Assuntos
Anemia Falciforme/genética , Cistationina beta-Sintase/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Protrombina/genética , Trombofilia/genética , Trombose/epidemiologia , Regiões 3' não Traduzidas/genética , Adulto , Alelos , Anemia Falciforme/epidemiologia , Criança , Estudos de Coortes , Cistationina beta-Sintase/fisiologia , Feminino , Frequência do Gene , Predisposição Genética para Doença , Guadalupe/epidemiologia , Humanos , Recém-Nascido , Estudos Longitudinais , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2) , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/fisiologia , Prevalência , Protrombina/fisiologia , Acidente Vascular Cerebral/epidemiologia , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/genética , Trombose/etiologia , Trombose/genéticaRESUMO
The precise excision of transposon Tn10 and a mini-Tn10 derivative, inserted in the gal or lac operons, was studied in dnaB252 and dnaE486 temperature-sensitive mutants of Escherichia coli. dnaB codes for a DNA replication helicase and dnaE for the alpha subunit of DNA polymerase III. Mutations in these genes were found to enhance, at the permissive temperature, the precise excision of both genetic elements. The increase factor was much more pronounced for the dnaB252 mutant with the transposons inserted in gal. The stimulated excision was only partially affected by a recA null mutation but was significantly reduced by introduction of recF null or ruvA mutations. A model involving template switching of the polymerase between the direct repeats flanking the transposons, on the same strand or between sister strands, could account for the observed results.
Assuntos
Proteínas de Bactérias , Replicação do DNA/genética , Elementos de DNA Transponíveis/genética , Escherichia coli/genética , Mutação , DNA Helicases/genética , DNA Polimerase III/genética , DnaB Helicases , Escherichia coli/metabolismo , Genes Bacterianos , Genótipo , Modelos Genéticos , TemperaturaRESUMO
Sae is a regulatory locus that activates the production of several exoproteins in Staphylococcus aureus. A 3.4-kb fragment of a S. aureus genomic library, screened with a probe adjacent to the transposon insertion of a sae::Tn551 mutant, was cloned into a bifunctional vector. This fragment was shown to carry the sae locus by restoration of exoprotein production in sae mutants. The sae locus was mapped to the SmaI-D fragment of the staphylococcal chromosome by pulse-field electrophoresis. Sequence analysis of the cloned fragment revealed the presence of two genes, designated saeR and saeS, encoding a response regulator and a histidine protein kinase, respectively, with high homology to other bacterial two-component regulatory systems.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Proteínas Quinases/genética , Staphylococcus aureus/genética , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Bacterianos/genética , Clonagem Molecular , Sequência Conservada , Biblioteca Genômica , Dados de Sequência Molecular , Mutagênese Insercional , Proteínas Quinases/biossíntese , Proteínas Quinases/química , Mapeamento por Restrição , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/metabolismoRESUMO
Mitomycin C (MMC) treatment or mutations in uvrD enhance the frequency of Tn10 precise excision. We have shown previously that several repair-recombination genes, such as recA, ruv and recF are involved in the induced excision process. In this study, we find that other genes belonging to the RecBC and RecF sexual recombination pathways also participate in this process since mutations in recB, sbcB or recO diminish, though to different degrees, the frequency of Tn10 precise excision induced by MMC treatment or by uvrD mutants. Pairwise combinations of some of these mutations were also tested for Tn10 induced precise excision; most of these double mutants showed additive effects in reducing the frequency of the excision process. The results of these studies suggest that recombinational-repair genes, particularly recF, sbcB and recO have different roles in the induced excision of Tn10 than in recombinational mating.
Assuntos
Proteínas de Bactérias/genética , DNA Helicases , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Exodesoxirribonucleases/genética , Recombinação Genética , Adenosina Trifosfatases/genética , Exodesoxirribonuclease V , Genes Bacterianos , Mitomicina/farmacologiaRESUMO
An avirulent mutant, designated RC122, was derived from Staphylococcus aureus bovine mastitis strain RC108 after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. Mutant RC122, which was isolated on the basis of reduced colony size, showed diminished virulence in mice (LD50 of RC122: 3.1 x 10(10) cfu vs LD50 of RC108: 2.3 x 10(7) cfu). Mutant RC122 grew more slowly than its parental strain and showed decreased production of several exoproteins, such as alpha- and beta-hemolysin, DNAse and coagulase. The production of its capsule was induced only under in vivo growth conditions. Clearance studies performed in the mouse kidney revealed that the kinetics of disappearance of the mutant was similar to that of its parental strain. Protection experiments carried out by intraperitoneal administration in mice showed that mutant RC122 conferred a good degree of protection from challenge with homologous and heterologous strains.
Assuntos
Mastite Bovina/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/patogenicidade , Animais , Bovinos , Feminino , Dose Letal Mediana , Camundongos , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/genética , Staphylococcus aureus/imunologia , VirulênciaRESUMO
It has been shown that the increased frequency of precise excision of Tn10 observed after UV or mitomycin C (MMC) treatment or with uvrD- mutants is recA-dependent. Previous work has also shown that expression of SOS genes is required for UV- or MMC-induced Tn10 precise excision. In order to determine if the increased excision of Tn10 in uvrD- mutants requires only expression of recA, or expression of other SOS genes, or both, we studied the precise excision of Tn10 in lexA3 (Ind-, SOS non-inducible) and lexA3 recAo98 (operator constitutive recA) mutants. The results of these experiments indicate that the induced excision of Tn10 in the uvrD- null mutant depends on the expression of recA rather than on any of the other genes repressed by LexA. The effect of a null recF mutation on the excision of Tn10 in a uvrD- mutant was also investigated and found to abolish the increased frequencies of this process. Similarly, the recF mutation was found to decrease markedly the increased precise excision of Tn10 induced by MMC in a uvrD+ isogenic strain. These observations indicate that recA and recF are involved in the increased frequencies of Tn10 excision exhibited by uvrD- mutants or after MMC treatment. It remains to be determined whether these two genes participate in these two induction processes in the same biochemical pathways.
