Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 enzyme complex nitrogenase, which reduces N2 to NH+4, involves two redox proteins, both irreversibly damaged by O2 (ref. 1). Enzyme activity therefore requires anaerobic conditions, a source of reductant and a large amount of ATP (approximately 16 ATPs per N2). In both aerobic and facultative anaerobic N2-fixing bacteria, nitrogenase synthesis is regulated by O2 and NH+4, but in the aerobes there are also processes to protect the enzyme from O2 damage. The mechanisms of repression by O2 and NH+4 seem to be independent in the organisms so far examined. In the facultative anaerobe, Klebsiella pneumoniae, O2 was shown to repress nitrogenase synthesis in an NH+4-constitutive strain. The fusion of the Escherichia coli lacZ gene into each transcriptional unit of the nitrogen fixation (nif) gene cluster in K. pneumoniae has facilitated studies with O2, because expression from the various nif promoters results in an O2-stable product (beta-galactosidase). Notably, the nifHDK operon (the nitrogenase structural genes) was more sensitive to O2 repression than the nifLA operon (regulatory genes). The characterization of mutants, reported here, indicates the involvement of a nif-regulatory gene product in the mechanism of O2 control of nitrogenase synthesis.
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PMID:Nitrogen fixation gene (nifL) involved in oxygen regulation of nitrogenase synthesis in K. pneumoniae. 701 40

The nifBQ transcriptional unit of Azotobacter vinelandii has been previously shown to be required for activity of the three nitrogenase systems, Mo nitrogenase, V nitrogenase, and Fe nitrogenase, present in this organism. We studied regulation of expression and the role of the nifBQ region by means of translational beta-galactosidase fusions to each of the five open reading frames: nifB, orf2 (fdxN), orf3 (nifO), nifQ, and orf5. Expression of the first three open reading frames was observed under all three diazotrophic conditions; expression of orf5 was never observed. Genes nifB and fdxN were expressed at similar levels. With Mo, expression of nifO and nifQ was approximately 20- and approximately 400-fold lower than that of fdxN, respectively. Without Mo, expression of nifB dropped three- to fourfold and that of nifQ dropped to the detection limit. However, expression of nifO increased threefold. The products of nifB, fdxN, nifO, and nifQ have been visualized in A. vinelandii as beta-galactosidase fusion proteins with the expected molecular masses. The NifB- fusion lacked activity for any of the three nitrogenase systems and showed an iron-molybdenum cofactor-deficient phenotype in the presence of Mo. The FdxN- mutation resulted in reduced nitrogenase activities, especially when V was present. Dinitrogenase activity in extracts was similarly affected, suggesting a role of FdxN in iron-molybdenum cofactor synthesis. The NifO(-)-producing mutation did not affect any of the nitrogenases under standard diazotrophic conditions. The NifQ(-)-producing mutation resulted in an increased (approximately 1,000-fold) Mo requirement for Mo nitrogenase activity, a phenotype already observed with Klebsiella pneumoniae. No effect of the NifQ(-)-producing mutation on V or Fe nitrogenase was found; this is consistent with its very low expression under those conditions. Mutations in orf5 had no effect on nitrogenase activity.
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PMID:Expression of the nifBfdxNnifOQ region of Azotobacter vinelandii and its role in nitrogenase activity. 849 13

From Azospirillum lipoferum (Al) FS, a nitrogen-fixing bacterium isolated from the rhizosphere of rice, we cloned and sequenced draT, encoding dinitrogenase reductase ADP-ribosyltransferase, and draG, encoding dinitrogenase reductase-activating glycohydrolase. The nucleotide sequences of draTG showed extensive similarity to the same genes from Azospirillum brasilense, Rhodospirillum rubrum and Rhodobacter capsulatus, and they are assumed to be co-transcribed as a single operon. When this draTG operon was introduced into Klebsiella oxytoca, this organism acquired the ability to respond to extracellular NH(+4) ions with reversible inhibition of nitrogenase activity, similar to that seen in Al FS. We constructed a plasmid containing a draT::lacZ gene fusion and found that beta-galactosidase activity was detected under microaerobic conditions, regardless of NH(+4) concentration, but not under aerobic conditions. This indicates that the transcription of draTG responds to the level of oxygen, but not to that of NH(+4) ions.
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PMID:Cloning, sequencing and transcriptional regulation of the draT and draG genes of Azospirillum lipoferum FS. 862 Oct 68

