RESUMO
Type IV pili (T4P) are ubiquitous and versatile bacterial cell surface structures involved in adhesion to host cells, biofilm formation, motility, and DNA uptake. In Gram-negative bacteria, T4P pass the outer membrane (OM) through the large, oligomeric, ring-shaped secretin complex. In the ß-proteobacterium Neisseria gonorrhoeae, the native PilQ secretin ring embedded in OM sheets is surrounded by an additional peripheral structure, consisting of a peripheral ring and seven extending spikes. To unravel proteins important for formation of this additional structure, we identified proteins that are present with PilQ in the OM. One such protein, which we name T4P secretin-associated protein (TsaP), was identified as a phylogenetically widely conserved component of the secretin complex that co-occurs with genes for T4P in Gram-negative bacteria. TsaP contains an N-terminal carbohydrate-binding lysin motif (LysM) domain and a C-terminal domain of unknown function. In N. gonorrhoeae, lack of TsaP results in the formation of membrane protrusions containing multiple T4P, concomitant with reduced formation of surface-exposed T4P. Lack of TsaP did not affect the oligomeric state of PilQ, but resulted in loss of the peripheral structure around the PilQ secretin. TsaP binds peptidoglycan and associates strongly with the OM in a PilQ-dependent manner. In the δ-proteobacterium Myxococcus xanthus, TsaP is also important for surface assembly of T4P, and it accumulates and localizes in a PilQ-dependent manner to the cell poles. Our results show that TsaP is a novel protein associated with T4P function and suggest that TsaP functions to anchor the secretin complex to the peptidoglycan.
Assuntos
Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Lipoproteínas/metabolismo , Neisseria gonorrhoeae/metabolismo , Western Blotting , Biologia Computacional , Eletroforese em Gel de Poliacrilamida , Proteínas de Fímbrias/isolamento & purificação , Lipoproteínas/genética , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Neisseria gonorrhoeae/genética , Peptidoglicano/metabolismo , Estrutura Terciária de ProteínaRESUMO
Corallococcus coralloides, like most other myxobacteria, undergoes a developmental program culminating in the formation of fruiting bodies. C. coralloides fruiting bodies are morphologically distinct from those of other fruiting myxobacteria for which full-length genome sequences are available. The genome sequence of the 10.0-Mb C. coralloides genome is presented herein.
Assuntos
Deltaproteobacteria/crescimento & desenvolvimento , Deltaproteobacteria/genética , Genoma Bacteriano , Sequência de Bases , Deltaproteobacteria/classificação , Deltaproteobacteria/isolamento & purificação , Dados de Sequência MolecularRESUMO
Genetic programs underlying multicellular morphogenesis and cellular differentiation are most often associated with eukaryotic organisms, but examples also exist in bacteria such as the formation of multicellular, spore-filled fruiting bodies in the order Myxococcales. Most members of the Myxococcales undergo a multicellular developmental program culminating in the formation of spore-filled fruiting bodies in response to starvation. To gain insight into the evolutionary history of fruiting body formation in Myxococcales, we performed a comparative analysis of the genomes and transcriptomes of five Myxococcales species, four of these undergo fruiting body formation (Myxococcus xanthus, Stigmatella aurantiaca, Sorangium cellulosum, and Haliangium ochraceum) and one does not (Anaeromyxobacter dehalogenans). Our analyses show that a set of 95 known M. xanthus development-specific genes--although suffering from a sampling bias--are overrepresented and occur more frequently than an average M. xanthus gene in S. aurantiaca, whereas they occur at the same frequency as an average M. xanthus gene in S. cellulosum and in H. ochraceum and are underrepresented in A. dehalogenans. Moreover, genes for entire signal transduction pathways important for fruiting body formation in M. xanthus are conserved in S. aurantiaca, whereas only a minority of these genes are conserved in A. dehalogenans, S. cellulosum, and H. ochraceum. Likewise, global gene expression profiling of developmentally regulated genes showed that genes that upregulated during development in M. xanthus are overrepresented in S. aurantiaca and slightly underrepresented in A. dehalogenans, S. cellulosum, and H. ochraceum. These comparative analyses strongly indicate that the genetic programs for fruiting body formation in M. xanthus and S. aurantiaca are highly similar and significantly different from the genetic program directing fruiting body formation in S. cellulosum and H. ochraceum. Thus, our analyses reveal an unexpected level of plasticity in the genetic programs for fruiting body formation in the Myxococcales and strongly suggest that the genetic program underlying fruiting body formation in different Myxococcales is not conserved. The evolutionary implications of this finding are discussed.
