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BACKGROUND: Staphylococcus aureus isolates expressing the Panton-Valentine Leukocidin (PVL) have been related to a wide range of diseases. Recently, pvl-positive community-associated methicillin-resistant S. aureus belonging to USA1100 (ST30/CC30/SCCmec IV) lineage has emerged in Brazilian hospitals. OBJECTIVE: The aim of this work was to sequence the genome of a pvl-positive USA1100 Vancomycin-Intermediate-Resistant S. aureus (VISA) isolate from Rio de Janeiro, Brazil. METHODS: The 13420 genome was sequenced using the HiSeq 2500 platform. The draft genome, plasmids annotation, and genome analysis were performed using RAST. Comparison of the relative pvl gene expression of six S. aureus isolates was performed by qRT-PCR. RESULTS: The isolate presented the ÏPVL phage codifying for the H2b PVL protein isoform, and another prophage carrying a PVL variant named lukF and lukS-PV.2. The 13420 genome presented a high number of virulence determinants, such as genes codifying for serine-protease proteins, enterotoxins (egc), the immune evasion cluster (IEC), adhesion proteins, spermine/spermidine acetyltransferase gene (blt), superantigen-like proteins, as well as the ica operon. Point mutations at vraS, tcaA, and tcaB genes were detected. Moreover, the PVL mRNA relative expression of the 13420 isolate was five times higher than mRNA PVL levels of the USA300/ST8 reference strain. CONCLUSION: We described for the first time the genome sequence of a VISA isolate harboring two pvl-associated genes and other virulence factors that may improve the USA1100/ST30 lineage fitness and impact its pathogenicity and spreading at Brazilian hospitals.
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The human microbiome is a collection of microorganisms that inhabit every surface of the body that is exposed to the environment, generally coexisting peacefully with their host. These microbes have important functions, such as producing vitamins, aiding in maturation of the immune system, and protecting against pathogens. We have previously shown that a small-molecule extract from the human fecal microbiome has a strong repressive effect on Salmonella enterica serovar Typhimurium host cell invasion by modulating the expression of genes involved in this process. Here, we describe the characterization of this biological activity. Using a series of purification methods, we obtained fractions with biological activity and characterized them by mass spectrometry. These experiments revealed an abundance of aromatic compounds in the bioactive fraction. Selected compounds were obtained from commercial sources and tested with respect to their ability to repress the expression of hilA, the gene encoding the master regulator of invasion genes in Salmonella We found that the aromatic compound 3,4-dimethylbenzoic acid acts as a strong inhibitor of hilA expression and of invasion of cultured host cells by Salmonella Future studies should reveal the molecular details of this phenomenon, such as the signaling cascades involved in sensing this bioactive molecule.IMPORTANCE Microbes constantly sense and adapt to their environment. Often, this is achieved through the production and sensing of small extracellular molecules. The human body is colonized by complex communities of microbes, and, given their biological and chemical diversity, these ecosystems represent a platform where the production and sensing of molecules occur. In previous work, we showed that small molecules produced by microbes from the human gut can significantly impair the virulence of the enteric pathogen Salmonella enterica Here, we describe a specific compound from the human gut that produces this same effect. The results from this work not only shed light on an important biological phenomenon occurring in our bodies but also may represent an opportunity to develop drugs that can target these small-molecule interactions to protect us from enteric infections and other diseases.
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Ecologia , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , alfa-Defensinas/metabolismo , Animais , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Humanos , Interleucina-17/imunologia , Interleucina-17/metabolismo , Intestinos/imunologia , Metaloproteinase 7 da Matriz/genética , Metaloproteinase 7 da Matriz/metabolismo , Metagenoma , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , alfa-Defensinas/genética , alfa-Defensinas/imunologiaRESUMO
Individual species of bacteria and yeast present in the food of wild fruit flies work together to provide the nutrients needed for larval growth.
