Most biological nitrogen fixation is catalyzed by molybdenum-dependent nitrogenase, an enzyme complex comprising two component proteins that contains three different metalloclusters. Diazotrophs contain a common core of nitrogen fixation nif genes that encode the structural subunits of the enzyme and components required to synthesize the metalloclusters. However, the complement of nif genes required to enable diazotrophic growth varies significantly amongst nitrogen fixing bacteria and archaea. In this study, we identified a minimal nif gene cluster consisting of nine nif genes in the genome of Paenibacillus sp. WLY78, a gram-positive, facultative anaerobe isolated from the rhizosphere of bamboo. We demonstrate that the nif genes in this organism are organized as an operon comprising nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV and that the nif cluster is under the control of a σ70 (σA)-dependent promoter located upstream of nifB. To investigate genetic requirements for diazotrophy, we transferred the Paenibacillus nif cluster to Escherichia coli. The minimal nif gene cluster enables synthesis of catalytically active nitrogenase in this host, when expressed either from the native nifB promoter or from the T7 promoter. Deletion analysis indicates that in addition to the core nif genes, hesA plays an important role in nitrogen fixation and is responsive to the availability of molybdenum. Whereas nif transcription in Paenibacillus is regulated in response to nitrogen availability and by the external oxygen concentration, transcription from the nifB promoter is constitutive in E. coli, indicating that negative regulation of nif transcription is bypassed in the heterologous host. This study demonstrates the potential for engineering nitrogen fixation in a non-nitrogen fixing organism with a minimum set of nine nif genes.
Vyšlo v časopise: A Minimal Nitrogen Fixation Gene Cluster from sp. WLY78 Enables Expression of Active Nitrogenase in. PLoS Genet 9(10): e32767. doi:10.1371/journal.pgen.1003865 Kategorie: Research Article prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003865
1. FalkowskiPG (1997) Evolution of the nitrogen cycle and its influence on the biological sequestration of CO2 in the ocean. Nature 387 : 272–275.
2. MerrickM, DixonR (1984) Why don't plants fix nitrogen? Trends Biotechnol 2 : 162–166.
3. BeattyPH, GoodAG (2011) Future prospects for cereals that fix nitrogen. Science 333 : 416–417.
4. RubioLM, LuddenPW (2008) Biosynthesis of the iron-molybdenum cofactor of nitrogenase. Annu Rev Microbiol 62 : 93–111.
5. RobertsCP, MacnellT, MacnellD, BrillWJ (1978) Regulation and characterization of protein products coded by the nif (nitrogen fixation) genes of Klebsiella pneumoniae. J Bacteriol 136 : 267–279.
6. GaviniN, TungturS, PulakatL (2006) Peptidyl-prolylcis/trans isomerase-independent functional NifH mutant of Azotobacter vinelandii. J Bacteriol 188 : 6020–6025.
7. HuY, RibbeMW (2011) Biosynthesis of nitrogenase FeMoco. Coord Chem Rev 255 : 1218–1224.
8. ArnoldW, RumpA, KlippW, PrieferUB, PühlerA (1988) Nucleotide sequence of a 24,206-base-pair DNA fragment carrying the entire nitrogen fixation gene cluster of Klebsiella pneumoniae. J Mol Biol 203 : 715–738.
9. SetubalJC, dos SantosP, GoldmanBS, ErtesvåH, EspinG (2009) Genome Sequence of Azotobacter vinelandii, an obligate aerobe specialized to support diverse anaerobic metabolic processes. J Bacteriol 191 : 4534–4545.
10. DodsworthJA, LeighJA (2006) Regulation of nitrogenase by 2-oxoglutarate-reversible, direct binding of a PII-like nitrogen sensor protein to dinitrogenase. Proc Natl Acad Sci USA 103 : 9779–9784.
11. Dos SantosPC, FangZ, MasonSW, Setubal JC. DixonR (2012) Distribution of nitrogen fixation and nitrogenase-like sequences amongst microbial genomes. BMC Genomics 13 : 162.
