The inactive MoFe protein (NifB-Kp1) of the nitrogenase from nifB mutants of Klebsiella pneumoniae. Its interaction with FeMo-cofactor and the properties of the active MoFe protein formed.

The inactive MoFe protein (NifB-Kp1) of nitrogenase from nifB mutants of Klebsiella pneumoniae may be activated by addition of the iron-molybdenum cofactor (FeMoco) extracted from active MoFe protein (Kp1). However, when apparently saturated with FeMoco, our preparations of NifB-Kp1 yielded activated protein, Kp1-asm, with a specific activity that was at best only 40% of that expected. This was not due to degradation of Kp1-asm, NifB-Kp1 or FeMoco during the activation reaction. Nor could activation be enhanced by addition of other nif-gene products or other proteins. Whereas fully active Kp1 contains 2 FeMoco/molecule, apparent saturation of our NifB-Kp1 preparations required the binding of only 0.4-0.65 FeMoco/molecule. By using chromatography Kp1-asm could be largely resolved from NifB-Kp1 that had not been activated. However, we were unable to isolate fully active MoFe protein (i.e. Kp1-asm containing 2 FeMoco/molecule) from solutions of NifB-Kp1 activated with FeMoco. The maximum activity/ng-atom of total Mo obtained for our purified Kp1-asm was approximately half the maximum activity for FeMoco. Since all NifB-Kp1 preparations contained some Mo, we suggest that FeMoco activated only those NifB-Kp1 molecules already containing one atom of (non-FeMoco) Mo, thus forming Kp1-asm with 2 Mo but only 1 FeMoco/molecule. Kp1-asm was identical with normal Kp1 in terms of its Mr, stability, e.p.r. signals, pattern of substrate reductions, CO inhibition and ATP/2e ratio. In addition, for preparations of differing specific activity, there was a constant and identical relationship between the e.p.r. signal intensity (from FeMoco) and the activity of both Kp1 and Kp1-asm. Assuming the above hypothesis on the structure of Kp1-asm, these data demonstrate that the two FeMoco sites in wild-type Kp1 operate independently.     

Structure-function relationships in the alpha subunit of Klebsiella pneumoniae nitrogenase MoFe protein from analysis of nifD mutants.

Crude extracts of wild-type, nitrogenase-derepressed Klebsiella pneumoniae fractionated by nondenaturing gel electrophoresis contain, in addition to the major form of the MoFe protein, two minor variants of lower electrophoretic mobility. Of seven Nif- mutants of K. pneumoniae with nonpolar point mutations in nifD (encoding the alpha subunit of Kp1), three exhibit a wild-type-like electrophoretic pattern, whereas in the remaining four, the slowest-migrating form becomes the predominant species. Amino acid substitutions in mutants of the first type are located in the N terminus of NifD and include Gly-85 to Arg (UN1661), Glu-121 to Lys (UN1649), and Gly-161 to Asp (UN1683). Mutations of the second type are Gly-186 to Asp (UN1648), Gly-195 to Glu (UN1680), Ser-443 to Pro (UN1793), and Gly-455 to Asp (UN1650). Six of the mutated residues show interspecies conservation, three are close to conserved cysteines, and two are located next to conserved histidines. Based on evidence pointing to the possibility that the lowest-mobility form lacks the iron-molybdenum cofactor, these results provide insights into the functional significance of specific sites in the alpha subunit of the MoFe protein.     

Low-temperature magnetic-circular-dichroism spectroscopy of the iron-molybdenum cofactor and the complementary cofactor-less MoFe protein of Klebsiella pneumoniae nitrogenase

The major metal clusters of the MoFe protein, Kpl , of Klebsiella pneumoniae nitrogenase were characterized separately by low-temperature magnetic-circular-dichroism spectroscopy. The spectra and magnetization curves of the extracted iron-molybdenum cofactor, FeMoco , and of 'P' clusters in NifB - Kpl , the inactive, FeMoco -less, MoFo protein from an nifB mutant, were measured and compared with those of the holoprotein. (When FeMoco and NifB - Kpl are combined, active Kpl is formed.) Reduced NifB - Kpl had a spectrum with a weak, paramagnetic, component superimposed on a diamagnetic background. The paramagnetic component was assigned to a contaminating, e.p.r.-active, species. Thionine-oxidized NifB - Kpl had a spectrum and magnetization properties very similar to those of thionine-oxidized Kpl , demonstrating that the 'P' clusters are not significantly affected by the absence of the FeMoco clusters. The spectra of reduced isolated FeMoco had similar magnetization curves but sharper features and higher intensities than those of this centre in dithionite-reduced Kpl . Furthermore, a shoulder near 580 nm in the Kpl spectrum was absent from that of FeMoco . This may be due to the loss of a ligand or to a change in symmetry of the FeMoco cluster on extraction.     

