Nitrogenase Of Klebsiella Research Articles

A possible link between Crohn’s disease and ankylosing spondylitis via Klebsiella infections

Crohn's disease (CD) is an immune-mediated gastrointestinal inflammatory disease, which could arise from an interplay between genetic and environmental factors. Klebsiella microbes were suggested to have a vital role in the initiation and perpetuation of the disease through the mechanism of molecular mimicry. This proposition is based on the results of various studies where significantly elevated levels of antibodies against the whole bacteria or preparations from Klebsiella microbes and antibodies to collagen types I, III, IV, and V were detected in patients with CD and patients with ankylosing spondylitis (AS). Molecular similarities were found between Klebsiella nitrogenase and HLA-B27 genetic markers and between Klebsiella pullulanase and collagen fibers types I, III, and IV. Furthermore, significantly positive correlations and cross-reactivity binding activities were observed between anti-Klebsiella and anticollagen antibodies among patients with CD and AS. Early treatment of CD patients with anti-Klebsiella measures is proposed, which may involve the use of antibiotics and low starch diet together with other traditionally used immunomodulatory, immunosuppressive, or biologic agents.

The Potential Use of Antibacterial Peptide Antibody Indices in the Diagnosis of Rheumatoid Arthritis and Ankylosing Spondylitis

Both rheumatoid arthritis (RA) and ankylosing spondylitis (AS) are potentially disabling arthritic disorders for which as yet no highly sensitive and reliable diagnostic laboratory markers are available. The objective of this study was to evaluate the levels of antibodies against Proteus and Klebsiella antigenic peptides in an endeavor to develop diagnostic indices for the identification of patients with RA and AS, respectively. Sera from 50 patients with RA, 34 patients with AS, and 38 healthy subjects were screened for antibodies against "ESRRAL" and "IRRET" synthetic amino acid peptides obtained from Proteus hemolysin and urease (HU) as well as against "QTDRED" and "DRDE" peptides from Klebsiella nitrogenase and pullulanase (NP) proteins, respectively. Multiplication of the 2 antibodies against each organism produced indices for RA-HU and AS-NP. Significantly increased levels of anti-HU antibodies (P<0.0001) were observed in patients with RA when compared with patients with AS or with healthy control subjects. Patients with AS were found to have significantly elevated levels of anti-NP (P<0.0001) antibodies when compared with patients with RA or with healthy subjects. Furthermore, all patients with RA were found to have values of anti-HU antibody (RA-HU) index above 95% confidence limit (CL) of the mean of healthy control subjects; meanwhile, all patients with AS were having values of anti-NP antibody (AS-NP) index above the 95% CL of the mean of healthy control subjects (100% sensitivity). However, the specificity of the RA-HU index in RA and the AS-NP index in patients with AS were 92% and 95%, respectively. The use of the RA-HU or AS-NP diagnostic index as a sole marker or in combination with other autoantibody markers could be used in the identification of patients with RA or AS, respectively. Longitudinal investigations starting with patients with early disease will be needed.

Construction and characterization of double mutants in nitrogenase ofKlebsiella pneumoniae

Two mutants in nitrogenase ofKlebsiella pneumoniae are constructed by site-directed mutagenesis and gene replacement procedure, which express the nitrogenases with Lysine and Glutamine substituting for α-Glutamine 190 and α-Histidine 194 respectively (Kp-Q α190 K and Kp-H α194 Q). The above two substitutions are respectively introduced into anifV mutant (expressing a citrate-containing nitrogenase) and sequentially two double mutants are obtained (Kp-Q α190 K-nifV − and Kp-H α194 Q-nifV −). All four mutants exhibit strict Nif-phenotype under the N2-fixation condition and fail to grow diazotrophically. Altered nitrogneases are effectively depressed and the C2H2 reduction analysis shows that the double substitutions in Kp-Q α190 K-nifV abolish cell C2H2 reduction activity, but Kp-H α194 Q-nifV − cells maintain a C2H2 reduction activity at 10% of that of wild type. Whole cell C2D2 reduction by all four mutants in comparison to the wild type andnifV mutant is also detected. The results show that only single α-Gln194 substitution does not perturb the stereospecificity of protonation of C2D2. These results indicate that the α-Glutamine 190 and its combination with homocitrate are essential to the catalytic activity of nitrogenase and it is proposed that α-Glutamine 190 and its combination with homocitrate are involved in the proton and/or electron transfer to FeMoco. The nitrogenases from these double mutants will be useful in further analysis of the entry of the proton and/or electron to FeMoco and the substrate binding sites.

