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.     

Expression ofKlebsiella pneumoniae nif genes inProteus mirabilis

Self-transmissible plasmids carryinghis andnif genes fromKlebsiella pneumoniae have been introduced into threehis mutants ofProteus mirabilis: strains 5006-1, WR19 and WR20. Expression ofhis by the transconjugants was unequivocal, if slightly temperature-sensitive, but none was Nif⁺ when tested for acetylene reduction in anaerobic glucose medium using inocula from rich or glucose-minimal aerobic agar cultures. Succinate or pyruvate in place of glucose, low glucose, lower temperature or elevated Na₂MoO₄ did not allownif expression and no nitrogenase MoFe-protein peptide was detected immunologically after exposure to conditions in which diazotrophic enterobacteria, normal or genetically constructed, derepressnif.One strain,P. mirabilis WR19, carrying thehis nif Km^r plasmid pMF250 was examined in detail. Thenif activator genenifA was introduced on the plasmid pCK1. Such derivatives remained Nif^- when tested, after aerobic growth on rich agar media, with normal or low glucose, with succinate or with elevated Mo. However, pre-conditioning by aerobic growth on glucose-minimal agar led to subsequent anaerobic expression ofnif in glucose medium from pMF250 in WR19 carrying pCK1. NH ₄ ⁺ or proline could serve as N-source in the glucose-minimal agar. Maximum activity was about 5% of that ofK. pneumoniae in our assay conditions. Material cross-reacting with anti-serum to the nitrogenase MoFe protein was formed. Nitrogenase activity was not switched off by NH ₄ ⁺ .P. mirabilis WR19 (pCK1) showed NH ₄ ⁺ -constitutive temperature-sensitive kanamycin resistance (anif-related phenotype of this plasmid) in aerobic glucose minimal medium. Expression ofnif inP. mirabilis WR19 (pCK1, pMF250) was NH ₄ ⁺ -repressible despite the constitutivenifA character of pCK1 and introduction of thentrA ⁺ plasmid pMM17 did not alter this phenotype. However, pCK1 did not give rise to NH ₄ ⁺ -constitutive diazotrophy in the wild-typeK. pneumoniae M5al. A construct of WR19 carrying pMF250 and constitutiventrC plasmid (pMD45) remained Nif^- even after pre-growth on glucose-minimal media.We conclude (a) thatP. mirabilis forms a gene product functionally equivalent to that ofntrA inK. pneumoniae, (b) that it forms no functional equivalent of thentrC product in our growth conditions. The need for pre-conditioning on aerobic glucose media remains perplexing.Non-common abbreviation NFDM Nitrogen-free-Davis-Mingioli medium     

DNA sequence analysis of spontaneous histidine mutations in a polA1 strain of Escherichia coli K12 suggests a specific role of the GTGG sequence

Summary Spontaneously arising histidine mutations in an Escherichia coli K12 strain deficient for DNA polymerase I were analysed at the DNA sequence level. We screened approximately 150000 colonies and isolated 106 histidine auxotrophs. Of these, 98 were unstable hisC mutations; 12 representative mutants analysed were shown to have arisen by the excision of a single quadruplet repeat in the sequence 5-GCTGGCTGGCTGGCTG-3. Of the eight mutations at other sites, three hisA deletions and one hisD deletion occurred as a consequence of misalignment of tandemly repeated pentamers (hisD) or decamers (hisA). A single hisA point mutation was found to be a missense mutation. Two extended deletions, covering the his operon were not analysed. We could not identify the hisC deletion by sequencing. We conclude that polA1 is a strong imitator that induces mutations mostly of the minus frameshift and deletion type by a Streisinger-type of mispairing in repetitive DNA sequences. Finally, the possible role of a 5-GTGG-3 sequence and its inverted or direct complements, which are found in the vicinity of all the deletions and frameshifts, is discussed.     

