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1.
With 0.5% substrate present in mineral medium, cells of Alcaligenes eutrophus H 16 were able to grow heterotrophically at the expense of guanine, hypoxanthine and xanthine, but not of adenine as sole sources of carbon and nitrogen. An increase in cell counts, however, was observed at lower adenine concentrations (0.1%). Similarly, adenine was only respired if present at low concentrations. Higher amounts of adenine were inhibitory to the utilization of adenine, guanine, hypoxanthine, xanthine, allantoin and glyoxylate, but not to that of fructose or glycerate. The adenine-dependent inhibition of adenine utilization was not overcome by the addition of thiamine, uridine or cytidine. The enzyme glyoxylate carboligase, usually formed in presence of metabolisable purines and of allantoin, was synthesized only at low adenine concentrations. Higher amounts were inhibitory even with allantoin present as additional substrate. According to these resutls, the utilization of purine derivatives and of allantoin as sources of carbon and energy is repressed by adenine in cells of A. eutrophus H 16. 相似文献
2.
The uptake of the radioactive ammoniumanalogue 14C-methylammonium1 was measured in heterotrophically grown cells of Alcaligenes eutrophus H16 in order to study the mechanism of NH
4
+
uptake. MA gradients of up to 200 were built up by a substrate-specific and energy-dependent system which showed a K
m of 35–111 M and a V
max of 0.4–1.8 nmol MA/min per mg protein. The involved carrier exhibited a higher affinity towards NH
4
+
than towards CH3NH
3
+
indicating that ammonium rather than MA was its natural substrate. Cold shock with hypotonic but not with hypertonic solutions caused the efflux of almost the entire accumulated MA. Osmotic shock did not affect the uptake reaction, suggesting that no periplasmic binding proteins were involved. Indirect observations indicate the membrane potential as driving force for MA uptake. High rates of uptake were observed in cells grown under nitrogen deficiency or with nitrate as nitrogen source. The uptake rate decreased during growth at high ammonium concentrations indicating that biosynthesis of nitrogenous compounds was supported by passive diffusion of NH3. The data suggest that the formation of the carrier is subject to nitrogen control.Non-standard abbreviations CCCP
Carbonylcyanide-m-chlorphe-nylhydazone
- MA
methylammonium
- pCMB
para-chlormercuribenzoate 相似文献
3.
Molecular and functional properties of DNA topoisomerase I isolated from a hydrogen-oxidizing bacterium, Alcaligenes eutrophus H16, were investigated. Under native conditions the enzyme forms a monomer with a relative molar mass of 98.500. A rod-like shape of the molecule was derived from the calculated frictional coefficient. The isoelectric point of the enzyme was determined to be in the range of 7.6–8.0. The enzyme activity is strictly Mg2+ dependent with an optimum at 3 mM Mg2+. The pH optimum ranges within 7.5–9.0. A. eutrophus DNA topoisomerase I activity is inhibited by M13 ssDNA, high ionic strength, polyamines, heparin and by a number of intercalating drugs.Abbreviations DTT
dithiothreitol
- BSA
bovine serum albumin
- EDTA
ethylenediaminetetraacetic acid
- SDS
sodium dodecyl sulfate
- Tris
tris(hydroxymethyl)aminomethane
- PMSF
phenylmethanesulfonyl fluoride
- PAGE
polyacrylamide gel electrophoresis 相似文献
4.
