Nucleotide sequence of a 24,206-base-pair DNA fragment carrying the entire nitrogen fixation gene cluster of Klebsiella pneumoniae

Arnold, Walter; Rump, Andreas; Klipp, Werner; Priefer, Ursula B.; Pühler, Alfred

doi:10.1016/0022-2836(88)90205-7

Cited by 273 publications

(171 citation statements)

References 53 publications

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“…Assignment of genes was based on deduced amino acid sequence identity to K. pneumoniae nif genes (Table 1; Fig. 3, 4, and 7) (2,5,53). The assignment of ORF1, -2, and -3 as nifU, nifS, and nifV, respectively, is consistent with our complementation results.…”

Section: Resultssupporting

confidence: 81%

“…In this organism, a maximum of 20 specific nif genes are required for N2 fixation. They are organized as a contiguous cluster spanning approximately 24 kb containing seven or eight transcriptional units (2,16,57). The nifH, nifD, and nifK genes respectively encode the Fe-protein (component 2) and the a and a subunits of the MoFe-protein (component 1) that together form the nitrogenase enzyme * Corresponding author.…”

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confidence: 99%

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Nucleotide sequence and genetic analysis of the Azotobacter chroococcum nifUSVWZM gene cluster, including a new gene (nifP) which encodes a serine acetyltransferase

et al. 1991

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Nucleotide sequence was obtained for a region of 7,099 bp spanning the nifU, nifS, nifV, niJW, nijZ, and nijM genes from Azotobacter chroococcum. Chromosomal mutations constructed at several sites within the locus confirmed a requirement for this region for expression of the molybdenum nitrogenase in this organism. The genes are tightly clustered and ordered as in Kkebsiella pneumoniae except for two additional open reading frames (ORFs) between nitfV and niJW. The arrangement of genes in A. chroococcum closely matches that described for Azotobacter vinelandii. The polypeptide encoded by ORF4 immediately downstream from nifV is 41% identical over 186 amino acids to the product of the cysE gene from Escherichia coli, which encodes serine acetyltransferase (SAT), a key enzyme in cysteine biosynthesis. Plasmids which potentially express ORF4 complemented E. coli JM39, a cysteine auxotroph which lacks SAT. SAT activity was detected in crude extracts of one such complemented strain. A strain of A. chroococcum carrying a chromosomal disruption of ORF4 grew normally with ammonium as the N source but more slowly than the parental strain when N2 was the sole N source. These data suggest that ORF4 encodes a nif-specific SAT required for optimizing expression of nitrogenase activity. ORF4 was assigned the name nijP. nijP may be required to boost rates of synthesis or intracellular concentrations of cysteine or methionine. Sequence identity between nitfV and leuA gene products suggests that nifV may catalyze a condensation reaction analogous to that carried out by isopropylmalate synthase (LEUA) but in which acetyl coenzyme and a-ketoglutarate are substrates for the formation of homocitrate, the proposed product of NIFV activity.Members of the genus Azotobacter are free-living, obligately aerobic, heterotrophic diazotrophs found in soil and freshwater environments. They are especially interesting from the point of view of N2 fixation because, depending on the species, they possess two or three genetically distinct nitrogenase systems whose expression is determined by metal availability (8). The genetics of N2 fixation in these organisms is complicated not only by their possession of alternative nitrogenases but also by their tolerance of 02 when fixing N2 (61). Our studies have concentrated on Azotobacter chroococcum MCD1, a derivative of ATCC 4412. This organism makes a typical molybdenum-dependent nitrogenase when molybdenum is available (77) which is replaced in the absence of molybdenum and presence of vanadium by a vanadium-dependent nitrogenase (60).Many of the genes required for N2 fixation were first defined in the facultative anaerobe Klebsiella pneumoniae M5A1, which possesses a molybdenum nitrogenase system only and fixes N2 in anaerobic or, at best, microaerobic environments. In this organism, a maximum of 20 specific nif genes are required for N2 fixation. They are organized as a contiguous cluster spanning approximately 24 kb containing seven or eight transcriptional units (2,16,57). The nifH, nifD, and nifK g...

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Section: Resultssupporting

confidence: 81%

mentioning

confidence: 99%

Nucleotide sequence and genetic analysis of the Azotobacter chroococcum nifUSVWZM gene cluster, including a new gene (nifP) which encodes a serine acetyltransferase

et al. 1991

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“…Packing of the nif genes in A1501, and the difference in G ϩ C content from the rest of the genome, favor the hypothesis that the 49-kb nif region is a genomic island. The K. pneumoniae 24-kb nif gene cluster comprises the tightest organization of nif genes identified to date (22). In all other systems studied, additional genes associated with core nif genes have been identified, but in most cases, their role in nitrogen fixation remains hypothetical (20).…”

Section: Discussionmentioning

confidence: 99%

Nitrogen fixation island and rhizosphere competence traits in the genome of root-associated Pseudomonas stutzeri A1501

