Temperature control of nitrogen fixation in Klebsiella pneumoniae

At growth temperatures above 37°C, Klebsiella pneumoniae does not grow in a medium containing N₂ or NO ₃ ^- as nitrogen sources. However, both the growth in the presence of other nitrogen sources as well as the in vitro nitrogenase activity are not affected at this temperature. The inability to fix N₂ at high temperature is due to the failure of the cells to synthesize nitrogenase and other nitrogen fixation (nif) gene encoded proteins. When cells grown under nitrogen fixing conditions at 30°C were shifted to 39°C, there was a rapid decrease of the rate of de novo biosynthesis of nitrogenase (component 1), nitrogenase reductase (component 2), and the nifJ gene product. There was no degradation of nitrogenase at the elevated temperature since preformed enzyme remained stable over a period of at least 3 h at 39°C. Thus, temperature seems to represent a third control system, besides NH ₄ ⁺ and O₂, governing the expression of nif genes of K. pneumoniae.     

Complementation analysis of Klebsiella pneumoniae mutants defective in nitrogen fixation.

Summary A series of mutants defective in nitrogen fixation (nif) were isolated in Klebsiella pneunoniae strain M5a1. The nif mutations were either located on plasmid pRD1 or on the K. pneumoniae chromosome. A total of 37 plasmid mutants and 28 chromosomal mutants were employed in complementation tests using the acetylene reduction technique. Most mutants could be assigned to one of seven nif cistrons: nifA, nifB, nifD, nifE, nifF, nifH, and nifK.Complementation analysis of two nif deletion mutants confirmed transductional evidence that these strains carry nifB-A-F deletions. One deletion mutant had, in contrast to previous transductional analysis, a functional nifK cistron and presumably is deleted for nifB-A-F-E.Examination of the biochemical phenotype of several mutants suggests that the nifA product has a regulatory function, and nifK, nifD and nifH are most probably the structural genes for nitrogenase.     

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.     

Pleiotropic effect of his gene mutations on nitrogen fixation in Klebsiella pneumoniae

Several his mutations were found to influence nitrogen fixation in Klebsiella pneumoniae: hisB, hisC, and hisD mutants had 50% of wild-type levels of nitrogenase activity when supplied with 30 μg or less histidine/ml although this concentration did not limit protein synthesis and the mutants retained a Nif⁺ plate phenotype. A hisA mutation had a similar but more dramatic effect. At low concentrations of histidine the hisA mutant strain had only 5% of the nitrogenase activity found at high histidine concentration or in a his⁺ strain, and was also Nif^- on low histidine agar plates. Addition of adenine restored nitrogenase activity in the hisA but not the hisB, hisC, or hisD mutants. Low levels of intracellular ATP, a consequence of hisG enzyme activity, correlated with loss of nitrogen-fixing ability in the hisA mutant which failed to sustain nif gene expression under these conditions. Synthesis of other major cell proteins was relatively unaffected indicating that nif gene expression is selectively regulated by the energy status of the organism.     

Complementation analysis of glnA-linked mutations which affect nitrogen fixation in Klebsiella pneumoniae

Summary A number of mutants have been isolated which affect regulation of the nitrogen fixation (nif) gene cluster in Klebsiella pneumoniae and all of which are linked to glnA, the structural gene for glutamine synthetase (G.S.). These mutants were classified on the basis of their G.S. and nitrogenase activities in conditions of nitrogen limitation and excess. The plasmid R68.45 was then used to generate a number of R-primes carrying the glnA region of the K. pneumoniae chromosome. One of these R-primes (pGE10) was subsequently used in complementation analysis and by isolation of transposon-induced insertion mutations in pGE10 we have demonstrated the existence of a gene, glnG, closely linked to glnA. Mutations in glnG have a similar phenotype to glnG mutants described in Escherichia coli (Pahel and Tyler 1979) and Salmonella typhimurium (Kustu et al. 1979) in that they substantially reduce G.S. activity but are not glutamine auxotrophs. GlnG mutants have very low nitrogenase activity indicating that the glnG product may be involved in regulation of the nif gene cluster in K. pneumoniae.     