Construction and characterization of double mutants in nitrogenase of Klebsiella pneumoniae

Construction and characterization of double mutants in nitrogenase of Klebsiella pneumoniae

Protonation and substitution reactions of Fe–S ‘basket’ clusters including extracted FeMo-cofactor of nitrogenase

In an extension to our earlier work on the mechanisms of reactions of Fe–S-based clusters, we now report studies on the substitution kinetics of structurally more ‘open’ Fe–S clusters. In particular, we concentrate on protonation of the ‘basket’ cluster [Fe 6S 6Cl 2(PEt 3) 4]. Protonation at a μ n -S can either accelerate or inhibit the rate of substitution of the cluster depending on: the number of protons added and whether substitution is associative or dissociative. Analysis of the kinetics allows us to calculate the p K a values of the protonated clusters and comparison with a variety of other Fe–S-based clusters shows that, irrespective of the structure and charge, the first (p K a 1=17.9–18.9) is rather insensitive to the cluster ligands whilst the second (p K a 2=13.6–16.9) is markedly more sensitive. Analogous studies on extracted FeMo-cofactor [MoFe 7S 9(R-homocitrate)(NMF) 2] (NMF= N-methylformamide) from the nitrogenase of Klebsiella pneumoniae shows the same general protonation/substitution characteristics as synthetic Fe–S-based clusters.

Nitrogenase of Klebsiella pneumoniae: kinetics of formation of the transition-state complex and evidence for an altered conformation of MoFe protein lacking a FeMoco centre.

We have investigated the kinetics of inactivation of Mo-nitrogenase isolated from Klebsiella pneumoniae when it forms an inhibited putative transition-state complex on incubation with ADP and AlF4-. In the presence of excess Kp2 (Fe protein of the Mo-nitrogenase of K. pneumoniae), the kinetics were found to depend on the Mo content of Kp1 (the MoFe protein of Mo-nitrogenase of K. pneumoniae). The residual nitrogenase activity versus time of incubation using Kp1 preparations containing integral, i.e. one or two Mo atoms per molecule of Kp1, were essentially monophasic, but significantly different rates of inactivation were observed. In contrast, the progress curves for preparations of Kp1 with non-integral Mo content were biphasic, suggesting the presence of two discrete catalytically active species of Kp1. The best fit to the observed data was obtained with a two-exponential expression, the amplitude of which was consistent with the Mo content, provided that the fast phase of the reaction was assigned to a Kp1 species containing one, and the slow phase to a species containing two Mo atoms per alpha2beta2 tetramer. This analysis provides the first evidence for the existence of a catalytically active Kp1 species containing a single Mo atom. These data also indicate that MoFe protein which does not have all FeMoco binding sites occupied has an altered conformation compared with a fully loaded protein, and that the Fe protein reacts with these conformations at different rates to form the stable, but inhibited transition-state complex.

The in-vivo identification of the MoFe protein (FeMo cofactor) of nitrogenase in Klebsiella pneumoniae and of the Mo-storage protein in Azotobacter vinelandii via the nuclear quadrupole interaction of [formula omitted

The expression of the MoFe protein of nitrogenase in Klebsiella pneumoniae was identified in vivo via the nuclear quadrupole interaction (NQI) of 99 Mo(β −) 99Tc using perturbed angular correlations of γ-rays. The NQI parameters were: ω approx. 360 Mrad/s and η approx. 1. In addition, the NQI of the ‘Mo-storage protein’ in Azotobacter vinelandii cells which had been grown in the presence of NH 4 + (13 mM), i.e. under conditions of strict repression of nitrogenase synthesis, was determined: ω approx. 190 Mrad/s, η approx. 0.25. Under these conditions, the characteristic signal of the MoFe protein (FeMo cofactor) was absent.

