B. burgdorferi lipoproteins localize to the surface, while outer membrane lipoproteins facing the periplasm are rare. This analysis underscores the dominant contribution of lipoproteins to the spirochete's rather complex and adaptable host-pathogen interface, and it encourages further functional exploration of its lipoproteome.

Diverse C-Terminal Sequences Involved in Flavobacterium johnsoniae Protein Secretion

Abstract: Flavobacteriumjohnsoniae and many related bacteria secrete proteins across the outer membrane using the type IX secretion system (T9SS). Proteins secreted by T9SSs have amino-terminal signal peptides for export across the cytoplasmic membrane by the Sec system and carboxy-terminal domains (CTDs) targeting them for secretion across the outer membrane by the T9SS. Most but not all T9SS CTDs belong to the family TIGR04183 (type A CTDs). We functionally characterized diverse CTDs for secretion by the F. johnsoniae T9SS. Attachment of the CTDs from F. johnsoniae RemA, AmyB, and ChiA to the foreign superfolder green fluorescent protein (sfGFP) that had a signal peptide at the amino terminus resulted in secretion across the outer membrane. In each case, approximately 80 to 100 amino acids from the extreme carboxy termini were needed for efficient secretion. Several type A CTDs from distantly related members of the phylum Bacteroidetes functioned in F. johnsoniae, supporting the secretion of sfGFP by the F. johnsoniae T9SS. F. johnsoniae SprB requires the T9SS for secretion but lacks a type A CTD. It has a conserved C-terminal domain belonging to the family TIGR04131, which we refer to as a type B CTD. The CTD of SprB was required for its secretion, but attachment of C-terminal regions of SprB of up to 1,182 amino acids to sfGFP failed to result in secretion. Additional features outside the C-terminal region of SprB may be required for its secretion.IMPORTANCE Type IX protein secretion systems (T9SSs) are common in but limited to members of the phylum Bacteroidetes Most proteins that are secreted by T9SSs have conserved carboxy-terminal domains that belong to the protein domain family TIGR04183 (type A CTDs) or TIGR04131 (type B CTDs). Here, we identify features of T9SS CTDs of F. johnsoniae that are required for protein secretion and demonstrate that type A CTDs from distantly related members of the phylum function with the F. johnsoniae T9SS to secrete the foreign protein sfGFP. In contrast, type B CTDs failed to target sfGFP for secretion, suggesting a more complex association with the T9SS.

Comprehensive Arrayed Transposon Mutant Library of Klebsiella pneumoniae Outbreak Strain KPNIH1

Abstract: Klebsiella pneumoniae and other carbapenem-resistant members of the family Enterobacteriaceae are a major cause of hospital-acquired infections, yet the basis of their success as nosocomial pathogens is poorly understood. To help provide a foundation for genetic analysis of K. pneumoniae, we created an arrayed, sequence-defined transposon mutant library of an isolate from the 2011 outbreak of infections at the U.S. National Institutes of Health Clinical Center. The library is made up of 12,000 individually arrayed mutants of a carbapenemase deletion parent strain and provides coverage of 85% of the predicted genes. The library includes an average of 2.5 mutants per gene, with most insertion locations identified and confirmed in two independent rounds of Sanger sequencing. On the basis of an independent transposon sequencing assay, about half of the genes lacking representatives in this "two-allele" library are essential for growth on nutrient agar. To validate the use of the library for phenotyping, we screened candidate mutants for increased antibiotic sensitivity by using custom phenotypic microarray plates. This screening identified several mutations increasing sensitivity to β-lactams (in acrB1, mcrB, ompR, phoP1, and slt1) and found that two-component regulator cpxAR mutations increased multiple sensitivities (to an aminoglycoside, a fluoroquinolone, and several β-lactams). Strains making up the two-allele mutant library are available through a web-based request mechanism.IMPORTANCE K. pneumoniae and other carbapenem-resistant members of the family Enterobacteriaceae are recognized as a top public health threat by the Centers for Disease Control and Prevention. The analysis of these major nosocomial pathogens has been limited by the experimental resources available for studying them. The work presented here describes a sequence-defined mutant library of a K. pneumoniae strain (KPNIH1) that represents an attractive model for studies of this pathogen because it is a recent isolate of the major sequence type that causes infection, the epidemiology of the outbreak it caused is well characterized, and an annotated genome sequence is available. The ready availability of defined mutants deficient in nearly all of the nonessential genes of the model strain should facilitate the genetic dissection of complex traits like pathogenesis and antibiotic resistance.