Nucleotide sequence of the Klebsiella pneumoniae nifD gene and predicted amino acid sequence of the alpha-subunit of nitrogenase MoFe protein.

The nucleotide sequence of the Klebsiella pneumoniae nifD gene is presented and together with the accompanying paper [Holland, Zilberstein, Zamir & Sussman (1987) Biochem. J. 247, 277-285] completes the sequence of the nifHDK genes encoding the nitrogenase polypeptides. The K. pneumoniae nifD gene encodes the 483-amino acid-residue nitrogenase alpha-subunit polypeptide of Mr 54156. The alpha-subunit has five strongly conserved cysteine residues at positions 63, 89, 155, 184 and 275, some occurring in a region showing both primary sequence and potential structural homology to the K. pneumoniae nitrogenase beta-subunit. A comparison with six other alpha-subunit amino acid sequences has been made, which indicates a number of potentially important domains within alpha-subunits.     

A quantitative approach to sequence comparisons of nitrogenase MoFe protein alpha- and beta-subunits including the newly sequenced nifK gene from Klebsiella pneumoniae.

The nucleotide sequence was determined for part of the Klebsiella pneumoniae nif gene cluster containing the 3' end of the nifD gene and the entire length of the nifK gene (encoding the alpha- and beta-subunits of the nitrogenase MoFe protein respectively), as well as the putative start of the nifY gene, a gene of as yet unknown function. A broad-based comparison of a number of MoFe protein alpha-subunits, beta-subunits and alpha-versus beta-subunits was carried out by the use of a computer program that simultaneously aligns three protein sequences according to the mutation data matrix of Dayhoff. A new kind of quantitative statistical measure of the similarity between the aligned sequences was obtained by calculating and plotting standardized similarity scores for overlapping segments along the aligned proteins. This calculation determines if a test sequence is similar to the consensus sequence of two other proteins that are known to be related to each other. The different beta-subunits compared were found to be significantly similar along most of their sequence, with the exception of two relatively short regions centred around residues 225 and 300, which contain insertions/deletions. The overall pattern of similarity between different alpha-subunits exhibits resemblance to the overall pattern of similarity between different beta-subunits, including regions of low similarity centred around residues 225 and 340. Comparison of alpha-subunits with beta-subunits showed that a region of significant similarity between the two types of subunits was located approximately between residues 120 and 180 in both subunits, but other parts of the proteins were only marginally similar. These results provide insights into likely tertiary structural features of the MoFe protein subunits.     

Purification and characterization of Klebsiella pneumoniae cytotoxins.

Isolation, purification and characterization of 3 new cytotoxins of a K. pneumoniae strain isolated from ready to eat pork sausage are reported. Purification process involved extraction of cytotoxins with polymyxin B sulphate, salt precipitation, gel filtration and anion exchange chromatography. Klebsiella cytotoxin (KCT) I, a glycoprotein of about 65 kDa was verocytotoxic, enterotoxic and dermonerotic. KCT II was erythemogenic, verocytotoxic and enterotoxic protein of co 55 kDa, while KCT III was about double in MW (110 kDa) hadverocytotoxicity but neither enterotoxicity nor dermatotoxicity. KCT I and II caused granulation, conglomeration, shrinkage, detachment and lysis of MDBK and Vero cells, while KCT III induced enlargement, vacuolation, granulation, multinucleolation and syncytia formation in exposed cells. All the three cytotoxins induced specific neutralizing antibodies and cytotoxins were detectable in nanogram quantities with enzyme-linked immunosorbant assay using homologous antibodies. None of the anticytotoxin cross-reacted with either heterologous Klebsiella cytotoxins or with verocytotoxic preparations of Shigella dysenteriae.     

