Structural analysis of the genes encoding the molybdenum-iron protein of nitrogenase in the Parasponia rhizobium strain ANU289 (original) (raw)
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Nitrogenase reductase: A functional multigene family in Rhizobium phaseoli
Proceedings of the National Academy of Sciences, 1985
The complete coding sequence of the nitrogenase reductase gene (nifH) is present in three different regions of a Rhizobium phaseoli symbiotic plasmid. Homology between two of the regions containing nijH coding sequences extends over 5 kilobases. These in turn share 1.3 kilobases of homology with the third region. The nucleotide sequences of the three nitrogenase reductase genes were found to be identical. Site-directed insertion mutagenesis indicated that none of the three genes is indispensable for nitrogen fixation during symbiosis with Phaseolus vulgaris. This implies that at least two of the reiterated genes can be functionally expressed.
Applied and Environmental Microbiology, 2004
Rhizobium etli , as well as some other rhizobia, presents nitrogenase reductase ( nifH ) gene reiterations. Several R. etli strains studied in this laboratory showed a unique organization and contained two complete nifHDK operons (copies a and b) and a truncated nifHD operon (copy c). Expression analysis of lacZ fusion demonstrated that copies a and b in strain CFN42 are transcribed at lower levels than copy c, although this copy has no discernible role during nitrogen fixation. To increase nitrogenase production, we constructed a chimeric nifHDK operon regulated by the strong nifHc promoter sequence and expressed it in symbiosis with the common bean plant ( Phaseolus vulgaris ), either cloned on a stably inherited plasmid or incorporated into the symbiotic plasmid (pSym). Compared with the wild-type strain, strains with the nitrogenase overexpression construction assayed in greenhouse experiments had, increased nitrogenase activity (58% on average), increased plant weight (32% on a...
Applied and Environmental Microbiology, 2004
Enhance Rhizobium etli Accumulation in -Hydroxybutyrate β Abolishing Poly-Promoter Region and nifH Engineering the http://aem.asm.org/content/70/6/3272 Updated information and services can be found at: These include: REFERENCES http://aem.asm.org/content/70/6/3272#ref-list-1 at: This article cites 36 articles, 22 of which can be accessed free CONTENT ALERTS more» articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to: on June 7, 2014 by guest http://aem.asm.org/ Downloaded from on June 7, 2014 by guest
Gene, 1989
The genes encoding the structural components of nitrogenase, niffrl, n#D and nifX, from the fast-growing, broad-host-range ~hi~obium strain ANU240 have been identified and characterized. They are duplicated and linked in an operon nifHDK in both copies. Sequence analysis of the nifH gene from each copy, together with partial sequence analysis of the nz$D and nzjiY genes, and restriction endonuclease analysis suggested that the duplication is precise. Comparison of the Fe-protein sequence from strain ANU240 with that from other nitrogen-fixing organisms revealed that, despite its broad host range and certain physiological properties characteristic of Bradyrhizobium strains, ANU240 is more closely related to the narrow-host-range Rhizobium strains than to the broad-host-range Brudyrhizobium strains. The promoter regions of both copies of the nif genes contain the consensus sequence characteristic of nif promoters, and functional analysis of the two promoters suggested that both nif operons are transcribed in noduIes.
The molecular genetics of nitrogen fixation in the bradyrhizobium sp. (parasponia) strain ANU289
1986
DNA sequences which encode the iron-molybdenum protein of nitrogenase in the Bradyrhizobium sp. lV (Parasponia) strain ANU289 have been identified, cloned and sequenced to complete the first characterization of nitrogenase in a Rhizobium species. Other sequences have also been identified, cloned and analyzed to reveal six additional regions likely to be necessary for the symbiotic, nitrogen-fixing phenotype of this strain. In strain ANU289, the genes for the two sub-units of the molybdenum-iron protein, nifD and nifK, were isolated by homology to cloned Klebsiella pneumoniae genes. The separation of these genes from the gene encoding the ironprotein of nitrogenase, nifH, was determined to be 20 kb. The role of these genes in nitrogen-fixation was verified by use of a s~ecific, site-directed mutagenesis system which has been developed. Characterization of the nifD and nifK genes reveals the protein structure of their product s to be highly conserved between other nitrogen-fixing orga...
Molecular Microbiology, 1989
The nifA gene has been identified between the f/xXand nifB genes in the clover microsymbiont Rhizobium leguminosarum biovar trifolii {R.I. bv. trifolii) strain ANU843. Expression of the nifA gene is induced in the symbiotic state and site-directed mutagenesis experiments indicate that nifA expression is essential for symbiotic nitrogen fixation. Interestingly, the predicted R.I. bv. trifolii ti\fA protein lacks an /V-terminal domain that is present in the homologous proteins from R.I. bv. viciae, Rhizobium meliloti, Bradyrhizobium Japonicum, Klebsiella pneumoniae and all other documented NifA proteins. This indicates that this A/-terminal domain is not essential for NifA function in R.I. bv. trifolii.