Assuntos
Myxococcales/genética , Esporos Bacterianos/fisiologia , Perfilação da Expressão Gênica , Genoma Bacteriano , Myxococcales/fisiologia , Proteobactérias/genéticaRESUMO
OBJECTIVE: To describe the process, efficacy and safety of intravenous thrombolysis for acute ischaemic stroke in an emergency department (ED) setting with remote specialist support through structured telephone consultation. DESIGN: Retrospective case series. SETTING: Three EDs within a single stroke service in northern England. PARTICIPANTS: Patients with acute stroke given intravenous thrombolytic therapy between 6 September 2007 and 1 October 2010. OUTCOME MEASURES: Combined death and dependency at 90 days (0-2 on the modified Rankin Scale for a good outcome vs 3-6 for a poor outcome), door-to-needle time, neurological impairment and presence of treatment related haemorrhage. RESULTS: 192 patients received intravenous thrombolysis. 94/178 (53%) were treated after remote specialist assessment. Data available from 178 patients showed similar proportions with a good outcome after each mode of assessment (56% in person and 48% by telephone). The median door-to-needle time was 8 min faster in the group assessed in person (65 vs 73 min by telephone) but there was no difference in neurological outcome or symptomatic haemorrhage. After review in person, the stroke specialist tended to treat patients with a higher median modified Rankin Scale (1 vs 0 by telephone). CONCLUSION: In a single stroke service the clinical outcomes of treatment with intravenous thrombolysis were similar whether assessment was performed after specialist review in person or via a telemedicine service consisting of ED staff training, telephone consultation and remote review of brain imaging by a stroke specialist.
Assuntos
Isquemia Encefálica/diagnóstico , Isquemia Encefálica/terapia , Consulta Remota , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/terapia , Terapia Trombolítica , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos Clínicos , Árvores de Decisões , Serviço Hospitalar de Emergência , Inglaterra , Feminino , Fibrinolíticos/administração & dosagem , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Telefone , Fatores de Tempo , Resultado do TratamentoRESUMO
A synbiotic containing Lactiplantibacillus plantarum [American Type Culture Collection (ATCC) strain identifier 202195] and fructooligosaccharide was reported to reduce the risk of sepsis in young infants in rural India. Here, the whole genome of two isolates of L. plantarum ATCC 202195, which were deposited to the ATCC approximately 20 years apart, were sequenced and analyzed to verify their taxonomic and strain-level identities, identify potential antimicrobial resistant genes and virulence factors, and identify genetic characteristics that may explain the observed clinical effects of L. plantarum ATCC 202195. Minimum inhibitory concentrations for selected antimicrobial agents were determined using broth dilution and gradient strip diffusion techniques. The two L. plantarum ATCC 202195 isolates were genetically identical with only three high-quality single nucleotides polymorphisms identified, and with an average nucleotide identity of 99.99%. In contrast to previously published reports, this study determined that each isolate contained two putative plasmids. No concerning acquired or transferable antimicrobial resistance genes or virulence factors were identified. Both isolates were sensitive to several clinically important antibiotics including penicillin, ampicillin and gentamicin, but resistant to vancomycin. Genes involved in stress response, cellular adhesion, carbohydrate metabolism and vitamin biosynthesis are consistent with features of probiotic organisms.
Assuntos
Lactobacillus plantarum/efeitos dos fármacos , Lactobacillus plantarum/genética , Antibacterianos/farmacologia , Genoma Bacteriano/efeitos dos fármacos , Genômica , Índia , Lactobacillus plantarum/metabolismo , Testes de Sensibilidade Microbiana , Plasmídeos/efeitos dos fármacos , Probióticos , Simbióticos , Fatores de Virulência/genéticaRESUMO
Social behavior in the bacterium Myxococcus xanthus relies on contact-dependent activities involving cell-cell and cell-substratum interactions. To identify outer membrane proteins that have a role in these activities, we profiled the outer membrane proteome of growing and starving cells using two strategies. First, outer membrane proteins were enriched by biotinylation of intact cells using the reagent NHS (N-hydroxysuccinimide)-PEO(12) (polyethylene oxide)-biotin with subsequent membrane solubilization and affinity chromatography. Second, the proteome of outer membrane vesicles (OMV) was determined. Comparisons of detected proteins show that these methods have different detection profiles and together provide a comprehensive view of the outer membrane proteome. From 362 proteins identified, 274 (76%) were cell envelope proteins including 64 integral outer membrane proteins and 85 lipoproteins. The majority of these proteins were of unknown function. Among integral outer membrane proteins with homologues of known function, TonB-dependent transporters comprise the largest group. Our data suggest novel functions for these transporters. Among lipoproteins with homologues of known function, proteins with hydrolytic functions comprise the largest group. The luminal load of OMV was enriched for proteins with hydrolytic functions. Our data suggest that OMV have functions in predation and possibly in transfer of intercellular signaling molecules between cells.