Azotobacter is a diazotrophic bacterium that harbors three genetically distinct nitrogenases referred to as nif, vnf, and anf systems. The nifM is an accessory gene located in the nif gene cluster and is transcriptionally regulated by the NifA. However, Azotobacter mutants that lack NifA are known to synthesize functional NifM and this accessory protein is known to be needed for the activity of nitrogenase-2 and nitrogenase-3. To determine how the transcription of nifM is regulated when Azotobacter is grown under conditions in which nitrogenase-2 or nitrogenase-3 is expressed, we generated an Azotobacter vinelandii strain that carries a nifM:lacZ-kanamycin resistance gene cassette in its chromosome. In this strain the nifM open reading frame was disrupted by the presence of a lacZ-kanamycin resistance gene cassette so that it could not produce active NifM. Moreover, the lacZ gene was placed under the transcriptional control elements of the nifM gene so that the lacZ expression could be used as a marker to determine the extent of expression of the nifM gene under different growth conditions. Our results show that this strain was unable to grow in Burk's nitrogen-free medium supplemented with either molybdenum or vanadium or lacking both metals suggesting that in the absence of functional NifM none of the nitrogenases were active. It was also found that the nifM expression was differentially regulated when the A. vinelandii cells were grown under conditions that activate nitrogenase-2 and nitrogenase-3, as determined by liquid beta-galactosidase activity measurements. These results suggest that the transcriptional activators, VnfA and AnfA, may regulate the nifM expression.
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PMID:Regulated expression of the nifM of Azotobacter vinelandii in response to molybdenum and vanadium supplements in Burk's nitrogen-free growth medium. 1052 62

A hybrid promoter consisting of the in tandem fusion of the Tn5 nptII and the Klebsiella pneumoniae nifH promoters was constructed to study the functionality of the nif genes transcriptional activator NifA from Bradyrhizobium japonicum in two different host bacteria. beta-galactosidase experiments in E. coli revealed that the hybrid nptII-nifH promoter can behave as a constitutive or a NifA-inducible promoter depending on the aeration conditions. Expression of the B. japonicum NifA from the hybrid nptII-nifH promoter (plasmid pBPF204) induced "in trans" lacZ transcription from the Azotobacter chroococcum nifH promoter in E. coli and A. diazotrophicus cells grown at low pO2. Similarly, the plasmid pBPF204 increased nitrogenase activity in A. diazotrophicus cells grown under microaerobic conditions. Based on these results, we suggest that the B. japonicum NifA could function as an efficient O2-sensitive transcriptional activator of nif genes in genetically distant diazotrophic bacteria.
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PMID:Functional Bradyrhizobium japonicum NifA expression under a hybrid nptII-nifH promoter in E. coli and Acetobacter diazotrophicus SRT4. 1093 42

Expression of Bradyrhizobium japonicum wild-type strain USDA110 nirK, norC and nosZ denitrification genes in soybean root nodules was studied by in situ histochemical detection of beta-galactosidase activity. Similarly, P(nirK)-lacZ, P(norC)-lacZ, and P(nosZ)-lacZ fusions were also expressed in bacteroids isolated from root nodules. Levels of beta-galactosidase activity were similar in both bacteroids and nodule sections from plants that were solely N(2)-dependent or grown in the presence of 4 mM KNO(3). These findings suggest that oxygen, and not nitrate, is the main factor controlling expression of denitrification genes in soybean nodules. In plants not amended with nitrate, B. japonicum mutant strains GRK308, GRC131, and GRZ25, that were altered in the structural nirK, norC and nosZ genes, respectively, showed a wild-type phenotype with regard to nodule number and nodule dry weight as well as plant dry weight and nitrogen content. In the presence of 4 mM KNO(3), plants inoculated with either GRK308 or GRC131 showed less nodules, and lower plant dry weight and nitrogen content, relative to those of strains USDA110 and GRZ25. Taken together, the present results revealed that although not essential for nitrogen fixation, mutation of either the structural nirK or norC genes encoding respiratory nitrite reductase and nitric oxide reductase, respectively, confers B. japonicum reduced ability for nodulation in soybean plants grown with nitrate. Furthermore, because nodules formed by each the parental and mutant strains exhibited nitrogenase activity, it is possible that denitrification enzymes play a role in nodule formation rather than in nodule function.
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PMID:Expression of nir, nor and nos denitrification genes from Bradyrhizobium japonicum in soybean root nodules. 1503 54