Yan

Yang

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

325

263

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The capacity to fix nitrogen is widely distributed in phyla of Bacteria and Archaea but has long been considered to be absent from the Pseudomonas genus. We report here the complete genome sequencing of nitrogen-fixing root-associated Pseudomonas stutzeri A1501. The genome consists of a single circular chromosome with 4,567,418 bp. Comparative genomics revealed that, among 4,146 protein-encoding genes, 1,977 have orthologs in each of the five other Pseudomonas representative species sequenced to date. The genome contains genes involved in broad utilization of carbon sources, nitrogen fixation, denitrification, degradation of aromatic compounds, biosynthesis of polyhydroxybutyrate, multiple pathways of protection against environmental stress, and other functions that presumably give A1501 an advantage in root colonization. Genetic information on synthesis, maturation, and functioning of nitrogenase is clustered in a 49-kb island, suggesting that this property was acquired by lateral gene transfer. New genes required for the nitrogen fixation process have been identified within the nif island. The genome sequence offers the genetic basis for further study of the evolution of the nitrogen fixation property and identification of rhizosphere competence traits required in the interaction with host plants; moreover, it opens up new perspectives for wider application of root-associated diazotrophs in sustainable agriculture.genome sequencing ͉ root-associated diazotroph

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“…The ⍀ fragment is flanked in inverted orientation by transcriptional and translational termination signals [41].) Plasmid pJES875 carries a deletion of nifLA that encodes only the first 113 amino acids of the N-terminal domain of NifL and the last 6 amino acids of the C terminus of NifA (2). Finally, the deletion in plasmid pJES875 was transferred to the chromosome of K. pneumoniae UN4495.…”

Section: Methodsmentioning

confidence: 99%

Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae

Schmitz

Kustu

1996

J Bacteriol

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In Klebsiella pneumoniae, products of the nitrogen fixation nifLA operon regulate transcription of the other nif operons. NifA activates transcription by 54 -holoenzyme. In vivo, NifL antagonizes the action of NifA under aerobic conditions or in the presence of combined nitrogen. In contrast to a previous report, we show that depletion of iron (Fe) from the growth medium with the chelating agent o-phenanthroline (20 M) mimics aerobiosis or combined nitrogen in giving rise to inhibition of NifA activity even under anaerobic, nitrogenlimiting conditions. Adding back Fe in only twofold molar excess over phenanthroline restores NifA activity, whereas adding other metals fails to do so. By using strains that lack NifL, we showed that NifA activity itself does not require Fe and is not directly affected by phenanthroline. In the free-living diazotroph Klebsiella pneumoniae, expression of nitrogen fixation (nif) genes is regulated by the products of the nifLA operon (3,8,14,32). NifA activates transcription of all nif genes (except nifLA) by the alternative holoenzyme form of RNA polymerase 54 -holoenzyme. NifA binds to an upstream activation sequence (34) and contacts promoterbound 54 -holoenzyme by means of a DNA loop (9). It catalyzes the ATP-dependent isomerization of closed complexes between 54 -holoenzyme and a nif promoter to transcriptionally productive open complexes (25,35). NifL, which is a negative regulator, inhibits NifA activity in vivo in response to molecular oxygen and/or combined nitrogen (22,32).NifL from K. pneumoniae is composed of two domains separated by a hydrophilic interdomain linker (Q-linker [12]). We showed recently that the C-terminal domain is sufficient to inhibit transcriptional activation by NifA in vitro and in vivo (37). Thus, the inhibitory function of NifL appears to lie in its C-terminal domain, which presumably interacts with NifA. The N-terminal domain of NifL contains a region of homology (ϳ30% amino acid identity over 130 residues) to the product of the bat gene from Halobacterium halobium (19,47,50). Since Bat is an oxygen-responsive activator of the synthesis of bacterio-opsin, it has been proposed that the region of homology may be involved in oxygen sensing. The mechanism(s) by which NifL senses oxygen and/or combined nitrogen is not understood.The N-terminal domain of NifL contains one CysXXCys motif (Cys-184-Ala-Asp-Cys-187). The similarity of this motif to sequences involved in binding metal clusters in proteins such as ferrodoxins and rubredoxins (1, 4, 24) suggested a possible metal-binding role of this region in NifL (21). On the basis of this prediction, Henderson et al. (21) examined the influence of metal ion deficiency on NifL activity. They concluded that iron (Fe) deficiency reduced the inhibitory activity of NifL expressed at high levels in Escherichia coli.To further study the function of a possible metal ion or iron-sulfur (Fe-S) cluster in oxygen-sensing by NifL, we examined the effect of Fe deficiency on NifL inhibition of NifA activity in vivo in a stra...

show abstract

Nucleotide sequence of a 24,206-base-pair DNA fragment carrying the entire nitrogen fixation gene cluster of Klebsiella pneumoniae

Cited by 273 publications

References 53 publications

Nucleotide sequence and genetic analysis of the Azotobacter chroococcum nifUSVWZM gene cluster, including a new gene (nifP) which encodes a serine acetyltransferase

Nucleotide sequence and genetic analysis of the Azotobacter chroococcum nifUSVWZM gene cluster, including a new gene (nifP) which encodes a serine acetyltransferase

Nitrogen fixation island and rhizosphere competence traits in the genome of root-associated Pseudomonas stutzeri A1501

Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae

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