RESUMO
In Escherichia coli, the phn operon encodes proteins responsible for the uptake and breakdown of phosphonates. The C-P (carbon-phosphorus) lyase enzyme encoded by this operon which catalyzes the cleavage of C-P bonds in phosphonates has been recalcitrant to biochemical characterization. To advance the understanding of this enzyme, we have cloned DNA from Rhizobium (Sinorhizobium) meliloti that contains homologues of the E. coli phnG, -H, -I, -J, and -K genes. We demonstrated by insertional mutagenesis that the operon from which this DNA is derived encodes the R. meliloti C-P lyase. Furthermore, the phenotype of this phn mutant shows that the C-P lyase has a broad substrate specificity and that the organism has another enzyme that degrades aminoethylphosphonate. A comparison of the R. meliloti and E. coli phn genes and their predicted products gave new information about C-P lyase. The putative R. meliloti PhnG, PhnH, and PhnK proteins were overexpressed and used to make polyclonal antibodies. Proteins of the correct molecular weight that react with these antibodies are expressed by R. meliloti grown with phosphonates as sole phosphorus sources. This is the first in vivo demonstration of the existence of these hitherto hypothetical Phn proteins.
Assuntos
Proteínas de Bactérias , Genes Bacterianos , Liases/genética , Família Multigênica , Óperon , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/metabolismo , Sequência de Aminoácidos , Anticorpos , Clonagem Molecular , Primers do DNA , Escherichia coli/genética , Cinética , Liases/biossíntese , Liases/metabolismo , Dados de Sequência Molecular , Peso Molecular , Organofosfonatos/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation of two cell types called the prespore and the mother cell. The process is induced by nutrient deprivation and culminates with the formation of a mature spore, which is released by lysis of the mother cell. We have studied commitment to sporulation with several different assays. The results indicate that commitment occurs soon after the formation of the asymmetrically positioned division septum that separates the prespore and the mother cell. This is earlier than the previously postulated point of commitment, prespore engulfment by the mother cell. Commitment coincides approximately with activation of the early prespore- and mother-cell-specific sigma factors, sigma(E) and sigma(F).
Assuntos
Bacillus subtilis/fisiologia , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Meios de Cultura , Genes Bacterianos , Muramidase/metabolismo , Mutação , Fator sigma/metabolismo , Esporos Bacterianos/genética , Esporos Bacterianos/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismoRESUMO
Inbred Drosophila melanogaster stocks were surveyed for alpha-glucosidases with nondenaturing gel electrophoresis using a fluorogenic substrate to stain the gels. The glucosidase most active under these conditions is polymorphic. We established that the polymorphism is genetic in origin and that the glucosidase was not likely to be a previously characterized enzyme. The gene encoding the enzyme was mapped cytogenetically to 33 A1-2- 33A8-B1, confirming that this is an enzyme not yet reported in D. melanogaster. The enzyme was partially purified by elution from nondenaturing gels, which enabled us to establish that it has optimal activity at pH 6 and interacts most strongly with alpha-1-4 glucosides. A developmental and tissue survey suggested that this enzyme could have a purely digestive role or be involved in carbohydrate metabolism inside the organism. We propose that this enzyme is involved in either starch digestion or glycogen metabolism.
Assuntos
Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , alfa-Glucosidases/genética , alfa-Glucosidases/isolamento & purificação , Animais , Sequência de Carboidratos , Mapeamento Cromossômico , Cruzamentos Genéticos , Feminino , Glucosídeos/química , Glucosídeos/metabolismo , Concentração de Íons de Hidrogênio , Masculino , Dados de Sequência Molecular , Polimorfismo Genético , Especificidade por Substrato , alfa-Glucosidases/metabolismoRESUMO
An analysis of the released oligosaccharides from a membrane glycoprotein preparation of third instar larvae (3rdIL), and purified larval serum protein 2 (LSP2) from Drosophila melanogaster was performed. Sequential exoglycosidase digestion in combination with high-resolution gel permeation chromatography and partial acetolysis indicated the presence of two series of oligomannosides; one of these series was unusual and characterized by the presence of a core alpha 1-6 linked fucose, the other was a typical mammalian oligomannose series containing the following isomers -D1, -D2, -D12, -D123 and -CD123 as well as the unprocessed Man9GlcNAc2 structure. Conventional oligomannose could only be detected in the LSP2 sample. This study opens the way to use powerful molecular and classical genetic techniques to analyse the control and functional significance of glycosylation in higher organisms.
Assuntos
Drosophila melanogaster/química , Glicoproteínas de Membrana/química , Oligossacarídeos/química , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Glicosilação , Manose/química , Manose/isolamento & purificação , Glicoproteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Oligossacarídeos/isolamento & purificaçãoRESUMO
The presence of a glycan of the same molecular size as the lipid linked precursor oligosaccharide (Glc3Man9GlcNAc2) of the N-linked protein glycosylation pathway in mammalian cells has been detected in a glycolipid fraction of cultured Drosophila melanogaster cells. Oligosaccharide sequencing studies were consistent with the existence of a glucosylated high mannose containing structure, which may be the common precursor for N-linked protein glycosylation in insect cells.
