RESUMO
Vibrio cholerae remains a major global health threat, disproportionately impacting parts of the world without adequate infrastructure and sanitation resources. In aquatic environments, V. cholerae exists both as planktonic cells and as biofilms, which are held together by an extracellular matrix. V. cholerae biofilms have been shown to be hyperinfective, but the mechanism of hyperinfectivity is unclear. Here we show that biofilm-grown cells, irrespective of the surfaces on which they are formed, are able to markedly outcompete planktonic-grown cells in the infant mouse. Using an imaging technique designed to render intestinal tissue optically transparent and preserve the spatial integrity of infected intestines, we reveal and compare three-dimensional V. cholerae colonization patterns of planktonic-grown and biofilm-grown cells. Quantitative image analyses show that V. cholerae colonizes mainly the medial portion of the small intestine and that both the abundance and localization patterns of biofilm-grown cells differ from that of planktonic-grown cells. In vitro biofilm-grown cells activate expression of the virulence cascade, including the toxin coregulated pilus (TCP), and are able to acquire the cholera toxin-carrying CTXФ phage. Overall, virulence factor gene expression is also higher in vivo when infected with biofilm-grown cells, and modulation of their regulation is sufficient to cause the biofilm hyperinfectivity phenotype. Together, these results indicate that the altered biogeography of biofilm-grown cells and their enhanced production of virulence factors in the intestine underpin the biofilm hyperinfectivity phenotype.
Assuntos
Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Regulação para Cima , Vibrio cholerae/genética , Fatores de Virulência/genética , Animais , Toxina da Cólera , Modelos Animais de Doenças , Fímbrias Bacterianas , Intestinos/diagnóstico por imagem , Intestinos/microbiologia , Intestinos/patologia , Camundongos , Fenótipo , Vibrio cholerae/crescimento & desenvolvimento , Virulência/genéticaRESUMO
MOTIVATION: We present a probabilistic model called a Joint Intervention Network (JIN) for inferring interactions among a chosen set of regulator genes. The input to the method are expression changes of downstream indicator genes observed under the knock-out of the regulators. JIN can use any number of perturbation combinations for model inference (e.g. single, double, and triple knock-outs). RESUITS/CONCLUSIONS: We applied JIN to a Vibrio cholerae regulatory network to uncover mechanisms critical to its environmental persistence. V. cholerae is a facultative human pathogen that causes cholera in humans and responsible for seven pandemics. We analyzed the expression response of 17 V. cholerae biofilm indicator genes under various single and multiple knock-outs of three known biofilm regulators. Using the inferred network, we were able to identify new genes involved in biofilm formation more accurately than clustering expression profiles.
Assuntos
Epistasia Genética , Modelos Genéticos , Modelos Estatísticos , Biofilmes/crescimento & desenvolvimento , Biometria , Redes Reguladoras de Genes , Genes Bacterianos , Humanos , Fenótipo , Vibrio cholerae/genética , Vibrio cholerae/patogenicidade , Vibrio cholerae/fisiologiaAssuntos
Biofilmes/crescimento & desenvolvimento , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Vibrio cholerae/genética , Vibrio cholerae/fisiologia , DNA Complementar/genética , Genoma Bacteriano , Humanos , Fases de Leitura Aberta , Fenótipo , Reação em Cadeia da Polimerase , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Vibrio cholerae/classificação , Vibrio cholerae/patogenicidadeRESUMO
The rugose colonial variant of Vibrio cholerae O1 El Tor produces an exopolysaccharide (EPS(ETr)) that enables the organism to form a biofilm and to resist oxidative stress and the bactericidal action of chlorine. Transposon mutagenesis of the rugose variant led to the identification of vpsR, which codes for a homologue of the NtrC subclass of response regulators. Targeted disruption of vpsR in the rugose colony genetic background yielded a nonreverting smooth-colony morphotype that produced no detectable EPS(ETr) and did not form an architecturally mature biofilm. Analysis of two genes, vpsA and vpsL, within the vps cluster of EPS(ETr) biosynthesis genes revealed that their expression is induced above basal levels in the rugose variant, compared to the smooth colonial variant, and requires vpsR. These results show that VpsR functions as a positive regulator of vpsA and vpsL and thus acts to positively regulate EPS(ETr) production and biofilm formation.
