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991.
Regulation of nitrogenase activity by oxygen in Azospirillum brasilense and Azospirillum lipoferum. 总被引:10,自引:6,他引:4 下载免费PDF全文
The nitrogenase activity of the microaerophilic bacteria Azospirillum brasilense and A. lipoferum was completely inhibited by 2.0 kPa of oxygen (approximately 0.02 atm of O2) in equilibrium with the solution. The activity could be partially recovered at optimal oxygen concentrations of 0.2 kPa. In contrast to the NH4+ switch off, no covalent modification of the nitrogenase reductase (Fe protein) was involved, as demonstrated by Western-blotting and 32P-labeling experiments. However, the inhibition of the nitrogenase activity under anaerobic conditions was correlated with covalent modification of the Fe protein. In contrast to the NH4+ switch off, no increase in the cellular glutamine pool and no modification of the glutamine synthetase occurred under anaerobic switch-off conditions. Therefore, a redox signal, independent of the nitrogen control of the cell, may trigger the covalent modification of the nitrogenase reductase of A. brasilense and A. lipoferum. 相似文献
992.
Regulation of two nickel-requiring (inducible and constitutive) hydrogenases and their coupling to nitrogenase in Methylosinus trichosporium OB3b. 总被引:4,自引:3,他引:1 下载免费PDF全文
Two uptake hydrogenases were found in the obligate methanotroph Methylosinus trichosporium OB3b; one was constitutive, and a second was induced by H2. Both hydrogenases could be assayed by measuring methylene blue reduction anaerobically or by coupling their activity to nitrogenase acetylene reduction activity in vivo in an O2-dependent reaction. The H2 concentration for half-maximal activity of the inducible and constitutive hydrogenases in both assays was 0.01 and 0.5 bar (1 and 50 kPa), respectively, making it easy to distinguish these enzymes from one another both in vivo and in vitro. Hydrogen uptake was shown to be coupled to ATP synthesis in methane-starved cells. Methane, methanol, formate, succinate, and glucose all repressed the H2-mediated synthesis of the inducible hydrogenase. Furthermore, this enzyme was only expressed in N-starved cultures and was repressed by NH4+ and NO3-; synthesis of the constitutive hydrogenase was not affected by excess N in the growth medium. In nickel-free, EDTA-containing medium, the activities of these two enzymes were negligible; however, both enzyme activities appeared rapidly following the addition of nickel to the culture. Chloramphenicol, when added along with nickel, had no effect on the rapid appearance of either the constitutive or inducible activity, indicating that nickel is not required for synthesis of the hydrogenase apoproteins. These observations all suggest that these hydrogenases are nickel-containing enzymes. Finally, both hydrogenases were soluble and could be fractionated by 20% ammonium sulfate; the constitutive enzyme remained in the supernatant solution, while the inducible enzyme was precipitated under these conditions. 相似文献
993.
NAD-linked aldehyde dehydrogenase for aerobic utilization of L-fucose and L-rhamnose by Escherichia coli. 总被引:7,自引:6,他引:1 下载免费PDF全文
Mutant analysis revealed that complete utilization of L-fucose and L-rhamnose by Escherichia coli requires the activity of a common NAD-linked aldehyde dehydrogenase which converts L-lactaldehyde to L-lactate. Mutations affecting this activity mapped to the ald locus at min 31, well apart from the fuc genes (min 60) encoding the trunk pathway for L-fucose dissimilation (as well as L-1,2-propanediol oxidoreductase) and the rha genes (min 88) encoding the trunk pathway for L-rhamnose dissimilation. Mutants that grow on L-1,2-propanediol as a carbon and energy source also depend on the ald gene product for the conversion of L-lactaldehyde to L-lactate. 相似文献
994.
Method for localization of cloned DNA fragments on the Escherichia coli chromosome. 总被引:3,自引:3,他引:0 下载免费PDF全文
In exponentially growing cultures of Escherichia coli strains carrying the dnaC28 mutation, DNA replication can be synchronized by temperature changes (R. L. Rodriguez, M. S. Dalbey, and C. I. Davern, J. Mol. Biol. 74:599-604, 1973). We used this synchronization procedure and DNA-DNA hybridization to develop a technique for the localization of cloned chromosomal fragments on the genetic map. Because of the bidirectional nature of replication in E. coli, our method gave two possible positions (one on each replication arm). However because of the precision obtained for each position (+/- 1 map unit), the final mapping with various genetic techniques was greatly facilitated. Using this technique and a simple chromosomal mobilization test, we located at 93.2 +/- 1 min a cloned DNA fragment carrying an extragenic suppressor of dnaA46, a thermosensitive mutation in the dnaA initiation gene. Further analysis showed that the groES (mopA) and groEL (mopB) genes, both located at 94.2 min on the standard map, were indeed carried by the cloned suppressor fragment. 相似文献
995.