Ralf Kömen Davide Zannoni W. John Ingledew Karin Schmidt 《Archives of microbiology》1991,155(4):382-390
The electron transport system of autotrophically grown Alcaligenes eutrophus H16 has been investigated by spectroscopic and thermodynamic approaches. The results have been interpreted as evidence that isolated membranes contain a branched respiratory chain composed of three c-type haems (E
m,7=+160 mV, + 170 mV, and + 335 mV), five b-type haems (E
m,7=+ 5 mV, + 75 mV, + 205 mV, + 300 mV, and + 405 mV), two (possibly three) a-type haems [E
m,7= + 255 mV, + 350 mV, (+ 420 mV)], and nne d-type haem. EPR-analysis of the signals at g=1.93, g=2.02, and g=1.90 revealed the presence of iron-sulphur centres diagnostic of complexes I (NADH dehydrogenase), II (succinate dehydrogenase), and III (ubiquinol/cytochrome c oxidoreductase). The low potential b haems (+ 5 mV and + 75 mV) plus the Rieske protein (g=1.90, E
m,7=+ 280 mV), thought to be part of an orthodox bc
1 complex, were present in low amounts as compared to their counterparts in membranes from Paracoccus denitrificans.CO-difference spectra in the presence of either succinate, NADH, hydrogen, ascorbate/TMPD, and/or dithionite as reductants, suggested the existance of four different oxidases composed by bo-, cb-, a-, and d-type haems.It is concluded that in contrast to other chemolithotrophes, e.g. P. denitrificans, autotrophic growth of Alcaligenes eutrophus utilizes a respiratory system in which the bc
1 complex containing pathway is only partially involved in electron transport.Abbreviations Cytochrome c-551, number
wavelength in nm
- Cytochrome c
270, number
mid-point potential in mV
-
E
m,7
mid-point potential of an oxidation-reduction couple at pH 7.0
- KP
buffer, potassium phosphate-buffer
- OD
optical density at 436 nm, 1 cm light path
- TMPD
N,N,N,N-tetramethyl-p-phenylenediamine 相似文献
5.
Properties and regulation of anthranilate synthase from Alcaligenes eutrophus H 16 were investigated. Anthranilate synthase was partially purified from crude extracts by affinity chromatography on tryptophan-substituted Sepharose, and was used for kinetic measurements. During the purification procedure the enzyme was stabilized by 50 mM l-glutamine or during chromatography on DEAE-cellulose and Sephadex G-200 with 30% glycerol, respectively.The glutamine dependent activity of anthranilate synthase was examined; it showed little change between pH 8.4 and pH 9.1. The Arrhenius plot was broken and the activation energy, H, calculated therefrom amounted to 8.9 kcal/mole up to 30°C and 5.5 kcal/mole at higher temperatures. The molecular weight determined by gelfiltration on Sephadex G-200 and by sucrose density gradient centrifugation resulted in 158000 and 126000, respectively. The K
m
-values for the two substrates chorismate and glutamine were found to be 5 M and 560 M, respectively.Anthranilate synthase was strongly inhibited by l-tryptophan; the only amino acid that affected enzyme activity. Homotropic interactions for chorismate (Hill coefficient n=1.4) were obtained in the presence of l-tryptophan. 50% inhibition were caused by 10 M l-tryptophan at 100 M chorismate. The inhibition with respect to l-glutamine was noncompetitive.Anthranilate synthase was not associated to phosphoribosyl transferase and easily separable from the latter by different chromatographic methods.Abbreviation TEA
triethanolamine 相似文献
6.
Transport of nickel ions was studied in Alcaligenes eutrophus. Two transport systems for nickel ions exist to satisfy the nickel demand for the lithotrophic hydrogen metabolism. A major nickel transport activity exhibited an apparent affinity constant (K
m) of 17 M nickel chloride. This activity was competitively inhibited by Mg2+, Mn2+, Zn2+, and Co2+. A minor nickel transport activity was determined in the presence of high (0.8 mM) magnesium. This activity was not inhibited by Zn2+ or Mn2+; its K
m was determined to be 0.34 M nickel chloride. These kinetics suggested a second transport system in A. eutrophus. The membrane potential of A. eutrophus was decreased upon the addition of ammonium ions leading to a decreased nickel transport. This inhibition could be reversed by fructose or by hydrogen indicating an energy dependent nickel transport. Protonophores inhibited the nickel transport. However, inhibitors of ATP synthase like dicyclohexylcabodimide or venturicidin had little or no effect on nickel transport. These data indicated that the transport was coupled to the proton motive force. 相似文献
7.