Assuntos
Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Polissacarídeos Bacterianos/biossíntese , Vibrio cholerae/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Clonagem Molecular , Elementos de DNA Transponíveis , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Marcação de Genes , Genes Reguladores , Dados de Sequência Molecular , Mutagênese Insercional , Fenótipo , Polissacarídeos Bacterianos/genética , Análise de Sequência de DNA , Transdução de Sinais , Transcrição Gênica , Vibrio cholerae/classificação , Vibrio cholerae/crescimento & desenvolvimento , Vibrio cholerae/metabolismoRESUMO
Vibrio cholerae O1 has figured prominently in the history of infectious diseases as a cause of periodic global epidemics, an affliction of refugees in areas of social strife and as the disease first subjected to modern epidemiological analysis during the classic investigations of John Snow in mid-19th century London [1]. Thus, publication of the entire genome sequence of V. cholerae O1 (biotype El Tor) in Nature [2] by a consortium of investigators from The Institute for Genomic Research, the University of Maryland and Harvard Medical School is properly regarded as an historic event that will trigger a paradigm shift in the study of this organism.
Assuntos
Cromossomos Bacterianos/genética , Genoma Bacteriano , Vibrio cholerae/genética , Vibrio cholerae/fisiologia , Cólera/epidemiologia , Cólera/microbiologia , Perfilação da Expressão Gênica , Genes Bacterianos/genética , Genômica , HumanosRESUMO
The Sac3 gene product of Chlamydomonas positively and negatively regulates the responses of the cell to sulfur limitation. In wild-type cells, arylsulfatase activity is detected only during sulfur limitation. The sac3 mutant expresses arylsulfatase activity even when grown in nutrient-replete medium, which suggests that the Sac3 protein has a negative effect on the induction of arylsulfatase activity. In contrast to its effect on arylsulfatase activity, Sac3 positively regulates the high-affinity sulfate transport system-the sac3 mutant is unable to fully induce high-affinity sulfate transport during sulfur limitation. We have complemented the sac3 mutant and cloned a cDNA copy of the Sac3 gene. The deduced amino acid sequence of the Sac3 gene product is similar to the catalytic domain of the yeast Snf1 family of serine/threonine kinases and is therefore classified as a Snf1-related kinase (SnRK). Specifically, Sac3 falls within the SnRK2 subfamily of kinases from vascular plants. In addition to the 11 subdomains common to Snf1-like serine/threonine kinases, Sac3 and the plant kinases have two additional subdomains and a highly acidic C-terminal region. The role of Sac3 in the signal transduction system that regulates the responses of Chlamydomonas to sulfur limitation is discussed.
Assuntos
Chlamydomonas reinhardtii/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Enxofre/metabolismo , Sequência de Aminoácidos , Animais , Arilsulfatases/metabolismo , Domínio Catalítico , Chlamydomonas reinhardtii/enzimologia , Chlamydomonas reinhardtii/genética , Meios de Cultura , Homeostase , Dados de Sequência Molecular , Mutagênese , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Polimorfismo Genético , Proteínas Serina-Treonina Quinases/química , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The rugose colony variant of Vibrio cholerae O1, biotype El Tor, is shown to produce an exopolysaccharide, EPSETr, that confers chlorine resistance and biofilm-forming capacity. EPSETr production requires a chromosomal locus, vps, that contains sequences homologous to carbohydrate biosynthesis genes of other bacterial species. Mutations within this locus yield chlorine-sensitive, smooth colony variants that are biofilm deficient. The biofilm-forming properties of EPSETr may enable the survival of V. cholerae O1 within environmental aquatic habitats between outbreaks of human disease.
Assuntos
Cloro/farmacologia , Genes Bacterianos , Família Multigênica , Polissacarídeos Bacterianos/biossíntese , Vibrio cholerae/fisiologia , Biofilmes , Resistência Microbiana a Medicamentos/genética , Variação Genética , Humanos , Dados de Sequência Molecular , Polissacarídeos Bacterianos/genética , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/genética , Vibrio cholerae/ultraestrutura , Microbiologia da ÁguaRESUMO
Vibrio cholerae is known to persist in aquatic environments under nutrient-limiting conditions. To analyze the possible involvement of the alternative sigma factor encoded by rpoS, which is shown to be important for survival during nutrient deprivation in several other bacterial species, a V. cholerae rpoS homolog was cloned by functional complementation of an Escherichia coli mutant by using a wild-type genomic library. Sequence analysis of the complementing clone revealed an 1.008-bp open reading frame which is predicted to encode a 336-amino-acid protein with 71 to 63% overall identity to other reported rpoS gene products. To determine the functional role of rpoS in V. cholerae, we inactivated rpoS by homologous recombination. V. cholerae strains lacking rpoS are impaired in the ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and carbon starvation. These results suggest that rpoS may be required for the persistence of V. cholerae in aquatic habitats. In addition, the rpoS mutation led to reduced production or secretion of hemagglutinin/protease. However, rpoS is not critical for in vivo survival, as determined by an infant mouse intestinal competition assay.
