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B Friedrich

Publications and source records attributed to B Friedrich.

At least 91 records · Page 5Linked to original sources
PubMed1992

Hydrogenase mutants of Alcaligenes eutrophus H16 show alterations in the electron transport system.

Mutations in the genes coding for the soluble and the membrane-bound hydrogenase of Alcaligenes eutrophus strain H16 significantly affected the expression of respiratory chain components. In lithoautotrophically grown wild type cells electron flow mainly proceeded via the cytochrome c oxidases. Mutants defective in the membrane-bound hydrogenase contained a 2- to 3-fold higher cytochrome a content than the wild type and cytochrome c oxidase of the aa3-type was preferentially used by these cells for substrate oxidation. Mutants impaired in the soluble hydrogenase revealed slow growth on hydrogen, presumably due to inefficient reverse electron flow mechanisms which provide the cells with NADH for autotrophic CO2-fixation. In this class of mutants the two quinol oxidases of the o- and d-type in addition to the co-type oxidase were the predominant electron-transport branches.

Alcaligenes↗
PubMed1992

Derived amino acid sequences of the nosZ gene (respiratory N2O reductase) from Alcaligenes eutrophus, Pseudomonas aeruginosa and Pseudomonas stutzeri reveal potential copper-binding residues. Implications for the CuA site of N2O reductase and cytochrome-c oxidase.

The nosZ genes encoding the multicopper enzyme nitrous oxide reductase of Alcaligenes eutrophus H16 and the type strain of Pseudomonas aeruginosa were cloned and sequenced for structural comparison of their gene products with the homologous product of the nosZ gene from Pseudomonas stutzeri [Viebrock, A. & Zumft, W. G. (1988) J. Bacteriol. 170, 4658-4668] and the subunit II of cytochrome-c oxidase (COII). Both types of enzymes possess the CuA binding site. The nosZ genes were identified in cosmid libraries by hybridization with an internal 1.22-kb PstI fragment (NS220) of nosZ from P. stutzeri. The derived amino acid sequences indicate unprocessed gene products of 70084 Da (A. eutrophus) and 70695 Da (P. aeruginosa). The N-terminal sequences of the NosZ proteins have the characteristics of signal peptides for transport. A homologous domain, extending over at least 50 residues, is shared among the three derived NosZ sequences and the CuA binding region of 32 COII sequences. Only three out of nine cysteine residues of the NosZ protein (P. stutzeri) are invariant. Cys618 and Cys622 are assigned to a binuclear center, A, which is thought to represent the CuA site of NosZ and is located close to the C terminus. Two conserved histidines, one methionine, one aspartate, one valine and two aromatic residues are also part of the CuA consensus sequence, which is the domain homologous between the two enzymes. The CuA consensus sequence, however, lacks four strictly conserved residues present in all COII sequences. Cys165 is likely to be a ligand of a second binuclear center, Z, for which we assume mainly histidine coordination. Of 23 histidine residues in NosZ (P. stutzeri), 14 are invariant, 7 of which are in regions with a degree of conservation well above the 50% positional identity between the Alcaligenes and Pseudomonas sequences. Conserved tryptophan residues are located close to several potential copper ligands. Trp615 may contribute to the observed quenching of fluorescence when the CuA site is occupied.

Alcaligenes↗
PubMed1992

Nucleotide sequence of the rpoN (hno) gene region of Alcaligenes eutrophus: evidence for a conserved gene cluster.

The nucleotide sequence of the rpoN gene, formerly designated hno, and flanking DNA regions of the aerobic hydrogen bacterium Alcaligenes eutrophus has been determined; rpoN codes for the RNA polymerase sigma factor sigma 54 involved in nitrogen regulation and diverse physiological functions of gram-negative bacteria. In A. eutrophus hydrogen metabolism is under control of rpoN. The Tn5-Mob insertion in a previously isolated pleiotropic mutant was mapped within the rpoN gene. The derived amino acid sequence of the A. eutrophus RpoN protein shows extensive homology to the RpoN proteins of other organisms. Sequencing revealed four other open reading frames: one upstream (ORF280) and three downstream (ORF130, ORF99 and ORF greater than 54) of the rpoN gene. A similar arrangement of homologous ORFs is found in the rpoN regions of other bacteria and is indicative of a conserved gene cluster.