Assuntos
Proteínas da Membrana Bacteriana Externa/análise , Myxococcus xanthus/metabolismo , Proteoma/análise , Proteômica/métodos , Aderência Bacteriana , Biotinilação , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Cromatografia de Afinidade , Microscopia Eletrônica , Myxococcus xanthus/fisiologia , Myxococcus xanthus/ultraestrutura , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND: Myxococcus xanthus is a Gram negative bacterium that can differentiate into metabolically quiescent, environmentally resistant spores. Little is known about the mechanisms involved in differentiation in part because sporulation is normally initiated at the culmination of a complex starvation-induced developmental program and only inside multicellular fruiting bodies. To obtain a broad overview of the sporulation process and to identify novel genes necessary for differentiation, we instead performed global transcriptome analysis of an artificial chemically-induced sporulation process in which addition of glycerol to vegetatively growing liquid cultures of M. xanthus leads to rapid and synchronized differentiation of nearly all cells into myxospore-like entities. RESULTS: Our analyses identified 1 486 genes whose expression was significantly regulated at least two-fold within four hours of chemical-induced differentiation. Most of the previously identified sporulation marker genes were significantly upregulated. In contrast, most genes that are required to build starvation-induced multicellular fruiting bodies, but which are not required for sporulation per se, were not significantly regulated in our analysis. Analysis of functional gene categories significantly over-represented in the regulated genes, suggested large rearrangements in core metabolic pathways, and in genes involved in protein synthesis and fate. We used the microarray data to identify a novel operon of eight genes that, when mutated, rendered cells unable to produce viable chemical- or starvation-induced spores. Importantly, these mutants displayed no defects in building fruiting bodies, suggesting these genes are necessary for the core sporulation process. Furthermore, during the starvation-induced developmental program, these genes were expressed in fruiting bodies but not in peripheral rods, a subpopulation of developing cells which do not sporulate. CONCLUSIONS: These results suggest that microarray analysis of chemical-induced spore formation is an excellent system to specifically identify genes necessary for the core sporulation process of a Gram negative model organism for differentiation.
Assuntos
Perfilação da Expressão Gênica , Myxococcus xanthus/genética , Esporos Bacterianos/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Glicerol/farmacologia , Myxococcus xanthus/citologia , Myxococcus xanthus/efeitos dos fármacos , Myxococcus xanthus/fisiologia , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/fisiologiaRESUMO
The Gram-negative metal ion-reducing bacterium Shewanella oneidensis MR-1 is motile by means of a single polar flagellum. We identified two potential stator systems, PomAB and MotAB, each individually sufficient as a force generator to drive flagellar rotation. Physiological studies indicate that PomAB is sodium-dependent while MotAB is powered by the proton motive force. Flagellar function mainly depends on the PomAB stator; however, the presence of both stator systems under low-sodium conditions results in a faster swimming phenotype. Based on stator homology analysis we speculate that MotAB has been acquired by lateral gene transfer as a consequence of adaptation to a low-sodium environment. Expression analysis at the single cell level showed that both stator systems are expressed simultaneously. An active PomB-mCherry fusion protein effectively localized to the flagellated cell pole in 70-80% of the population independent of sodium concentrations. In contrast, polar localization of MotB-mCherry increased with decreasing sodium concentrations. In the absence of the Pom stator, MotB-mCherry localized to the flagellated cell pole independently of the sodium concentration but was rapidly displaced upon expression of PomAB. We propose that selection of the stator occurs at the level of protein localization in response to sodium concentrations.