Assuntos
Proteínas de Bactérias/genética , Fator sigma/genética , Vibrio cholerae/fisiologia , Microbiologia da Água , Sequência de Aminoácidos , Animais , Clonagem Molecular , Expressão Gênica , Teste de Complementação Genética , Intestinos/microbiologia , Metaloendopeptidases/biossíntese , Camundongos , Dados de Sequência Molecular , Mutagênese Insercional , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Vibrio cholerae/patogenicidadeRESUMO
A Chlamydomonas reinhardtii adenosine triphosphate (ATP) sulfurylase cDNA clone (pATS1) was selected by complementing a mutation in the ATP sulfurylase gene (cysD) of Escherichia coli. E. coli cysD strains harboring pATS1 grow on medium containing sulfate as the sole sulfur source and exhibit ATP sulfurylase activity. The amino acid sequence of the C. reinhardtii ATP sulfurylase, derived from the nucleotide sequence of the complementing gene (ATS1), is 25 to 40% identical to that of ATP sulfurylases in other eukaryotic organisms and has a putative transit peptide at its amino terminus. ATP sulfurylase mRNA was present when cells were grown in sulfur-replete medium, but accumulated to higher levels when the cells were exposed to sulfur-limiting conditions. Furthermore, sulfur-stress-induced accumulation of the ATS1 transcript was reduced in a strain defective in SAC1, a gene that is critical for acclimation to sulfur-limited growth.
Assuntos
Chlamydomonas reinhardtii/genética , Regulação Enzimológica da Expressão Gênica , Sulfato Adenililtransferase/biossíntese , Enxofre/deficiência , Aclimatação , Sequência de Aminoácidos , Animais , DNA Complementar/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Biblioteca Gênica , Teste de Complementação Genética , Genoma , Dados de Sequência Molecular , Mutação , RNA Mensageiro/análise , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Sulfato Adenililtransferase/genéticaRESUMO
The sac1 mutant of Chlamydomonas reinhardtii is aberrant in most of the normal responses to sulfur limitation; it cannot synthesize arylsulfatase, does not take up sulfate as rapidly as wild-type cells, and does not synthesize periplasmic proteins that normally accumulate during sulfur-limited growth. Here, we show that the sac1 mutant dies much more rapidly than wild-type cells during sulfur deprivation; this emphasizes the vital role of the acclimation process. The loss of viability of the sac1 mutant during sulfur deprivation is only observed in the light and is mostly inhibited by DCMU. During sulfur-stress, wild-type cells, but not the sac1 mutant, downregulate photosynthesis. Thus, death of the sac1 mutant during sulfur deprivation is probably a consequence of its inability to downregulate photosynthesis. Furthermore, since SAC1 is necessary for the downregulation of photosynthesis, the process must be highly controlled and not simply the result of a general decrease in protein synthesis due to sulfur limitation. Genomic and cDNA copies of the SAC1 gene have been cloned. The deduced amino acid sequence of Sac1 is similar to an Escherichia coli gene that may involved in the response of E.coli to nutrient deprivation.
Assuntos
Proteínas de Bactérias/metabolismo , Chlamydomonas reinhardtii/metabolismo , Proteínas de Membrana , Proteínas de Membrana Transportadoras , Enxofre/deficiência , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Sequência de Bases , Chlamydomonas reinhardtii/genética , Clonagem Molecular , Regulação da Expressão Gênica , Teste de Complementação Genética , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Fenótipo , Homologia de Sequência de AminoácidosRESUMO
We have characterized sulfate transport in the unicellular green alga Chlamydomonas reinhardtii during growth under sulfur-sufficient and sulfur-deficient conditions. Both the Vmax and the substrate concentration at which sulfate transport is half of the maximum velocity of the sulfate transport (K1/2) for uptake were altered in starved cells: the Vmax increased approximately 10-fold, and the K1/2 decreased approximately 7-fold. This suggests that sulfur-deprived C. reinhardtii cells synthesize a new, high-affinity sulfate transport system. This system accumulated rapidly; it was detected in cells within 1 h of sulfur deprivation and reached a maximum by 6 h. A second response to sulfur-limited growth, the production of arylsulfatase, was apparent only after 3 h of growth in sulfur-free medium. The enhancement of sulfate transport upon sulfur starvation was prevented by cycloheximide, but not by chloramphenicol, demonstrating that protein synthesis on 80S ribosomes was required for the development of the new, high-affinity system. The transport of sulfate into the cells occurred in both the light and the dark. Inhibition of ATP formation by the antibiotics carbonylcyanide m-chlorophenylhydrazone and gramicidin-S and inhibition of either F- or P-type ATPases by N,N-dicyclohexylcarbodiimide and vanadate completely abolished sulfate uptake. Furthermore, nigericin, a carboxylate ionophore that exchanges H+ for K+, inhibited transport in both the light and the dark. Finally, uptake in the dark was strongly inhibited by valinomycin. These results suggest that sulfate transport in C. reinhardtii is an energy-dependent process and that it may be driven by a proton gradient generated by a plasma membrane ATPase.
