Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of mutations in the -10, -35, and Fnr box regions of the narGHJI promoter of Escherichia coli were determined by assaying the expression of beta-galactosidase from narG::lacZ fusion plasmids under aerobic and anaerobic conditions. A 1-base change in the -10 hexamer completely abolished expression, whereas a 3-base change to create the consensus TATAAT resulted in significant aerobic as well as anaerobic expression. A mutation in the putative -35 hexamer did not affect anaerobic expression but reduced aerobic expression from the construction with the -10 consensus sequence. A mutation in the Fnr box severely reduced anaerobic expression but did not affect aerobic expression. When the complete 5' region of the nar operon including the NarL box was present, nitrate stimulated both aerobic and anaerobic expression. Stimulation of expression by nitrate occurred in an fnr mutant but not in a narL mutant. We conclude that the rate of transcription of the nar operon is dependent on two distinct modes of transcription. One mode, which occurs at low levels, depends on the -10 and -35 hexamer sequences and is dramatically enhanced by changing the -10 sequence to the consensus TATAAT. The second depends on the -10 and Fnr box sequences but is independent of the -35 sequence. This second mode occurs at a very high level under anaerobic conditions when Fnr is activated and is also enhanced by changing the -10 sequence to the consensus TATAAT. NarL, activated by nitrate, stimulated both modes of transcription, indicating that it does not act through Fnr but that it directly affects the interaction of RNA polymerase with the promoter.
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PMID:Role of alternative promoter elements in transcription from the nar promoter of Escherichia coli. 173 6

In the nitrate reductase system of Escherichia coli, the maximal expression of the nar operon is obtained under anaerobiosis in the presence of nitrate. Mudl (Ap,lac) insertion mutants, which only grew on lactose anaerobically if supplemented with nitrate were mapped at the chlC locus at min 27 of the map. In these fusion strains which lack benzyl viologen dependent nitrate reductase (NR) activity as well as the formiate-linked NR activity, the synthesis of beta-galactosidase reflects the regulation of the wild type nar operon at the transcriptional level. From these strains, two classes of spontaneous regulatory mutants were isolated: class I mutants which synthesized beta-galactosidase in anaerobiosis in the absence of nitrate and class II mutants in which the synthesis of that enzyme was partially independent of nitrate and it was no longer repressed by oxygen. The class I regulatory mutation was tightly linked to the nar operon as shown by bacteriophage P1 transductions. It probably affects either a closely linked cis-active element or a gene coding for a negative regulatory protein.
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PMID:Isolation and characterization of mutants affected in the expression of the nar operon Escherichia coli. 213 55

The nar operon, which encodes the three subunits of nitrate reductase in Escherichia coli, is fully induced under anaerobic conditions with nitrate. Two distinct regulatory domains have been delineated in the 5' region of the operon which respond respectively to positive induction by the fnr gene product under anaerobic conditions and to positive induction by the narL gene product in the presence of nitrate (S.F. Li, T. Rabi, and J.A. DeMoss, J. Bacteriol. 164:25-32). To characterize these two regulatory regions, we determined the DNA sequence for a 500-base-pair (bp) region extending upstream from the first structural gene of the nar operon. Analysis of subsequent subclones of the operon established that the 5' limit of the nar operon lies between 215 and 260 bp upstream from the translational start site of the first structural gene. The region required for induction by the fnr gene product is located within 160 bp from the translation start site, while the region responding to induction by nitrate extends an additional 100 bp upstream. Protein fusions of lacZ with the N-terminal sequence of the narG gene were constructed so that beta-galactosidase formation was under the control of the nar promoter and one or both regulatory domains. Analysis of strains bearing these fusion plasmids indicated that the expression of the hybrid proteins paralleled that of nitrate reductase by the parent plasmids, demonstrating that the regulatory signals did not extend significantly into the first structural gene. The transcriptional start site and the level of the transcription were determined by the S1 mapping procedure. One major transcript was identified which initiated -50 bp from the translational start site of the first structural gene. The synthesis of the transcript was repressed aerobically, was fully induced by nitrate anaerobically, and was greatly reduced in an Fnr- mutant. Possible regulatory sequences were identified in the 200-bp regulatory region extending upstream from the transcription start site.
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PMID:Promoter region of the nar operon of Escherichia coli: nucleotide sequence and transcription initiation signals. 330 46

The nar promoter of Escherichia coli is maximally induced under anaerobic or microaerobic conditions in the presence of nitrate. We previously demonstrated in batch experiments that the intact nar promoter of E. coli cloned into a pBR322-based plasmid serves as a high-level expression system in a nar mutant of E. coli (Lee et al., 1996b). In this study, we extend characterization of the nar promoter expression system to the fed-batch culture mode, which is widely used in industrial-scale fermentation. From these experiments, it was found that the specific beta-galactosidase activity expressed from the lacZ gene fused to the nar promoter was maximal when host cells were grown under aerobic conditions [dissolved oxygen, (DO) = 80%] to absorbance at 600 nm (OD600) = 35 before induction of the nar promoter by lowering DO to 1-2% with alternating microaerobic and aerobic conditions. Approximately 15 h after induction, the OD600 of the culture reached 135 and the specific beta-galactosidase activity increased to 40,000 Miller units, equivalent to approximately 35% of the total cellular proteins. The specific beta-galactosidase activity before induction was approximately 1,000 Miller units, giving an induction ratio of approximately 40. Based on these results, we conclude that the nar promoter provides a convenient and effective high level expression system under conditions of fed-batch culture.
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PMID:Fed-batch cultivation of an oxygen-dependent inducible promoter system, the nar promoter in Escherichia coli with an inactivated nar operon. 1009 53