Genome Scale Mutational Analysis of Geobacter sulfurreducens Reveals Distinct Molecular Mechanisms for Respiration and Sensing of Poised Electrodes versus Fe(III) Oxides.

Abstract: Geobacter sulfurreducens generates electrical current by coupling intracellular oxidation of organic acids to the reduction of proteins on the cell surface that are able to interface with electrodes. This ability is attributed to the bacterium's capacity to respire other extracellular electron acceptors that require contact, such as insoluble metal oxides. To directly investigate the genetic basis of electrode-based respiration, we constructed Geobacter sulfurreducens transposon-insertion sequencing (Tn-Seq) libraries for growth, with soluble fumarate or an electrode as the electron acceptor. Libraries with >33,000 unique insertions and an average of 9 insertions/kb allowed an assessment of each gene's fitness in a single experiment. Mutations in 1,214 different genomic features impaired growth with fumarate, and the significance of 270 genes unresolved by annotation due to the presence of one or more functional homologs was determined. Tn-Seq analysis of -0.1 V versus standard hydrogen electrode (SHE) electrode-grown cells identified mutations in a subset of genes encoding cytochromes, processing systems for proline-rich proteins, sensory networks, extracellular structures, polysaccharides, and metabolic enzymes that caused at least a 50% reduction in apparent growth rate. Scarless deletion mutants of select genes identified via Tn-Seq revealed a new putative porin-cytochrome conduit complex (extABCD) crucial for growth with electrodes, which was not required for Fe(III) oxide reduction. In addition, four mutants lacking components of a putative methyl-accepting chemotaxis-cyclic dinucleotide sensing network (esnABCD) were defective in electrode colonization but grew normally with Fe(III) oxides. These results suggest that G. sulfurreducens possesses distinct mechanisms for recognition, colonization, and reduction of electrodes compared to Fe(III) oxides.IMPORTANCE Since metal oxide electron acceptors are insoluble, one hypothesis is that cells sense and reduce metals using the same molecular mechanisms used to form biofilms on electrodes and produce electricity. However, by simultaneously comparing thousands of Geobacter sulfurreducens transposon mutants undergoing electrode-dependent respiration, we discovered new cytochromes and chemosensory proteins supporting growth with electrodes that are not required for metal respiration. This supports an emerging model where G. sulfurreducens recognizes surfaces and forms conductive biofilms using mechanisms distinct from those used for growth with metal oxides. These findings provide a possible explanation for studies that correlate electricity generation with syntrophic interspecies electron transfer by Geobacter and reveal many previously unrecognized targets for engineering this useful capability in other organisms.

EssD, a Nuclease Effector of the Staphylococcus aureus ESS Pathway.

Abstract: Specialized secretion systems of bacteria evolved for selective advantage, either killing microbial competitors or implementing effector functions during parasitism. Earlier work characterized the ESAT-6 secretion system (ESS) of Staphylococcus aureus and demonstrated its contribution to persistent staphylococcal infection of vertebrate hosts. Here, we identify a novel secreted effector of the ESS pathway, EssD, that functions as a nuclease and cleaves DNA but not RNA. EssI, a protein of the DUF600 family, binds EssD to block its nuclease activity in the staphylococcal cytoplasm. An essD knockout mutant or a variant lacking nuclease activity, essDL546P, elicited a diminished interleukin-12 (IL-12) cytokine response following bloodstream infection of mice, suggesting that the effector function of EssD stimulates immune signaling to support the pathogenesis of S. aureus infections. IMPORTANCE Bacterial type VII or ESAT-6-like secretion systems (ESS) may have evolved to modulate host immune responses during infection, thereby contributing to the pathogenesis of important diseases such as tuberculosis and methicillin-resistant S. aureus (MRSA) infection. The molecular mechanisms whereby type VII secretion systems achieve their goals are not fully elucidated as secreted effectors with biochemical functions have heretofore not been identified. We show here that MRSA infection relies on the secretion of a nuclease effector that cleaves DNA and contributes to the stimulation of IL-12 signaling during infection. These results identify a biological mechanism for the contribution of the ESS pathway toward the establishment of MRSA disease.