RESUMO
Intraspecies and intergenus complementation analysis were utilized to demonstrate that photosynthesis genes are clustered in distantly related purple photosynthetic bacteria. Specifically, we show that the linkage order for genes involved in bacteriochlorophyll and carotenoid biosynthesis in Rhodospirillum centenum are arranged essentially as in Rhodobacter capsulatus and Rhodobacter sphaeroides. In addition, the location and relative distance observed between the puf and puh operons which encode for light harvesting and reaction-centre structural genes are also conserved between these species. Conservation of the photosynthesis gene cluster implies either that there are structural or regulatory constraints that limit rearrangement of the photosynthesis gene cluster or that there may have been lateral transfer of the photosynthesis gene cluster among different species of phototrophic bacteria.
Assuntos
Bacterioclorofilas/genética , Carotenoides/biossíntese , Fotossíntese , Rhodobacter/genética , Rhodospirillum/genética , Mapeamento Cromossômico , Cromossomos Bacterianos , Genes Bacterianos , Teste de Complementação Genética , Ligação Genética , Óperon , Filogenia , Rhodobacter capsulatus/genética , Rhodobacter sphaeroides/genéticaRESUMO
Rhodospirillum centenum resembles typical nonsulfur photosynthetic bacteria in a number of respects, including its ability to grow either anaerobically as a phototroph or aerobically as a heterotroph. We demonstrate, however, that R. centenum is unusual in its ability to synthesize a functional photosynthetic apparatus regardless of the presence of molecular oxygen. Aerobically expressed photopigments were shown to be functionally active, as demonstrated by the ability of heterotrophically grown cells to grow photosynthetically, without a lag, when suddenly placed under anaerobic conditions. An R. centenum mutant that has acquired the ability to repress synthesis of photopigments in the presence of oxygen was also characterized. Both the wild type and the oxygen-repressed mutant of R. centenum were found to exhibit high light intensity repression of photopigment biosynthesis. The latter result suggests that R. centenum contains separate regulatory circuits for controlling synthesis of its photochemical apparatus by light intensity and oxygen.
Assuntos
Oxigênio/metabolismo , Pigmentos Biológicos/biossíntese , Rhodospirillum/metabolismo , Luz , Microscopia Eletrônica , Fotossíntese , Rhodospirillum/crescimento & desenvolvimento , Rhodospirillum/ultraestrutura , Análise EspectralRESUMO
A genetic system has been developed for studying bacterial photosynthesis in the recently described nonsulfur purple photosynthetic bacterium Rhodospirillum centenum. Nonphotosynthetic mutants of R. centenum were obtained by enrichment for spontaneous mutations, by ethyl methanesulfonate mutagenesis coupled to penicillin selection on solid medium, and by Tn5 transposition mutagenesis with an IncP plasmid vector containing a temperature-sensitive origin of replication. In vivo and in vitro characterization of individual strains demonstrated that 38 strains contained mutations that blocked bacteriochlorophyll a biosynthesis at defined steps of the biosynthetic pathway. Collectively, these mutations were shown to block seven of eight steps of the pathway leading from protoporphyrin IX to bacteriochlorophyll a. Three mutants were isolated in which carotenoid biosynthesis was blocked early in the biosynthetic pathway; the mutants also exhibited pleiotropic effects on stability or assembly of the photosynthetic apparatus. Five mutants failed to assemble a functional reaction center complex, and seven mutants contained defects in electron transport as shown by an alteration in cytochromes. In addition, several regulatory mutants were isolated that acquired enhanced repression of bacteriochlorophyll in response to the presence of molecular oxygen. The phenotypes of these mutants are discussed in relation to those of similar mutants of Rhodobacter and other Rhodospirillum species of purple photosynthetic bacteria.