Alcaligenes↗
PubMed1992

A gene complex coding for the membrane-bound hydrogenase of Alcaligenes eutrophus H16.

One of the key enzymes in the chemolithoautotrophic metabolism of Alcaligenes eutrophus H16 is a dimeric, membrane-associated hydrogenase. The genetic determinants of this enzyme are located on the endogenous megaplasmid pHG1 (G. Eberz, C. Hogrefe, C. Kortlüke, A. Kamienski, and B. Friedrich, J. Bacteriol. 168:636-641, 1986). Complementation studies showed that the information required for the formation of active membrane-bound hydrogenase occupies more than 7.5 kb of megaplasmid DNA. We cloned and sequenced this region and identified the genes encoding the two hydrogenase subunits (hoxK and hoxG). The nucleotide sequence contains nine additional closely spaced open reading frames. Immunoelectron microscopy showed that the gene product of one of these open reading frames (hoxM) is involved in the process leading to the attachment of hydrogenase to the membrane. Other open reading frames may encode additional processing functions and components of a hydrogenase-linked electron transport chain. Analysis of Tn5-B21-mediated transcriptional fusions provided evidence that the structural genes and accessory functions belong to at least three coordinately regulated transcriptional units.

Alcaligenes↗
PubMed1992

Maturation of membrane-bound hydrogenase of Alcaligenes eutrophus H16.

The formation of the catalytically active membrane-bound hydrogenase (MBH) of Alcaligenes eutrophus H16 requires the genes for the small and large subunits of the enzyme (hoxK and hoxG, respectively) and an accompanying set of accessory genes (C. Kortl ke, K. Horstmann, E. Schwartz, M. Rohde, R. Binsack, and B. Friedrich, J. Bacteriol. 174:6277-6289, 1992). Other genes located in the adjacent pleiotropic region are also required. In the absence of these genes, MBH is synthesized but is catalytically inactive. Immunological analyses revealed that cells containing active MBH produced the small and large subunits of the enzyme in two distinct conformations each; only one of each, presumably the immature form, occurred in cells devoid of MBH activity. The results suggest that the conversion of the two subunits into the catalytically active membrane-associated heterodimer depends on specific maturation processes mediated by hox genes.

Alcaligenes↗
PubMed1992

Expression of bacterial mercuric ion reductase in Saccharomyces cerevisiae.

The gene merA coding for bacterial mercuric ion reductase was cloned under the control of the yeast promoter for alcohol dehydrogenase I in the yeast-Escherichia coli shuttle plasmid pADH040-2 and transformed into Saccharomyces cerevisiae AH22. The resulting transformant harbored stable copies of the merA-containing hybrid plasmid, displayed a fivefold increase in the MIC of mercuric chloride, and synthesized mercuric ion reductase activity.

Alcohol Dehydrogenase↗
PubMed1991

Construction and properties of a triprotein containing the high-affinity nickel transporter of Alcaligenes eutrophus.

The high-affinity nickel transporter of Alcaligenes eutrophus H16 is encoded by gene hoxN, which maps within the hydrogenase gene cluster of megaplasmid pHG1. A tripartite gene fusion was constructed, consisting of (i) the Escherichia coli lacZ gene for beta-galactosidase, (ii) a segment encoding an endoproteolytically cleavable peptide, and (iii) the A. eutrophus gene hoxN. An E. coli strain harboring this construct (plasmid pCH307) efficiently produced the corresponding triprotein upon induction. A broad-host-range derivative of pCH307 was shown to complement an A. eutrophus HoxN- mutant.

Alcaligenes↗
PubMed1991

Cloning, nucleotide sequence, and heterologous expression of a high-affinity nickel transport gene from Alcaligenes eutrophus.