Symbiotic Autoregulation of nifA Expression in Rhizobium leguminosarum bv. viciae
Journal of Bacteriology, 2004
NifA is the general transcriptional activator of nitrogen fixation genes in diazotrophic bacteria. In Rhizobium leguminosarum bv. viciae UPM791, the nifA gene is part of a gene cluster (orf71 orf79 fixW orf5 fixABCX nifAB) separated by 896 bp from an upstream and divergent truncated duplication of nifH (⌬nifH). Symbiotic expression analysis of genomic nifA::lacZ fusions revealed that in strain UPM791 nifA is expressed mainly from a 54 -dependent promoter (P nifA1 ) located upstream of orf71. This promoter contains canonical NifA upstream activating sequences located 91 bp from the transcription initiation site. The transcript initiated in P nifA1 spans 5.1 kb and includes nifA and nifB genes. NifA from Klebsiella pneumoniae was able to activate transcription from P nifA1 in a heterologous Escherichia coli system. In R. leguminosarum, the P nifA1 promoter is essential for effective nitrogen fixation in symbiosis with peas. In its absence, partially efficient nitrogen-fixing nodules were produced, and the corresponding bacteroids exhibited only low levels of nifA gene expression. The basal level of nifA expression resulted from a promoter activity originating upstream of the fixX-nifA intergenic region and probably from an incomplete duplication of P nifA1 located immediately upstream of fixA.
Proceedings of The National Academy of Sciences, 1997
Rhizobium leguminosarum bv. viciae expresses an uptake hydrogenase in symbiosis with peas (Pisum sativum) but, unlike all other characterized hydrogenoxidizing bacteria, cannot express it in free-living conditions. The hydrogenase-specific transcriptional activator gene hoxA described in other species was shown to have been inactivated in R. leguminosarum by accumulation of frameshift and deletion mutations. Symbiotic transcription of hydrogenase structural genes hupSL originates from a ؊24͞؊12 type promoter (hupS p ). A regulatory region located in the ؊173 to ؊88 region was essential for promoter activity in R. leguminosarum. Activation of hupS p was observed in Klebsiella pneumoniae and Escherichia coli cells expressing the K. pneumoniae nitrogen fixation regulator NifA, and in E. coli cells expressing R. meliloti NifA. This activation required direct interaction of NifA with the essential ؊173 to ؊88 regulatory region. However, no sequences resembling known NifA-binding sites were found in or around this region. NifA-dependent activation was also observed in R. etli bean bacteroids. NifAdependent hupS p activity in heterologous hosts was also absolutely dependent on the RpoN -factor and on integration host factor. Proteins immunologically related to integration host factor were identified in R. leguminosarum. The data suggest that hupS p is structurally and functionally similar to nitrogen fixation promoters. The requirement to coordinate nitrogenase-dependent H 2 production and H 2 oxidation in nodules might be the reason for the loss of HoxA in R. leguminosarum and the concomitant NifA control of hup gene expression. This evolutionary acquired control would ensure regulated synthesis of uptake hydrogenase in the most common H 2 -rich environment for rhizobia, the legume nodule.
Mol Gen Genet, 1981
The ability to identify" genes that specify nitrogenase (mfgenes) in Rhizobium depends on the close homology between them and the corresponding n/f genes of Klebsiella pneumoniae (Nuti et al. 1979; Ruvkun and Ausubel 1980). Rhizobium plasmids of high molecular weight (> 100 Md) were separated on agarose gels, transferred to nitrocellulose filters and tested for their ability to hybridise with radioactively labelled pSA30, containing the nifKDH region of K. pneumoniae. Five large plasmids, each present in different strains of R. leguminosarum or R. phasemi, were found to hybridise. Each of these plasmids had previously been shown to determine other symbiotic functions such as nodulation ability. The n/f genes on three different plasmids appeared to be in conserved DNA regions since they were within an EcoRI restriction fragment of the same size.
Journal of …, 1987
We show here that Rhizobium meliloti, the nitrogen-fixing endosymbiont of alfalfa (Medicago sativa), has a regulatory gene that is structurally homologous to previously characterized ntrC genes in enteric bacteria. DNA sequence analysis showed that R. meliloti ntrC is homologous to previously sequenced ntrC genes from Klebsiella pneumoniae and Bradyrhizobium sp. (Parasponia) and that an ntrB-like gene is situated directly upstream from R. meliloti ntrC. Similar to its counterparts in K. pneumoniae and Escherichia coli, R. meliloti ntrC is expressed when the cells are grown in nitrogen-limiting media. In addition, R. meliloti ntrC is required for growth on media containing nitrate as the sole nitrogen source and for the ex planta transcription of several R. meliloti nif genes. On the other hand, root nodules elicited by R. melioti ntrC mutants fix nitrogen as well as nodules elicited by wild-type R. meliloti. These latter results indicate that R. meliloti has separate regulatory pathways for activating nif gene expression ex planta and during symbiotic nitrogen fixation.