Different organization of nif genes in nonheterocystous and heterocystous cyanobacteria

Summary Labeled probes carrying the Anabaena PCC 7120 nitrogenase (nifK and nifD) and nitrogenase reductase (nifH) genes were hybridized to Southern blots of DNA from diverse N₂-fixing cyanobacteria in order to test a previous observation of different nif gene organization in nonheterocystous and heterocystous strains. The nif probes showed no significant hybridization to DNA from a unicellular cyanobacterium incapable of N₂ fixation. All nonheterocystous cyanobacteria examined (unicellular and filamentous) had a contiguous nifKDH gene cluster whereas all of the heterocystous strains showed separation of nifK from contiguous nifDH genes. These findings suggest that nonheterocystous and heterocystous cyanobacteria have characteristic and fundamentally different nif gene arrangements. The noncontiguous nif gene pattern, as shown with two Het^- mutants, is independent of phenotypic expression of heterocyst differentiation and aerobic N₂-fixation. Thus nif arrangement could be a useful taxonomic marker to distinguish between phenotypically Het^- heterocystous cyanobacteria and phylogenetically unrelated nonheterocystous strains.     

Nitrogen fixation genes (nif K,D,H) in the filamentous nonheterocystous cyanobacteriumPlectonema boryanum do not rearrange

The organisation of the structural genes for nitrogen fixation (nif K,D and H) in a nonheterocystous, filamentous cyanobacteriumPlectonema boryanum has been examined in comparison with a heterocystous cyanobacterium,Anabaena torulosa. DNA from repressed (fix-) cultures ofA. torulosa showed a discontinuousnif region spread over approximately 18 kb, an arrangement typical of the vegetative cells of heterocystous cyanobacteria. The region contained a contiguousnif DH separated fromnif K. by nearly 11 kb DNA. The intervening 11 kb DNA harboured the genexis A involved in the rearrangement ofnif K,D,H to form a cluster during differentiation of heterocysts. DNA fromPlectonema boryanum had a small, contiguousnif KDH cluster spanning a region of approximately 4 kb. DNA homologous to the 11 kb excison with its residentxis A was not present.Nif hybridisation patterns of restriction digests of the DNA isolated from repressed (fix^-) or induced (fix^--) cultures ofP. boryanum were completely identical. These results unequivocally demonstrate that in the nonheterocystous cyanobacterium, unlike in the heterocystous strains, no gene rearrangement, either within thenif KDII cluster or in its vicinity, accompanies the expression of nitrogenase activity.     

Isolation and Characterization of Frankia sp. Strain FaC1 Genes Involved in Nitrogen Fixation

Genomic DNA was isolated from Frankia sp. strain FaC1, an Alnus root nodule endophyte, and used to construct a genomic library in the cosmid vector pHC79. The genomic library was screened by in situ colony hybridization to identify clones of Frankia nitrogenase (nif) genes based on DNA sequence homology to structural nitrogenase genes from Klebsiella pneumoniae. Several Frankia nif clones were isolated, and hybridization with individual structural nitrogenase gene fragments (nifH, nifD, and nifK) from K. pneumoniae revealed that they all contain the nifD and nifK genes, but lack the nifH gene. Restriction endonuclease mapping of the nifD and nifK hybridizing region from one clone revealed that the nifD and nifK genes in Frankia sp. are contiguous, while the nifH gene is absent from a large region of DNA on either side of the nifDK gene cluster. Additional hybridizations with gene fragments derived from K. pneumoniae as probes and containing other genes involved in nitrogen fixation demonstrated that the Frankia nifE and nifN genes, which play a role in the biosynthesis of the iron-molybdenum cofactor, are located adjacent to the nifDK gene cluster.     

Organization of the nif genes in cyanobacteria in symbiotic association with Azolla and Anthoceros

The sizes of endonuclease digestion fragments of DNA from cyanobacteria in symbiotic association with Azolla caroliniana or Anthoceros punctatus, or in free-living culture, were compared by Southern hybridization using cloned nitrogenase (nif) genes from Anabaena sp. PCC 7120 as probes. The restriction fragment pattern produced by cyanobacteria isolated from A. caroliniana by culture through symbiotic association with Anthoceros differed from that of the major symbiotic cyanobacterium freshly separated from A. caroliniana. The results indicate that minor cyanobacterial symbionts occur in association with Azolla and that the dominant symbiont was not cultured in the free-living state. Both the absence of hybridization to an xisA gene probe and the mapping of restriction fragments indicated a contiguous nifHDK organization in all cells of the symbiont in association with Azolla. On the other hand, in the cultured isolate from Azolla and in Nostoc sp. 7801, the nifD and nifK genes are nominally separated by an interval of unknown length, compatible with the interruption of the nifHDK operon by a DNA element as observed in Anabaena sp. PCC 7120. In the above cultured strains, restriction fragments consistent with a contiguous nifHDK operon were also present at varying hybridization intensities, especially in Nostoc sp. 7801 grown in association with Anthoceros, presumably due to gene rearrangement in a fraction of the cells.Non-standard abbreviations bp base pairs - kb kilobase pairs - kd kilodaltons     

