Paul Woodley

Sequence and molecular analysis of the nifL gene of Azotobacter vinelandii.

In both Klebsiella pneumoniae and Azotobacter vinelandii the nifL gene, which encodes a negative regulator of nitrogen fixation, lies immediately upstream of nifA. We have sequenced the A. vinelandii nifL gene and found that it is more homologous in its C‐terminal domain to the histidine protein kinases (HPKs) than Is K. pneumoniae NifL. In particular A. vinelandii NifL contains a conserved histidine at a position shown to be phosphorylated in other systems. Both NifL proteins are homologous in their N‐termini to a part of the Halobacterium halobium bat gene product; Bat is involved in regulation of bacterio‐opsin, the expression of which is oxygen sensitive. The same region showed homology to the haembinding N‐terminai domain of the Rhizobium meliloti fixL gene product, an oxygen‐sensing protein. Like K. pneumoniae NifL, A. vinelandii NifL is shown here to prevent expression of nif genes in the presence of NH+4 or oxygen. The sequences found homologous in the C‐terminal regions of NifL, FixL and Bat might therefore be involved in oxygen binding or sensing. An in‐frame deletion mutation in the nifL coding region resulted in loss of repression by NH+4 and the mutant excreted high amounts of ammonia during nitrogen fixation, thus confirming a phenotype reported earlier for an insertion mutation. In addition, nifLA are cotranscribed in A. vinelandii as in K. pneumoniae, but expression from the A. vinelandii promoter requires neither RpoN nor NtrC.

Genome size and complexity in Azotobacter chroococcum

All of eight strains of Azotobacter chroococcum examined contained between two and six plasmids ranging from 7 to more than 200 MDal in size. Strain MCC-1, a derivative of NCIMB 8003, was cured of various of the four largest of its five plasmids and the phenotypes of the strains compared. all fixed nitrogen and exhibited uptake hydrogenase activity. No differences were observed in carbon source utilization or antibiotic, heavy metal or UV resistance. The genome sizes of two strains of A. chroococcum were determined by two-dimensional electrophoresis. Strain CW8, an isolate from local soil containing two small plasmids of 6 and 6.5 MDAl contained unique DNA sequences equivalent to 1.78 x 10(6) (+/- 20%) bp (1.2 x 10(9) Dal). In strain MDC-1, a derivative of MCC-1, containing a 190 MDal and 7 MDal plasmid, the genome size was 1.94 x 10(6) (+/- 20%) bp. In exponential batch cultures, both contained 20 to 25 genome equivalents per cell. MCD-1 exhibited complex UV kill kinetics with a marked plateau of resistance; CW8 showed a simple response inconsistent with the possibility of organization of its DNA into identical chromosome copies capable of independent segregation.

Second gene (nifH*) coding for a nitrogenase iron protein in Azotobacter chroococcum is adjacent to a gene coding for a ferredoxin-like protein.

Azotobacter chroococcum MCD1 contains a cluster of nitrogen fixation (nif) genes coding for the structural polypeptides for nitrogenase (nifH for the Fe‐protein and nifD and nifK for the MoFe protein) and a second sequence in the genome homologous to nifH. DNA fragments bearing this second nifH‐like sequence were cloned and the DNA sequence around the homologous region determined. Two open reading frames were identified in this region. One codes for a protein of 289 amino acid residues and is highly homologous to other Fe‐proteins but is different from the gene adjacent to the nifDK genes in A. chroococcum. This putative gene we call nifH*. The following open reading frame codes for a protein of 63 amino acids, nine of which are cysteine residues. The protein is homologous to the small low‐potential ferredoxins found in anaerobic bacteria, and in particular those from Chlorobium limicola. Linkage between a structural gene for nitrogenase and a small ferredoxin has not previously been observed. Sequence analysis suggests that the two genes form an operon. Transcription of the ferredoxin gene on a 1320‐bp transcript was only detectable under conditions in which A. chroococcum MCD1155, which carries a chromosomal deletion of 6.3 kb removing the entire nifHDK cluster, is capable of fixing N2, i.e. in media containing no added molybdenum or high levels of NH3. The size of the observed transcript agrees well with the predicted size for a transcript encoding nifH* and the ferredoxin genes. Expression of the nifH* promoter was not significantly activated in Escherichia coli even when nifA, the positive activator of nif genes in Klebsiella pneumoniae, was supplied in multiple copies. The results are discussed in relation to an alternative pathway for N2 fixation in A. chroococcum.

