RESUMO
Symbiotic associations are ubiquitous in the microbial world and have a major role in shaping the evolution of both partners. One of the most interesting mutualistic relationships exists between protozoa and methanogenic archaea in the fermentative forestomach (rumen) of ruminant animals. Methanogens reside within and on the surface of protozoa as symbionts, and interspecies hydrogen transfer is speculated to be the main driver for physical associations observed between the two groups. In silico analyses of several rumen methanogen genomes have previously shown that up to 5% of genes encode adhesin-like proteins, which may be central to rumen interspecies attachment. We hypothesized that adhesin-like proteins on methanogen cell surfaces facilitate attachment to protozoal hosts. Using phage display technology, we have identified a protein (Mru_1499) from Methanobrevibacter ruminantiumâ M1 as an adhesin that binds to a broad range of rumen protozoa (including the genera Epidinium and Entodinium). This unique adhesin also binds the cell surface of the bacterium Butyrivibrio proteoclasticus, suggesting a broad adhesion spectrum for this protein.
Assuntos
Adesinas Bacterianas/metabolismo , Proteínas Arqueais/metabolismo , Cilióforos/microbiologia , Hidrogênio/metabolismo , Metano/metabolismo , Methanobrevibacter/metabolismo , Rúmen/microbiologia , Adesinas Bacterianas/genética , Animais , Proteínas Arqueais/genética , Bovinos , Cilióforos/fisiologia , Methanobrevibacter/classificação , Methanobrevibacter/genética , Methanobrevibacter/isolamento & purificação , Rúmen/parasitologiaRESUMO
SsoPox, a bifunctional enzyme with organophosphate hydrolase and N-acyl homoserine lactonase activities from the hyperthermophilic archaeon Sulfolobus solfataricus, was overexpressed and purified from recombinant Pseudomonas putida KT2440 with a yield of 9.4 mg of protein per liter of culture. The enzyme has a preference for N-acyl homoserine lactones (AHLs) with acyl chain lengths of at least 8 carbon atoms, mainly due to lower K(m) values for these substrates. The highest specificity constant obtained was for N-3-oxo-decanoyl homoserine lactone (k(cat)/K(m) = 5.5 × 10(3) M(-1)·s(-1)), but SsoPox can also degrade N-butyryl homoserine lactone (C(4)-HSL) and N-oxo-dodecanoyl homoserine lactone (oxo-C(12)-HSL), which are important for quorum sensing in our Pseudomonas aeruginosa model system. When P. aeruginosa PAO1 cultures were grown in the presence of SsoPox-immobilized membranes, the production of C(4)-HSL- and oxo-C(12)-HSL-regulated virulence factors, elastase, protease, and pyocyanin were significantly reduced. This is the first demonstration that immobilized quorum-quenching enzymes can be used to attenuate the production of virulence factors controlled by quorum-sensing signals.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Percepção de Quorum , Sulfolobus solfataricus/enzimologia , 4-Butirolactona/análogos & derivados , 4-Butirolactona/química , 4-Butirolactona/metabolismo , Hidrolases de Éster Carboxílico/genética , Eletroforese em Gel de Poliacrilamida , Enzimas Imobilizadas/metabolismo , Expressão Gênica , Lactonas/química , Lactonas/metabolismo , Elastase Pancreática/biossíntese , Peptídeo Hidrolases/biossíntese , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Hidrolases de Triester Fosfórico/metabolismo , Reação em Cadeia da Polimerase , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/patogenicidade , Piocianina/biossíntese , Análise de Sequência de DNA , Transdução de Sinais , Sulfolobus solfataricus/genética , Fatores de Virulência/biossínteseRESUMO
Raspberry ringspot virus (RpRSV) is an important virus that infects horticultural crops including grapevine, cherry, berry fruit and rose. The genome sequences of RpRSV are highly diverse between isolates and this makes the design of a PCR-based detection method difficult. In this study, a TaqMan real-time RT-PCR assay was developed for the rapid and sensitive detection of RpRSV. Primers and probes targeting the most conserved region of the movement protein gene were designed to amplify a 229 bp fragment of RpRSV RNA-2. The assay was able to amplify all RpRSV isolates tested. The detection limit of the RpRSV target region was estimated to be 61-98 copies, depending on the RpRSV strain. The sensitivity was about 100 times greater than the conventional RT-PCR assay using the same primers as the real-time RT-PCR assay. A comparison with published conventional RT-PCR assays indicated that both published assays lacked reliability and sensitivity, as neither were able to amplify all RpRSV isolates tested, and both were at least 1000 times less sensitive than the novel TaqMan real-time RT-PCR assay. The assay can also be run as a duplex reaction with the nad5 plant internal control primers and probe to simultaneously verify the PCR competency of the samples. The amplicon obtained with the real-time RT-PCR assay is suitable for direct sequencing if it is necessary to further confirm the RpRSV identity or determine the RpRSV strain.