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Drosophila melanogaster , Microbiota , Animais , Drosophila , NutrientesRESUMO
The Vibrio parahaemolyticus Scr system modulates decisions pertinent to surface colonization by affecting the cellular level of cyclic dimeric GMP (c-di-GMP). In this work, we explore the scope and mechanism of this regulation. Transcriptome comparison of ΔscrABC and wild-type strains revealed expression differences with respect to â¼100 genes. Elevated c-di-GMP repressed genes in the surface-sensing regulon, including those encoding the lateral flagellar and type III secretion systems and N-acetylglucosamine-binding protein GpbA while inducing genes encoding other cell surface molecules and capsular polysaccharide. The transcription of a few regulatory genes was also affected, and the role of one was characterized. Mutations in cpsQ suppressed the sticky phenotype of scr mutants. cpsQ encodes one of four V. parahaemolyticus homologs in the CsgD/VpsT family, members of which have been implicated in c-di-GMP signaling. Here, we demonstrate that CpsQ is a c-di-GMP-binding protein. By using a combination of mutant and reporter analyses, CpsQ was found to be the direct, positive regulator of cpsA transcription. This c-di-GMP-responsive regulatory circuit could be reconstituted in Escherichia coli, where a low level of this nucleotide diminished the stability of CpsQ. The molecular interplay of additional known cps regulators was defined by establishing that CpsS, another CsgD family member, repressed cpsR, and the transcription factor CpsR activated cpsQ. Thus, we are developing a connectivity map of the Scr decision-making network with respect to its wiring and output strategies for colonizing surfaces and interaction with hosts; in doing so, we have isolated and reproduced a c-di-GMP-sensitive regulatory module in the circuit.
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Aderência Bacteriana , GMP Cíclico/análogos & derivados , Regulação Bacteriana da Expressão Gênica , Transcrição Gênica , Vibrio parahaemolyticus/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , GMP Cíclico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/fisiologia , Deleção de Genes , Perfilação da Expressão Gênica , Ligação Proteica , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismoRESUMO
Infection with Salmonella enterica serovar Typhi in humans causes the life-threatening disease typhoid fever. In the laboratory, typhoid fever can be modeled through the inoculation of susceptible mice with Salmonella enterica serovar Typhimurium. Using this murine model, we previously characterized the interactions between Salmonella Typhimurium and host cells in the gallbladder and showed that this pathogen can successfully invade gallbladder epithelial cells and proliferate. Additionally, we showed that Salmonella Typhimurium can use bile phospholipids to grow at high rates. These abilities are likely important for quick colonization of the gallbladder during typhoid fever and further pathogen dissemination through fecal shedding. To further characterize the interactions between Salmonella and the gallbladder environment, we compared the transcriptomes of Salmonella cultures grown in LB broth or physiological murine bile. Our data showed that many genes involved in bacterial central metabolism are affected by bile, with the citric acid cycle being repressed and alternative respiratory systems being activated. Additionally, our study revealed a new aspect of Salmonella interactions with bile through the identification of the global regulator phoP as a bile-responsive gene. Repression of phoP expression could also be achieved using physiological, but not commercial, bovine bile. The biological activity does not involve PhoPQ sensing of a bile component and is not caused by bile acids, the most abundant organic components of bile. Bioactivity-guided purification allowed the identification of a subset of small molecules from bile that can elicit full activity; however, a single compound with phoP inhibitory activity could not be isolated, suggesting that multiple molecules may act in synergy to achieve this effect. Due to the critical role of phoP in Salmonella virulence, further studies in this area will likely reveal aspects of the interaction between Salmonella and bile that are relevant to disease.
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Proteínas de Bactérias/metabolismo , Ácidos e Sais Biliares/farmacologia , Bile , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/metabolismo , Animais , Proteínas de Bactérias/genética , Bile/química , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Bovinos , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise Serial de Proteínas , Salmonella enterica/genéticaRESUMO
During the colonization of hosts, bacterial pathogens are presented with many challenges that must be overcome for colonization to occur successfully. This requires the bacterial sensing of the surroundings and adaptation to the conditions encountered. One of the major impediments to the pathogen colonization of the mammalian gastrointestinal tract is the antibacterial action of bile. Salmonella enterica serovar Typhimurium has specific mechanisms involved in resistance to bile. Additionally, Salmonella can successfully multiply in bile, using it as a source of nutrients. This accomplishment is highly relevant to pathogenesis, as Salmonella colonizes the gallbladder of hosts, where it can be carried asymptomatically and promote further host spread and transmission. To gain insights into the mechanisms used by Salmonella to grow in bile, we studied the changes elicited by Salmonella in the chemical composition of bile during growth in vitro and in vivo through a metabolomics approach. Our data suggest that phospholipids are an important source of carbon and energy for Salmonella during growth in the laboratory as well as during gallbladder infections of mice. Further studies in this area will generate a better understanding of how Salmonella exploits this generally hostile environment for its own benefit.
