Organization of nitrogen fixation genes in a nonheterocystous, filamentous cyanobacterium

Abstract The nonheterocystous, filamentous cyanobacterium, Plectonema boryanum fixes nitrogen only under microaerophilic conditions. The organization of nitrogen fixation genes ( nifH, D, K ) in Plectonema was determined by using cloned fragments from the Anabaena nif genes as probes in Southern hybridizations. Regions of Plectonema DNA were homologous to Anabaena nifH, nifD , and nifK genes, and the resulting pattern of hybridization was used to construct a map of nifH, D, K DNA isolated from Plectonema cells grown under non-nitrogen fixing conditions (combined nitrogen and O₂ present). The nifH and nifD genes are on the same 3 kbp Hin dIII fragment, and nifK is on a 1 kbp Hin dIII fragment. All three nif fragments are adjacent to one another on a 12 kbp Cla I fragment.     

Cloning of the nifHDK genes and their organisation in the heterocystous cyanobacterium Mastigocladus laminosus

Abstract The heterocystous, nitrogen-fixing cyanobacterium Mastigocladus laminosus UTEX 1931 has an adjacent arrangement of the nifH, nifD and nifK genes, apparently similar to Fischerella sp. 27929, but unlike Anabaena 7120. In addition, unlike Fischerella sp. 27929, M. laminosus UTEX 1931 contains an additional nifH -like sequence located approximately 10 kb from the nifHDK gene cluster.     

The structural nif genes of the cyanobacteria Gloeothece sp. and Calothrix sp. share homology with those of Anabaena sp., but the Gloeothece genes have a different arrangement.

Probes carrying the Anabaena sp. strain PCC 7120 nitrogenase reductase (nifH) and nitrogenase (nifK and nifD) genes were hybridized to Southern blots of DNA from the unicellular, aerobic nitrogen-fixing cyanobacterium Gloeothece sp. strain PCC 6909 and from the filamentous cyanobacterium Calothrix sp. strain PCC 7601. These data suggest that the Gloeothece sp. nif structural proteins must be similar to those of other diazotrophs and that the ability for aerobic nitrogen fixation does not reside in the nif protein complex. We also found that the nif structural genes of Gloeothece sp. are clustered, whereas those of Calothrix sp. are arranged more like those of Anabaena sp.     

Molecular cloning of nif DNA from Azotobacter vinelandii.

Two clones which contained nif DNA were isolated from a clone bank of total EcoRI-digested Azotobacter vinelandii DNA. The clones carrying the recombinant plasmids were identified by use of the 32P-labeled 6.2-kilobase (kb) nif insert from pSA30 (which contains the Klebsiella pneumoniae nifK, nifD, and nifH genes) as a hybridization probe. Hybridization analysis with fragments derived from the nif insert of pSA30 showed that the 2.6-kb insert from one of the plasmids (pLB1) contains nifK whereas the 1.4-kb insert from the other plasmid (pLB3) contains nifD. Marker rescue tests using genetic transformation indicated that the 2.6-kb A. vinelandii nif fragment contains the wild-type alleles for the nif-6 and nif-38 mutations carried by Nif- strains UW6 and UW38. The 1.4-kb insert contains the wild-type allele for the nif-10 mutation carried by Nif- strain UW10.     

Structural and functional analysis of nitrogenase genes from the broad-host-range Rhizobium strain ANU240

The genes encoding the structural components of nitrogenase, nifH, nifD and nifK, from the fast-growing, broad-host-range Rhizobium 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 nifD and nifK 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 Bradyrhizobium 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 nodules.     

DNA hybridization analysis of the nif region of two methylotrophs and molecular cloning of nif-specific DNA.

DNA isolated from two diazotrophic methylotrophs, the obligate methanotroph Methylosinus sp. strain 6 and the methanol autotroph Xanthobacter sp. H4-14, hybridized to DNA fragments encoding nitrogen fixation (nif) genes from Klebsiella pneumoniae. This interspecific nif homology was limited to DNA fragments encoding the nitrogenase structural proteins (nifH, nifD, and nifK) and specific methylotroph DNA sequences. The hybridization patterns obtained with the two methylotrophs were dissimilar, indicating that the nif region of methylotrophs is not physically conserved. By using the K. pneumoniae nif structural genes as a probe, a fragment of nif DNA from each methylotroph was cloned and characterized. The DNA fragment from Methylosinus sp. 6 encoded two polypeptides of 57,000 and 34,000 molecular weight.     

A DNA fragment hybridizing to a nif probe in Rhodobacter capsulatus is homologous to a 16S rRNA gene

We have sequenced the Rhodobacter capsulatus nifH and nifD genes. The nifH gene, which codes for the dinitrogenase reductase protein, is 894 bp long and codes for a polypeptide of predicted Mr 32,412. The nifD gene, which codes for the alpha subunit of dinitrogenase, is 1,500 bp long and codes for a protein of predicted Mr 56,113. A 776-bp BglII-XhoI fragment containing only nif sequences was used as a hybridization probe against R. capsulatus genomic DNA. Two HindIII fragments, 11.8 kb and 4.7 kb in length, hybridize to this probe. Both fragments have been cloned from a cosmid library. The 11.8-kb fragment contains the nifH, D and K genes, as previously demonstrated (Scolnik and Haselkorn, 1984). In this paper we present evidence that suggests that the 4.7-kb HindIII fragment contains a gene coding for 16S rRNA, and that although homology between nif and this fragment can be observed in filter hybridization experiments, a second copy of the nif structural genes seems not to be present in this region.     