High-affinity nickel transport in Alcaligenes eutrophus H16 is mediated by a function designated hoxN. hoxN lies within the hydrogenase gene cluster of megaplasmid pHG1. An insertional mutation at the hoxN locus led to an increased nickel requirement. In this mutant (strain HF260) both autotrophic growth on hydrogen and wild-type level of urease, a nickel-containing enzyme, were dependent on high concentration of nickel in the medium. Studies with a heterologous in vivo expression system revealed that the hoxN locus encodes two proteins with Mr = 30,000 and 28,000. Only the larger polypeptide was essential for nickel transport. The hoxN locus was cloned on a 2.2-kilobase pair fragment. Nucleotide sequence analysis of the hoxN locus revealed an open reading frame with a coding capacity for a protein of 33.1 kDa. The insertion leading to the Nic- phenotype of strain HF260 maps within this open reading frame indicating that it does in fact have coding function. The deduced amino acid sequence of the hoxN gene has several features typical of a hydrophobic integral membrane protein. Alkaline phosphatase fusion proteins produced by insertion of the transposon TnphoA into hoxN gave significant levels of alkaline phosphatase activity indicating that protein HoxN contains periplasmic domains. Taken together, our results suggest that gene hoxN encodes the high-affinity nickel transporter of A. eutrophus.

Alcaligenes↗
PubMed1991

Determinants encoding resistance to several heavy metals in newly isolated copper-resistant bacteria.

Three copper-resistant, gram-negative bacteria were isolated and characterized. Of the three strains, Alcaligenes denitrificans AH tolerated the highest copper concentration (MIC = 4 mM CuSO(4)). All three strains showed various levels of resistance to other metal ions. A. denitrificans AH contains sequences which cross-hybridized with the mer (mercury resistance) determinant of Tn21 and the czc (cobalt, zinc, and cadmium resistance), cnr (cobalt and nickel resistance), and chr (chromate resistance) determinants of A. eutrophus CH34. DNA-DNA hybridization with probes prepared from A. eutrophus CH34 and Tn21 revealed the presence of chr-, cnr-, and mer-like sequences on the 200-kb plasmid pHG27 and of czc, cnr, and mer homologs located on the chromosome. The second strain, classified as Alcaligenes sp. strain PW, carries czc, cnr, and mer homologs on the 240-kb plasmid pHG29-c and a chr determinant on the 290-kb plasmid pHG29-a; a third plasmid, the 260-kb large plasmid pHG29-b, is cryptic. In contrast to the Alcaligenes strains, which were isolated from metal-contaminated water, Pseudomonas paucimobilis CD was isolated from the air. This strain harbors two cryptic plasmids: the 210-kb large plasmid pHG28-a and the 40-kb plasmid pHG28-b. Southern analysis revealed no homology between the metal ion resistance determinants of A. eutrophus CH34 and P. paucimobilis CD.

Journal Article↗
PubMed1991

Humoral and cellular defense against intestinal murine infection with Yersinia enterocolitica.

The role of phagocytes and the complement system as potential host defense mechanisms against bacterial infection were studied in mice with two isogenic strains of Yersinia enterocolitica serotype O8 differing in pathogenicity because of differences in plasmid content. Complement depletion in mice by intraperitoneal injection of cobra venom factor did not affect the course of colonization of the intestinal tissue by each strain, indicating that in mice complement is not essential for the elimination of these bacteria. This conclusion is supported by the fact that fresh murine serum had no bactericidal effect in vitro either on the pathogenic or on the nonpathogenic strain. However, in the intestinal tissue as well as in the peritoneal cavity, only the pathogenic, plasmid-bearing Y. enterocolitica strain survived, while the nonpathogenic, plasmidless strain was rapidly eliminated. Since elimination from the peritoneal cavity is due to phagocytosis by polymorphonuclear leukocytes and macrophages, resistance to phagocytosis in vivo seems to be the decisive factor determining the virulence of pathogenic Y. enterocolitica strains.

Animals↗
PubMed1991

Three trans-acting regulatory functions control hydrogenase synthesis in Alcaligenes eutrophus.