Deletion analysis of the nitrogen fixation regulatory gene,nifL of Klebsiella pneumoniae

A number of in-frame deletions have been constructed in the Klebsiella pneumoniae regulatory gene nifL. The effects of each nifL mutation on NifA-mediated expression from the nifH promoter of K. pneumoniae have then been assessed with respect to both nitrogen and oxygen control. These experiments indicate that, in contrast to the situation with the homologous regulatory proteins NtrB and NtrC, NifA activity is not impaired in the absence of NifL. We conclude that the only function of NifL is to inactivate NifA in response to an increase in the nitrogen or oxygen status of the cell.     

Perturbation of nifT expression in Klebsiella pneumoniae has limited effect on nitrogen fixation.

In the nitrogenase system of Klebsiella pneumoniae, nifT is located between nifDK, the structural genes for dinitrogenase, and nifY, whose product is involved in nitrogenase maturation. It is, therefore, a reasonable hypothesis that the NifT protein might also have a role in the maturation of nitrogenase. However, the phenotypic characterization of nifT and nifT-overexpressing strains for effects on the regulation, maturation, and activity of nitrogenase identified no properties that were distinct from those of the wild type. We conclude that the K. pneumoniae NifT protein is not essential for nitrogen fixation under the conditions examined.     

Glutamine synthetase mutations which affect expression of nitrogen fixation genes in Klebsiella pneumoniae.

Previous studies have implicated glutamine synthetase (L-glutamate:ammonia ligase [adenosine diphosphate for-ing], EC 6.6.1.2) as a major controlling element of the nitrogen fixation (nif) genes in Klebsiella pneumoniae. We report here the isolation of a new class of K. pneumoniae mutants which exhibit altered patterns of nif and hut (histidine utlization) regulation. The expression of nif in these mutants, which were isolated as Gln+ (glutamine nonrequiring) revertants of a particular glnA mutation, is extremely sensitive to ammonia repression. These mutants have a Nif- Hut- phenotype at external ammonia concentrations at which wild-type strains are Nif+ Hut+. On the other hand, these mutants can be fully derepressed for nif at very low ammonia concentrations. We adopted the nomenclature "GlnR- (Nif- Hut-)" to facilitate discussion of the phenotype of these mutant strains. The mutations in these strains which confer the GlnR- phenotype map at or near glnA, the structural gene for glutamine synthetase.     

Expression of Klebsiella pneumoniae nitrogen fixation genes in nitrate reductase mutants of Escherichia coli.

Nitrate reductase (nar) A, B and E mutants of Escherichia coli with plasmids carrying Klebsiella pneumoniae nitrogen fixation (nif) genes reduced acetylene independently of added molybdate, but nar D mutants showed pleiotropic dependence on the concentration of added molybdate for expression of both nar and nif. No complementation of nar mutations by nif occurred; nitrite but not nitrate repressed nif in nar hosts. Derepression of nif occurred in molybdenum-deficient nar D (nif) strains since nitrogenase peptides were present. nifB mutants, thought to have a lesion in the pathway of molybdenum to nitrogenase, as well as nif deletion mutants, had normal nitrate reductase activity.     

Characterisation of mutations in the Klebsiella pneumoniae nitrogen fixation regulatory gene nifL which impair oxygen regulation

The nifL gene product of Klebsiella pneumoniae inhibits the activity of the positive activator protein NifA in response to increased levels either of fixed nitrogen or of oxygen in the medium. In order to demonstrate that the responses to these two effectors are discrete we have subjected nifL to hydroxylamine mutagenesis and isolated nifL mutants that are impaired in their ability to respond to oxygen but not to fixed nitrogen. Two such mutations were sequenced and shown to be single base pair changes located in different parts of nifL. The amino acid sequence of NifL shows limited homology to the histidine protein kinases which comprise the sensing component of bacterial two-component regulatory systems. In the light of the location of one of the oxygen-insensitive mutations (Leu294Phe) we have reassessed this homology and we suggest that the Gln273-Leu317 region of NifL may facilitate interactions between NifL and NifA.Abbreviations X-gal 5-bromo-4-chloro-3-indolyl--D-galactopy-ranoside - USAs upstream activator sequences     