Nitrogen fixation andnif gene organization in branched heterocystous cyanobacteria: Variation in the presence ofxisA

Five branched heterocystous cyanobacteria (Scytonematopsis sp.,Scytonema sp.,Tolypothrix ceylonica, Mastigocladus sp. andFischerella sp.) were examined for their pattern of induction of nitrogenase activity andnif gene organization. All the forms showed the onset of nitrogenase activity after 12 h which could be correlated with the appearance of proheterocysts. The highest activity was exhibited byT. ceylonica. Hybridization studies revealed the presence of thenifD gene but the absence of thexisA gene inMastigocladus sp. andScytonematopsis sp. Interestingly,Tolypothrix sp. andScytonema sp. DNA samples hybridized withxisA. Hence no uniformity seems to exist regarding the presence ofxisA and the relatednif gene organization in branched heterocystous cyanobacteria. This investigation throws light on the primitive character and phylogenetic relatedness of branched forms to the coccoid/colonial forms. It also provides evidence for the proposition that stigonematacean cyanobacteria may not represent the most advanced cyanobacterial forms; rather they may link the coccoid and filamentous forms.     

Nitrogen fixation in strains ofAzotobacter chroococcum bearing deletions of a cluster of genes coding for nitrogenase

Strains of the obligately aerobic nitrogen fixing organismAzotobacter chroococcum were constructed which contained defined chromosomal deletions in which the nitrogenase structural genenifHDK cluster (nifH for the polypeptide of the Fe-protein component of nitrogenase andnifD andnifK for the alpha and beta subunits respectively of the MoFe-protein component of the enzyme) was replaced by a kanamycin resistance gene. N₂ fixation was nevertheless observed in deletion strains though only in a molybdenum-deficient medium or in spontaneously arising tungstate-resistant derivatives. In comparison with the parent strain growing in molybdenum-sufficient medium, diazotrophic growth was slow and the nitrogenase activity in vivo was characterised by disproportionately low rates of C₂H₂-reduction compared to H₂-evolution and relative insensitivity of H₂-evolution to inhibition by C₂H₂. The findings show reiteration of functional structural genes for nitrogenase inA. chroococcum consistent with our previous observation of twonifH genes in this organism and detection in this work of a secondnifK-like sequence in the genomes of both parent and deletion strains whenA. chroococcum nifK DNA was used as a probe.     

Analysis of codon usage in genes for nitrogen fixation from phylogenetically diverse diazotrophs

Summary A cluster analysis based on codon usage in genes for biological nitrogen fixation (nif genes) grouped diazotrophs into three distinct classes: anaerobes, cyanobacteria, and aerobes. In thenif genes ofKlebsiella pneumoniae there was no evidence for selection pressure in favor of highly translatable codons. However, in the nitrogen regulatory operonglnAntrBntrC of enteric bacteria the stoichiometrically high level of glutamine synthetase may be facilitated by the presence of efficiently translatable codons inglnA. Thenif genes of the cyanobacteriumAnabaena showed codon selection in favor of translational efficiency. Computation of codon adaptation indices for expression in heterologous systems indicated that the reading frames most suitable for expression ofnif genes inEscherichia coli, Bacillus subtilis, andSaccharomyces cerevisiae were present in azotobacters, clostridia, and cyanobacteria, respectively. In codon-usage-based cluster analysis, type 3 nitrogenase genes ofAzotobacter vinelandii grouped along with type 1 and type 2 genes. This is in contrast to the nucleotide sequence-based multiple alignment in which type 3 nitrogenase genes ofA. vinelandii have been reported to cluster with entirely unrelated diazotrophs such as methanogens and clostridia. This may be indicative of lateral transfer ofnif genes among widely divergent taxons. The chromosomal- and plasmid-locatednif genes of rhizobia also cluster separately in nucleotide sequence-based analysis but showed similar codon usage. These analyses suggested that the phylogeny ofnif genes drawn on the basis of nucleotide sequence homology was not masked by the taxon-specific pressure on codon usage.     