In a previous work (Kömen et al. 1991) it has been concluded that membrane fragments isolated from autotrophically grown Alcaligenes eutrophus H16 contain several iron-sulphur centres along with haems of a-, b-, c-, and d-type. These redox components have been proposed to be part of a branched respiratory chain leading to multiple membrane bound oxidases. Here, some of the respiratory activities catalyzed by membrane fragments from wild type cells of A. eutrophus (H16) and, for comparison, Paracoccus denitrificans, have been investigated through the use of electron transport inhibitors. Cyanide (CN-) titration curves indicated that in A. eutrophus H16 oxidation of succinate and H2 preferentially proceeds via the cytochrome c oxidase(s) branch (I 50=2 · 10-5 M) whereas the NADH dependent respiration started being inhibited at higher CN- concentrations (I 50=5 · 10-4 M). In membranes isolated from both, cells harvested at late growth-phase (OD 12) and from a mutant deficient in cytochrome c oxidase activity (A. eutrophus RK1), respiration was insensitive to low CN- concentrations (< 10-4 M), and it was sustained by the high catalytic activities of two quinol oxidases. These alternative oxidases of b- (formally o-) and d-type showed different sensitivities to KCN (I 50=10-3 M and 10-2 M, respectively). Interestingly, the cytochrome c oxidase(s) dependent respiration of H16 membranes was insensitive to antimycin A but largely inhibited by myxothiazol (10-6 M). This, and previous work (Kömen et al. 1991), suggest that although the respiratory chain of A. eutrophus is endowed with a putative bc 1 complex, its biochemical nature and role in respiration of this organism are apparently different from those of P. denitrificans. The peculiarity of the respiratory chain of A. eutrophus is confirmed by the rotenone insensitivity of the NADH oxidation in both protoplasts and membrane fragments from wild type and soluble hydrogenase deficient cells (HF14 and HF160). A tentative model of the respiratory chain of autotrophically grown A. eutrophus is presented. 相似文献
8.
Walther Johannssen Holger Gerberding Manfred Rohde Christiane Zaborosch Frank Mayer 《Archives of microbiology》1991,155(3):303-308
The soluble NAD-dependent hydrogenase (hydrogen-NAD oxidoreductase, EC 1.12.1.2), consisting of four non-identical subunits, was isolated from Alcaligenes eutrophus H16 and from Nocardia opaca 1b and analyzed by a HPLC gel permeation technique and electron microscopy. The tetrameric enzyme particles from both origins, as determined from negatively stained electron microscopic samples, were found to be elongated and very similar in shape and size. The A. eutrophus enzyme was measured in more detail. It exhibited dimensions of 12.7 nm by 5.5 nm (axial ratio 2.3:1). Dissociation into smaller particles and unspecific aggregation combined with partial inactivation were observed in the presence of the inhibitor NADH. Kept in buffer without added nickel, the enzyme was partially dissociated. Reassociation of tetramers without restored enzyme activity was achieved by addition of 0.5 mM NiCl2. A working model for the structural organization of the tetrameric enzyme particle is presented. 相似文献
9.
The soluble, NAD+-reducing hydrogenase in intact cells of Alcaligenes eutrophus was inactivated by oxygen when electron donors such as hydrogen or pyruvate were available. The sole presence of either oxygen or oxidizable substrates did not lead to inactivation of the enzyme. Inactivation occurred similarly under autotrophic growth conditions with hydrogen, oxygen and carbon dioxide. The inactivation followed first order reaction kinetics, and the half-life of the enzyme in cells exposed to a gas atmosphere of hydrogen and oxygen (8:2, v/v) at 30° C was 1.5 h. The process of inactivation did not require ATP-synthesis. There was no experimental evidence that the inactivation is a reversible process catalyzed by a regulatory protein. The possibility is discussed that the inactivation is due to superoxide radical anions (O
2
-
) produced by the hydrogenase itself. 相似文献
10.
Alcaligenes eutrophus and three other hydrogen bacteria exposed to plasmid-curing agents generated autotrophic-minus mutants at high frequency. These mutants were blocked in the metabolism of H2 as an energy source and had normal levels of enzymes involved in CO2 fixation. The loss of hydrogenase activity in A. eutrophus was accompanied by the loss or alteration of a plasmid that had molecular weight of approximately 200×106. Mobilization of this plasmid from wild-type A. eutrophus strains into cured hydrogenase-minus derivatives restored hydrogenase function. It is concluded that A. eutrophus contains a large plasmid required for hydrogen metabolism and thereby autotrophic growth.Abbreviations Aut
autotrophic
- Hup
hydrogen uptake
- NTG
N-methyl-N-nitro-N-nitrosoguanidine
- RuBP
ribulose bisphosphate
- RuMP
ribulose monophosphate
- Kan
kanamycin
- Nal
nalidixic acid
- Rif
rifampicin
- Tet
tetracycline 相似文献
11.
