Using Synthetic Biology to Distinguish and Overcome Regulatory and Functional Barriers Related to Nitrogen Fixation (original) (raw)
Related papers
Nature, 1980
Gene fusions in which the lac genes are under the control of each promoter in the Klebsiella pneumoniae nitrogen fixation (nit) gene cluster have been constructed. These fusions have been used to examine positive control of the cluster and the response of individual genes to repression by ammonia and oxygen. De-repression of nif transcriptional units is coordinate and molybdate is required for maximal expression of the s tr u ctural gene operon, which is autogenously regulated.
Using synthetic biology to increase nitrogenase activity
Microbial cell factories, 2016
Nitrogen fixation has been established in protokaryotic model Escherichia coli by transferring a minimal nif gene cluster composed of 9 genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV) from Paenibacillus sp. WLY78. However, the nitrogenase activity in the recombinant E. coli 78-7 is only 10 % of that observed in wild-type Paenibacillus. Thus, it is necessary to increase nitrogenase activity through synthetic biology. In order to increase nitrogenase activity in heterologous host, a total of 28 selected genes from Paenibacillus sp. WLY78 and Klebsiella oxytoca were placed under the control of Paenibacillus nif promoter in two different vectors and then they are separately or combinationally transferred to the recombinant E. coli 78-7. Our results demonstrate that Paenibacillus suf operon (Fe-S cluster assembly) and the potential electron transport genes pfoAB, fldA and fer can increase nitrogenase activity. Also, K. oxytoca nifSU (Fe-S cluster assembly) and nifFJ (elec...
Promoter mutations that allow nifA-independent expression of the nitrogen fixation nifHDKY operon
Proceedings of the National Academy of Sciences, 1983
The nifHDKY operon of KlebsieUa pneumoniae encodes 'for structural polypeptides of nitrogenase and requires the nifA gene product for transcription. Mutations that allow transcription of the niJHDKY operon in absence of the nifA gene product were characterized in plasmids containing the regulatory region of nifHDKY and nifH fused in:phase to lacZ.-B-Galactosidase activity served as a measure for nifHWexpression. Most mutations were located in the nif regulatory region and included insertion sequence 2 (IS2) insertions, a sequence duplication, and a base substitution. In Escherichia coli, 3-pgalactosidase activity expressed from the mutant plasmids in the absence of MfA was 6-30% of the.nifA-activated, parental level. Expression from most mutant plasmids was further increased by nifA. InK. pneumoniae, IS2-containing plasmids expressed low levels of S-galactosidase and responded poorly, if at all, to activation by nifA, whereas expression from other mutant types was similar to that observed in E. coli. Nucleotide sequence analysis of two mutants indicated that sequences within 41 base pairs upstream to the nifH coding sequence were involved in nif-specific regulation. The results suggest that an inverted repeat element in this region, which could theoretically form.a cruciform structure in the DNA, is involved in the transcriptional control of the nifHDKY operon.
Journal of bacteriology, 1978
Two hundred and thirty-five Nif- strains of Klebsiella pneumoniae were characterized by two-dimensional polyacrylamide gel electrophoresis. Forty-two of these strains were tested further by in vitro acetylene reduction assays. By these techniques, nine nif-coded polypeptides were identified, and eight of these were assigned to specific nif genes. Nitrogenase component I required nifK and nifD, which coded for the beta and alpha subunits, and nifB, -E, and -N were required for the iron-molybdenum cofactor, which is a part of the active site of nitrogenase. nifH coded for the structural protein of component II, and nifM and nifS products seemed to be necessary for the synthesis of an active component II. There were two genes, nifF and nifJ, that were required for N2 fixation in vivo but not for N2 fixation in vitro. There were at least two cases (nifE and nifN, nifK and nifD) of two proteins that seemed to require each other for stability in vivo. Regulation of N2 fixation is apparent...
MGG Molecular & General Genetics, 1981
Some restriction endonuclease fragments of mf DNA, when carried on small multicopy plasmids, inhibited n/f expression in Klebsiella pneumoniae. A study of this inhibitory effect revealed, (1) that overproduction of the n/JL gene product inhibited transcription of two n/f operons examined, n/fJ and n/JHDKY and, (2) that when transcription was initiated from the promoter of the n0~IDKY operon on multicopy plasmids there was a corresponding decrease in the transcription rates of the chromosomally located mfJ and mJHDKY but not the n/fLA operon. Studies of transcription in vivo also showed that the tufA gene product was essential for transcription initiation from the m)q-IDKY and n/fBQ promoters. These results, taken with earlier observations (see Discussion) provide evidence that the mJL and nifA gene products are respectively a repressor and activator of mftranscription initiation from all n/f promoters except that of the mJLA operon.
MGG Molecular & General Genetics, 1982
Recombinant DNA plasmids containing inserts from the glnA region of Escherichia coli were used to study the expression of gln, hut, and n/f operons in a regulation defective mutant (Gln-Hut-Nif-) of Klebsiella pneumoniae, KP5060. Genes adjacent to the C-terminal end ofglnA on the E. coli chromosome were able to derepress hut and n/f operons in K. pneumoniae in the absence of glnA product. However, complete derepression of n/f operons required inclusion of the segment adjacent to the N-terminal end of the glnA region of the E. coli chromosome along with the C-terminal end segment. In the absence of functional glnA, such a fully derepressed strain expressed mf and hut constitutively indicating a role for the catalytic activity of glutamine synthetase in repression of the genes under nitrogen control.
Kinetics of nif Gene Expression in a Nitrogen-Fixing Bacterium
Journal of Bacteriology, 2014
Nitrogen fixation is a tightly regulated trait. Switching from N 2 fixation-repressing conditions to the N 2 -fixing state is carefully controlled in diazotrophic bacteria mainly because of the high energy demand that it imposes. By using quantitative real-time PCR and quantitative immunoblotting, we show here how nitrogen fixation ( nif ) gene expression develops in Azotobacter vinelandii upon derepression. Transient expression of the transcriptional activator-encoding gene, nifA , was followed by subsequent, longer-duration waves of expression of the nitrogenase biosynthetic and structural genes. Importantly, expression timing, expression levels, and NifA dependence varied greatly among the nif operons. Moreover, the exact concentrations of Nif proteins and their changes over time were determined for the first time. Nif protein concentrations were exquisitely balanced, with FeMo cofactor biosynthetic proteins accumulating at levels 50- to 100-fold lower than those of the structura...
Klebsiella pneumoniae nif-lac fusions are expressed in Agrobacterium tumefaciens C58
MGG Molecular & General Genetics, 1987
Plasmids containing hybrid genes, in which different KlebsielIa pneumoniae nif (nitrogen-fixation) promoters were fused with the structural part of the Escherichia coli lac operon, were introduced into a double auxotrophic derivative of Agrobacterium tumefaciens C58. A study of their expression in the new host was made simple by the inherent inability of A. tumefaciens C58 to produce fl-galactosidase unless provided with the wild-type lac operon of E. coli. As shown by quantitative measurements of the enzyme, all K. pneumoniae promoters were expressed well in A. tumefaciens C58, even under conditions known to repress them. It also has been shown that the activity of K. pneumoniae nifA is essential for the expression of nifHDK even when introduced into A. tumefaciens. After entering the new host the plasmids, the nif genes and the fusion alleles contained in them, remained stable. Possible mechanisms responsible for the constitutive behaviour of n/f promoters in A. tumefaciens are discussed.