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Bile/metabolismo , Bile/microbiologia , Metabolômica , Fosfolipídeos/metabolismo , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/metabolismo , Animais , Carbono/metabolismo , Metabolismo Energético , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The interplay between pathogens and their hosts has been studied for decades using targeted approaches, such as the analysis of mutants and host immunological responses. Although much has been learned from such studies, they focus on individual pathways and fail to reveal the global effects of infection on the host. To alleviate this issue, high-throughput methods, such as transcriptomics and proteomics, have been used to study host-pathogen interactions. Recently, metabolomics was established as a new method to study changes in the biochemical composition of host tissues. We report a metabolomic study of Salmonella enterica serovar Typhimurium infection. Our results revealed that dozens of host metabolic pathways are affected by Salmonella in a murine infection model. In particular, multiple host hormone pathways are disrupted. Our results identify unappreciated effects of infection on host metabolism and shed light on mechanisms used by Salmonella to cause disease and by the host to counter infection.
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Interações Hospedeiro-Parasita/fisiologia , Metabolômica/métodos , Salmonelose Animal/metabolismo , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Análise de Fourier , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The importance of the mammalian intestinal microbiota to human health has been intensely studied over the past few years. It is now clear that the interactions between human hosts and their associated microbial communities need to be characterized in molecular detail if we are to truly understand human physiology. Additionally, the study of such host-microbe interactions is likely to provide us with new strategies to manipulate these complex systems to maintain or restore homeostasis in order to prevent or cure pathological states. Here, we describe the use of high-throughput metabolomics to shed light on the interactions between the intestinal microbiota and the host. We show that antibiotic treatment disrupts intestinal homeostasis and has a profound impact on the intestinal metabolome, affecting the levels of over 87% of all metabolites detected. Many metabolic pathways that are critical for host physiology were affected, including bile acid, eicosanoid, and steroid hormone synthesis. Dissecting the molecular mechanisms involved in the impact of beneficial microbes on some of these pathways will be instrumental in understanding the interplay between the host and its complex resident microbiota and may aid in the design of new therapeutic strategies that target these interactions.
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Antibacterianos/farmacologia , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Animais , Eicosanoides/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Mucosa Intestinal/efeitos dos fármacos , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Estreptomicina/farmacologiaRESUMO
Humans live in symbiosis with a diverse community of microorganisms, which has evolved to carry out many specific tasks that benefit the host, including protection against invading pathogens. Within the chemical diversity of the gastrointestinal tract, small molecules likely constitute chemical cues for the communication between the microbiota and pathogens. Therefore, we sought to investigate if molecules produced by the human gut microbiota show biological activity against the human pathogen Vibrio cholerae. To probe the effects of the gut metabolome on V. cholerae, we investigated its response to small-molecule extracts from human feces, from a complex bacterial community cultivated in vitro, and from culture supernatants of Enterocloster citroniae, Bacteroides thetaiotaomicron, and Bacteroides vulgatus. Using RNA sequencing, we determined the impact of the human gut metabolome on V. cholerae global gene expression. Among the genes downregulated in the presence of the fecal extract, the most overrepresented functional category was cell motility, which accounted for 39% of repressed genes. Repression of V. cholerae motility by the fecal extract was confirmed phenotypically, and E. citroniae extracts reproduced this phenotype. A complex in vitro microbial community led to increased motility, as did extracts from B. vulgatus, a species present in this community. Accordingly, mucin penetration was also repressed by fecal and E. citroniae extracts, suggesting that the phenotypes observed may have implications for host colonization. Together with previous studies, this work shows that small molecules from the gut metabolome may have a widespread, significant impact on microbe-microbe interactions established in the gut environment.