Assuntos
Flagelos/metabolismo , Proteínas Motores Moleculares/metabolismo , Shewanella/metabolismo , Sódio/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/genética , Flagelos/genética , Teste de Complementação Genética , Proteínas Motores Moleculares/genética , Filogenia , Deleção de Sequência , Shewanella/genética , Especificidade por Substrato , Transcrição GênicaRESUMO
Proteins of two-component systems (TCS) have essential functions in the sensing of external and self-generated signals in bacteria and in the generation of appropriate output responses. Accordingly, in Myxococcus xanthus, TCS are important for normal motility and fruiting body formation and sporulation. Here we analyzed the M. xanthus genome for the presence and genetic organization of genes encoding TCS. Two hundred seventy-two TCS genes were identified, 251 of which are not part of che gene clusters. We report that the TCS genes are unusually organized, with 55% being orphan and 16% in complex gene clusters whereas only 29% display the standard paired gene organization. Hybrid histidine protein kinases and histidine protein kinases predicted to be localized to the cytoplasm are overrepresented among proteins encoded by orphan genes or in complex gene clusters. Similarly, response regulators without output domains are overrepresented among proteins encoded by orphan genes or in complex gene clusters. The most frequently occurring output domains in response regulators are involved in DNA binding and cyclic-di-GMP metabolism. Our analyses suggest that TCS encoded by orphan genes and complex gene clusters are functionally distinct from TCS encoded by paired genes and that the connectivity of the pathways made up of TCS encoded by orphan genes and complex gene clusters is different from that of pathways involving TCS encoded by paired genes. Experimentally, we observed that orphan TCS genes are overrepresented among genes that display altered transcription during fruiting body formation. The systematic analysis of the 25 orphan genes encoding histidine protein kinases that are transcriptionally up-regulated during development showed that 2 such genes are likely essential for viability and identified 7 histidine protein kinases, including 4 not previously characterized that have important function in fruiting body formation or spore germination.
Assuntos
Regulação Bacteriana da Expressão Gênica , Myxococcus xanthus/genética , Transdução de Sinais , Biologia Computacional , Perfilação da Expressão Gênica , Genes Bacterianos , Genoma Bacteriano , Histidina Quinase , Locomoção/genética , Myxococcus xanthus/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/fisiologia , Análise de Sequência de DNA , Esporos Bacterianos/genéticaRESUMO
BACKGROUND AND PURPOSE: Surface neuromuscular electrical stimulation (sNMES) after stroke aims to improve upper limb function and reduce shoulder pain, but current evidence of effectiveness is inconclusive. We have undertaken a randomized controlled trial to evaluate sNMES to the shoulder after acute stroke. METHODS: One hundred seventy-six patients, within 10 days of stroke onset, were randomized to receive sNMES or placebo in addition to stroke unit care. The primary outcome measure was upper limb function measured by the Action Research Arm Test (ARAT) 3 months after stroke. Secondary outcome measures included other measures of upper limb function, upper limb impairment, pain, disability, and global health status. Outcome assessments were blinded. RESULTS: There was no difference in arm function between groups in terms of the primary outcome measure. The median ARAT at 3 months was 50 in the intervention group and 55.5 in the control group (P=0.068). Significant differences were seen at 3 months in favor of the control group for other measures of arm function and impairment: grasp and gross movement subsections of the ARAT, Frenchay Arm Test, and the arm subsection of the Motricity Index. Secondary analysis suggested that these differences were most marked in subjects with severe initial upper limb weakness. CONCLUSIONS: A 4-week program of sNMES to the shoulder after acute stroke does not improve functional outcome and may worsen arm function in severely impaired stroke patients. "Routine" use of sNMES to the proximal affected upper limb after acute stroke cannot be recommended.
Assuntos
Terapia por Estimulação Elétrica/métodos , Ombro , Acidente Vascular Cerebral/fisiopatologia , Acidente Vascular Cerebral/terapia , Doença Aguda , Idoso , Idoso de 80 Anos ou mais , Terapia por Estimulação Elétrica/efeitos adversos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/fisiologia , Recuperação de Função Fisiológica , Ombro/fisiologia , Acidente Vascular Cerebral/epidemiologia , Fatores de Tempo , Resultado do TratamentoRESUMO
Hallmarks of the myxobacteria include the formation of spore-filled fruiting bodies in response to starvation and synthesis of secondary metabolites. Myxococcus stipitatus forms morphologically highly distinct fruiting bodies and produces secondary metabolites with antibiotic or cytotoxic activities. Here, we present the 10.35-Mb genome sequence of M. stipitatus strain DSM 14675.