The MoFe protein of the complex metalloenzyme nitrogenase folds as a heterotetramer containing two copies each of the homologous alpha and beta subunits, encoded by the nifD and the nifK genes respectively. Recently, the functional expression of a fusion NifD-K protein of nitrogenase was demonstrated in Azotobacter vinelandii, strongly implying that the MoFe protein is flexible as it could accommodate major structural changes, yet remain functional [M.H. Suh, L. Pulakat, N. Gavini, J. Biol. Chem. 278 (2003) 5353-5360]. This finding led us to further explore the type of interaction between the fused MoFe protein units. We aimed to determine whether an interaction exists between the two fusion MoFe proteins to form a homodimer that is equivalent to native heterotetrameric MoFe protein. Using the Bacteriomatch Two-Hybrid System, translationally fused constructs of NifD-K (fusion) with the full-length lambdaCI of the pBT bait vector and also NifD-K (fusion) with the N-terminal alpha-RNAP of the pTRG target vector were made. To compare the extent of interaction between the fused NifD-K proteins to that of the beta-beta interactions in the native MoFe protein, we proceeded to generate translationally fused constructs of NifK with the alpha-RNAP of the pTRG vector and lambdaCI protein of the pBT vector. The strength of the interaction between the proteins in study was determined by measuring the beta-galactosidase activity and extent of ampicillin resistance of the colonies expressing these proteins. This analysis demonstrated that direct protein-protein interaction exists between NifD-K fusion proteins, suggesting that they exist as homodimers. As the interaction takes place at the beta-interfaces of the NifD-K fusion proteins, we propose that these homodimers of NifD-K fusion protein may function in a similar manner as that of the heterotetrameric native MoFe protein. The observation that the extent of protein-protein interaction between the beta-subunits of the native MoFe protein in BacterioMatch Two-Hybrid System is comparable to the extent of protein-protein interaction observed between the NifD-K fusion proteins in the same system further supports this idea.
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PMID:Functional NifD-K fusion protein in Azotobacter vinelandii is a homodimeric complex equivalent to the native heterotetrameric MoFe protein. 1620 90

Nitrogen-fixing bacteria were isolated from the rhizosphere of different crops of Korea. A total of 16 isolates were selected and characterized. Thirteen of the isolates produced characteristics similar to those of the reference strains of Azospirillum, and the remaining 3 isolates were found to be Enterobacter spp. The isolates could be categorized into 3 groups based on their ARDRA patterns, and the first 2 groups comprised Azospirillum brasilense and Azospirillum lipoferum. The acetylene reduction activity (ARA) of these isolates was determined for free cultures and in association with wheat roots. There was no correlation between pure culture and plant-associated nitrogenase activity of the different strains. The isolates that showed higher nitrogenase activities in association with wheat roots in each group were selected and sequenced. Isolates of Azospirillum brasilense CW301, Azospirillum brasilense CW903, and Azospirillum lipoferum CW1503 were selected to study colonization in association with wheat roots. We observed higher expression of beta-galactosidase activity in A. brasilense strains than in A. lipoferum strains, which could be attributed to their higher population in association with wheat roots. All strains tested colonized and exhibited the strongest beta-galactosidase activity at the sites of lateral roots emergence.
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PMID:Wheat root colonization and nitrogenase activity by Azospirillum isolates from crop plants in Korea. 1633 34