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Bactérias/química , Bactérias/metabolismo , Microbioma Gastrointestinal , Metaboloma , Vibrio cholerae/crescimento & desenvolvimento , Adulto , Bactérias/classificação , Bactérias/genética , Fezes/química , Fezes/microbiologia , Feminino , Regulação Bacteriana da Expressão Gênica , Humanos , Interações Microbianas , Plâncton/genética , Plâncton/fisiologia , Vibrio cholerae/genética , Vibrio cholerae/fisiologiaRESUMO
Although treatable with antibiotics, tuberculosis is a leading cause of death. Mycobacterium tuberculosis antibiotic resistance is becoming increasingly common and disease control is challenging. Conventional drug susceptibility testing takes weeks to produce results, and treatment is often initiated empirically. Therefore, new methods to determine drug susceptibility profiles are urgent. Here, we used mass-spectrometry-based metabolomics to characterize the metabolic landscape of drug-susceptible (DS), multidrug-resistant (MDR) and extensively drug-resistant (XDR) M. tuberculosis. Direct infusion mass spectrometry data showed that DS, MDR, and XDR strains have distinct metabolic profiles, which can be used to predict drug susceptibility and resistance. This was later confirmed by Ultra-High-Performance Liquid Chromatography and High-Resolution Mass Spectrometry, where we found that levels of ions presumptively identified as isoleucine, proline, hercynine, betaine, and pantothenic acid varied significantly between strains with different drug susceptibility profiles. We then confirmed the identification of proline and isoleucine and determined their absolute concentrations in bacterial extracts, and found significantly higher levels of these amino acids in DS strains, as compared to drug-resistant strains (combined MDR and XDR strains). Our results advance the current understanding of the effect of drug resistance on bacterial metabolism and open avenues for the detection of drug resistance biomarkers.
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Antituberculosos/farmacologia , Tuberculose Extensivamente Resistente a Medicamentos/metabolismo , Metaboloma/fisiologia , Metabolômica/métodos , Mycobacterium tuberculosis/metabolismo , Tuberculose Extensivamente Resistente a Medicamentos/tratamento farmacológico , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificaçãoRESUMO
Bacteria communicate through the production of diffusible signal molecules termed autoinducers. The molecules are produced at basal levels and accumulate during growth. Once a critical concentration has been reached, autoinducers can activate or repress a number of target genes. Because the control of gene expression by autoinducers is cell-density-dependent, this phenomenon has been called quorum sensing. Quorum sensing controls virulence gene expression in numerous micro-organisms. In some cases, this phenomenon has proven relevant for bacterial virulence in vivo. In this article, we provide a few examples to illustrate how quorum sensing can act to control bacterial virulence in a multitude of ways. Several classes of autoinducers have been described to date and we present examples of how each of the major types of autoinducer can be involved in bacterial virulence. As quorum sensing controls virulence, it has been considered an attractive target for the development of new therapeutic strategies. We discuss some of the new strategies to combat bacterial virulence based on the inhibition of bacterial quorum sensing systems.
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Escherichia coli/patogenicidade , Percepção de Quorum , Staphylococcus aureus/patogenicidade , Proteínas de Bactérias/metabolismo , Liases de Carbono-Enxofre/metabolismo , Regulação Bacteriana da Expressão Gênica , Peptídeos Cíclicos , VirulênciaRESUMO
We show that dimethyl sulfoxide (DMSO) inhibits Salmonella hilA expression and that this inhibition is stronger under anaerobiosis. Because DMSO can be reduced to dimethyl sulfide (DMS) during anaerobic growth, we hypothesized that DMS was responsible for hilA inhibition. Indeed, DMS strongly inhibited the expression of hilA and multiple Salmonella pathogenicity island 1 (SPI-1)-associated genes as well as the invasion of cultured epithelial cells. Because DMSO and DMS are widespread in nature, we hypothesize that this phenomenon may contribute to environmental sensing by Salmonella.