Nitrogen fixation in wheat provided by Klebsiella pneumoniae 342

In this report, all of the criteria necessary for the demonstration of nitrogen fixation in wheat (Triticum aestivum L.), the world's most important crop, are shown upon inoculation with a nitrogen-fixing bacterium, Klebsiella pneumoniae 342 (Kp342). Kp342 relieved nitrogen (N) deficiency symptoms and increased total N and N concentration in the plant. Nitrogen fixation was confirmed by 15N isotope dilution in the plant tissue and in a plant product, chlorophyll. All of these observations were in contrast to uninoculated plants, plants inoculated with a nitrogen-fixing mutant of Kp342, and plants inoculated with dead Kp342 cells. Nitrogenase reductase was produced by Kp342 in the intercellular space of the root cortex. Wild-type Kp342 and the nifH mutant colonized the interior of wheat roots in equal numbers on a fresh weight basis. The nitrogen fixation phenotype described here was specific to cv. Trenton. Inoculation of cvs. Russ or Stoa with Kp342 resulted in no relief of nitrogen deficiency symptoms.     

Clostridial pyruvate oxidoreductase and the pyruvate-oxidizing enzyme specific to nitrogen fixation in Klebsiella pneumoniae are similar enzymes

The chemical characterization, EPR properties, and mechanism of pyruvate:flavodoxin (ferredoxin) oxidoreductase from Klebsiella pneumoniae and Clostridium thermoaceticum have been investigated. A simple, specific, and sensitive assay and an efficient purification (based on the high affinity of these enzymes for a dye attached to agarose) are reported. The observed iron content of 8 atoms/subunit is twice that reported by others, whereas the contents of lipoate and flavin are less than 0.1 mol/subunit, in agreement with previous reports. Spectroscopic evidence suggests that the iron is present in Fe4S4(2+,1+) clusters. Reduction of the enzyme requires the presence of CoA as well as 1.1 pyruvate/subunit, which is very nearly the theoretical amount required the reduce two Fe4S(2+,1+) clusters. In the absence of CoA, stoichiometric amounts of pyruvate are decarboxylated, but the Fe/S centers are not reduced. We conclude that the K. pneumoniae and C. thermoaceticum enzymes are adapted to rapid reduction of low potential 1-e- carriers, similar to the pyruvate oxidoreductase of Halobacterium (Kerscher, L., and Oesterhelt, D. (1977) FEBS Lett. 83, 197-201), but different in that an Fe/S center-radical pair is used in the latter enzyme in place of the pair of Fe4S4 centers we find. The K. pneumoniae and C. thermoaceticum oxidoreductases appear to be mechanistically closely related to the Clostridium acidiurici enzyme (Uyeda, K., and Rabinowitz, J. C. (1971) J. Biol. Chem. 246, 3111-3119), differing as a class from the lipoate-containing, pyridine nucleotide-reducing enzyme present in aerobes (Reed, L. J. (1974) Accts. Chem. Res. 2, 740-746). The function of the Klebsiella enzyme is to supply electrons to nitrogenase. This is accomplished in vitro with purified components via a nif-specific flavodoxin or other low potential 1-e- carriers such as viologen dyes or ferredoxins. The in vivo molar ratio of nitrogenase to the physiological reduction system, estimated from activity measurements of individual components in crude extracts, was 0.4:0.03:2:1 pyruvate oxidoreductase:flavodoxin:nitrogenase component II:nitrogenase component I.     

Regulation of nitrogen metabolism in glutamine auxotrophs of Klebsiella pneumoniae

We examined the regulation of nitrogen metabolism in four classes (glnA, glnB, glnF, and glnG) of Gln- auxotrophs of Klebsiella pneumoniae. These studies indicate that glutamine synthetase does not directly mediate the physiological response to NH4+ in this organism. We present evidence suggesting that the effect of NH4+ on the expression of genes involved in nitrogen metabolism involves the products of the glnF and glnG genes.