Complete nucleotide sequence of the Azotobacter vinelandii nitrogenase structural gene cluster

DNA fragments coding for the structural genes for Azotobacter vinelandii nitrogenase have been isolated and sequenced. These genes, nifH, nifD and nifK, code for the iron (Fe) protein and the alpha and beta subunits of the molybdenum-iron (MoFe) protein, respectively. They are arranged in the order: promoter:nifH:nifD:nifK. There are 129 nucleotides separating nifH and nifD and 101 nucleotides separating nifD and nifK. The amino acid (aa) sequences deduced from the nucleotide sequences are discussed in relation to the prosthetic group-binding regions of the nifHDK-encoded polypeptides.     

Expression of the Anabaena sp. strain PCC 7120 xisA gene from a heterologous promoter results in excision of the nifD element.

An 11-kilobase-pair element interrupts the nifD gene in vegetative cells of Anabaena sp. strain PCC 7120. The nifD element normally excises only from the chromosomes of cells that differentiate into nitrogen-fixing heterocysts. The xisA gene contained within the element is required for the excision. Shuttle vectors containing the Escherichia coli tac consensus promoter fused to various 5' deletions of the xisA gene were constructed and conjugated into Anabaena sp. strain PCC 7120 cells. Some of the expression plasmids resulted in excision of the nifD element in a high proportion of vegetative cells. Excision of the element required deletion of an xisA 5' regulatory region which presumably blocks expression in Anabaena sp. strain PCC 7120 vegetative cells but not in E. coli. Strains lacking the nifD element grew normally in medium containing a source of combined nitrogen and showed normal growth and heterocyst development in medium lacking combined nitrogen. The xisA gene was shown to be the only Anabaena gene required for the proper rearrangement in E. coli of a plasmid containing the borders of the nifD element.     

Isolation and complementation of mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen.

Mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen were isolated by mutagenesis with UV irradiation, followed by a period of incubation in yellow light and then by penicillin enrichment. A cosmid vector, pRL25C, containing replicons functional in Escherichia coli and in Anabaena species was constructed. DNA from wild-type Anabaena sp. strain PCC 7120 was partially digested with Sau3AI, and size-fractionated fragments about 40 kilobases (kb) in length were ligated into the phosphatase-treated unique BamHI site of pRL25C. A library of 1,054 cosmid clones was generated in E. coli DH1 bearing helper plasmid pDS4101. A derivative of conjugative plasmid RP-4 was transferred to this library by conjugation, and the library was replicated to lawns of mutant Anabaena strains with defects in the polysaccharide layer of the envelopes of the heterocysts. Mutant EF116 was complemented by five cosmids, three of which were subjected to detailed restriction mapping; a 2.8-kb fragment of DNA derived from one of the cosmids was found to complement EF116. Mutant EF113 was complemented by a single cosmid, which was also restriction mapped, and was shown to be complemented by a 4.8-kb fragment of DNA derived from this cosmid.     

Cloning and expression in Escherichia coli of the gene encoding a novel L-2,4-diaminobutyrate decarboxylase of Acinetobacter baumannii

Abstract The gene encoding L-2,4-diaminobutyrate decarboxylase (DABA DC) was cloned from Acinetobacter baumannii ATCC 19606. The gene was evidently under the control of its own promoter. Interestingly, the host carrying this clone also produced an appreciable amount of 1,3-diaminopropane. Restriction mapping and subsequent subcloning of the cloned insert localized the DABA DC gene within a 2.45-kb SphI/Eco RI fragment. For endogenous production of DAP, a 1.75-kb Eco RI/ Pst I region downstream from the DABA DC gene was further required. Southern blot hybridization revealed some heterogeneity in the DABA DC genes among other Acinetobacter species.     

Analysis of genes encoding an alternative nitrogenase in the archaeon Methanosarcina barkeri 227

Methanosarcina barkeri 227 possesses two clusters of genes potentially encoding nitrogenases. We have previously demonstrated that one cluster, called nif2, is expressed under molybdenum (Mo)-sufficient conditions, and the deduced amino acid sequences for nitrogenase structural genes in that cluster most closely resemble those for the Mo nitrogenase of the gram-positive eubacterium Clostridium pasteurianum. The previously cloned nifH1 from M. barkeri shows phylogenetic relationships with genes encoding components of eubacterial Mo-independent eubacterial alternative nitrogenases and other methanogen nitrogenases. In this study, we cloned and sequenced nifD1 and part of nifK1 from M. barkeri 227. The deduced amino acid sequence encoded by nifD1 from M. barkeri showed great similarity with vnfD gene products from vanadium (V) nitrogenases, with an 80% identity at the amino acid level with the vnfD gene product from Anabaena variabilis. Moreover, there was a small open reading frame located between nifD1 and nifK1 with clear homology to vnfG, a hallmark of eubacterial alternative nitrogenases. Stimulation of diazotrophic growth of M. barkeri 227 by V in the absence of Mo was demonstrated. The unusual complement of nif genes in M. barkeri 227, with one cluster resembling that from a gram-positive eubacterium and the other resembling a eubacterial V nitrogenase gene cluster, suggests horizontal genetic transfer of those genes.