Molecular mimicry: Any role in the pathogenesis of spondyloarthropathies?

Ankylosing spondylitis and reactive arthritis are seronegative spondyloarthropathies, which are strongly associated with HLA-B27. Despite intensive investigation, the basis for this association is not clear. However, in recent years one favored hypothesis to explain this linkage has been that of molecular mimicry, i.e., sharing of linear or conformational epitopes common to microbial antigens and host structures. During the past few years several examples of molecular mimicry between HLA-B27 and microbial antigens have been described. Heat shock proteins, among others, have been considered as target candidates for autoimmune phenomena, because of the high degree of homology between bacterial and mammalian species. Reactive arthritis triggered by Yersinia or Salmonella provides a unique model for studying the pathogenetic mechanisms underlying human inflammatory joint diseases in general, because the arthritogenic microbes are known and well-characterized. We have described two bacterial proteins that share amino acid homology with HLA-B27, namely YadA (Yersinia adhesin) and OmpH, outer surface proteins of Yersinia and Salmonella, respectively. Notably, the area of identity of these amino acid sequences is located in the same place on the HLA-B27 molecule as a hexapeptide identical between Klebsiella nitrogenase and HLA-B27, and a pentapeptide shared by a Shigella flexneri protein and HLA-B27. We have investigated immune responses to a panel of synthetic peptides based on the HLA-B27-homologous portions of pathogen-specific antigens in patients with reactive arthritis and ankylosing spondylitis. One third of the patients have antibodies to the synthetic peptides. However, instead of recognizing the HLA-B27-homologous portion, the antibodies are directed against the flanking sequences of the synthetic peptides. The concept of the role of molecular mimicry between HLA-B27 and microbial antigens in the pathogenesis of spondyloarthropathies is discussed, with a conclusion that no convincing evidence for its significance exists at the present.

Klebsiella pneumoniae nitrogenase: pre-steady-state absorbance changes show that redox changes occur in the MoFe protein that depend on substrate and component protein ratio; a role for P-centres in reducing dinitrogen?

The pre-steady-state absorbance changes that occur during the first 0.6 s of reaction of the nitrogenase of Klebsiella pneumoniae can be simulated by associating redox changes with the different states of the MoFe protein described by our published kinetic model for nitrogenase [Lowe and Thorneley (1984) Biochem. J. 224, 877-886]. When the substrate is changed, from H+ to C2H2 (acetylene) or N2, or the nitrogenase component protein ratio is altered, these pre-steady-state absorbance changes are affected in a manner that is quantitatively predicted by our model. The results, together with parallel e.p.r. studies, are interpreted as showing that the P-clusters become oxidized when the MoFe protein is in the state where bound N2 is irreversibly committed to being reduced and is protonated to the hydrazido(2-) level.

Energy transduction by nitrogenase: binding of MgADP to the MoFe protein is dependent on the oxidation state of the iron-sulphur 'P' clusters.

