RESUMO
Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight, a serious disease of rice. A virulence- and xylanase-deficient mutant of Xoo was isolated following ethyl methane sulfonate (EMS) mutagenesis. A cosmid clone that restored virulence and xylanase secretion was obtained from a genomic library by functional complementation. Transposon mutagenesis and marker exchange studies revealed genes on the cloned DNA that were required for xylanase production and virulence. Sequence analysis with transposon-specific primers revealed that these genes were homologues of xps F and xps D, which encode components of a protein secretion system in Xanthomonas campestris pv. campestris. Enzyme assays showed xylanase accumulation in the periplasmic space and cytoplasm of the xps F mutant and the complementing clone restored transport to the extracellular space.
Assuntos
Proteínas de Membrana Transportadoras , Oryza/microbiologia , Xanthomonas/genética , Xilosidases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Elementos de DNA Transponíveis/genética , Escherichia coli/genética , Metanossulfonato de Etila/farmacologia , Dados de Sequência Molecular , Mutagênese , Alinhamento de Sequência , Análise de Sequência de DNA , Análise de Sequência de Proteína , Virulência/genética , Xanthomonas/enzimologia , Xanthomonas/patogenicidade , Xilano Endo-1,3-beta-Xilosidase , Xilosidases/deficiência , Xilosidases/metabolismoRESUMO
The gene encoding extracellular xylanase (xynA) was amplified as a 770 bp DNA fragment from Bacillus subtilis 168 chromosomal DNA by PCR. The genes encoding endo-beta-1,4-glucanase (eglS) and endo-beta-1,3-1,4-glucanase (bglS) were isolated from a genomic library of B. subtilis 168. The sequences of xynA and eglS were identical to those of the xylanase and cellulase genes from B. subtilis PAP115. Integrative plasmids containing DNA fragments with deletions in the coding region of the genes were constructed and used to replace the chromosomal eglS, bglS and xynA genes of B. subtilis 168. Strains without any detectable activity against xylan (Xyn-), carboxymethylcellulose (Egl-) or mixed linked beta-1,3-1,4-glucan (Egl- Bgl-) were obtained. The genes were mapped at 170 degrees (eglS), 175 degrees (xynA) and 340 degrees (bglS) on the B. subtilis chromosome.
Assuntos
Bacillus subtilis/genética , Celulase/genética , Genes Bacterianos/genética , Glicosídeo Hidrolases/genética , Xilosidases/genética , Bacillus subtilis/enzimologia , Sequência de Bases , Celulase/deficiência , Clonagem Molecular , Endo-1,4-beta-Xilanases , Teste de Complementação Genética , Ligação Genética , Glucanos/metabolismo , Glicosídeo Hidrolases/deficiência , Hidrólise , Dados de Sequência Molecular , Mutagênese , Mapeamento por Restrição , Análise de Sequência de DNA , Xilanos/metabolismo , Xilosidases/deficiênciaAssuntos
Doença de Gaucher/enzimologia , Glicosídeo Hidrolases/deficiência , Xilosidases/deficiência , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Doença de Gaucher/genética , Glicosídeo Hidrolases/metabolismo , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Baço/enzimologia , Xilosidases/metabolismoRESUMO
Biochemical investigations were performed on autopsy tissues obtained from an 11-year-old girl who died with the juvenile, subacute neuropathic form of Gaucher disease. In addition to the expected deficiency of glucocerebrosidase activity, extracts of both liver and kidney from this individual displayed a profound (greater than or equal to 90%) deficiency of "soluble" beta-glucosidase, beta-xylosidase, and beta-galactosidase activities. Fibroblasts obtained from this individual also contained markedly reduced levels of beta-xylosidase activity but normal levels of beta-D-fucosidase and beta-galactosidase activity. Because the soluble beta-glucosidase, beta-xylosidase, and a portion of the beta-galactosidase activities from control human liver all cochromatographed on a gel filtration column of Sephadex G-200, it is suggested that these activities all reside in a single enzyme, analogous to the situation described in a number of nonhuman, mammalian tissues. This demonstration of multiple glycosidase deficiencies in addition to the deficiency of glucocerebrosidase in a case of subacute neuropathic Gaucher disease suggests that other biochemical aberrations, in addition to a deficiency of glucocerebrosidase, might contribute to pathology in some cases of Gaucher disease.