12. CurattiL, HernandezJA, IgarashiRY, SobohB, ZhaoD (2007) In vitro synthesis of the iron–molybdenum cofactor of nitrogenase from iron, sulfur, molybdenum, and homocitrate using purified proteins. Proc Natl Acad Sci USA 104 : 17626–17631.
13. DixonR, PostgateJ (1972) Genetic transfer of nitrogen fixation from Klebsiella pneumoniae to Escherichia coli. Nature 237 : 102–103.
14. XieJB, BaiLQ, WangLY, ChenSF (2012) Phylogeny of 16S rRNA and nifH genes and regulation of nitrogenase by oxygen and ammonium in the genus Paenibacillus. Microbiology 81 : 5–6.
15. ZhaoH, XieB, ChenSF (2006) Cloning and sequencing of nifBHDKENX genes of Paenibacillus massiliensis T7 and its nif promoter analysis. China C Life Sci 49 : 115–122.
16. OhCJ, KimHB, KimJ, KimWJ, LeeH, et al. (2012) Organization of nif gene cluster in Frankia sp. EuIK1 strain, a symbiont of Elaeagnus umbellate. Arch Microbiol 194 : 29–34.
17. WelshEA, LibertonM, StockelJ, SatoH, LohT (2008) The genome of Cyanothece 51142, a unicellular diazotrophic cyanobacterium important in the marine nitrogen cycle. Proc Natl Acad Sci USA 105 : 15094–15099.
18. Enkh-AmgalanJ, KawasakiH, Oh-okaH, SekiT (2006) Cloning and characterization of a novel gene involved in nitrogen fixation in Heliobacterium chlorum, a possible regulatory gene. Arch Microbiol 186 : 327–337.
19. KesslerPS, BlankC, LeighJA (1998) The nif gene operon of the methanogenic archaeon Methanococcus maripaludis. J Bacteriol 180 : 1504–1511.
20. YehHY, ChenTC, LiouKM, HsuHT, ChungKM (2011) The core-independent promoter-specific interaction of primary sigma factor. Nucleic Acids Res 39 : 913–925.
21. JarmerH, LarsenTS, KroghA, SaxildHH, BrunakS (2001) Sigma A recognition sites in the Bacillus subtilisgenome. Microbiology 147 : 2417–2424.
22. YamadaM, KuboM, MiyakeT, SakaguchiR, HigoY, et al. (1991) Promoter sequence analysis in Bacillus and Escherichia, construction of strong promoters in E. coli. Gene 99 : 109–114.
23. Barriuso-IglesiasM, BarreiroC, FlechosoF, MartínJF (2006) Transcriptional analysis of the F0F1 ATPase operon of Corynebacterium glutamicum ATCC 13032 reveals strong induction by alkaline pH. Microbiology 152 : 11–21.
24. DilworthMJ (1966) Acetylene reduction by nitrogen fixing preparations from Clostridium pasteurianum. Biochem Biophys Acta 127 : 285–294.
25. SchöllhornR, BurrisRH (1967) Acetylene as a competitive inhibitor of N-2 fixation. Proc Natl Acad Sci USA 58 : 213–216.
26. MontoyaJP, VossM, KahlerP, CaponeDG (1996) A simple, high-precision, high-sensitivity tracer assay for N2 fixation. Appl Environ Microbiol 62 : 986–993.
27. BochnerBR, GadzinskiP, PanomitrosE (2011) Phenotype microarrays for high-throughput phenotypic yesting and assay of gene function. Genome Res 11 : 1246–1255.
28. McLeanP, DixonR (1981) Requirement of nifV gene for production of wild-type nitrogenase enzyme in Klebsiella pneumoniae. Nature 292 : 655–656.
29. HooverTR, ImperialJ, LuddenPW, ShahVK (1988) Homocitrate cures the NifV-phenotype in Klebsiella pneumoniae. J Bacteriol 170 : 1978–1989.