Hydrolysis of MgATP to MgADP is essential for nitrogenase action. There is good evidence for binding of both nucleotides to the Fe protein of nitrogenase, but data indicating their binding to the MoFe protein have been controversial [see Miller and Eady (1989) Biochem. J. 263, 725-729]. The binding of MgADP to the MoFe protein of nitrogenase of Klebsiella pneumoniae was investigated by non-equilibrium gel-filtration column methods. No binding of MgADP to the dithionite-reduced protein could be detected. Treatment of the MoFe protein with phenosafranine [midpoint potential (Em) -270 mV] did not affect the activity, and oxidized the 'P' clusters but not the iron-molybdenum cofactor (FeMoco) centres. This oxidized species bound 3.9 mol of MgADP with a binding pattern characteristic of low rates of ligand dissociation. These observations suggest that the variability in published data on nucleotide binding to the MoFe protein is related to poor control of the protein oxidation level. Our data, coupled with the observation that 'P' clusters become oxidized during reduction of N2 [Lowe, Fisher and Thorneley (1993) Biochem. J., in the press], led us to propose that the ADP binding sites are transiently filled during enzyme turnover by hydrolysis of ATP originally bound to the Fe protein, and that hydrolysis occurs on a bridging site on the MoFe-Fe-protein complex.

Molecular mimicry in the pathogenesis of spondyloarthropathies. A critical appraisal of cross-reactivity between microbial antigens and HLA-B27.

We describe an amino acid homology between a virulence plasmid encoded outer membrane protein of Yersinia, YadA (previously called Yop1) and HLA-B27. This tetrapeptide is also included in the hexapeptide, earlier found to be identical between Klebsiella nitrogenase and HLA-B27. The synthetic peptide based on the HLA-B27 homologous portion of the YadA does not stimulate lymphocytes obtained from HLA-B27+ patients with Yersinia-triggered reactive arthritis or from controls. One-third of the yersiniosis patients have antibodies against the synthetic peptide. Instead of recognizing the HLA-B27 homologous portion, the antibodies are directed against the left flanking sequence of the synthetic peptide. Similar results were obtained regarding antibody response to Klebsiella nitrogenase derived synthetic peptide included in the panel of controls; the response was not restricted to patients with reactive arthritis nor was it specifically directed against the sequence shared by HLA-B27 and Klebsiella pneumoniae nitrogenase. The present results do not support the role of molecular mimicry or cross-reactive antibodies in the pathogenesis of spondyloarthropathies.

Autoantibodies to the HLA-B27 sequence cross-react with the hypothetical peptide from the arthritis-associated Shigella plasmid.

We previously reported elevated serum antibody levels to a peptide representing the HLA-B27 polymorphic region (B27 peptide) in HLA-B27(+) ankylosing spondylitis (AS) patients. A plasmid (pHS-2) isolated from arthritogenic Shigella flexneri strains had been shown to encode an amino acid sequence homologous to HLA-B27. Rabbit antibody to this sequence (pHS-2 peptide) strongly cross-reacted with B27 peptide and, to a much lesser extent, with Klebsiella nitrogenase peptide. Serum antibody levels to pHS-2 peptide were studied in 160 spondylarthropathy patients. 12 of 115 (10.4%) AS patients, 2 of 45 (4.4%) patients with Reiter's syndrome or reactive arthritis as well as 6 of 147 (4.1%) normal controls were shown to have elevated anti-pHS-2 peptide antibodies. Antibody levels to B27 and pHS-2 peptides were significantly correlated in 134 HLA-B27(+) patients (r = 0.333, P less than 0.001). 13 of 15 affinity-purified anti-B27 peptide antibodies from patients strongly cross-reacted with pHS-2 peptide, whereas only 3 weakly cross-reacted to nitrogenase peptide. Leucine appeared to be a critical residue for this cross-reaction. AS patients' anti-B27 peptide antibodies reacted with HLA-B27 transfected L cells. These results may suggest that pHS-2 peptide more efficiently "mimics" B27 peptide than does nitrogenase peptide. Involvement of pHS-2 in pathogenesis of spondylarthropathy through molecular mimicry mechanisms requires further study.

Cross‐reactive epitope with Klebsiella pneumoniae nitrogenase in articular tissue of HLA–B27+ patients with ankylosing spondylitis

Synovial tissues from patients with ankylosing spondylitis or reactive arthritis were examined by an immunoperoxidase technique, using antisera to synthetic peptides representing antigens shared between HLA-B27.1 and Klebsiella pneumoniae nitrogenase. With either antiserum, all HLA-B27+ patients with synovial inflammation showed strong immunoperoxidase staining in synovial lining cells, vascular endothelium, and infiltrating inflammatory cells. These findings indicate that antigens showing cross-reactivity between HLA-B27.1 and Klebsiella nitrogenase epitopes are strongly expressed within inflamed synovial tissues of HLA-B27+ patients.

