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.     

Repression of nitrogen fixation in Klebsiella pneumoniae at high temperature

Clostridium perfringens 11268 CDR (Rifr Tcs), the strain transformed in our experiments, was generated by curing a spontaneous, rifampicin-resistant mutant of C. perfringens 11268 (Rifr Tcr). High-temperature growth yielded tetracycline-sensitive, rifampicin-resistant cells which no longer contained pCW3, a 42.8-kilobase plasmid. The tetracycline-sensitive, rod-shaped cell was then converted to an L-phase variant by growth in the presence of penicillin G (10 micrograms/ml) and 0.4 M sucrose. After several passages, the antibiotic was removed from the medium, and cells continued to grow as L-phase variants. Another large plasmid, pJU124 (38.8 kilobases), which confers tetracycline resistance, was used for transformation. Transformation of L-phase variants of C. perfringens 11268 CDR (Rifr Tcs) was mediated by polyethylene glycol. Transformation frequency is a nonlinear function of DNA concentration. Restriction analysis showed that the plasmid isolated from the transformants was identical to that supplied. Stable L-phase variants do not revert to rod-shaped cells, but autoplasts can be both transformed and reverted.     

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.     

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.     

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.     

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.     

Nitrogen fixation specific regulatory genes of Klebsiella pneumoniae and Rhizobium meliloti share homology with the general nitrogen regulatory gene ntrC of K. pneumoniae.

We have determined the complete nucleotide sequences of three functionally related nitrogen assimilation regulatory genes from Klebsiella pneumoniae and Rhizobium meliloti. These genes are: 1) The K. pneumoniae general nitrogen assimilation regulatory gene ntrC (formerly called glnG), 2) the K. pneumoniae nif-specific regulatory gene nifA, and 3) an R. meliloti nif-specific regulatory gene that appears to be functionally analogous to the K. pneumoniae nifA gene. In addition to the DNA sequence data, gel-purified K. pneumoniae nifA protein was used to determine the amino acid composition of the nifA protein. The K. pneumoniae ntrC and nifA genes code for proteins of 52,259 and 53,319 d respectively. The R. meliloti nifA gene codes for a 59,968 d protein. A central region within each polypeptide, consisting of approximately 200 amino acids, is between 52% and 58% conserved among the three proteins. Neither the amino termini nor the carboxy termini show any conserved sequences. Together with data that shows that the three regulatory proteins activate promoters that share a common consensus sequence in the -10 (5'-TTGCA-3') and -23 (5'-CTGG-3') regions, the sequence data presented here suggest a common evolutionary origin for the three regulatory genes.     

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.     

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     

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.     

The purification, characterization and role of the d-type cytochrome oxidase of Klebsiella pneumoniae during nitrogen fixation

Klebsiella pneumoniae synthesized only b-type and d-type cytochromes under the wide range of growth conditions tested, and reaction with CO revealed two potential oxidases. The o-type oxidase was produced only in the presence of O2 and appeared to be repressed by glucose. The d-type oxidase was, by contrast, produced only in the absence of measurable O2 (less than 1 microM), and was the only oxidase expressed in nitrogen-fixing conditions. It was extracted from the membrane, purified and shown to be similar to that from E. coli in being a heterodimer (subunits of Mr 52,000 and 35,000), in containing two distinguishable b haems and haem d (one or two molecules per molecule of oxidase), and in being able to react with O2 to give a stable oxygenated intermediate. The purified d-type cytochrome oxidase had a very high affinity for O2 (Km 20 nM; measured by the spectral properties of leghaemoglobin). It is proposed that this provides a role for this oxidase in lowering the O2 concentration to allow nitrogenase synthesis and function, and to provide a terminal oxidase to permit electron-transport-coupled ATP synthesis which supports the increase in efficiency of nitrogen fixation observed under microaerobic conditions.     

Interaction between the fumarate reductase system of Escherichia coli and the nitrogen fixation genes of Klebsiella pneumoniae.

For phenotypic expression of nif+Kp genes in Escherichia coli K-12, the anaerobic electron transport system to fumarate must be functional. The role of the fumarate reduction system is to energize the membrane and thus provide the energy necessary for nitrogen fixation.