RESUMO
Pseudomonas oryzihabitans is an uncommon pathogen that may cause catheter-associated infections, particularly in immunocompromised patients. Although it has been isolated from environment, the source of human infection is not well documented. In the present study, 14 isolates of P. oryzihabitans were recovered over a 28-month period from a karstified chalk aquifer, allowing to advance that distributed natural water could be a source of contamination. Microbiological analyses showed that the bacterium was mainly associated with suspended particulate matters. To investigate the clonality of P. oryzihabitans environmental isolates, 16S rRNA gene sequencing, antibiogram and randomly amplified polymorphic DNA (RAPD) typings were performed. Results demonstrated (i) the presence of at least three clones within the aquifer and (ii) that the presence of the bacterium in groundwater is not only the result of a biofilm bloom but also of an exogenous contamination.
Assuntos
Silicatos de Alumínio/química , Biofilmes , Carbonato de Cálcio/química , Pseudomonas/genética , Pseudomonas/patogenicidade , Argila , Processos Heterotróficos , Microscopia Eletrônica de Varredura , Polimorfismo Genético , Pseudomonas/isolamento & purificação , Pseudomonas/fisiologia , Purificação da ÁguaRESUMO
The celA gene encoding a beta-1,4 endoglucanase (CelA) from Myxococcus xanthus has been cloned in Escherichia coli and sequenced. The C-terminal region of CelA displayed a high level of similarity with the catalytic domain of several Egl belonging to the glycosyl hydrolases family 6 (CenA from Cellulomonas fimi, CelA from Microbispora bispora, E2 from Thermonospora fusca, CasA from Streptomyces KSM9 and CelA1 from Streptomyces halstedii) and less similarity to the cellobiohydrolases of the fungi Trichoderma reesei and Agaricus bisporus. Using PCR amplification we found in another myxobacterium, Stigmatella aurantiaca, a part of a glycosyl hydrolase belonging to the same family. The N-terminal part of CelA displayed significant similarities with the cellulose-binding domain of other cellulases belonging to a rare subset of family II, such as the avicelase I from Streptomyces reticuli, both tandem repeats N1 and N2 of the cellulase CenC from Cellulomonas fimi, and the N-terminal part of the Egl E1 from Thermonospora fusca. Analyses of the multiple alignments and reconstruction of phylogenetic trees strongly suggest that both domains of CelA were acquired by independent horizontal transfers between Gram+ soil bacteria and scavenging myxobacteria followed by domain shuffling.
Assuntos
Actinomyces/metabolismo , Celulase/genética , Myxococcus xanthus/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Catálise , Clonagem Molecular , DNA Bacteriano , Escherichia coli/genética , Transferência Genética Horizontal , Dados de Sequência Molecular , Myxococcus xanthus/enzimologia , Myxococcus xanthus/metabolismo , Filogenia , Homologia de Sequência de AminoácidosRESUMO
The sequence of a 1955-bp TaqI DNA fragment from Myxococcus xanthus was determined. This fragment contains two complete genes, designated prtA and prtB. The prtA and prtB ORFs extend over 828 and 798 bp, respectively. They are separated only by 3 nt and appear to be present in a polycistronic transcriptional unit. A typical lipoprotein signal sequence is present at the N terminus of the two deduced polypeptides. The aa sequence of PrtA shows a high degree of identity to the region adjacent to the Ser residue belonging to the catalytic triad of serine proteases from Staphylococcus aureus and Enterococcus faecalis. It also exhibits features characteristic of trypsin-like serine proteases in that it contains the same pattern of variable and conserved regions. The deduced aa sequence of PrtB reveals a signature zinc-binding consensus motif (HEXXHXXGXXH/Met-turn) characteristic of the class of metalloproteases called metzincins. Plasmids containing prtA, prtB, or both were constructed. Protease activity studies of Escherichia coli clones containing these plasmids showed that both genes are necessary for this activity, whatever their cis or trans position. As prtB produces a putative membrane-bound lipoprotein of 266 aa, the protease activation must occur at the membrane level.
Assuntos
Endopeptidases/genética , Genes Bacterianos , Metaloendopeptidases/genética , Myxococcus xanthus/enzimologia , Serina Endopeptidases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , Endopeptidases/química , Lipoproteínas/genética , Metaloendopeptidases/química , Dados de Sequência Molecular , Myxococcus xanthus/genética , Relação Estrutura-AtividadeRESUMO
The inducibility of two promoter systems, one heterologous and one homologous, has been assessed in the Gram-negative bacterium Myxococcus xanthus. The heterologous system involved the hybrid tac promoter and the presence of lacIq, the lac repressor from Escherichia coli. This system is inducible in its natural host with isopropyl-beta-D-thiogalactopyranoside (IPTG). The homologous promoter system involves the light-inducible carQRS promoter, which is normally involved in the expression of the regulators of the light-inducible light-protective carotenoid synthesis regulon in M. xanthus. In each case, promoter activity and strength was assayed using the E. coli gene lacZ. In our constructs, which were present in a single copy in the M. xanthus chromosome, the carQRS promoter yielded at least a 47-fold increase in beta-galactosidase production upon light induction, whilst IPTG increased by 8-fold the amount of enzyme produced under the control of the ptac-lacIq system. Regulation by the latter was significantly higher than that obtained with the unmodified lacZ promoter.