Vanadium nitrogenase of Azotobacter chroococcum. MgATP-dependent electron transfer within the protein complex.

The kinetics of MgATP-induced electron transfer from the Fe protein (Ac2V) to the VFe protein (AclV) of the vanadium-containing nitrogenase from Azotobacter chroococcum were studied by stopped-flow spectrophotometry at 23 degrees C at pH 7.2. They are very similar to those of the molybdenum nitrogenase of Klebsiella pneumoniae [Thorneley (1975) Biochem. J. 145, 391-396]. Extrapolation of the dependence of kobs. on [MgATP] to infinite MgATP concentration gave k = 46 s-1 for the first-order electron-transfer reaction that occurs with the Ac2V MgATPAclV complex. MgATP binds with an apparent KD = 230 +/- 10 microM and MgADP acts as a competitive inhibitor with Ki = 30 +/- 5 microM. The Fe protein and VFe protein associate with k greater than or equal to 3 x 10(7) M-1.s-1. A comparison of the dependences of kobs. for electron transfer on protein concentrations for the vanadium nitrogenase from A. chroococcum with those for the molybdenum nitrogenase from K. pneumoniae [Lowe & Thorneley (1984) Biochem. J. 224, 895-901] indicates that the proteins of the vanadium nitrogenase system form a weaker electron-transfer complex.

Klebsiella pneumoniae nitrogenase. Inhibition of hydrogen evolution by ethylene and the reduction of ethylene to ethane.

Ethylene (C2H4) inhibited H2 evolution by the Mo-containing nitrogenase of Klebsiella pneumoniae. The extent of inhibition depended on the electron flux determined by the ratio of Fe protein (Kp2) to MoFe protein (Kp1) with KiC2H4 = 409 kPa ([Kp2]/[Kp1] = 22:1) and KC2H4i = 88 kPa ([Kp1]/[Kp2] = 21:1) at 23 degrees C at pH 7.4. At [Kp2]/[Kp1] = 1:1, inhibition was minimal with C2H4 (101 kPa). Extrapolation of data obtained when C2H4 was varied from 60 to 290 kPa indicates that at infinite pressure of C2H4 total inhibition of H2 evolution should occur. C2H4 inhibited concomitant S2O4(2-) oxidation to the same extent that it inhibited H2 evolution. Although other inhibitors of total electron flux such as CN- and CH3NC uncouple MgATP hydrolysis from electron transfer, C2H4 did not affect the ATP/2e ratio. Inhibition of H2 evolution by C2H4 was not relieved by CO. C2H4 was reduced to C2H6 at [Kp2]/[Kp1] ratios greater than or equal to 5:1 in a reaction that accounted for no more than 1% of the total electron flux. These data are discussed in terms of the chemistry of alkyne and alkene reduction on transition-metal centres.

Nitrogenase of Klebsiella pneumoniae. Rhodanese-catalysed restoration of activity of the inactive 2Fe species of the Fe protein.

The inactive 2Fe species of the Fe protein of the nitrogenase of Klebsiella pneumoniae was generated by treating oxidized Fe protein (Kp2) with MgATP and chelator. Incubation of the 2Fe species of Kp2 with the sulphurtransferase rhodanese in the presence of thiosulphate, ferric citrate and reduced lipoate reproducibly restored activity. The extent of restoration of activity depended on the molar ratio of 2Fe Kp2 to rhodanese and was time-dependent. Re-activation did not occur in the reaction mixture lacking rhodanese.