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Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Células Epiteliais/microbiologia , Salmonella/efeitos dos fármacos , Sulfetos/farmacologia , Transativadores/antagonistas & inibidores , Anaerobiose , Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Salmonella/fisiologiaRESUMO
Salmonella species can cause typhoid fever and gastroenteritis in humans and pose a global threat to human health. In order to establish a successful infection, Salmonella utilize a large number of genes encoding a variety of virulence factors. Different animal models of infection have been used to better understand the mechanisms underlying each disease including cattle, rodents, and nematodes. To date, a number of different bacterial virulence factors have been identified using such animal models, most of which are secreted by two type three secretion systems (T3SS) encoded within Salmonella pathogenicity islands (SPI) 1 and 2. These proteins alter various host cell pathways, facilitating the invasion of epithelial cells during infection, as well as the survival and replication of Salmonella inside phagocytic cells. On the other hand, host genetics and resistance also play a role in the susceptibility to Salmonella infection. The natural resistance-associated macrophage protein 1 (Nramp1), for example, is critical for host defense, since mice lacking Nramp1 fail to control bacterial replication and succumb to low doses of S. Typhimurium. In this chapter, we analyze the different pathogen and host factors that play a role in the dynamic interaction between Salmonella and its host and their impact on disease.
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Trato Gastrointestinal/imunologia , Interações Hospedeiro-Patógeno/imunologia , Infecções por Salmonella/imunologia , Salmonella/patogenicidade , Animais , Proteínas de Transporte de Cátions/genética , Bovinos , Modelos Animais de Doenças , Trato Gastrointestinal/microbiologia , Ilhas Genômicas/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Camundongos , Salmonella/genética , Infecções por Salmonella/genética , Infecções por Salmonella/microbiologia , Virulência/genética , Fatores de Virulência/genéticaAssuntos
Salmonelose Animal/imunologia , Infecções por Salmonella/imunologia , Animais , Feminino , Humanos , MasculinoRESUMO
Bacteria have been long considered primitive organisms, with a lifestyle focused on the survival and propagation of single cells. However, in the past few decades it became obvious that bacteria can display sophisticated group behaviors. For instance, bacteria can communicate amongst themselves and with their hosts, by producing, sensing, and responding to chemical signals. By doing so, they can sense their surroundings and adapt as to increase their chances of survival and propagation. Here, we review the discovery of bacterial intercellular communication, some of the signaling molecules identified to date, the role of intercellular signaling in symbiotic and pathogenic relationships between bacteria and their hosts and its implications for the development of new therapeutic strategies against human disease.
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Fenômenos Fisiológicos Bacterianos , Percepção de Quorum , Transdução de Sinais , Interações Hospedeiro-Patógeno , HumanosRESUMO
Staphylococcus epidermidis is one of the leading causes of bloodstream infections, particularly in premature neonates, and biofilm formation is a major virulence factor. We characterized biofilm formation by 50â¯S. epidermidis neonatal isolates under osmotic stress and evaluated the expression of biofilm-associated genes. Phenotypical analyses of biofilm production were performed in culture medium with or without addition of NaCl or glucose. In control medium (no additions), most isolates (84%) were nonproducers or weak biofilm producers. Growth in NaCl-containing medium increased the number of moderate/strong producers, and this increase was even greater in medium containing glucose. Most of the protein-enriched biofilms (60%) could be observed only during growth in glucose, whereas 50% of the polysaccharide-enriched biofilms were observed during growth in NaCl. Studies that evaluate the conditions used to characterize biofilm production are important to help us understand the dynamics of this important virulence factor in S. epidermidis and their impact on neonatal infections.