Random Tn5 mutagenesis of the regulatory region of megaplasmid pHG1 of Alcaligenes eutrophus led to the identification of three distinct loci designated hoxA, hoxD, and hoxE. Sequencing of the hoxA locus revealed an open reading frame which could code for a polypeptide of 482 amino acids with a molecular mass of 53.5 kDa. A protein of comparable apparent molecular mass was detected in heterologous expression studies with a plasmid-borne copy of the hoxA gene. Amino acid alignments revealed striking homologies between HoxA and the transcriptional activators NifA and NtrC of Klebsiella pneumoniae and HydG of Escherichia coli. HoxA- mutants of A. eutrophus lacked both NAD-reducing soluble hydrogenase and membrane-bound hydrogenase. In HoxA- mutants, the synthesis of beta-galactosidase from a hoxS'-'lacZ operon fusion was drastically reduced, indicating that HoxA is essential for the transcription of hydrogenase genes. Mutants defective in hoxD and hoxE also lacked the catalytic activities of the two hydrogenases; however, in contrast to HoxA- mutants, they contained immunologically detectable NAD-reducing soluble hydrogenase and membrane-bound hydrogenase proteins, although at a reduced level. The low hydrogenase content in the HoxD- and HoxE- mutants correlated with a decrease in beta-galactosidase synthesized under the direction of a hoxS'-'lacZ operon fusion. Thus, hoxD and hoxE apparently intervene both in the regulation of hydrogenase synthesis and in subsequent steps leading to the formation of catalytically active enzymes.

Alcaligenes↗
PubMed1991

[The unstable thoracic wall: possibilities for treatment].

In the treatment of severe chest injuries with flail chest either positive-pressure mechanical ventilation (and tracheostomy) is necessary or the surgical stabilisation of the chest wall by osteosyntheses of the broken ribs. Fourteen patients (age 27-73) with severe chest injuries and paradoxical respiration were operated and the rib fractures stabilized by means of small reconstruction plates and cerclage wires. Three patients died due to other reasons. There were no serious complications. The results are reported. In severe chest injuries surgical treatment seems to be justified; it reduces the need of artificial respiration and can be recommended.

Bone Wires↗
PubMed1990

Purification and characterization of a 19-kilodalton intracellular protein. An activation-regulated putative protein kinase C substrate of T lymphocytes.

Activation of protein kinase C in T cells results in rapid phosphorylation of a 19-kDa intracellular protein termed 19K. We report the purification of 19K from human peripheral T cells and an internal 20-amino acid sequence determined from this protein. It is shown that 19K is a novel cytoplasmatic protein which is phosphorylated in vitro by partially purified protein kinase C. 19K-specific antibodies, raised by immunizing rabbits with purified protein, were used to show that the 19K is expressed, and phosphorylated in response to protein kinase C activation, in several cellular systems. These antibodies were also used to precipitate 19K from both [35S]methionine and 32Pi-labeled T cells. The data showed that 15 min of phorbol ester treatment has no effect on the rate of 19K synthesis but results in induction of 19K phosphorylation. However, we demonstrate, by Western blot analysis, that expression of 19K in primary peripheral T cells increased at least 10-fold over a period of 4 days after activation. The increase in 19K expression correlates with initiation of DNA synthesis, and in proliferating T cells 19K comprises approximately 0.2% of total cytoplasmatic protein. Thus, 19K is a novel putative protein kinase C substrate which is subject to activation associated up-regulation in human T cells.

Amino Acid Sequence↗
PubMed1990

Identification of distinct NAD-linked hydrogenase protein species in mutants and nickel-deficient wild-type cells of Alcaligenes eutrophus H16.

By crossed immunoelectrophoresis with antibodies against the NAD-linked hydrogenase the presence of three hydrogenase protein species was demonstrated in crude extracts of Alcaligenes eutrophus H16. Protein 1 (antigen 1) exhibited NAD-reducing activity and was shown to be identical with the native heterotetrameric enzyme. Protein 2 (antigen 2) was catalytically inactive in the antibody-precipitated form and corresponded to the beta subunit (56 kDa) of the holoenzyme. Protein 3 (antigen 3) was serologically distinct from antigen 2 and catalyzed NADH-oxidizing (diaphorase) activity, suggesting that it either consists of the alpha peptide or of the alpha and gamma subunits of the diaphorase dimer. Tandem immunoelectrophoresis revealed that antigen 2 was the predominant protein species in cells cultivated under nickel deficiency. Low concentrations of the diaphorase-active antigen 3 were also detected under these conditions. Extracts from mutants defective in the catalytic activity of NAD-reducing hydrogenase still contained the four polypeptides. This was shown by immunodiffusion and immunoblotting with antibodies raised against the individual subunits. However, as observed with nickel-deficient cells, no complete tetrameric protein could be identified, and the dominant subunit species (70-80%) was the beta peptide.

Alcaligenes↗
PubMed1990

Regulation of assimilatory nitrate reductase formation in Klebsiella aerogenes W70.