Ferric Uptake Regulator Fur Is Conditionally Essential in Pseudomonas aeruginosa

Abstract: In Pseudomonas aeruginosa, the ferric uptake regulator (Fur) protein controls both metabolism and virulence in response to iron availability. Differently from other bacteria, attempts to obtain fur deletion mutants of P. aeruginosa failed, leading to the assumption that Fur is an essential protein in this bacterium. By investigating a P. aeruginosa conditional fur mutant, we demonstrate that Fur is not essential for P. aeruginosa growth in liquid media, biofilm formation, and pathogenicity in an insect model of infection. Conversely, Fur is essential for growth on solid media since Fur-depleted cells are severely impaired in colony formation. Transposon-mediated random mutagenesis experiments identified pyochelin siderophore biosynthesis as a major cause of the colony growth defect of the conditional fur mutant, and deletion mutagenesis confirmed this evidence. Impaired colony growth of pyochelin-proficient Fur-depleted cells does not depend on oxidative stress, since Fur-depleted cells do not accumulate higher levels of reactive oxygen species (ROS) and are not rescued by antioxidant agents or overexpression of ROS-detoxifying enzymes. Ectopic expression of pch genes revealed that pyochelin production has no inhibitory effects on a fur deletion mutant of Pseudomonas syringae pv. tabaci, suggesting that the toxicity of the pch locus in Fur-depleted cells involves a P. aeruginosa-specific pathway(s).IMPORTANCE Members of the ferric uptake regulator (Fur) protein family are bacterial transcriptional repressors that control iron uptake and storage in response to iron availability, thereby playing a crucial role in the maintenance of iron homeostasis. While fur null mutants of many bacteria have been obtained, Fur appears to be essential in Pseudomonas aeruginosa for still unknown reasons. We obtained Fur-depleted P. aeruginosa cells by conditional mutagenesis and showed that Fur is dispensable for planktonic growth, while it is required for colony formation. This is because Fur protects P. aeruginosa colonies from toxicity exerted by the pyochelin siderophore. This work provides a functional basis to the essentiality of Fur in P. aeruginosa and highlights unique properties of the Fur regulon in this species.

Mutation and Suppressor Analysis of the Essential Lipopolysaccharide Transport Protein LptA Reveals Strategies To Overcome Severe Outer Membrane Permeability Defects in Escherichia coli.

