RESUMO
Analyses of Rhizobium meliloti trp auxotrophs suggest that anthranilate biosynthesis by the R. meliloti trpE(G) gene product is necessary during nodule development for establishment of an effective symbiosis. trpE(G) mutants, as well as mutants blocked earlier along this pathway in aromatic amino acid biosynthesis, form nodules on alfalfa that have novel defects. In contrast, R. meliloti trp mutants blocked later in the tryptophan-biosynthetic pathway form normal, pink, nitrogen-fixing nodules. trpE(G) mutants form two types of elongated, defective nodules containing unusually extended invasion zones on alfalfa. One type contains bacteroids in its base and is capable of nitrogen fixation, while the other lacks bacteroids and cannot fix nitrogen. The trpE(G) gene is expressed in normal nodules. Models are discussed to account for these observations, including one in which anthranilate is postulated to act as an in planta siderophore.
Assuntos
Genes Bacterianos , Mutagênese Insercional , Sinorhizobium meliloti/genética , Simbiose/genética , Triptofano/biossíntese , ortoaminobenzoatos/metabolismo , Deleção Cromossômica , Cromossomos Bacterianos , Clonagem Molecular , Elementos de DNA Transponíveis , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Escherichia coli/genética , Expressão Gênica , Glucuronidase/metabolismo , Medicago sativa/citologia , Medicago sativa/microbiologia , Fenótipo , Plasmídeos , Sinorhizobium meliloti/metabolismoRESUMO
The peptide-N4-(N-acetyl-beta-D-glucosaminyl) asparagine amidase F (PNGase F) gene from Flavobacterium meningosepticum was cloned into a high copy number Escherichia coli plasmid. Levels of PNGase F activity produced in cultures of the recombinant strain were up to 100-fold higher than those obtained in cultures of F. meningosepticum. The complete PNGase F gene sequence was determined. Comparison of the predicted amino acid sequence of pre-PNGase F to the N-terminal sequence of the native mature enzyme indicates that the protein is synthesized with a 40-amino acid signal sequence that is removed during secretion in F. meningosepticum. The recombinant PNGase F produced in E. coli is a mixture of products comprised predominantly of two proteins with molecular masses of 36.3 and 36.6 kDa. These proteins have a higher apparent molecular mass than the 34.7-kDa native enzyme. N-terminal amino acid sequencing demonstrated that these higher molecular mass products result from cleavage of the pre-PNGase F in E. coli upstream of the native N terminus. The PNGase F gene was engineered to encode a preenzyme that was processed in E. coli to give an N terminus identical to that of the native enzyme. Purified preparations of this form of recombinant PNGase F were shown to be suitable for glycoprotein analyses since they possess no detectable endo-beta-N-acetylglucosaminidase F, exoglycosidase, or protease activity.
Assuntos
Amidoidrolases/genética , Escherichia coli/genética , Flavobacterium/genética , Amidoidrolases/isolamento & purificação , Amidoidrolases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular/métodos , Flavobacterium/enzimologia , Expressão Gênica , Engenharia Genética , Dados de Sequência Molecular , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Plasmídeos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por RestriçãoRESUMO
Three groups of linked markers were mapped in Bacillus thuringiensis 4042B by using two-, three-, and four-factor crosses mediated by the temperate bacteriophages TP-13 and TP-18. The order of markers was (trp-11, trp-2)-(leu-1, leu-2)-his-1-(lys-1, lys-2)-cys-1 in the first group; met-1-(argCl, argOl)-met-2-(pyr-1, pyrA2) in the second group; and met-3-pur-1-(nal-1, nal-2)-str-1-(pur-2, pur-4)-pur-3 in the third group. Electron microscopic measurements of head sizes suggested that the volume of the TP-13 phage head is seven times greater than that of the TP-18 phage head. The TP-18 genome was shown by DNA restriction analysis to have a molecular mass of 36 megadaltons. TP-13 was useful for scanning large segments of the B. thuringiensis chromosome, and TP-18 was effective for ordering markers too closely linked for simple resolution with TP-13.