Tn5 was introduced into Alcaligenes eutrophus strain H1 by a suicide vector pSUP1011. Physical characterization of mutants obtained after Tn5 mutagenesis revealed a relatively high frequency of plasmid curing, or deletion of a 50 kb plasmid DNA segment. Results of Southern hybridization and chromosomal walking indicate that the same continuous stretch of plasmid DNA (designated as D region of plasmid) is deleted in four independent isolates. Moreover, the same deletion of plasmid DNA is also observed in a mitomycin C-generated mutant strain H1-4.Journal Paper No. J-12095 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2607, supported in part by a grant from the Iowa High Technology Council 相似文献
12.
Three nitrate reductase activities were detected in Alcaligenes eutrophus strain H16 by physiological and mutant analysis. The first (NAS) was subject to repression by ammonia and not affected by oxygen indicating a nitrate assimilatory function. The second (NAR) membrane-bound activity was only formed in the absence of oxygen and was insensitive to ammonia repression indicating a nitrate respiratory function. The third (NAP) activity of potential respiratory function occurred in the soluble fraction of cells grown to the stationary phase of growth. In contrast to NAR and NAS, expression of NAP did not require nitrate for induction and was independent of the rpoN gene product. Genes for the three reductases map at different loci. NAR and NAS are chromosomally encoded whereas NAP is a megaplasmid-borne activity in A. eutrophus. 相似文献
13.
Some structural and functional properties of ribosomes from the hydrogen-oxidizing bacterium Alcaligenes eutrophus were studied in order to investigate the background of expression of genetic information at the translational level. Ribosomal proteins from 30S subunits of A. eutrophus H16 were separated by two-dimensional gel electrophoresis into 21 spots, those from 50S subunits into 32 spots. While electrophoretic mobilities of several ribosomal proteins differed markedly from those of Escherichia coli, proteins sharing common immunological determinants with E. coli ribosomal proteins S1 and L7/L12 were found in A. eutrophus. Shifting from heterotrophic to autotrophic conditions of growth had no influence on the ribosomal protein pattern. Ribosomes of A. eutrophus had similar requirements for Mg2+ and poly(U) concentrations for optimum polyphenylalanine synthesis as those of E. coli. Protein synthesis elongation factors Tu from A. eutrophus and E. coli were immunologically similar. Efficiency of the A. eutrophus polyphenylalanine-synthesizing system was comparable to that of an analogous system derived from E. coli. This suggests that A. eutrophus could be employed for efficient expression of recombinant DNA. 相似文献
14.
Vera Weihs Karin Schmidt Beate Schneider Bärbel Friedrich 《Archives of microbiology》1989,151(6):546-550
A soluble flavohemoprotein (Fhp) was isolated to near homogeneity from heterotrophically grown cells of Alcaligenes eutrophus H16. Purified protein was used to raise polyclonal antibodies in rabbits. The anti-Fhp was employed to determine the content of Fhp in soluble extracts of wildtype and mutant strains of Alcaligenes. This analysis revealed that the formation of Fhp was strictly dependent on the presence of the individual megaplasmid, indigenous to A. eutrophus wild-type strains H16, H20 and N9A. Alcaligenes hydrogenophilus M50 did not contain Fhp; however, transfer of the A. eutrophus H16 specific plasmid pHG1 into this host, conferred Fhp-forming capacity. The fhp gene was isolated from a cosmid library of pHG1 DNA. A subcloned HindIII fragment of 3.27 kilobase pairs (kb) restored Fhp synthesis in plasmid-free mutants of A. eutrophus. Immunological studies showed that Fhp could also be expressed in the cloning organism Escherichia coli. 相似文献
15.
Martin Dominik Vollmer Karin Stadler-Fritzsche Michael Schlömann 《Archives of microbiology》1993,159(2):182-188
2,4-Dichlorophenoxyacetate (2,4-D) in Alcaligenes eutrophus JMP134 (pJP4) is degraded via 2-chloromaleylacetate as an intermediate. The latter compound was found to be reduced by NADH in a maleylacetate reductase catalyzed reaction. Maleylacetate and chloride were formed as products of 2-chloromaleylacetate reduction, the former being funnelled into the 3-oxoadipate pathway by a second reductive step. There was no indication for an involvement of a pJP4-encoded enzyme in either the reduction or the dechlorination reaction.Abbreviations 2,4-D
2,4-dichlorophenoxyacetate 相似文献
16.