Properties of the alfalfa–Rhizobium meliloti symbiotic nitrogenase enzyme system in vivo and in vitro

A comparison of hydrogen production and acetylene reduction by excised alfalfa nodules, nitrogenase proteins isolated from Rhizobium meliloti bacteroids, and nitrogenase proteins purified from Klebsiella pneumoniae revealed several differences in the catalytic properties of the in vivo and in vitro systems. In all cases, assays for nitrogenase activity were carried out at near-physiological enzyme concentrations. Intact nodules maintained high nitrogenase activity to much lower reaction temperatures than the isolated enzymes. The Q10 value for acetylene reduction was 1.4 for nodules, but exceeded 5 for isolated R. meliloti nitrogenase in the temperature range 12–28 °C. The reduction of substrates requiring more than two reducing equivalents by the isolated enzyme system was severely restricted by the effects of reaction temperature on electron flux, as shown by very low values for electron allocation to dinitrogen determined indirectly at 12 °C. Temperature-induced lags in the initial reaction velocity of the reconstituted Klebsiella nitrogenase system were not observed with the isolated, but unseparated, R. meliloti nitrogenase proteins when acetylene was the substrate. The respiration-supported nodule system was not saturated with oxygen under air at reaction temperatures between 12 and 25 °C. Measurements of the adenine nucleotide levels of whole nodules indicated that the observed effects of reaction temperature on nitrogenase in vitro may be partially compensated in vivo by increases in adenylate energy charge and marked decreases in the ratio of ADP/ATP which occur as the nodule temperature is lowered. From the results reported here, it is clear that the R. meliloti – alfalfa nitrogenase system is limited in activity by oxygen supply. However, electron flux is sufficient within the physiological temperature range to maintain the Mo–Fe protein in the catalytically active state.

Electron transfer to nitrogenase in Klebsiella pneumoniae. nifF gene cloned and the gene product, a flavodoxin, purified.

The nifF gene of Klebsiella pneumoniae was cloned into a multicopy plasmid in order to construct a strain that synthesizes and retains an elevated concentration of the gene product relative to the wild-type strain. Characterization of the isolated flavodoxin, which serves as an electron donor to nitrogenase, shows unambiguously that it is the product of the nifF gene.

Synthesis and Activity of Nitrogenase in Klebsiella pneumoniae Exposed to Low Concentrations of Oxygen

Effects of very low concentrations of dissolved O2 on nitrogenase activity in Klebsiella pneumoniae were studied in a stirred chamber system which enabled simultaneous measurements of steady-state O2 concentrations, O2 consumption and C2H2 reduction. A strain carrying a chromosomal nifH::lac fusion as well as the Nif+ plasmid pRD1, expressed nitrogenase activity with 80 nM-O2, a concentration known to inhibit nifH::lac expression by about 50% Thus nitrogenase activity in vivo was no more sensitive to O2 than expression of nifH::lac. When compared with anaerobic treatments, dissolved O2 near 30 nM apparently stimulated nitrogenase derepression and enhanced the activity of nitrogenase synthesized anaerobically. Thus, in this organism, N2 fixation occurs in microaerobic as well as anaerobic conditions.

Role of the nifQ gene product in the incorporation of molybdenum into nitrogenase in Klebsiella pneumoniae.

NifQ- mutants of Klebsiella pneumoniae are defective in nitrogen fixation due to an elevated requirement for molybdenum. When millimolar concentrations of molybdate were added to the medium, the effects of the nifQ mutations were suppressed. NifQ- mutants were not impaired in the uptake of molybdate, but molybdate accumulation was defective in these mutants. All of the nif-coded proteins were present in NifQ- cells derepressed in the absence of molybdenum. Molybdenum-activatable nitrogenase component I was found at the same level observed in the wild type. Molybdenum, thus, does not play a role in nif expression or in the short-term stability of nif-coded proteins. The defect in NifQ- mutants was in the incorporation of molybdenum into nitrogenase component I. The nifQ gene product acts together with the products of nifB, nifN, and nifE in the biosynthesis of the iron-molybdenum cofactor of nitrogenase.