Assuntos
Galactosidases/biossíntese , Isopropiltiogalactosídeo/farmacologia , Myxococcales/enzimologia , Estimulação Luminosa , Tioglicosídeos/farmacologia , beta-Galactosidase/biossíntese , Indução Enzimática/efeitos dos fármacos , Genética Microbiana , Técnicas In Vitro , Myxococcales/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacosRESUMO
We describe herein a molecular method for estimating the abundance of the cadA gene, which encodes a Cd2+/ATPase protein transporter, in bacterial DNA extracted from samples of environmental water. Competitive polymerase chain reaction (cPCR) may be the most appropriate technique for assessing the prevalence of the cadA gene in microbial communities in highly heterogeneous and polluted environments, such as the Seine estuary (France). We describe the development of this method: (i) the choice of two specific primers, based on the sequences encoding the cadmium binding site and the ion channel domains; (ii) the construction of a competitor sequence and assessment of its amplification efficiency; and (iii) the estimation of the copy number of the cadA gene. The cadA content in the bacterial community is expressed as the number of gene copies per ng of total DNA extracted, which is independent of the DNA extraction yield. This molecular procedure was improved to analyze cadA levels in bacterial DNA extracted from estuary water accidentally contaminated with cadmium. Results revealed a subsequent increase in the copy number of the cadA gene in the microbial community.
Assuntos
Adenosina Trifosfatases/genética , Bactérias/efeitos dos fármacos , Cádmio/farmacologia , Farmacorresistência Bacteriana/genética , Água Doce/microbiologia , Poluição Química da Água , Bactérias/genética , Bactérias/isolamento & purificação , Primers do DNA , DNA Bacteriano/análise , DNA Bacteriano/isolamento & purificação , Dosagem de Genes , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Expression in Escherichia coli of the Myxococcus xanthus gene celA, which encodes an extracellular endoglucanase, resulted in CelA being distributed between cytoplasm, periplasm and membrane. The presence of an adjacent open reading frame downstream from the full celA gene, or the absence of a putative lipoprotein signal sequence, confined CelA distribution to the periplasm and membrane, or to the cytoplasm and periplasm, respectively.
Assuntos
Celulase/genética , Escherichia coli/genética , Myxococcus xanthus/genética , Sequência de Aminoácidos , Celulase/metabolismo , Clonagem Molecular , Escherichia coli/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Myxococcus xanthus/química , Myxococcus xanthus/enzimologia , Fases de Leitura Aberta , Plasmídeos , Sinais Direcionadores de ProteínasRESUMO
An endoglucanase, CelA, is secreted by Myxococcus xanthus only during exponential growth. The production of this enzyme is decreased by mutations in 5 different genes (Exc +/- phenotype), three of which correspond to asg genes which regulate the production of an early cell-to-cell signal in development. Transcription of celA is decreased in two of these Exc +/- mutants, whereas a post-transcriptional step is affected in two other Exc- mutants. Thus, asg genes, in addition to regulating the onset of development, also regulate a gene (celA) that is expressed during exponential growth and that is not involved in development.
Assuntos
Celulase/genética , Regulação Bacteriana da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Myxococcus xanthus/genética , Fosfotransferases , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Northern Blotting , Caseínas/química , Celulase/biossíntese , Eletroforese , Dados de Sequência Molecular , Myxococcus xanthus/crescimento & desenvolvimento , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/biossíntese , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , RNA/química , RNA Mensageiro/biossíntese , Análise de Sequência de DNA , Transdução de Sinais , Transcrição Gênica/genéticaAssuntos
Iridoviridae , Lepidópteros/microbiologia , Animais , Antígenos Virais/análise , Linhagem Celular , Efeito Citopatogênico Viral , DNA Viral/análise , Hidrolases/metabolismo , Insetos/microbiologia , Iridoviridae/análise , Iridoviridae/enzimologia , Iridoviridae/fisiologia , Iridoviridae/ultraestrutura , Lipídeos/análise , Proteínas Virais/análise , Replicação ViralRESUMO
Synthesis of the polypeptides induced in CIV-infected cells was studied using radiolabelled methionine in a permissive cell line of Choristoneura fumiferana. Analysis of labelled cell extracts by high resolution polyacrylamide gel electrophoresis (PAGE) revealed the sequential appearance of at least 28 structural and non-structural polypeptides in permissive conditions. This was confirmed by analysis of protein synthesis in non-permissive cell lines of Lymantria dispar, Aedes albopictus or by the use of protein, RNA and DNA synthesis inhibitors in the permissive cell line.