Expression of klebsiella his and nif genes in serratia marcescens,Erwinia herbicola and Proteus mirabilis

Plasmid pRD1, an R plasmid of the P incompatibility group which carries his and nif genes from Klebsiella pneumoniae in addition to drug resistance markers derived from RP4, was transferred to His^- mutants of Serratia marcescens, Erwinia herbicola and Proteus mirabilis. His⁺ transconjugants were obtained at low but different frequencies according to recipient genus. Transconjugants all acquired the drug resistance, and were Nif⁺ in S. marcescens and E. herbicola, having acetylene-reducing activities of the same order of magnitude as the parent K. pneumoniae and fixing ¹⁵N₂. No evidence for nif expression in P. mirabilis transconjugants was obtained though the nif genes were present.     

Regulation of nitrogen fixation. Nitrogenase-derepressed mutants of Klebsiella pneumoniae

1. A new procedure is described for selecting nitrogenase-derepressed mutants based on the method of Brenchley et al. (Brenchley, J. E., Prival, M. J. and Magasanik, B. (1973) J. Biol. Chem. 248, 6122–6128) for isolating histidase-constitutive mutants of a non-N₂-fixing bacterium.2. Nitrogenase levels of the new mutants in the presence of NH₄⁺ were as high as 100% of the nitrogenase activity detected in the absence of NH₄⁺.3. Biochemical characterization of these nitrogen fixation (nif) derepressed mutants reveals that they fall into three classes. Three mutants (strains SK-24, 28 and 29), requiring glutamate for growth, synthesize nitrogenase and glutamine synthetase constitutively (in the presence of NH₄⁺). A second class of mutants (strains SK-27 and 37) requiring glutamine for growth produces derepressed levels of nitrogenase activity and synthesized catalytically inactive glutamine synthetase protein, as determined immunologically. A third class of glutamine-requiring, nitrogenase-derepressed mutants (strain SK-25 and 26) synthesizes neither a catalytically active glutamine synthetase enzyme nor an immunologically cross-reactive glutamine synthetase protein.4. F-prime complementation analysis reveals that the mutant strains SK-25, 26, 27, 37 map in a segment of the Klebsiella chromosome corresponding to the region coding for glutamine synthetase. Since the mutant strains SK-27 and SK-37 produce inactive glutamine synthetase protein, it is concluded that these mutations map within the glutamine synthetase structural gene.     

Identification of nifE-, nifN- and nifS-like genes in Bradyrhizobium japonicum

Summary The 17 kb region between the Bradyrhizobium japonicum nitrogenase genes (nifDK and nifH) was investigated for the presence of further nif or fix genes by site-directed insertion or deletion/replacement mutagenesis and interspecies hybridization. Mutant strains were tested for their ability to reduce acetylene in free-living, microaerobic culture (Nif phenotype) and in soybean root nodules (Fix phenotype). The presence of a gene, previously identified by hybridization with the Klebsiella pneumoniae nifB gene, was proved by isolation of a nifB insertion mutant which was completely Nif^- and Fix^-. Three other regions were found to be homologous to the K. pneumoniae genes nifE, nifN, and nifS, NifE and nifN insertion mutants were completely Nif^-/Fix^- whereas nifS mutants were leaky with 30% residual Fix activity. Taken together, the data show that the B. japonicum genome harbours a cluster of closely adjacent genes which are directly concerned with nitrogenase function.     