RESUMO
In bacteria, large G domain GTPases have well-established functions in translation, protein translocation, tRNA modification and ribosome assembly. In addition, bacteria also contain small Ras-like GTPases consisting of stand-alone G domains. Recent data have revealed that small Ras-like GTPases as well as large G domain GTPases in bacteria function in the regulation of cell polarity, signal transduction and possibly also in cell division. The small Ras-like GTPase MglA together with its cognate GAP MglB regulates cell polarity in Myxococcus xanthus, and the small Ras-like GTPase CvnD9 in Streptomyces coelicolor is involved in signal transduction. Similarly, the large GTPase FlhF together with the ATPase FlhG regulates the localization and number of flagella in polarly flagellated bacteria. Moreover, large dynamin-like GTPases in bacteria may function in cell division. Thus, the function of GTPases in bacteria may be as pervasive as in eukaryotes.
Assuntos
Polaridade Celular , GTP Fosfo-Hidrolases/metabolismo , Myxococcus xanthus/enzimologia , Myxococcus xanthus/fisiologia , Transdução de Sinais , Streptomyces coelicolor/enzimologia , Streptomyces coelicolor/fisiologia , Modelos BiológicosRESUMO
Most genes are conserved in mammals, but certain gene families have acquired large numbers of lineage-specific loci through repeated rounds of gene duplication, divergence, and loss that have continued in each mammalian group. One such family encodes KRAB-zinc finger (KRAB-ZNF) proteins, which function as transcriptional repressors. One particular subfamily of KRAB-ZNF genes, including ZNF91, has expanded specifically in primates to comprise more than 110 loci in the human genome. Genes of the ZNF91 subfamily reside in large gene clusters near centromeric regions of human chromosomes 19 and 7 with smaller clusters or isolated copies in other locations. Phylogenetic analysis indicates that many of these genes arose before the split between the New and Old World monkeys, but the ZNF91 subfamily has continued to expand and diversify throughout the evolution of apes and humans. Paralogous loci are distinguished by divergence within their zinc finger arrays, indicating selection for proteins with different regulatory targets. In addition, many loci produce multiple alternatively spliced transcripts encoding proteins that may serve separate and perhaps even opposing regulatory roles because of the modular motif structure of KRAB-ZNF genes. The tissue-specific expression patterns and rapid structural divergence of ZNF91 subfamily genes suggest a role in determining gene expression differences between species and the evolution of novel primate traits.
Assuntos
Evolução Molecular , Primatas/genética , Dedos de Zinco/genética , Animais , Cromossomos Humanos Par 19 , Cromossomos Humanos Par 7 , DNA Intergênico , Bases de Dados Factuais , Dosagem de Genes , Duplicação Gênica , Genoma Humano , Humanos , Fatores de Transcrição Kruppel-Like/genética , Família Multigênica , Filogenia , Mapeamento Físico do Cromossomo , Proteínas Repressoras/genética , Análise de Sequência de DNARESUMO
Krüppel-type zinc finger (ZNF) motifs are prevalent components of transcription factor proteins in all eukaryotes. KRAB-ZNF proteins, in which a potent repressor domain is attached to a tandem array of DNA-binding zinc-finger motifs, are specific to tetrapod vertebrates and represent the largest class of ZNF proteins in mammals. To define the full repertoire of human KRAB-ZNF proteins, we searched the genome sequence for key motifs and then constructed and manually curated gene models incorporating those sequences. The resulting gene catalog contains 423 KRAB-ZNF protein-coding loci, yielding alternative transcripts that altogether predict at least 742 structurally distinct proteins. Active rounds of segmental duplication, involving single genes or larger regions and including both tandem and distributed duplication events, have driven the expansion of this mammalian gene family. Comparisons between the human genes and ZNF loci mined from the draft mouse, dog, and chimpanzee genomes not only identified 103 KRAB-ZNF genes that are conserved in mammals but also highlighted a substantial level of lineage-specific change; at least 136 KRAB-ZNF coding genes are primate specific, including many recent duplicates. KRAB-ZNF genes are widely expressed and clustered genes are typically not coregulated, indicating that paralogs have evolved to fill roles in many different biological processes. To facilitate further study, we have developed a Web-based public resource with access to gene models, sequences, and other data, including visualization tools to provide genomic context and interaction with other public data sets.