Assuntos
Iridoviridae/metabolismo , Proteínas Virais/biossíntese , Animais , Células Cultivadas , Cicloeximida/farmacologia , Replicação do DNA , Dactinomicina/farmacologia , Insetos , Proteínas Estruturais Virais , Replicação ViralRESUMO
We report a procedure for the purification of Chilo iridescent virus (Iridovirus type 6), an evaluation of the purification procedure, and the results of analyses of the virion proteins by acrylamide gel electrophoresis. Purity was evaluated in three ways, i.e., by analysis of purified virions from artificial mixtures of infected and labeled uninfected larvae, electrophoresis at neutral pH, and electron-microscopic examination. Analysis of the polypeptides of purified CIV gave the following results: (i) after solubilization with SDS-B-mercaptoethanol, 16 polypeptides could be resolved in Coomassie brillant blue-stained electrophoretograms with molecular weights ranging from 18,000 to 115,000; (ii) after solubilization with SDS-urea, 26 polypeptides could be resolved with molecular weights ranging from 10,000 to 230,000 daltons.
Assuntos
Iridoviridae/ultraestrutura , Lepidópteros/microbiologia , Mariposas/microbiologia , Peptídeos/análise , Proteínas Virais/análise , Animais , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Proteínas Virais/isolamento & purificaçãoRESUMO
The surface antigens of Iridovirus type 6 (CIV) were characterized by crossed immunoelectrophoresis. Using different solubilization techniques, up to seven antigenic determinants corresponding to four viral structural polypeptides were identified.
Assuntos
Antígenos de Superfície/análise , Antígenos Virais/análise , Iridoviridae/imunologia , Proteínas Virais/imunologia , Epitopos , Imunoeletroforese Bidimensional , Iridoviridae/isolamento & purificação , Iridoviridae/ultraestrutura , Proteínas Virais/análise , Proteínas Estruturais ViraisRESUMO
The nephrology-dialysis department of the Havre's hospital has launched a project of certification ISO 9002 in 1996, based on reflections from the centers of dialysis Upper Normandy. The problems encountered were mainly the lack of ways, a bad documentary structure, false ideas on the quality and a bad perception from the client. With the help of a quality manager, il has enable the project to advance and finalized, obtaining the certification of activity. In june 2000 "Procedure of taking in charge all the patient's medical and para-medical cost in the center of the Nephrology-Dialysis service" of the GHH (Hospital Group of the Havre) has brought not only the certificate but notable improvements on the level of documentary management, the errors, relationship clients-suppliers and projects of collaboration with the other services of nephrology dialysis of France.
Assuntos
Certificação , Administração Hospitalar , Departamentos Hospitalares/normas , Nefrologia/normas , Garantia da Qualidade dos Cuidados de Saúde/organização & administração , Certificação/legislação & jurisprudência , Previsões , Controle de Formulários e Registros , França , Relações Interinstitucionais , Aceitação pelo Paciente de Cuidados de Saúde , Resolução de Problemas , Garantia da Qualidade dos Cuidados de Saúde/legislação & jurisprudência , Controle de Qualidade , Diálise Renal/normasRESUMO
A recombinant Myxococcus xanthus strain was constructed that constitutively produces two proteins from Escherichia coli, the cytoplasmic beta-galactosidase and the periplasmic pH 2.5 acid phosphatase (AppA protein). We have previously shown that during vegetative growth, AppA protein is partly accumulated in the periplasm of M. xanthus and partly released into the medium. We demonstrate here that during starvation-induced development, release of periplasmic AppA protein to the medium did not occur over a period of 20 h. This was coincident with, but not caused by, the arrest of the synthesis of the foreign proteins. We have shown that this lack of secretion could be attributed to starvation per se and did not depend on the ability of the cells to undergo development. Our findings suggest that protein secretion which occurs during the first hours of starvation-induced development might therefore take place via a different route from that which occurs in vegetative cells.
Assuntos
Proteínas de Bactérias/metabolismo , Myxococcus xanthus/metabolismo , Fosfatase Ácida/genética , Fosfatase Ácida/metabolismo , Proteínas de Bactérias/genética , Permeabilidade da Membrana Celular , Meios de Cultura , Escherichia coli/enzimologia , Escherichia coli/genética , Genes Bacterianos , Concentração de Íons de Hidrogênio , Óperon Lac , Myxococcus xanthus/genética , Myxococcus xanthus/crescimento & desenvolvimento , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
A cytoplasmic polyhedrosis virus (CPV) was isolated from Euxoa scandens and propagated in vitro in Lymantria dispar cells. The virions and polyhedra were analyzed by polyacrylamide gel electrophoresis. Polyhedra produced in vivo and in vitro contained six polypeptides with identical relative mobilities while nonoccluded viral particles produced in vivo were composed of four polypeptides. The RNA content of the purified virions extracted from infected midgut was resolved in ten segments with molecular weights similar to that of CPV type 5 RNA.