Genetic analysis of the het and nif genes in the blue-green alga Nostoc muscorum

Summary Two multiply marked complementary strains namely Het ⁺ Nif⁺ Str-R and Het ^- Nif^- Ery-R MSO-R were constructed and crossed under conditions counterselective for the Het ⁺ Nif⁺ Str-R parent and selective only for recombinants of Str-R and Ery-R or Str-R and MSO-R constitution. The results of the recombinant analysis with regard to the selected and unselected markers suggested that the Het ^- Nif^- Ery-R MSO-R parent acted as a recipient and the Het ⁺ Nif⁺ Str-R parent as donor of the genetic markers in the cross. The joint inheritance of Het ⁺ and Nif ⁺ unselected markers among the recombinants was found to occur more frequently than the inheritance of the Het ⁺ or Nif ⁺ markers alone. The observed joint inheritance of Het ⁺ and Nif ⁺ markers among the recombinants probably results from the inheritance of the regulatory gene(s) required for the activation of latent het and nif genes. This interpretation is fully supported by (a) the frequency distribution of unselected Het ⁺ and Nif ⁺ markers and (b) the reversion frequency of Het ^- Nif ^- strains to Het ⁺ Nif⁺ prototrophy. Accordingly the apparent close genetic linkage of het and nif genes is not due to their organization in a single operon but to their common regulation by regulatory gene(s) of a positive control nature. The Het ⁺ Nif⁺ wild type, mutant, revertant, and recombinant strains all appear similar in their NO ₃ ^- repression of both heterocyst and nitrogenase. The Het ⁺ Nif^- and Het ^- Nif⁺ recominants also show similar NO ₃ ^- repression of their heterocyst and nitrogenase respectively. The presence of only microaerobic acetylene reducing activity in Het ^- Nif⁺ recombinants clearly indicates the heterocyst to be an organ for protection of nitrogenase against oxygen toxicity.Abbreviations CFU Colony forming units - Ery erythromycin - Ery-R erythromycin resistance - het genotypic designation of genes required for heterocyst differentiation - Het phenotype designation of genes required for heterocyst differentiation - MSO l-Methionine-dl-sulfoximine - MSO-R MSO-resistance - N₂ medium Chu 10 medium without combined nitrogen - NH ₄ ⁺ medium basic mineral medium with ammonium nitrogen - nif genotype designation of genes required for N₂ fixation - Nif phenotype designation of genes required for N₂ fixation - NO ₃ ^- medium Chu 10 medium supplemented with KNO₃ - NTG N-methyl-N-nitro-N-nitrosoguanidine - r gene(s) regulatory gene(s) - Str streptomycin - Str-R streptomycin resistance - Str-S streptomycin sensitive     

Effect of Naturally Occurring nif Reiterations on Symbiotic Effectiveness in Rhizobium phaseoli

Most naturally occurring strains of Rhizobium phaseoli possess reiteration of the nif genes. Three regions contain nitrogenase structural genes in strain CFN42. Two of these regions (a and b) have copies of nifH, nifD, and nifK, whereas the third region (c) contains only nifH. Strains containing mutations in either nif region a or nif region b had significantly diminished symbiotic effectiveness compared with the wild-type strain on the basis of nodule mass, total nitrogenase activity per plant, nitrogenase specific activity, total nitrogen in the shoot, and percentage of nitrogen. A strain containing mutations in both nif region a and nif region b was totally ineffective. These data indicate that both nif region a and nif region b are needed for full symbiotic effectiveness in R. phaseoli.     

Genetic analysis of nitrogen fixation in a tropical fast-growing Rhizobium

The Rhizobium strain ORS571, which is associated with the tropical legume Sesbania rostrata, has the property of growing in the free-living state at the expense of ammonia or N₂ as sole nitrogen source. Five mutants, isolated as unable to form colonies on plates under conditions of nitrogen fixation, were studied. All of them, which appear as Fix^- in planta, are nif mutants. With mutant 5740, nitrogenase activity of the crude extract was restored by addition of pure Mo-Fe protein of Klebsiella pneumoniae. A 13-kb BamHI DNA fragment from the wild-type strain, which hybridized with a probe carrying the nifHDK genes of K. pneumoniae, was cloned in vector pRK290 to yield plasmid pRS1. The extent of homology between the probe and the BamHI fragment was estimated at 4 kb and hybridization with K. pneumoniae nifH, nifK, and possibly nifD was detected. The pRS1 plasmid was introduced into the sesbania rhizobium nif mutants. Genetic complementation was observed with strain 5740(pRS1) both in the free-living state and in planta. It thus appears that biochemistry and genetics of nitrogen fixation in this particular Rhizobium strain can be performed with bacteria grown under non-symbiotic conditions.     