Structural genes for the vanadium nitrogenase from Azotobacter chroococcum

Structural genes for the VFe‐protein (Ac1V) of the vanadium nitrogenase from Azotobacter chroococcum were cloned and sequenced. The VFe‐protein contains three subunit types with Mr of 53,793 (alpha), 52,724 (beta) and 13,274 (delta). alpha and beta subunits show 18 and 15% sequence identity respectively, with alpha and beta subunits of the MoFe‐protein of A.chroococcum molybdenum nitrogenase. The genes for the three subunits vnfD (alpha), vnfG (delta) and vnfK (beta) are contiguous and form an operon whose transcription is repressed in response to ammonia. The Fe‐protein component of the V‐nitrogenase (Ac2V) is the product of nifH* that we have previously cloned and sequenced. This gene was located 2.5 kb upstream of vnfD. A deletion in the vnfD, G and K gene cluster prevents V‐dependent nitrogen fixation. A strain defective in both V‐nitrogenase and Mo‐nitrogenase structural genes showed no residual nitrogen fixing capacity arguing against the presence of a third nitrogen fixation system in this organism.

Cloning and organisation of some genes for nitrogen fixation from Azotobacter chroococcum and their expression in Klebsiella pneumoniae

SummaryBy DNA hybridisation, restriction fragments of genomic DNA from Azotobacter chroococcum and A. vinelandii bearing sequences homologous to Klebsiella pneumoniae nitrogenase structural genes were detected. These were different in the two species and inconsistent with the arrangement of the homologous sequences as a contiguous cluster of unique genes. The use of a DNA probe specific for nifH showed that in A. chroococcum two nifH-like sequences were present in the genome. From gene libraries for A. chroococcum, several recombinant cosmid clones bearing nif genes were identified and physically mapped. One copy of the nifH-like sequences was closely linked to nifD and K, the order of genes being as for K. pneumoniae. This cluster was sub-cloned into the broad host-range vector pKT230. The resultant plasmid complemented for C2H2-reduction but not growth in N2 several Nif- mutants of A. vinelandii and K. pneumoniae and also abolished growth in N2 in Nif+ parents. The inhibition was ascribed to a short region adjacent to nifH, which probably corresponds to the promoter as its inhibitory affects were alleviated by provision of K. pneumoniae nifA in multiple copies. 3 sizes of transcripts are produced from the region containing nifH and nifD of A. chroococcum in cultures derepressing for nif. A region bearing homology to a fragment of the K. pneumoniae nif cluster bearing nifV was identified 15 Kb away from nifHDK in A. chroococcum however the order of genes is probably similar to that of K. pneumoniae.

Redundancy of the conserved His residue in Azotobacter vinelandii NifL, a histidine autokinase homologue which regulates transcription of nitrogen fixation genes.

The NifL protein of Azotobacter vinelandii inhibits NifA, the activator of nif (nitrogen fixation) transcription, in response to oxygen and fixed nitrogen. NifL shows strong homology in its C‐terminal domain to the histidine autokinase domains of the canonical two‐component sensor proteins, including the region around His‐304, which corresponds to the residue known to be phosphorylated in other systems. To examine the mechanism of sensory transduction by NifL, mutations encoding 10 substitutions for His‐304 were introduced into the A. vinelandii chromosome. Regulation of nif transcription was measured using acetylene reduction and RNA blots. The substitutions His‐304 → Arg and His‐304 → Pro impaired regulation by both fixed nitrogen and oxygen, but substitution of Ala, Phe, Ile, Lys, Asn, Ser, Thr, Val had no effect. None of the mutants, including His‐304 → Arg and His‐304 → Pro, excreted ammonium during diazotrophy, a phenotype of nifL deletion mutants, suggesting that the molecular basis of this effect differs from that responsible for the inhibition of nif transcription. The data show conclusively that phosphorylation of His‐304 is not essential for any of the known functions of A. vinelandii NifL. Homology to the family of histidine autokinases is therefore inadequate evidence for a mechanism of sensory transduction involving phosphorylation of the conserved histidine residue.