30. DixonR, KahnD (2004) Genetic regulation of biological nitrogen fixation. Nat Rev Microbiol 2 : 621–631.
31. HuergoLF, PedrosaFO, Muller-SantosM, ChubatsuLS, MonteiroRA, et al. (2012) PII signal transduction proteins: pivotal players in post-translational control of nitrogenase activity. Microbiology 158 : 176–190.
32. Masson-BoivinC, GiraudE, PerretX, BatutJ (2009) Establishing nitrogen-fixing symbiosis with legumes: how many rhizobium recipes? Trends Microbiol 17 : 458–66.
33. BoydES, AnbarAD, MillerS, HamiltonTL, LavinM, et al. (2011) A late methanogen origin for molybdenum-dependent nitrogenase. Geobiology 9 : 221–232.
34. Nieva-GómezD, RobertsGP, KlevickisS, BrillWJ (1980) Electron transport to nitrogenase in Klebsiella pneumoniae. Proc Natl Acad Sci USA 77 : 2555–5558.
35. HillS, KavanaghEP (1980) Roles of nifF and nifJ gene products in electron transport to nitrogenase in Klebsiella pneumoniae. J Bacteriol 141 : 470–475.
36. HowardKS, McLeanPA, HansenFB, LemleyPV, KoblanKS, et al. (1986) Klebsiella pneumoniae nifM gene product is required for stabilization and activation of nitrogenase iron protein in Escherichia coli. J Biol Chem 261 : 772–778.
37. ZhaoD, CurattiL, RubioLM (2007) Evidence for nifU and nifS participation in the biosynthesis of the iron-molybdenum cofactor of nitrogenase. J Bio Chem 282 : 37016–37025.
38. HarrisGS, WhiteTC, FloryJE, Orme-JohnsonWH (1990) Genes required for formation of the apoMoFe protein of Klebsiella pneumoniae nitrogenase in Escherichia coli. J Biol Chem 265 : 15909–15919.
39. ImperialJ, HooverTR, MaddenMS, LuddenPW, ShahVK (1989) Substrate reduction properties of dinitrogenase activated in vitro are dependent upon the presence of homocitrate or its analogs during iron-molybdenum cofactor synthesis. Biochemistry 28 : 7796–7799.
40. LakeMW, WuebbensMM, RajagopalanKV, SchindelinH (2001) Mechanism of ubiquitin activation revealed by the structure of a bacterial MoeB-MoaD complex. Nature 414 : 325–329.
41. LehmannC, BegleyTP, EalickSE (2006) Structure of the Escherichia coli ThiS-ThiF complex, a key component of the sulfur transfer system in thiamin biosynthesis. Biochemistry 45 : 9–11.
42. SchmitzJ, WuebbensMM, RajagopalanKV, LeimkühlerS (2007) Role of the C-terminal Gly-Gly motif of Escherichia coli MoaD, a molybdenum cofactor biosynthesis protein with a ubiquitin fold. Biochemistry 46 : 909–916.
43. Groot KormelinkT, KoendersE, HagemeijerY, OvermarsL, SiezenRJ, et al. (2012) Comparative genome analysis of central nitrogen metabolism and its control by GlnR in the class Bacilli. BMC Genomics 13 : 191.
44. LiR, LiY, KristiansenK, WangJ (2008) SOAP, short oligonucleotide alignment program. Bioinformatics 24 : 713–714.
45. DelcherAL, BratkeKA, PowersEC, SalzbergSL (2007) Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23 : 673–679.
46. LoweTM, EddySR (1997) tRNAscan-SE, a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25 : 0955–0964.
47. ZianniM, TessanneK, MerighiM, LagunaR, TabitaFR (2006) Identification of the DNA bases of a DNase I footprint by the use of dye primer sequencing on an automated capillary DNA analysis instrument. J Biomol Tech 17 : 103–13.
48. Miller JH (1972) Experiments in Molecular Genetics. New York: Cold Spring Harbor Laboratory Press.
Zvýšte si kvalifikáciu online z pohodlia domova
Nemáte účet? Registrujte sa
Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.