Purification and chemical characterization of type 1 pili isolated from Klebsiella pneumoniae

Type 1 pili were purified from a Klebsiella pneumoniae strain isolated from a human urinary tract infection. The pili were removed from the bacteria by mechanical shearing, precipitated out of solution by ammonium sulfate, solubilized in a deoxycholate-containing buffer, and finally purified by gel filtration. Chemical characterization of the isolated pili revealed a single protein subunit (pilin) which had a Mr = 21,500. Amino acid compositional analysis revealed a high content of residues that contribute significantly to secondary structure. Automated sequence analysis of the NH2-terminal region revealed a striking homology (79% identity) with type 1 pili of Escherichia coli. In contrast, NH2-terminal sequence comparison of K. pneumoniae pilin with other previously reported bacterial pilins showed no significant homology. No immunological cross-reactivity was detectable between E. coli and K. pneumoniae pili when tested by Ouchterlony double immunodiffusion or by rocket immunoelectrophoresis. The results of this study, when compared to other studies of bacterial pili, indicate that type 1 pili from members of the Enterobacteriaceae share morphological similarities and that their monomeric subunits are chemically similar. In addition, these results give strong evidence that the type 1 pilins of the enteric bacteria represent a separate class of homologous pilins.     

Nitrogenase MoFe protein subunits from Klebsiella pneumoniae expressed in foreign hosts. Characteristics and interactions

The expression of selected nitrogen fixation (nif) genes from Klebsiella pneumoniae in foreign hosts provides an approach to determine the pathway, minimal genetic requirements, and host dependence of nitrogenase assembly. In this study, we investigated the assembly of the alpha 2 beta 2 MoFe protein, responsible for substrate binding and reduction, by introducing nifD and nifK (encoding respectively, the alpha and beta subunits) into Escherichia coli and the yeast Saccharomyces cerevisiae. In E. coli, both genes were expressed from the nifHDKY operon; in yeast, the genes, separately fused to the yeast ADH1 promoter, were introduced on two different plasmids. Denaturing immunoblot analyses demonstrated the presence of significant amounts of NifD and NifK in both hosts. In E. coli, the level or perhaps modification of NifD depended on the growth medium of the bacteria. Nondenaturing, anaerobic immunoblot assays revealed in E. coli, nif-specific antigens of lower electrophoretic mobility than Kp1, which may represent assembly intermediates. In yeast, no putative assembled products were evident, and the predominant antigens corresponded to the monomeric forms of the polypeptides. These results indicate that, unlike NifH, the Fe protein subunit (Berman, J., Gershoni, J. M., and Zamir, A. (1985) J. Biol. Chem. 260, 5240-5243), NifD and NifK are insufficient for the assembly of an electrophoretically Kp1-like structure. Homodimerization of nifK and probably of nifD primary gene products does not appear to occur spontaneously and hence is unlikely to represent the initial step in the assembly. The difference between the two hosts suggests that the cellular environment or mode of expression could affect the interaction between the two subunits.     

The delta nifB (or delta nifE) FeMo cofactor-deficient MoFe protein is different from the delta nifH protein

We have examined three strains of Azotobacter vinelandii, which contain defined deletions within the nifH, nifB, or nifE genes. All three strains accumulate inactive FeMo cofactor-deficient forms of the MoFe protein of nitrogenase. These forms can be activated in vitro by addition of isolated FeMo cofactor in N-methylformamide. Although the phenotypes of these strains are superficially the same, our characterizations demonstrate that the FeMo cofactor-deficient MoFe protein synthesized by the delta nifH strain is quite different from that synthesized by either the delta nifB or delta nifE strains. These differences include the following: 1) the activation of the delta nifH protein requires MgATP, whereas the activation of the delta nifB and delta nifE proteins does not; 2) the delta nifH extracts can be activated with FeMo cofactor to wild-type levels of activity, whereas delta nifB and delta nifE extracts cannot; 3) the delta nifH protein is markedly less heat stable than the delta nifB and delta nifE proteins; and 4) the migration of the delta nifH protein on native gels is very different when compared with delta nifB and delta nifE, which look like each other. These data can be explained if the nifB and nifE gene products are only involved in FeMo cofactor biosynthesis, whereas the nifH gene product is involved in both the initial synthesis of FeMo cofactor and in the insertion of preformed FeMo cofactor into the MoFe protein. A model is presented that suggests that the FeMo cofactor-deficient MoFe protein synthesized by the delta nifH strain is the one that normally participates in MoFe protein assembly in wild-type cells.     