A nar promoter system (a modified nar promoter in a mutant host Escherichia coli (pMW618/W3110narL(-))), which is maximally induced under microaerobic conditions, was developed and characterized through batch and fed-batch culture to see whether the modified nar promoter can be used as an oxygen-dependent inducible promoter in the absence of nitrate ion. The modified nar promoter (pMW618) derived by mutations at -10 and -35 regions of the wild-type nar promoter does not require nitrate ion for the full induction, while a mutant host E. coli, W3110narL(-), does not express nitrate-dependent regulatory protein, NARL, from the host chromosome. In this study, it was found from fed-batch culture that the specific beta-galactosidase activity expressed from the lacZ gene fused to the modified nar promoter in the absence of nitrate ion was maximal when E. coli was grown under aerobic conditions (dissolved oxygen (DO) at 80%) to absorbance at 600 nm (OD(600)) of 35, and then the modified nar promoter was induced by lowering DO to 1-2% with alternating microaerobic and aerobic conditions. The maximal specific beta-galactosidase activity became 58,000 Miller at OD(600) of 160 with an induction ratio of 20. On the basis of these results, we conclude that the modified nar promoter system (pMW618/W3110narL(-)), requiring only reduction of DO for the full induction, provides a convenient and effective high-level expression system under conditions of fed-batch culture.
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PMID:Development and characterization of an oxygen-dependent inducible promoter system, the modified nar promoter in a mutant Escherichia coli. 1069 79

The nar promoters, whose transcription is maximally induced under microaerobic conditions in the presence of nitrate ion, were characterized in fed-batch culture to determine whether they can be used for metabolic engineering, by which overall production of valuable chemicals can be increased. For this purpose, we tested whether the expression level of a reporter gene, the lacZ gene from the nar promoter, could be maintained constant throughout the induction period by manipulation of dissolved oxygen (DO) levels at a given nitrate ion concentration. First, E. coli was grown under aerobic conditions (DO 80%) to absorbance at 600 nm (OD(600)) of 35, then the nar promoter was induced by reduction of DO to different levels, combined with different frequencies and duration of alternating microaerobic and aerobic conditions throughout the entire induction period. For a wild-type nar promoter (pMW61) in a mutant host E. coli with a mutation in the narG gene on the chromosome of the host (RK5265), it was possible to maintain production of beta-galactosidase activity per cell (specific beta-galactosidase activity) at a constant rate at 5000, 10,000, 15,000, and 20,000 Miller units, using different combinations of nitrate ion concentrations (0.1%, 0.5%, and 1%) and DO levels. In addition, it was possible to maintain production of specific beta-galactosidase activity at a constant rate at about 10,000 Miller units in the absence of nitrate ion when a nitrate-independent nar promoter (pMW618) in the narL(-) mutant of the W3110 E. coli strain (W3110narL(-)) was used. Based on these results, we conclude that the nar promoter system provides a convenient expression system for metabolic engineering as well as for maximal production of recombinant proteins under conditions of fed-batch culture.
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PMID:Characterization of an oxygen-dependent inducible promoter, the nar promoter of Escherichia coli, to utilize in metabolic engineering. 1146 Feb 48

The recombinant proteins produced from Escherichia coli as a host cell need to be made at as low a cost as possible because of the end of the monopoly following expiry of the patent on early pharmaceutical proteins, and thus expanding applications to non-pharmaceutical large-scale products. We review in this article how the various promoters used in recombinant E. coli could affect its protein products, especially with emphasis on relatively new oxygen-dependent nar promoters for beta-galactosidase production. Several studies carried out in the authors' laboratory show that the nar promoter does not require any chemicals except 1% nitrate and oxygen for protein production. And according to recent work with the modified strains it is possible to produce the enzyme (beta-galactosidase) even without the nitrate ions at 45% of its total protein content when its cell density reached OD = 176.
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PMID:Fed-batch cultures of Escherichia coli cells with oxygen-dependent nar promoter systems. 1199 Nov 78

The nar promoter of Escherichia coli, which is maximally induced under anaerobic conditions in the presence of nitrate, was characterized to see whether the nar promoter cloned onto pBR322 can be used as an inducible promoter. To increase the expression level, the nar promoter was expressed in E. coli where active nitrate reductase cannot be expressed from the nar operon on the chromosome. A plasmid with the lacZ gene expressing beta-galactosidase instead of the structural genes of the nar operon was used to simplify an assay of induction of the nar promoter. The following effects were investigated to find optimal conditions: methods of inducing the nar promoter, optimal nitrate and molybdate concentrations maximally inducing the nar promoter, the amount of expressed beta-galactosidase, and induction ratio (specific beta-galactosidase activity after maximal induction/specific beta-galactosidase activity before induction). The following results were obtained from the experiments: induction of the nar promoter was optimal when E. coli was grown in the presence of 1% nitrate at the beginning of culture; expression of beta-galactosidase was not affected by molybdate; the induction ratio was maximal, approximately 300, when the overnight culture was grown in the flask for 2.5 h (OD(600) is congruent to 1.3) before being transferred to the fermentor; the amount of beta-galactosidase per cell and per medium volume was maximal when E. coli was grown under aerobic conditions to OD(600) = 1.7; then the nar promoter was induced under microaerobic conditions made by lowering dissolved oxygen level (DO) to 1-2%. After approximately 6 h of induction, OD(600) became 3.2 and specific beta-galactosidase activity became 36,000 Miller units, equivalent to 35% of total cellular proteins, which was confirmed from sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
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PMID:Characterization of an oxygen-dependent inducible promoter system, the nar promoter, and Escherichia coli with an inactivated nar operon. 1862 30