A 365-base-pair (bp) DNA fragment, containing the promoter region of the nitrogenase reductase (nifH) gene from stem Rhizobium BTAi1, has been isolated and sequenced. The transcription initiation sites were localized at positions 152 (major initiation) and 114 (minor initiation) nucleotides upstream of the translation initiation codon. The 200-bp nucleotide sequence upstream of the nifH structural gene shows substantial homology to the corresponding nifH regions of cowpea Rhizobium (100%), Parasponia Rhizobium (89%), and Rhizobium japonicum (88%). The nifH promoter region of stem Rhizobium BTAi1 was fused to the lacZ gene of Escherichia coli. The fusion and a 1.6-kilobase DNA specifying neomycin phosphotransferase were inserted into a 3,4-kilobase fragment of stem Rhizobium chromosome, and the resulting construct was placed on a mobilizable vector, pREV1000. Stem Rhizobium transconjugants resistant to kanamycin were found to contain the nifH promoter region-lacZ fusion linked to the neomycin phosphotransferase gene at the site of chromosomal homology. Analysis of the DNA from stable transconjugants showed integration of a single copy of these sequences into the chromosome by a double-reciprocal crossover event. The transconjugants formed nitrogen-fixing nodules, indicating that the insertion occurred in a "nonessential" region of the stem Rhizobium chromosome. Transconjugant strain BTAi1000 grows on beta-galactosidase indicator plates under aerobic conditions as white colonies, whereas under microaerobic conditions (97% N(2)/3% O(2)), which derepress nitrogenase, the colonies turn blue within 15-24 hr. beta-Galactosidase activity in derepressed cultures of BTAi1000 showed a 200-fold increase in comparison to the wild-type strain, whereas stem nodules formed by BTAi1000 exhibited 15- to 20-fold higher beta-galactosidase values than wild-type nodules. Nitrogenase promoter-dependent expression of beta-galactosidase in stem nodules was inhibited by fixed nitrogen, suggesting that the nifH promoter-lacZ fusion is controlled coordinately in trans with the native nif region.
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PMID:Expression of beta-galactosidase controlled by a nitrogenase promoter in stem nodules of Aeschynomene scabra. 1659 14

Nitric oxide (NO) has gained interest as a major signaling molecule during plant development and in response to environmental cues. Formation of NO during symbiotic interactions has been reported, but the role and sources of NO in nodules remain unclear. In this work, the involvement of denitrification, performed by the symbiont Bradyrhizobium japonicum, in NO formation in soybean nodules in response to flooding conditions has been investigated by inoculating plants with napA-, nirK-, or norC-deficient mutants. Levels of nitrosylleghemoglobin (LbNO) in flooded nirK and norC nodules were significantly higher than those observed in wild-type nodules. In addition, nirK and norC nodules accumulated more nitrite and NO, respectively, than wild-type nodules. By contrast, levels of LbNO, nitrite, and NO in flooded napA nodules were lower than in wild-type nodules. These results suggest that LbNO formation in soybean nodules in response to flooding conditions is caused by nitrite and NO generated from periplasmic nitrate reductase (Nap) and also containing nitrite reductase (NirK) denitrification enzymes. Flooding caused a decrease of nifH expression and nitrogenase activity in wild-type and norC nodules but not in napA or nirK nodules. Incubation of wild-type and norC nodules with a NO scavenger counteracted the effect of flooding. Under free-living conditions, beta-galactosidase activity from a nifD'-'lacZ fusion decreased in a norC mutant, which also accumulated NO in the medium. These results suggest that NO formed by Cu-containing nitrite reductase in soybean nodules in response to flooding has a negative effect on expression of nitrogenase. We propose that Lb has a major role in detoxifying NO and nitrite produced by bacteroidal denitrification in response to flooding conditions.
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PMID:Production of nitric oxide and nitrosylleghemoglobin complexes in soybean nodules in response to flooding. 2036 76


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