耐盐氨基甲酸乙酯水解酶的分离纯化及酶学性质

氨基甲酸乙酯是发酵食品中存在的一种致癌物质,酶法去除发酵食品中的氨基甲酸乙酯是消除氨基甲酸乙酯危害的一种重要方法。从小鼠的胃部获得了一株产氨基甲酸乙酯水解酶的肺炎克雷伯氏菌,为了解该氨基甲酸乙酯水解酶的酶学性质,从肺炎克雷伯氏菌中提取获得氨基甲酸乙酯水解酶粗酶液,经硫酸铵沉淀、离子交换层析和凝胶过滤层析分离得到氨基甲酸乙酯水解酶纯酶。通过十二烷基硫酸钠聚丙烯酰胺电泳(SDS-PAGE)分析,估计该酶的分子量约为55 kDa。其水解氨基甲酸乙酯的Km值为74 mmol/L。酶反应的最适温度为55℃,最适pH为7.0。乙二胺四乙酸(EDTA)和二硫苏糖醇(DTT)对该酶有较强的激活作用,而Cu2+和Zn2+则有较强的抑制作用。该酶可耐受高浓度NaCl,对低浓度乙醇也有一定的耐受性,对于酱油中氨基甲酸乙酯的消除有一定的参考意义。     

Purification and characterization of a bacteriocin from Klebsiella pneumoniae 158

Klebocin, a bacteriocin produced by Klebsiella pneumoniae 158, was purified to homogeneity by ammonium sulphate fractionation and sequential DEAE-Sephacel and Sephadex G-150 column chromatography. The purified preparation had an Mr of approximately 40 000 on SDS-PAGE. Chemical analysis of the purified preparation showed it to be a protein, and it was sensitive to digestion by various proteolytic enzymes.     

Mol- mutants of Klebsiella pneumoniae requiring high levels of molybdate for nitrogenase activity.

Mol- mutants of Klebsiella pneumoniae requiring high levels of molybdate for nitrogenase and nitrate reductase activity were characterized. The effects of mol mutations on nitrogenase activity were very similar to those caused by nifQ mutations. Mol- mutants of K. pneumoniae appear to be equivalent to ChlD- mutants of Escherichia coli.     

Crystallographic analysis of the MoFe protein of nitrogenase from a nifV mutant of Klebsiella pneumoniae identifies citrate as a ligand to the molybdenum of iron molybdenum cofactor (FeMoco)

The x-ray crystal structure of NifV(-) Klebsiella pneumoniae nitrogenase MoFe protein (NifV(-) Kp1) has been determined and refined to a resolution of 1.9 A. This is the first structure for a nitrogenase MoFe protein with an altered cofactor. Moreover, it is the first direct evidence that the organic acid citrate is not just present, but replaces homocitrate as a ligand to the molybdenum atom of the iron molybdenum cofactor (FeMoco). Subsequent refinement of the structure revealed that the citrate was present at reduced occupancy.     

Nucleotide sequence of the gene coding for the nitrogenase iron protein from Klebsiella pneumoniae

We report the complete DNA sequence of the Klebsiella pneumoniae nifH gene, the gene which codes for component 2 (Fe protein or nitrogenase reductase) of the nitrogenase enzyme complex. The amino acid sequence of the K. pneumoniae nitrogenase Fe protein is deduced from the DNA sequence. The K. pneumoniae Fe protein contains 292 amino acids, has a Mr = 31,753, and contains 9 cysteine residues. We compare the amino acid sequence of the K. pneumoniae protein with available amino acid sequence data on nitrogenase Fe proteins from two other species, Clostridium pasteurianum and Azotobacter vinelandii. The C. pasteurianum Fe protein, for which the complete sequence is known, shows 67% homology with the K. pneumoniae Fe protein. Extensive regions of strong conservation (90-95%) are found, while other regions show relatively poor conservation (30-35%). It is suggested that these strongly conserved regions are of special importance to the function of this enzyme, and the findings are discussed in the light of evolutionary theories on the origin of nif genes.     