Nucleotide sequence and genetic analysis of the Rhodobacter capsulatus ORF6-nifU I SVW gene region: possible role of Nif W in homocitrate processing

DNA sequence analysis of a 3494-bp HindIII-Bc1I fragment of the Rhodobacter capsulatus nif region A revealed genes that are homologous to ORF6, nifU, nifS, nifV and nifW from Azotobacter vinelandii and Klebsiella pneumoniae. R. capsulatus nifU, which is present in two copies, encodes a novel type of NifU protein. The deduced amino acid sequences of NifU_I and NifU_II share homology only with the C-terminal domain of NifU from A. vinelandii and K. pneurnoniae. In contrast to nifA andnifB which are almost perfectly duplicated, the predicted amino acid sequences of the two NifU proteins showed only 39% sequence identity. Expression of the ORF6-nifU _ISVW operon, which is preceded by a putative σ⁵⁴-dependent promoter, required the function of NifA and the nif-specific rpoN gene product encoded by nifR4. Analysis of defined insertion and deletion mutants demonstrated that only nifS was absolutely essential for nitrogen fixation in R. capsulatus. Strains carrying mutations in nifV were capable of very slow diazotrophic growth, whereas ORF6, nifU _I and nifW mutants as well as a nifU _I/nifU_II, double mutant exhibited a Nif⁺ phenotype. Interestingly, R. capsulatus nifV mutants were able to reduce acetylene not only to ethylene but also to ethane under conditions preventing the expression of the alternative nitrogenase system. Homocitrate added to the growth medium repressed ethane formation and cured the NifV phenotype in R. capsulatus. Higher concentrations of homocitrate were necessary to complement the NifV phenotype of a polar nifV mutant (NifV^?NifW^?), indicating a possible role of NifW either in homocitrate transport or in the incorporation of this compound into the iron-molybdenum cofactor of nitrogenase.     

Characterization of the ntrBC genes of Azospirillam brasilense Sp7: Their involvement in the regulation of nitrogenase synthesis and activity

A 7.1 kb EcoRI fragment from Azospirillum brasilense, that hybridized with a probe carrying the ntrBC genes from Bradyrhizobium japonicum, was cloned. The nucleotide sequence of a 3.8 kb subfragment was established. This led to the identification of two open reading frames, encoding polypeptides of 401 and 481 amino acids, that were similar to NtrB and NtrC, respectively. A broad host range plasmid containing the putative Azospirillum ntrC gene was shown to restore nitrogen fixation under free-living conditions to a ntrC-Tn5 mutant of Azorhizobium caulinodans. Several Tn5 insertion mutants were isolated in the ntrBC coding region in A. brasilense. These mutants were prototrophic and Nif⁺. However, their nitrogenase activity was slightly lower than in the wild type and they were unable to grow on nitrate as sole nitrogen source. Under microaerobiosis and in the absence of ammonia, a nifA-lacZ fusion was expressed in the mutants at about 60% of the level in the wild type. In the presence of ammonia, the fusion was similarly expressed (60% of the maximum) both in the wild type and mutants. Addition of ammonia to a nitrogen-fixing culture of ntrBC mutants did not abolish nitrogenase activity, in contrast with the wild type. It thus appears that in Azospirillum the ntrBC genes are not essential for nitrogen fixation, although NtrC controls nifA expression to some extent. They are, however, required for the switch-off of nitrogenase activity.     

