The Pseudomonas stutzeri strain A1501 (formerly known as Alcaligenes faecalis) fixes nitrogen under microaerobic conditions in the free-living state and colonizes rice endophytically. The authors characterized a region in strain A1501, corresponding to most of the nif genes and the rnf genes, involved in electron transport to nitrogenase in Rhodobacter capsulatus. The region contained three groups of genes arranged in the same order as in Azotobacter vinelandii: (1) nifB fdx ORF3 nifQ ORF5 ORF6; (2) nifLA-rnfABCDGEF-nifY2/nafY; (3) ORF13 ORF12-nifHDK-nifTY ORF1 ORF2-nifEN. Unlike in A. vinelandii, where these genes are not contiguous on the chromosome, but broken into two regions of the genome, the genes characterized here in P. stutzeri are contiguous and present on a 30 kb region in the genome of this organism. Insertion mutagenesis confirmed that most of the nif and the rnf genes in A1501 were essential for nitrogen fixation. Using lacZ fusions it was found that nif and rnf gene expression was under the control of ntrBC, nifLA and rpoN and that the rnf gene products were involved in the regulation of the nitrogen fixation process.
AllenL. N.,
HansonR. S.1985; Construction of broad-host-range cosmid cloning vectors: identification of genes necessary for growth of Methylobacterium organophilum on methanol. J Bacteriol 161:955–962
ArnoldW.,
RumpA.,
KlippW.,
PrieferU. B.,
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
BergersenF. J.1991; Physiological control of nitrogenase and uptake hydrogenase. In Biology and Biochemistry of Nitrogen Fixation pp 76–102 Edited by
DilworthM. J.,
GlennA. R.
Amsterdam: Elsevier;
CarltonJ. M.,
AnguioliS. V.,
SuhB.41 other authors2002; Genome sequence and comparative analysis of the model rodent malaria parasite Plasmodium yoelii yoelii . Nature 419:512–519
DeanD. R.,
JacobsonM. R.1992; Biochemical genetics of nitrogenase. In Biological Nitrogen Fixation pp 763–834 Edited by
StaceyG.,
BurrisR. H.,
EvansH. J.
New York: Chapman & Hall;
de ZamaroczyM.,
DelormeF.,
ElmerichC.1989; Regulation of transcription and promoter mapping of the structural genes for nitrogenase ( nifHDK) of Azospirillum brasilense Sp7. Mol Gen Genet 220:88–94
DittaG.,
SchmidhauserT.,
YakobsonE.,
LuP.,
LiangX. W.,
FinlayD. R.,
GuineyD.,
HelinskiD. R.1985; Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid 13:149–153
DixonR.1998; The oxygen responsive NifL-NifA complex: a novel two-component regulatory system controlling nitrogenase synthesis in γ -Proteobacteria. Arch Microbiol 169:371–380
EgenerT.,
MartinD. E.,
SarkarA.,
Reinhold-HurekB.2001; Role of a ferredoxin gene cotranscribed with the nifHDK operon in N2 fixation and nitrogenase “switch-off” of Azoarcus sp. strain BH72. J Bacteriol 183:3752–3760
EgenerT.,
SarkarA.,
MartinD. E.,
Reinhold-HurekB.2002; Identification of a NifL-like protein in a diazotroph of the β -subgroup of the Proteobacteria, Azoarcus sp. strain BH72. Microbiology 148:3203–3212
ElmerichC.1991; Genetics and regulation of Mo-nitrogenase. In Biology and Biochemistry of Nitrogen Fixation pp 103–141 Edited by
DilworthM. J.,
GlennA. R.
Amsterdam: Elsevier;
GalimandM.,
PerroudB.,
DelormeF.,
PaquelinA.,
VieilleC.,
BozouklianH.,
ElmerichC.1989; Identification of DNA regions homologous to nitrogen fixation genes nifE , nifUS and fixABC in Azospirillum brasilense Sp7. J Gen Microbiol 135:1047–1059
HäseC.,
MekalanosJ. J.1999; Effects of changes in membrane sodium flux on virulence gene expression in Vibrio cholerae . Proc Natl Acad Sci U S A 96:3183–3187
JacobsonM. R.,
BrigleK. V.,
BennettL. T.,
SetterquistR. A.,
WilsonM. S.,
CashV. L.,
BeynonJ.,
NewtonW. E.,
DeanD. R.1989; Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii . J Bacteriol 171:1017–1027
JoergerR. D.,
BishopP. E.1988; Nucleotide sequence and genetic analysis of the nifB-nifQ region from Azotobacter vinelandii . J Bacteriol 170:1475–1487
JouanneauY.,
JeongH. S.,
HugoN.,
MeyerC.,
WillisonJ. C.1998; Overexpression in Escherichia coli of the rnf genes from Rhodobacter capsulatus : characterization of two membrane-bound iron-sulphur proteins. Eur J Biochem 251:54–64
KokotekW.,
LotzW.1989; Construction of a lacZ -kanamycin-resistance cassette, useful for site-directed mutagenesis and as a promoter probe. Gene 84:467–471