Dietmar Helmut Pieper Andrea Elisabeth Kuhm Karin Stadler-Fritzsche Peter Fischer Hans-Joachim Knackmuss 《Archives of microbiology》1991,156(3):218-222
2,4-Dichloro-cis,cis-muconate is established as ringcleavage product in the degradation of 3,5-dichlorocatechol by Alcaligenes eutrophus JMP 134. The formerly described isomerization of 2-chloro-trans- to 2-chlorocis-4-carboxymethylenebut-2-en-4-olide as an essential catabolic step could not be certified. 相似文献
17.
In Alcaligenes eutrophus H16 a pleiotropic DNA-region is involved in formation of catalytically active hydrogenases. This region lies within the hydrogenase gene cluster of megaplasmid pHG1. Nucleotide sequence determination revealed five open reading frames with significant amino acid homology to the products of the hyp operon of Escherichia coli and other hydrogenase-related gene products of diverse organisms. Mutants of A. eutrophus H16 carrying Tn5 insertions in two genes (hypB and hypD) lacked catalytic activity of both soluble (SH) and membrane-bound (MBH) hydrogenase. Immunological analysis showed that the mutants contained SH-and MBH-specific antigen. Growing the cells in the presence of 63Ni2+ yielded significantly lower nickel accumulation rates of the mutant strains compared to the wild-type. Analysis of partially purified SH showed only traces of nickel in the mutant protein suggesting that the gene products of the pleiotropic region are involved in the supply and/or incorporation of nickel into the two hydrogenases of A. eutrophus. 相似文献
18.
Friedrich Cornelius G. Suetin Sergej Lohmeyer Michael 《Archives of microbiology》1984,140(2-3):206-211
Archives of Microbiology - Qualitative and quantitative determination of proteins of the soluble hydrogenase (hydrogen: NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus H16 was done by... 相似文献
19.
The accumulation and efflux of 63Ni2+ ions were studied using two strains of the strictly respiratory bacterium Alcaligenes eutrophus, the wild type strain N9A and its transconjugant N9A-M243 which harbors plasmid pMOL28.1 encoding constitutive resistance to nickel. When 1 M 63Ni2+ is added to respiring cells, strain N9A accumulates high, but M243 only negligibly small amounts of nickel. When the cells were preincubated for about 20 h under anoxic conditions and were then exposed to 1 M 63Ni2+ anaerobically, both strains accumulated approximately the same amounts of nickel. Aeration of these preloaded cells resulted in rapid efflux by strain M243 but renewed uptake of nickel by N9A. 63Ni2+ uptake and efflux are highly sensitive to protonophores such as CCCP, FCCP and TCS but insensitive to DCCD (each at 20 M concentrations). Measurements on the effects of the inhibitors on biosynthetic processes requiring ATP either from substrate phosphorylation or from electron transport phosphorylation made sure that at the concentrations used the inhibitor effects were specific. Thus the results suggest that in the nickelresistant strain M243 under normal aerobic conditions two constitutive energy-dependent nickel transport systems can function concomitantly, a chromosomally-determined specific uptake system and a plasmid-mediated specific nickel efflux system. 相似文献
20.
Forty-four mutants of Alcaligenes eutrophus H 16 were isolated which grew poorly or not at all under autotrophic conditions. Four types were characterized with respect to their defects and their physiological properties. One mutant lacked both enzymes specific for autotrophic CO2 fixation, another one lacked both hydrogenases, and two mutants lacked either the membrane-bound or the soluble hydrogenase. Comparing the results of studies on these mutant types, the following conclusions were drawn: the lack of each hydrogenase enzyme could be partially compensated by the other one; the lack of membrane-bound hydrogenase did not affect autotrophic growth, whereas the lack of the soluble hydrogenase resulted in a decreased autotrophic growth rate. When pyruvate as well as hydrogen were supplied to the wild-type, the cell yield was higher than in the presence of pyruvate alone. Mutant experiments under these conditions indicated that either of both hydrogenases was able to add to the energy supply of the cell. Only the soluble hydrogenase was involved in the control of the rate of hydrogen oxidation by carbon dioxide; the mutant lacking this enzyme did not respond to the presence or absence of CO2. The suppression of growth on fructose by hydrogen could be mediated by either of both hydrogenases alone. 相似文献