Abstract: In Gram-negative bacteria, lipopolysaccharide (LPS) contributes to the robust permeability barrier of the outer membrane (OM), preventing the entry of toxic molecules, such as detergents and antibiotics. LPS is transported from the inner membrane (IM) to the OM by the Lpt multiprotein machinery. Defects in LPS transport compromise LPS assembly at the OM and result in increased antibiotic sensitivity. LptA is a key component of the Lpt machine that interacts with the IM protein LptC and chaperones LPS through the periplasm. We report here the construction of lptA41, a quadruple mutant in four conserved amino acids potentially involved in LPS or LptC binding. Although viable, the mutant displays increased sensitivity to several antibiotics (bacitracin, rifampin, and novobiocin) and the detergent SDS, suggesting that lptA41 affects LPS transport. Indeed, lptA41 is defective in Lpt complex assembly, and its lipid A carries modifications diagnostic of LPS transport defects. We also selected and characterized two phenotypic bacitracin-resistant suppressors of lptA41 One mutant, in which only bacitracin sensitivity is suppressed, harbors a small in-frame deletion in mlaA, which codes for an OM lipoprotein involved in maintaining OM asymmetry by reducing accumulation of phospholipids in the outer leaflet. The other mutant, in which bacitracin, rifampin, and SDS sensitivity is suppressed, harbors an additional amino acid substitution in LptA41 and a nonsense mutation in opgH, encoding a glycosyltransferase involved in periplasmic membrane-derived oligosaccharide synthesis. Characterization of the suppressor mutants highlights different strategies adopted by the cell to overcome OM defects caused by impaired LPS transport.IMPORTANCE Lipopolysaccharide (LPS) is the major constituent of the outer membrane (OM) of most Gram-negative bacteria, forming a barrier against antibiotics. LPS is synthesized at the inner membrane (IM), transported across the periplasm, and assembled at the OM by the multiprotein Lpt complex. LptA is the periplasmic component of the Lpt complex, which bridges IM and OM and ferries LPS across the periplasm. How the cell coordinates the processes involved in OM biogenesis is not completely understood. We generated a mutant partially defective in lptA that exhibited increased sensitivity to antibiotics and selected for suppressors of the mutant. The analysis of two independent suppressors revealed different strategies adopted by the cell to overcome defects in LPS biogenesis.

Spheres of Hope, Packets of Doom: the Good and Bad of Outer Membrane Vesicles in Interspecies and Ecological Dynamics.

Abstract: Outer membrane vesicles (OMVs) are proteoliposome nanoparticles ubiquitously produced by Gram-negative bacteria. Typically bearing a composition similar to those of the outer membrane and periplasm of the cells from which they are derived, OMVs package an array of proteins, lipids, and nucleic acids. Once considered inconsequential by-products of bacterial growth, OMVs have since been demonstrated to mediate cellular stress relief, promote horizontal gene transfer and antimicrobial activity, and elicit metazoan inflammation. Recently, OMVs have gained appreciation as critical moderators of interorganismal dynamics. In this review, we focus on recent progress toward understanding the functions of OMVs with regard to symbiosis and ecological contexts, and we propose potential avenues for future OMV studies.

Regulation of the Locus of Enterocyte Effacement in Attaching and Effacing Pathogens

Abstract: Attaching and effacing (AE) pathogens colonize the gut mucosa using a type three secretion system (T3SS) and a suite of effector proteins. The locus of enterocyte effacement (LEE) is the defining genetic feature of the AE pathogens, encoding the T3SS and the core effector proteins necessary for pathogenesis. Extensive research has revealed a complex regulatory network that senses and responds to a myriad of host- and microbiota-derived signals in the infected gut to control transcription of the LEE. These signals include microbiota-liberated sugars and metabolites in the gut lumen, molecular oxygen at the gut epithelium, and host hormones. Recent research has revealed that AE pathogens also recognize physical signals, such as attachment to the epithelium, and that the act of effector translocation remodels gene expression in infecting bacteria. In this review, we summarize our knowledge to date and present an integrated view of how chemical, geographical, and physical cues regulate the virulence program of AE pathogens during infection.

Replication Restart in Bacteria.

Abstract: In bacteria, replication forks assembled at a replication origin travel to the terminus, often a few megabases away. They may encounter obstacles that trigger replisome disassembly, rendering replication restart from abandoned forks crucial for cell viability. During the past 25 years, the genes that encode replication restart proteins have been identified and genetically characterized. In parallel, the enzymes were purified and analyzed in vitro, where they can catalyze replication initiation in a sequence-independent manner from fork-like DNA structures. This work also revealed a close link between replication and homologous recombination, as replication restart from recombination intermediates is an essential step of DNA double-strand break repair in bacteria and, conversely, arrested replication forks can be acted upon by recombination proteins and converted into various recombination substrates. In this review, we summarize this intense period of research that led to the characterization of the ubiquitous replication restart protein PriA and its partners, to the definition of several replication restart pathways in vivo, and to the description of tight links between replication and homologous recombination, responsible for the importance of replication restart in the maintenance of genome stability.