Further Analysis of Nitrogen Fixation (nif) Genes in Azotobacter chroococcum: Identification and Expression in Klebsiella pneumoniae of nifS, nifV, nifM and nifB Genes and Localization of nifE/N-, nifU-, nifA- and fix ABC-like Genes

The results presented extend previous investigations on the genetics of nitrogen fixation in Azotobacter chroococcum and indicate that nif- and fix-like DNA is located in at least five different regions of the genome. Region I contains functional copies of nifS,V and M, as well as nifH, D and K, all of which complemented mutants of Klebsiella pneumoniae. In addition, nifE- and/or nifN-like and nifU-like DNA is located in this region. The organization of the nif cluster in region I closely resembles that of K. pneumoniae. though spread over 22 kb as compared with 14 kb. Region II contains a functional nifB gene, which complemented a K. pneumoniae nifB mutant, and seems to be adjacent to ap nifA-like gene. Region III harbours nifH*, encoding a second nitrogenase Fe-protein. Region IV contains a reiteration of nifE- on and/or nifN-like sequences, and DNA homologous to Rhizobium meliloti fixABC is present in region V. The apparent complexity of nifDNA in A. chroococcum is probably related to the two systems for N2-fixation pr present in this organism.

Genetic and Physical Characterisation of nif and ntr Genes in Azotobacter chroococcum and A. vinelandii

Characterisation of genes affecting nitrogen fixation in Azotobacters has progressed rapidily using molecular methods: nif genes similar to those in K. pneumoniae have been revealed and also other genes whose function is related to the distinctive ability of Azotobacters to fix nitrogen under aerobic conditions or in the absence of detectable Mo. Recent advances in understanding the genetic basis for nitrogen fixation and its regulation in Azotobacters are described in this paper.

Regulation of Expression of Genes for Three Nitrogenases in Azotobacter Vinelandii

Three genetically distinct nitrogenases can be synthesized in A. vinelandii: conventional molybdenum nitrogenase encoded by nifHDK, a vanadium nitrogenase encoded by vnfHDGK, and a third enzyme which contains nei ther Mo nor V encoded by anfHDGK. A complex array of regulatory proteins controls expression of the three sets of nitrogenase structural genes. These include the three specific activators NIFA, VNFA, and ANFA; NIFL, which as in K. pneumoniae is necessary for ammonium repression of Mo ni trogenase genes; and NFRX which is required for expression from the nifH and anfH promoters. The nfrX gene has been found to be similar to an E. coli gene, glnD, which encodes uridylyl transferase. A model is presented showing the way these interacting regulatory products are thought to regulate synthesis of the three nitrogenases. Two other observations are of more practical significance. Firstly, the A. vinelandii nifL mutants excreted more than SmM ammonium when fixing nitrogen. Secondly, Mo fully repressed the alternative nitrogenase genes at 30°, repressed much less at 20°, and not at all at 14°. This result suggests that A. vinelandii has the capacity to synthesize non-molybdenum nitrogenases regardless of environmental Mo content at temperate soil temperatures.

Aspects of Genetics of Azotobacters

There have been a number of studies on the genetics of N2-fixation in Azotobacter. Early reports describe the isolation and biochemical characterization of several classes of mutants defective in N2-fixation or normal regulation of the genes (nif) responsible (Wyss, Wyss 1950; Green et al, 1953; Fisher, Brill 1969; Sorger, Trofimenkof, 1970; Shah et al, 1973; Gordon, Brill, 1972; Shah et al, 1974). Some of the mutations were mapped by transformation to provide an approximate linkage map (Bishop, Brill, 1977). Further progress was hampered by a lack of good genetic systems for this genus. Also, Nif mutants aside, the wide variety of mutants useful for such work were and remain elusive (see Roberts, Brill, 1981), a problem attributed to a high level of genetic redundancy in the form of an unusually high chromosome copy number (Sadoff et al, 1979).