Assuntos
Proteínas de Bactérias/genética , DNA Helicases , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Recombinases Rec A/genética , Adenosina Trifosfatases/genética , Escherichia coli/efeitos dos fármacos , Mitomicina/farmacologia , Mutação , Resposta SOS em GenéticaRESUMO
Agr and sar are known regulatory loci of Staphylococcus aureus that control the production of several extracellular and cell-wall-associated proteins. A pleiotropic insertional mutation in S. aureus, designated sae, that leads to the production of drastically diminished levels of alpha- and beta-hemolysins and coagulase and slightly reduced levels of protein A has been described. The study of the expression of the genes coding for these exoproteins in the sae::Tn551 mutant (carried out in this work by Northern blot analyses) revealed that the genes for alpha- and beta-hemolysins (hla and hlb) and coagulase (coa) are not transcribed and that the gene for protein A (spa) is transcribed at a somewhat reduced level. These results indicate that the sae locus regulates these exoprotein genes at the transcriptional level. Northern blot analyses also show that the sae mutation does not affect the expression of agr or sar regulatory loci. An sae::Tn551 agr::tetM double mutant has been phenotypically characterized as producing reduced or null levels of alpha-, beta-, and delta-hemolysins, coagulase, and high levels of protein A. Northern blot analyses carried out in this work with the double mutant revealed that hla, hlb, hld, and coa genes are not transcribed, while spa is transcribed at high levels. The fact that coa is not expressed in the sae agr mutant, as in the sae parental strain, while spa is expressed at the high levels characteristic of the agr parental strain, suggests that sae and agr interact in a complex way in the control of the expression of the genes of several exoproteins.
Assuntos
Coagulase/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Proteínas Hemolisinas/metabolismo , Proteína Estafilocócica A/metabolismo , Staphylococcus aureus/genética , Transativadores , Proteínas de Bactérias/genética , Northern Blotting , Coagulase/genética , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Proteínas Hemolisinas/genética , Mutagênese Insercional , Plasmídeos , Proteína Estafilocócica A/genética , Staphylococcus aureus/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Transdução GenéticaRESUMO
A vaccine was developed against bovine mastitis based on inactivated, highly encapsulated Staphylococcus aureus cells; a crude extract of Staph. aureus exopolysaccharides; and inactivated, unencapsulated Staph, aureus and Streptococcus spp. cells. This vaccine was tested on 30 heifers during a 7-mo period. The 30 heifers were randomly assigned to three groups of 10 heifers each. The prepartum group received two injections of the vaccine at 8 and 4 wk before calving, and the postpartum group received two injections at 1 and 5 wk after calving. The control group received two injections of a placebo at 8 and 4 wk before calving. The vaccine or the placebo was administered subcutaneously in the brachiocephalicus muscle of the neck. The frequencies of intramammary infections caused by Staph. aureus were reduced from 18.8% for heifers in the control group to 6.7 and 6.0% for heifers in the prepartum and postpartum groups, respectively. This protective effect was maintained for at least 6 mo. The relative risk of mastitis caused by Staph. aureus was 0.31 and 0.28 for heifers in the prepartum and postpartum groups, respectively, compared with that for heifers in the control group. The results of the trial indicated the effectiveness of the vaccine in decreasing the incidence of intrammammary infections caused by Staph. aureus. A slight but nonsignificant increase occurred in fat production in the milk of vaccinated cows. The vaccine had no observable effect on somatic cell count or streptococcal infections.
Assuntos
Vacinas Bacterianas , Mastite Bovina/prevenção & controle , Animais , Argentina , Bovinos , Feminino , Lipídeos/biossíntese , Mastite Bovina/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Leite/metabolismo , Coelhos , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/imunologia , Infecções Estreptocócicas/prevenção & controle , Streptococcus/imunologiaRESUMO
A vaccine against bovine mastitis was developed. The vaccine was based on inactivated, highly encapsulated Staphylococcus aureus cells; a crude extract of Staph. aureus exopolysaccharides; and inactivated unencapsulated Staph. aureus and Streptococcus spp. cells. In this study, the vaccine was evaluated in 164 cows from two commercial dairies (A and B) during a 4-mo period. Two doses of the vaccine were administered subcutaneously to 82 cows in the brachiocephalicus muscle of the neck within a 4-wk interval. The results of this trial revealed significantly fewer intramammary infections caused by Staph. aureus at various levels of severity (clinical, subclinical, and latent) in cows that were vaccinated. The odds ratios of all types of intrammammary infections caused by Staph. aureus for dairies A and B, which were determined by a logistic model, were 1.84 and 1.89, respectively, for quarters of vaccinated cows and quarters of control cows. The colony counts for Staph. aureus in milk from infected quarters of vaccinated cows were significantly lower than those in milk from infected quarters of control cows. Also, the somatic cell counts per milliliter in milk from vaccinated cows were significantly decreased when the initial somatic cell count was < 500,000 cells/ml at the start of the trial. The vaccine had no observable effect on fat production in milk or on streptococcal infections.