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Biofilmes/crescimento & desenvolvimento , Pressão Osmótica , Staphylococcus epidermidis/fisiologia , Biofilmes/efeitos dos fármacos , Meios de Cultura/química , DNA Bacteriano/genética , Expressão Gênica , Glucose/farmacologia , Humanos , Recém-Nascido , Fenótipo , Cloreto de Sódio/farmacologia , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/efeitos dos fármacosRESUMO
During the last decades it has become increasingly clear that the microbes that live on and in humans are critical for health. The communities they form, termed microbiomes, are involved in fundamental processes such as the maturation and constant regulation of the immune system. Additionally, they constitute a strong defense barrier to invading pathogens, and are also intricately linked to nutrition. The parameters that affect the establishment and maintenance of these microbial communities are diverse, and include the genetic background, mode of birth, nutrition, hygiene, and host lifestyle in general. Here, we describe the characterization of the gut microbiome of individuals living in the Amazon, and the comparison of these microbial communities to those found in individuals from an urban, industrialized setting. Our results showed striking differences in microbial communities from these two types of populations. Additionally, we used high-throughput metabolomics to study the chemical ecology of the gut environment and found significant metabolic changes between the two populations. Although we cannot point out a single cause for the microbial and metabolic changes observed between Amazonian and urban individuals, they are likely to include dietary differences as well as diverse patterns of environmental exposure. To our knowledge, this is the first description of gut microbial and metabolic profiles in Amazonian populations, and it provides a starting point for thorough characterizations of the impact of individual environmental conditions on the human microbiome and metabolome.
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In Vibrio parahaemolyticus, scrC participates in controlling the decision to be a highly mobile swarmer cell or a more adhesive, biofilm-proficient cell type. scrC mutants display decreased swarming motility over surfaces and enhanced capsular polysaccharide production. ScrC is a cytoplasmic membrane protein that contains both GGDEF and EAL conserved protein domains. These domains have been shown in many organisms to respectively control the formation and degradation of the small signaling nucleotide cyclic dimeric GMP (c-di-GMP). The scrC gene is part of the three-gene scrABC operon. Here we report that this operon influences the cellular nucleotide pool and that c-di-GMP levels inversely modulate lateral flagellar and capsular polysaccharide gene expression. High concentrations of this nucleotide prevent swarming and promote adhesiveness. Further, we demonstrate that ScrC has intrinsic diguanylate cyclase and phosphodiesterase activities, and these activities are controlled by ScrAB. Specifically, ScrC acts to form c-di-GMP in the absence of ScrA and ScrB; whereas ScrC acts to degrade c-di-GMP in the presence of ScrA and ScrB. The scrABC operon is specifically induced by growth on a surface, and the analysis of mutant phenotypes supports a model in which the phosphodiesterase activity of ScrC plays a dominant role during surface translocation and in biofilms.
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Proteínas de Bactérias/metabolismo , GMP Cíclico/farmacologia , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/metabolismo , Vibrio parahaemolyticus/crescimento & desenvolvimento , Aderência Bacteriana , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , GMP Cíclico/metabolismo , Dimerização , Proteínas de Escherichia coli , Flagelos/genética , Flagelos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutação , Óperon , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Fósforo-Oxigênio Liases/química , Fósforo-Oxigênio Liases/genética , Fósforo-Oxigênio Liases/metabolismo , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismoRESUMO
Vibrio fischeri quorum sensing involves the LuxI and LuxR proteins. The LuxI protein generates the quorum-sensing signal N-3-oxohexanoyl-l-homoserine lactone (3OC6-HSL), and LuxR is a signal-responsive transcriptional regulator which activates the luminescence (lux) genes and 17 other V. fischeri genes. For activation of the lux genes, LuxR binds to a 20-base-pair inverted repeat, the lux box, which is centered 42.5 base pairs upstream of the transcriptional start of the lux operon. Similar lux box-like elements have been identified in only a few of the LuxR-activated V. fischeri promoters. To better understand the DNA sequence elements required for LuxR binding and to identify binding sites in LuxR-regulated promoters other than the lux operon promoter, we have systematically mutagenized the lux box and evaluated the activity of many mutants. By doing so, we have identified nucleotides that are critical for promoter activity. Interestingly, certain lux box mutations allow a 3OC6-HSL-independent LuxR activation of the lux operon promoter. We have used the results of the mutational analysis to create a consensus lux box, and we have used this consensus sequence to identify LuxR binding sites in 3OC6-HSL-activated genes for which lux boxes could not be identified previously.