Assuntos
Nepovirus/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Rubus/virologia , Primers do DNA/genética , Limite de Detecção , Nepovirus/classificação , Doenças das Plantas/virologia , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Zero-valent nickel compounds are organometallic chemicals that are used in synthetic applications and may also occur as intermediates in nickel-catalyzed hydrogenation reactions used in food processing. Few studies have been performed on their possible genotoxic actions. We have tested two commercially available examples of this class of compounds. Solubility and stability were examined. Mutagenicity testing did not confirm a previous report that bis(1,5-cyclooctadiene)nickel is positive in the Ames assay. No stimulation of lipid peroxidation was observed in studies of bovine erythrocytes exposed in vitro. Our results do not indicate that zero-valent nickel compounds have genotoxic effects.
Assuntos
Eritrócitos/efeitos dos fármacos , Mutagênicos/toxicidade , Níquel/toxicidade , Compostos Organometálicos/toxicidade , Animais , Bovinos , Eritrócitos/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Testes de Mutagenicidade , Mutagênicos/química , Níquel/química , Compostos Organometálicos/química , Solubilidade , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Metasecretome is a collection of cell-surface and secreted proteins that mediate interactions between microbial communities and their environment. These include adhesins, enzymes, surface structures such as pili or flagella, vaccine targets or proteins responsible for immune evasion. Traditional approaches to exploring matasecretome of complex microbial communities via cultivation of microorganisms and screening of individual strains fail to sample extraordinary diversity in these communities, since only a limited fraction of microorganisms are represented by cultures. Advances in culture-independent sequence analysis methods, collectively referred to as metagenomics, offer an alternative approach that enables the direct analysis of collective microbial genomes (metagenome) recovered from environmental samples. This protocol describes a method, metasecretome phage display, which selectively displays the metasecretome portion of the metagenome. The metasecretome library can then be used for two purposes: (1) to sequence the entire metasecretome (using PacBio technology); (2) to identify metasecretome proteins that have a specific function of interest by affinity-screening (bio-panning) using a variety of methods described in other chapters of this volume.
Assuntos
Clonagem Molecular/métodos , Biblioteca Gênica , Metagenoma , Metagenômica/métodos , Biblioteca de PeptídeosRESUMO
Microbial surface and secreted proteins (the secretome) contain a large number of proteins that interact with other microbes, host and/or environment. These proteins are exported by the coordinated activities of the protein secretion machinery present in the cell. A group of bacteriophage, called filamentous phage, have the ability to hijack bacterial protein secretion machinery in order to amplify and assemble via a secretion-like process. This ability has been harnessed in the use of filamentous phage of Escherichia coli in biotechnology applications, including screening large libraries of variants for binding to "bait" of interest, from tissues in vivo to pure proteins or even inorganic substrates. In this review we discuss the roles of secretome proteins in pathogenic and non-pathogenic bacteria and corresponding secretion pathways. We describe the basics of phage display technology and its variants applied to discovery of bacterial proteins that are implicated in colonization of host tissues and pathogenesis, as well as vaccine candidates through filamentous phage display library screening. Secretome selection aided by next-generation sequence analysis was successfully applied for selective display of the secretome at a microbial community scale, the latter revealing the richness of secretome functions of interest and surprising versatility in filamentous phage display of secretome proteins from large number of Gram-negative as well as Gram-positive bacteria and archaea.
RESUMO
[This corrects the article on p. 429 in vol. 7, PMID: 27092113.].