Klebsiella aerogenes W70 could grow aerobically with nitrate or nitrite as the sole nitrogen source. The assimilatory nitrate reductase and nitrite reductase responsible for this ability required the presence of either nitrate or nitrite as an inducer, and both enzymes were repressed by ammonia. The repression by ammonia, which required the NTR (nitrogen regulatory) system (A. Macaluso, E. A. Best, and R. A. Bender, J. Bacteriol. 172:7249-7255, 1990), did not act solely at the level of inducer exclusion, since strains in which the expression of assimilatory nitrate reductase and nitrite reductase was was independent of the inducer were also susceptible to repression by ammonia. Insertion mutations in two distinct genes, neither of which affected the NTR system, resulted in the loss of both assimilatory nitrate reductase and nitrite reductase. One of these mutants reverted to the wild type, but the other yielded pseudorevertants at high frequency that were independent of inducer but still responded to ammonia repression.

Ammonia↗
PubMed1990

Cloning and nucleotide sequences of the genes for the subunits of NAD-reducing hydrogenase of Alcaligenes eutrophus H16.

The genes hoxF, -U, -Y, and -H which encode the four subunit polypeptides alpha, gamma, delta, and beta of the NAD-reducing hydrogenase (HoxS) of Alcaligenes eutrophus H16, were cloned, expressed in Pseudomonas facilis, and sequenced. On the basis of the nucleotide sequence, the predicted amino acid sequences, and the N-terminal amino acid sequences, it was concluded that the structural genes are tightly linked and presumably organized as an operon, denoted hoxS. Two pairs of -24 and -12 consensus sequences resembling RpoN-activatable promoters lie upstream of hoxF, the first of the four genes. Primer extension experiments indicate that the second promoter is responsible for hoxS transcription. hoxF and hoxU code for the flavin-containing dimer (alpha and gamma subunits) of HoxS which exhibits NADH:oxidoreductase activity. A putative flavin-binding region is discussed. The 26.0-kilodalton (kDa) gamma subunit contains two cysteine clusters which may participate in the coordination of two [4F3-4S]centers. The genes hoxY and hoxH code for the small 22.9-kDa delta subunit and the nickel-containing 54.8-kDa beta subunit, respectively, of the hydrogenase dimer of HoxS. The latter dimer exhibits several conserved regions found in all nickel-containing hydrogenases. The roles of these regions in coordinating iron and nickel are discussed. Although the deduced amino acid sequences of the delta and beta subunits share some conserved regions with the corresponding polypeptides of other [NiFe] hydrogenases, the overall amino acid homology is marginal. Nevertheless, significant sequence homology (35%) to the corresponding polypeptides of the soluble methylviologen-reducing hydrogenase of Methanobacterium thermoautotrophicum was found. Unlike the small subunits of the membrane-bound and soluble periplasmic hydrogenases, the HoxS protein does not appear to be synthesized with an N-terminal leader peptide.

Alcaligenes↗
PubMed1989

Purification of the F420-reducing hydrogenase from Methanosarcina barkeri (strain Fusaro).

The 8-hydroxy-5-deazaflavin (coenzyme F420)-reducing and methyl-viologen-reducing hydrogenase of the anaerobic methanogenic archaebacterium Methanosarcina barkeri strain Fusaro has been purified 64-fold to apparent electrophoretic homogeneity. The purified enzyme had a final specific activity of 11.5 mumol coenzyme F420 reduced.min-1.mg protein-1 and the yield was 4.8% of the initial deazaflavin-reducing activity. The hydrogenase exists in two forms with molecular masses of approximately 845 kDa and 198 kDa. Both forms reduce coenzyme F420 and methyl viologen and are apparently composed of the same three subunits with molecular masses of 48 kDa (alpha), 33 kDa (beta) and 30 kDa (gamma). The aerobically purified enzyme was catalytically inactive. Conditions for anaerobic reductive activation in the presence of hydrogen, 2-mercaptoethanol and KCl or methyl viologen were found to yield maximal hydrogenase activity. Determination of the apparent Km of coenzyme F420 and methyl viologen gave values of 25 microM and 3.3 mM, respectively. The respective turnover numbers of the high molecular mass form of the hydrogenase are 353 s-1 and 9226 s-1.

Archaea↗