The Uptake Hydrogenase in the Unicellular Diazotrophic Cyanobacterium Cyanothece sp. Strain PCC 7822 Protects Nitrogenase from Oxygen Toxicity

Cyanothece sp. strain PCC 7822 is a unicellular, diazotrophic cyanobacterium that can produce large quantities of H₂ when grown diazotrophically. This strain is also capable of genetic manipulations and can represent a good model for improving H₂ production from cyanobacteria. To this end, a knockout mutation was made in the hupL gene (ΔhupL), and we determined how this would affect the amount of H₂ produced. The ΔhupL mutant demonstrated virtually no nitrogenase activity or H₂ production when grown under N₂-fixing conditions. To ensure that this mutation only affected the hupL gene, a complementation strain was constructed readily with wild-type properties; this indicated that the original insertion was only in hupL. The mutant had no uptake hydrogenase activity but had increased bidirectional hydrogenase (Hox) activity. Western blotting and immunocytochemistry under the electron microscope indicated that the mutant had neither HupL nor NifHDK, although the nif genes were transcribed. Interestingly, biochemical analysis demonstrated that both HupL and NifH could be membrane associated. The results indicated that the nif genes were transcribed but that NifHDK was either not translated or was translated but rapidly degraded. We hypothesized that the Nif proteins were made but were unusually susceptible to O₂ damage. Thus, we grew the mutant cells under anaerobic conditions and found that they grew well under N₂-fixing conditions. We conclude that in unicellular diazotrophs, like Cyanothece sp. strain PCC 7822, the HupLS complex helps remove oxygen from the nitrogenase, and that this is a more important function than merely oxidizing the H₂ produced by the nitrogenase.     

Using Synthetic Biology to Distinguish and Overcome Regulatory and Functional Barriers Related to Nitrogen Fixation

Biological nitrogen fixation is a complex process requiring multiple genes working in concert. To date, the Klebsiella pneumoniae nif gene cluster, divided into seven operons, is one of the most studied systems. Its nitrogen fixation capacity is subject to complex cascade regulation and physiological limitations. In this report, the entire K. pneumoniae nif gene cluster was reassembled as operon-based BioBrick parts in Escherichia coli. It provided ∼100% activity of native K. pneumoniae system. Based on the expression levels of these BioBrick parts, a T7 RNA polymerase–LacI expression system was used to replace the σ⁵⁴-dependent promoters located upstream of nif operons. Expression patterns of nif operons were critical for the maximum activity of the recombinant system. By mimicking these expression levels with variable-strength T7-dependent promoters, ∼42% of the nitrogenase activity of the σ⁵⁴-dependent nif system was achieved in E. coli. When the newly constructed T7-dependent nif system was challenged with different genetic and physiological conditions, it bypassed the original complex regulatory circuits, with minor physiological limitations. Therefore, we have successfully replaced the nif regulatory elements with a simple expression system that may provide the first step for further research of introducing nif genes into eukaryotic organelles, which has considerable potentials in agro-biotechnology.     

Deletion mutants of nitrogen fixation in Klebsiella pneumoniae: Mapping of a cluster of nif genes essential for nitrogenase activity

Deletions of the nitrogen fixation (nif) region of the Klebsiella genome were isolated by selecting for resistance to virulent phages whose resistance loci are adjacent to nif. The extent of the various deletions was monitored by assaying several different enzymes or gene products coded for by this segment of DNA. Three classes of deletion mutants were detected: (1) gluconate-6-phosphate dehydrogenase minus (gnd^?), histidine minus but histidinol dehydrogenase plus (his^?, his D⁺), nitrogenase plus (nif⁺), shikimate utilization plus (shu⁺); (2) gnd^?, his D^?, nif^?, shu⁺; (3) gnd^?, his D^?, nif^?, shu^?. From these studies we conclude that the cluster of nif genes essential for nitrogenase activity is located on the genetic linkage map of Klebsiella between his and shu; the gene order in this region in thus phage-resistance locus (rfb?), gnd, his operon, nif, shu. Genetic analysis substantiates the finding that the nif cluster is located proximally to the operator end of the his operon.     