Purification and characterization of the 3-hydroxybenzoate-6-hydroxylase from Klebsiella pneumoniae

Abstract We isolated 3-hydroxybenzoate-6-hydroxylase (E.C. 1.14.13.), an inducible enzyme that catalyzed the para -hydroxylation of 3-hydroxybenzoate (3-HBA) to 2,5-dihydroxybenzoate, from Klebsiella pneumoniae . Although the enzyme was found to be mainly induced by its substrate, a coordinated induction of 3-hydroxybenzoate hydroxylase and gentisate dioxygenase was also observed in the presence of the product of the reaction. The purified enzyme was a monomer with a molecular mass of 42 000. It contained FAD as a prosthetic group, utilized NADH or NADPH with similar efficiencies and its activity was inhibited by Cu²⁺, Fe²⁺ and Hg²⁺. Other properties, such as induction mechanism and kinetic parameters were also studied. Moreover, for the first time the amino acid composition of a 3-hydroxybenzoate-6-hydroxylase was determined.     

The molybdenum--iron protein of Klebsiella pneumoniae nitrogenase. Evidence for non-identical subunits from peptide ''mapping''.

The molybdenum- and iron-containing protein components of nitrogenase purified from Klebsiella pneumoniae, Azotobacter vinelandii, Azotobacter chroococcum and Rhizobium japonicum bacteroids all gave either one or two protein-staining bands after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, depending on the commercial brand of sodium dodecyl sulphate used. The single band obtained with K. pneumoniae Mo-Fe protein when some commercial brands of sodium dodecyl sulphate were used in the preparation of the electrode buffer was resolved into two bands by the addition of 0.01% (v/v) dodecanol to the buffer. Protein extracted from the two bands obtained after electrophoresis of K. pneumoniae Mo-Fe protein gave unique and distinct peptide 'maps' after tryptic digestion. Undissociated Mo-Fe protein contained both sets of tryptic peptides. These data are consistent with Mo-Fe protein from K. pneumoniae being composed of non-identical subunits. Amino acid analyses of the subunit proteins revealed some clear differences in amino acid content, but the two subunits showed close compositional relatedness, with a different index [Metzer, H., Shapiro, M.B., Mosiman, J.E. & Vinton, J.G. (1968) Nature (London) 219, 1166-1168] of 4.7.     

Purification and biochemical characterization of gentisate 1,2-dioxygenase from Klebsiella pneumoniae M5a1

Abstract Type strains and 62 clinical isolates of Prevotella intermedia and Prevotella nigrescens were typed with the use of genomic DNA fingerprints and rRNA gene probes. The strains were further serotyped with monoclonal antibodies and characterized with SDS-PAGE, enzymatic activities, hemolysis and hemagglutination and coaggregation with Streptococcus and Actinomyces spp. P. intermedia and P. nigrescens were found to have distinct ribotype patterns which correspond to previously defined serotypes I and II/III, respectively. No clear phenotypic difference related to hemolysis, hemagglutination and coaggregation with Streptococcus and Actinomyces species, or expression of aminopeptides and lipase was found between P. intermedia and P. nigrescens .     

Isolation and characterization of prototrophic relaxed mutants of Klebsiella pneumoniae

Summary A new selection procedure has been developed for isolating prototrophic relaxed mutants of Klebsiella pneumoniae. Two mutants were isolated. One of them showed a fully relaxed phenotype, while the other one behaved in a semi-relaxed way.The wild-type strain, as well as the rel mutants exerted similar patterns to their E. coli counterparts in RNA, protein, ppGpp and pppGpp accumulation during amino starvation, carbon source shift-down and nitrogen starvation. Both mutants became stringent after introducing an F-factor carrying the relA ⁺ allele from Escherichia coli. The relaxed phenotype could be recovered by curing the F-factor. Some of the pleiotropic consequences of rel mutations found in E. coli are present in the Klebsiella mutants also while some of them are absent.The mutants are defective in dinitrogen fixation after the exhaustion of limiting ammonium from the culture medium. However, their merodiploid derivatives, carrying the E. coli relA ^- allele, showed the wild-type level of nitrogenase activity under the same conditions.Fellow of the 6th International Training Course jointly sponsored by UNDP/UNESCO Hungarian Academy of Sciences. Present address: Akademie der Wissenschaften der DDR, Forschungszentrum für Molekularbiologie und Medizin, Zentralinstitut für Mikrobiologie und Experimentelle Therapie Jena, Beuthenberg Str. 11, DDR-69 Jena