996.
Overexpression and DNA-binding properties of the mer-encoded regulatory protein from plasmid NR1 (Tn21). 总被引:13,自引:10,他引:3 下载免费PDF全文
A Heltzel D Gambill W J Jackson P A Totis A O Summers 《Journal of bacteriology》1987,169(7):3379-3384
In plasmid NR1 the expression of genes involved in mercury resistance (Tn21) is regulated by the trans-acting product of the merR gene. An in vivo T7 RNA polymerase-promoter overexpression system was used to detect a protein of approximately 16,000 daltons encoded by the merR reading frame. Overexpressed MerR constituted about 5% of labeled proteins. An in vitro MerR-mer-op (mer-op is the mer operator and promoter region) gel electrophoresis binding assay established that the binding site for MerR was located between the putative -35 and -10 sequences of the promoter for the mer structural genes. A nonsense mutation in the carboxyl half of MerR resulted in the loss of biological function and the loss of in vitro mer-op binding properties. 相似文献
997.
Translocation of Vibrio harveyi N,N''-diacetylchitobiase to the outer membrane of Escherichia coli. 总被引:3,自引:0,他引:3 下载免费PDF全文
The gene encoding N,N'-diacetylchitobiase (chitobiase) of the chitinolytic marine bacterium Vibrio harveyi has been isolated. While expression of the chitobiase gene (chb) was inducible by N,N'-diacetylchitobiose in V. harveyi, it was expressed constitutively when cloned in Escherichia coli, suggesting that controlling elements are not closely linked to chb. Chitobiase was found in the membrane fraction of E. coli cells containing plasmids with the cloned V. harveyi chb gene. When membranes of such cells were separated on Osborn gradients, chitobiase activity was found mainly in the outer membrane band. Translocation of the enzyme to the outer membrane was accompanied by cleavage of a signal peptide. A fusion protein, in which 22 amino acids from the amino terminus of prechitobiase were replaced with 21 amino acids from the pUC19 lacZ amino terminus, was not processed, and 99% of the activity was located in the cytoplasmic fraction. A homology to six amino acids surrounding the lipoprotein processing and modification site was found near the amino terminus of prechitobiase. 相似文献
998.
999.
Isolation of the outer membrane and characterization of the major outer membrane protein from Spirochaeta aurantia. 总被引:15,自引:10,他引:5 下载免费PDF全文
A M Kropinski T R Parr Jr B L Angus R E Hancock W C Ghiorse E P Greenberg 《Journal of bacteriology》1987,169(1):172-179
The outer membrane of Spirochaeta aurantia was isolated after cells were extracted with sodium lauryl sarcosinate and was subsequently purified by differential centrifugation and KBr isopycnic gradient centrifugation. The purified outer membrane was obtained in the form of carotenoid-containing vesicles. Four protein species with apparent molecular weights of 26,000 (26K), 36.5K, 41K, and 48.5K were readily observed as components of the vesicles. The 36.5K protein was the major polypeptide and constituted approximately 90% of the outer membrane protein observed on sodium dodecyl sulfate-polyacrylamide gels. Under mild denaturing conditions the 36.5K major protein exhibited an apparent molecular weight of approximately 90,000. This, together with the results of protein cross-linking studies, indicates that the 36.5K polypeptide has an oligomeric conformation in the native state. Reconstitution of solubilized S. aurantia outer membrane into lipid bilayer membranes revealed the presence of a porin, presumably the 36.5K protein, with an estimated channel diameter of 2.3 nm based on the measured single channel conductance of 7.7 nS in 1 M KCl. 相似文献
1000.
Characterization of the Erwinia carotovora pelB gene and its product pectate lyase. 总被引:8,自引:3,他引:5 下载免费PDF全文
The pelB gene encodes pectate lyase B, one of three pectate lyases identified in Erwinia carotovora EC. Pectate lyase B was purified from Escherichia coli containing the pelB gene on a recombinant plasmid. The activity of the protein was optimal at a pH of 8.3. The amino acid composition, N-terminal amino acid sequence, and C-terminal peptide sequence were determined and compared with the polypeptide sequence deduced from the DNA sequence of pelB. Purified pectate lyase B started at amino acid 23 of the predicted sequence, suggesting that a 22-amino-acid leader peptide had been removed. Pectate lyase B of E. carotovora EC and pectate lyase B of E. chrysanthemi EC16 contain 352 and 353 amino acids, respectively (N. T. Keen, S. Tanaki, W. Belser, D. Dahlbeck, and B. Staskawicz, J. Bacteriol. 168:595-606, 1986). The two proteins are 72% homologous on the basis of DNA sequence data, and 75% of the amino acids are identical. 相似文献