The hisD-hisC gene border of the Salmonella typhimurium histidine operon

Summary We have sequenced the hisD-hisC gene border of the Salmonella typhimurium histidine operon. The translation termination codon of the hisD gene overlaps with the translation initiation codon of the hisC gene in the manner . The Shine-Dalgarno sequence of the hisC gene is contained entirely within hisD and there is no intercistronic space since all of the bases are utilized in coding. Two mutations that alter the hisD-hisC gene border are analyzed. Both mutations simultaneously abolish the termination codon of hisD and modify the initiation codon of hisC. One of the mutations changes the hisC initiation codon from AUG to AUU. The AUU codon is 10 to 20% as efficient as AUG for initiation of translation of the hisC gene. The mutant hisC ribosome binding site is compared to the ribosome binding site of the Escherichia coli infC gene which has been reported to contain an AUU initiation codon. The role of overlapping termination/initiation codons in regulating translation of polycistronic mRNAs in bacterial operons is discussed.     

Characterization of anf genes specific for the alternative nitrogenase and identification of nif genes required for both nitrogenases in Rhodobacter capsulatus

To identify Rhodobacter capsulatus nif genes necessary for the alternative nitrogenase, strains carrying defined mutations in 32 genes and open reading frames of nif region A, B or C were constructed. The ability of these mutants to grow on nitrogen-free medium with molybdenum (Nif phenotype) or in a nifHDK deletion background on medium without molybdenum (Anf phenotype) was tested. Nine nif genes and nif-associated coding regions are absolutely essential for the alternative nitrogenase. These genes comprise nifV and nifB, the nif-specific ntr system (nifR1, R2, R4) and four open reading frames, which exhibit no homology to known genes. In addition, a significantly reduced activity of both the alternative nitrogenase and the molybdenum-dependent nitrogenase was found for fdxN mutants. By random Tn5 mutagenesis of a nifHDK deletion strain 42 Anf^? mutants were isolated. Southern hybridization experiments demonstrated that 17 of these Tn5 mutants were localized in at least 13 different restriction fragments outside of known nif regions. Ten different Anf^? Tn5 mutations are clustered on a 6 kb DNA fragment of the chromosome designated anf region A. DNA sequence analysis revealed that this region contained the structural genes of the alternative nitrogenase (anfHDGK). The identification of several Tn5 insertions mapping outside of anf region A indicated that at least 10 genes specific for the alternative nitrogenase are present in R. capsulatus.     

Differential response of NaCl-Resistant mutants of the cyanobacterium Nostoc muscorum to salinity and osmotic stresses

Summary In the parent strain of Nostoc muscorum, the percentage survival, nitrogenase activity and oxygenic photosynthesis were severely impaired by NaCl (ionic) and sucrose (non-ionic) stresses. Spontaneously occurring NaCl-Resistant mutant clones of the cyanobacterium N. muscorum were found to exhibit differential responses under ionic and non-ionic stresses. One of the mutants (NaCl-R) was found to show resistance in terms of percentage survival, nitrogenase activity and oxygenic photosynthesis under saline (ionic) as well as osmotic (non-ionic) stresses and showing compatible solute strategy for such adaptation. Another mutant (Na⁺-R) was found to show resistance only to salinity stress and showed an enhanced Na⁺-efflux system driven by H⁺. The Na⁺-R mutant differed from the NaCl-R mutant strain in the sense that it was sucrose sensitive.     

Genetic fusions that place the lactose genes under histidine operon control

The genes of the Salmonella histidine operon (his) have been placed on an F′ pro lac plasmid using genetic methods that rely on recombinational homology provided by Tn10 transposon insertions. The position and orientation of the transposed his genes permit subsequent deletion mutations to form operon fusions that put the lac genes under his operon control. Strains carrying such fusions show co-ordinate regulation of histidinol dehydrogenase and beta-galactosidase expression. While all of the operon fusions have an intact hisD gene, complementation testing and deletion mapping reveal that the genes downstream of hisD are deleted to varying extents. The beta-galactosidase produced by these operon fusions is itself a fused protein containing the amino terminus of one or another of the his enzymes. Two of the operon fusions having join-points in the hisB gene retain histidinol phosphate phosphatase activity and may produce a bifunctional protein having beta-galactosidase as well as the phosphatase activity. The methods that have been used to isolate these his-lac fusions should be applicable to other genetic systems.     

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.