Bacterial Life and Dinitrogen Fixation at a Gypsum Rock

The organisms of a bluish-green layer beneath the shards of a gypsum rock were characterized by molecular techniques. The cyanobacterial consortium consisted almost exclusively of Chroococcidiopsis spp. The organisms of the shards expressed nitrogenase activity (C₂H₂ reduction) aerobically and in light. After a prolonged period of drought at the rock, the cells were inactive, but they resumed nitrogenase activity 2 to 3 days after the addition of water. In a suspension culture of Chroococcidiopsis sp. strain PCC7203, C₂H₂ reduction required microaerobic conditions and was strictly dependent on low light intensities. Sequencing of a segment of the nitrogenase reductase gene (nifH) indicated that Chroococcidiopsis possesses the alternative molybdenum nitrogenase 2, expressed in Anabaena variabilis only under reduced O₂ tensions, rather than the widespread, common molybdenum nitrogenase. The shards apparently provide microsites with reduced light intensities and reduced O₂ tension that allow N₂ fixation to proceed in the unicellular Chroococcidiopsis at the gypsum rock, unless the activity is due to minute amounts of other, very active cyanobacteria. Phylogenetic analysis of nifH sequences tends to suggest that molybdenum nitrogenase 2 is characteristic of those unicellular or filamentous, nonheterocystous cyanobacteria fixing N₂ under microaerobic conditions only.     

携带Paenibacillus polymyxa WLY78固氮基因簇(nif)的重组大肠杆菌Escherichia coli78-7转录组分析

[目的]来自Paenibacillus polymyxa WLY78的固氮基因簇（nifBHDKEfNXhesAnifV）可以转化入Escherichia coli中表达并使重组大肠杆菌合成有固氮活性的固氮酶。本文拟通过对重组大肠杆菌E.coli 78-7的转录组分析以提高其固氮能力。[方法]对固氮条件（无氧无NH₄⁺）和非固氮条件（空气和100 mmol/L NH₄⁺）培养的重组大肠杆菌E.coli 78-7进行转录组分析。[结果]nif基因在两种培养条件下显著表达,说明在重组大肠杆菌中可规避原菌中氧气和NH₄⁺对nif基因的负调控。对于固氮过程必需的非nif基因,如参与钼、硫、铁元素转运的mod、cys和feoAB,这些基因在两种培养条件下表达水平有差异。而参与铁硫簇合成的suf和isc基因簇在两条件下表达水平差异巨大。此外,参与氮代谢的基因在固氮条件下显著上调。[结论]重组大肠杆菌中与固氮相关的非nif基因在该菌的固氮过程中具有较大影响,本文对在异源宿主中调高固氮酶活性研究具有重要意义。     

念珠藻属植物nifH基因的适应性进化分析

念珠藻(Nostoc)固氮过程关键在于固氮酶的催化,而固氮酶复合物中的铁蛋白(NifH)是由高度保守的nifH基因编码的,该基因是进化史上现存最古老的功能基因之一。该研究选取念珠藻属及近缘类群的nifH基因序列共40条,采用最大似然法构建系统发育树;运行PAML4.9软件,对nifH基因编码蛋白进行生物信息学分析,并使用分支模型、位点模型和分支-位点模型检测该基因的选择位点,探讨nifH基因的适应性进化特征。结果表明:(1)最大似然树显示内类群中该研究物种共分为6个分支(A、B、C、D、E和F),其中D和E是2个大的分支,每个大分支中又各包含2个特殊的小分支A、F和B、C,其中F分支包含新疆古尔班通古特沙漠采集到的9株念珠藻,A分支包含F分支及该研究测定序列的4株葛仙米,B分支包含本研究测定序列的4株地皮菜和3株未定种的念珠藻,C分支包含NCBI数据库中下载的5株念珠藻、鱼腥藻序列和本研究测定序列的1株念珠藻。(2)在所分析的3种进化模型中,仅通过分支-位点模型检测出14个统计学上显著的正选择位点,即1F、2S、3S、4T、5A、6F、7F、8I、9S、10C、17I、27Y、29D和31R位点,表明念珠藻属植物的nifH基因发生了适应性变化,分支-位点模型是研究藻类基因适应性进化较好的模型。     

Overlapping sequences of Klebsiella pneumoniae nifDNA cloned and characterized.

Summary A HindIII (17.0 kb) and an EcoRl restriction fragment (6.9 kb) of Klebsiella pneumoniae nif DNA were cloned on two small amplifiable plasmids, pCM1 and pSA30 respectively. These plasmids between them carry 14 of the 15 known Klebsiella nif genes. The operon for the three structural genes for nitrogenase, nifpHDK, is carried on pSA30: four and five of the remaining six operons are on pCRA37 and pCM1 respectively. All of the nif genes were assigned to endonculease restriction fragments of DNA using the Southern blotting technique (Southern, 1975) with total DNA of nif insertion mutants and radioactive plasmid DNA which contained cloned nif DNA sequences. Their locations were consistent with the genetic map of nif genes. The estimated size of the nif gene cluster was 24 kb.