KumagaiH.,
FujiwaraT.,
MatsubaraH.,
SaekiK.1997; Membrane localization, topology, and mutual stabilization of the rnfABC gene products in Rhodobacter capsulatus and implications for a new family of energy-coupling NADH oxidoreductases. Biochemistry 36:5509–5521
LeeS.,
RethA.,
MeletzusD.,
SevillaM.,
KennedyC.2000; Characterization of a major cluster of nif , fix and associated genes in a sugarcane endophyte, Acetobacter diazotrophicus . J Bacteriol 182:7088–7091
LiangY. Y.,
KaminskiP. A.,
ElmerichC.1991; Identification of a nifA -like regulatory gene of Azospirillum brasilense Sp7 expressed under conditions of nitrogen fixation and in the presence of air and ammonia. Mol Microbiol 5:2735–2744
LiangY. Y.,
ArsèneF.,
ElmerichC.1993; Characterization of the ntrBC genes of Azospirillum brasilense Sp7: their involvement in the regulation of nitrogenase synthesis and activity. Mol Gen Genet 240:188–196
LinM.,
SmallaK.,
HeuerH.,
van ElsasJ. D.2000; Effect of an Alcaligenes faecalis inoculant strain on bacterial communities in flooded microcosms planted with rice seedlings. Appl Soil Ecol 15:211–225
LovelessT. M.,
BishopP. E.1999; Identification of genes unique to Mo-independent nitrogenase systems in diverse diazotrophs. Can J Microbiol 45:312–317
LovellC. R.,
PicenoY. M.,
QuattroJ. M.,
BagwellC. E.2000; Molecular analysis of diazotroph diversity in the rhizosphere of the smooth cordgrass, Spartina alterniflora . Appl Environ Microbiol 66:3814–3822
MerrickM. J.1992; Regulation of nitrogen fixation genes in free-living and symbiotic bacteria. In Biological Nitrogen Fixation pp 835–876 Edited by
StaceyG.,
BurrisR. H.,
EvansH. J.
New York: Chapman and Hall;
Reyes-RamirezF.,
LittleR.,
DixonR.2001; Role of Escherichia coli nitrogen regulatory genes in the nitrogen response of the Azotobacter vinelandii NifL-NifA complex. J Bacteriol 183:3076–3082
RubioL. M.,
RangarajP.,
HomersM. J.,
RobertsG. P.,
LuddenP. W.2002; Cloning and mutational analysis of the γ gene from Azotobacter vinelandii defines a new family of proteins capable of metallocluster binding and protein stabilization. J Biol Chem 277:14299–14305
RudnickP.,
KunzC.,
GunatilakaM. K.,
HinesE. R.,
KennedyC.2002; Role of GlnK in NifL-mediated regulation of NifA activity in Azotobacter vinelandii . J Bacteriol 184:812–820
SaezL. P.,
GarciaP.,
Martinez-LuqueM.,
KlippW.,
BlascoR.,
CastilloF.2001; Role of draTG and rnf genes in reduction of 2,4-dinitrophenol by Rhodobacter capsulatus . J Bacteriol 183:1780–1783
SambrookJ.,
FritschE. F.,
ManiatisT.1989Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
SchmelM.,
JahnA.,
Meyer zu VilsendorfA.,
HenneckeS.,
MasepohlB.,
SchupplerM.,
MarxerM.,
OelzeJ.,
KlippW.1993; Identification of a new class of nitrogen fixation genes in Rhodobacter capsulatus : a putative membrane complex involved in electron transport to nitrogenase. Mol Gen Genet 241:602–615
SiddavattamD.,
SteiblH. D.,
KreutzerR.,
KlingmullerW.1995; Regulation of nif gene expression in Enterobacter agglomerans : nucleotide sequence of the nifLA operon and influence of temperature and ammonium on its transcription. Mol Gen Genet 249:629–636
SimonR.,
PrieferU.,
PühlerA.1983; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Biotechnology 1:784–791
ToukdarianA.,
KennedyC.1986; Regulation of nitrogen metabolism in Azotobacter vinelandii : isolation of ntr and glnA genes and construction of ntr mutants. EMBO J 5:399–407
VermeirenH.,
HaiW. L.,
VanderleydenJ.1998; Colonization and nifH expression on rice roots by Alcaligenes faecalis A15. In Nitrogen Fixation with Non-Legumes pp 167–177 Edited by
MalikK. A.,
MirzaM. S.,
LadhaJ. K.
Dordrecht: Kluwer;
YoungJ. P. W.1992; Phylogenetic classification of nitrogen-fixing organisms. In Biological Nitrogen Fixation pp 43–86 Edited by
StaceyG.,
BurrisR. H.,
EvansH. J.
New York: Chapman & Hall;
ZehrJ.,
McReynoldsL. A.1989; Use of degenerate oligonucleotides for amplification of the nifH gene from the marine cyanobacterium Trichodesmium thiebautii . Appl Environ Microbiol 55:2522–2526
ZhangY.,
PohlmannE. L.,
LuddenP. W.,
RobertsG. P.2000; Mutagenesis and functional characterization of the glnB , glnA , and nifA genes from the photosynthetic bacterium Rhodospirillum rubrum . J Bacteriol 182:983–992