Isolation of mutants of Alcaligenes eutrophus unable to derepress the fermentative alcohol dehydrogenase

Eight representative strains of Alcaligenes eutrophus, two strains of Alcaligenes hydrogenophilus and three strains of Paracoccus denitrificans were examined for their ability to use different alcohols and acetoin as a carbon source for growth. A. eutrophus strains N9A, H16 and derivative strains were unable to grow on ethanol or on 2,3-butanediol. Alcohol-utilizing mutants derived from these strains, isolated in this study, can be categorized into two major groups: Type I-mutants represented by strain AS1 occurred even spontaneously and were able to grow on 2,3-butanediol (t _d=2.7–6.4 h) and on ethanol (t _d=15–50 h). The fermentative alcohol dehydrogenase was present on all substrates tested, indicating that this enzyme in vivo is able to oxidize 2,3-butanediol to acetoin which is a good substrate for wild type strains. Type II-mutants represented by strain AS4 utilize ethanol as a carbon source for growth (t _d=3–9 h) but do not grow on butanediol. In these mutants the fermentative alcohol dehydrogenase is only present in cells cultivated under conditions of restricted oxygen supply, but a different NAD-dependent alcohol dehydrogenase is present in ethanol grown cells. Cells grown on ethanol, acetoin or 2,3-butanediol synthesized in addition two proteins exhibiting NAD-dependent acetaldehyde dehydrogenase activity and acetate thiokinase. An acylating acetaldehyde dehydrogenase (EC 1.2.1.10) was not detectable. Applying the colistin- and pin point-technique for mutant selection to strain AS1, mutants, which lack the fermentative alcohol dehydrogenase even if cultivated under conditions of restricted oxygen supply, were isolated; the growth pattern served as a readily identifiable phenotypic marker for the presence or absence of this enzyme.     

A multifunctional fermentative alcohol dehydrogenase from the strict aerobe Alcaligenes eutrophus: purification and properties

A NAD (P)-linked alcohol dehydrogenase was isolated from the soluble extract of the strictly respiratory bacterium Alcaligenes eutrophus N9A. Derepression of the formation of this enzyme occurs only in cells incubated under conditions of restricted oxygen supply for prolonged times. The purification procedure included precipitation by cetyltrimethylammonium bromide and ammonium sulfate and subsequent chromatography on DEAE-Sephacel, Cibacron blue F3G-A Sepharose and thiol-Sepharose. The procedure resulted in a 120-fold purification of a multifunctional alcohol dehydrogenase exhibiting dehydrogenase activities for 2,3-butanediol, ethanol and acetaldehyde and reductase activities for diacetyl, acetoin and acetaldehyde. During purification the ratio between 2,3-butanediol dehydrogenase and ethanol dehydrogenase activity remained nearly constant. Recovering about 20% of the initial 2,3-butanediol dehydrogenase activity, the specific activity of the final preparation was 70.0 U X mg protein-1 (2,3-butanediol oxidation) and 2.8 U X mg protein-1 (ethanol oxidation). The alcohol dehydrogenase is a tetramer of a relative molecular mass of 156000 consisting of four equal subunits. The determination of the Km values for different substrates and coenzymes as well as the determination of the pH optima for the reactions catalyzed resulted in values which were in good agreement with the fermentative function of this enzyme. The alcohol dehydrogenase catalyzed the NAD (P)-dependent dismutation of acetaldehyde to acetate and ethanol. This reaction was studied in detail, and its possible involvement in acetate formation is discussed. Among various compounds tested for affecting enzyme activity only NAD, NADP, AMP, ADP, acetate and 2-mercaptoethanol exhibited significant effects.     

Phospholipid bound to the flavohemoprotein from Alcaligenes eutrophus.

The structurally characterized flavohemoprotein from Alcaligenes eutrophus (FHP) contains a phospholipid-binding site with 1-16 : 0-2-cyclo-17 : 0-diacyl-glycerophospho-ethanolamine and 1-16 : 0-2-cyclo-17 : 0-diacyl-glycerophospho-glycerol as the major occupying compounds. The structure of the phospholipid is characterized by its compact form, due to the -sc/beta/-sc conformation of the glycerol and the nonlinear arrangement of the sn-1- and sn-2-fatty acid chains. The phospholipid-binding site is located adjacent to the heme molecule at the bottom of a large cavity. The fatty acid chains form a large number of van der Waal's contacts with nonpolar side chains, whereas the glycerophosphate moiety, which points towards the entrance of the channel, is linked to the protein matrix by polar interactions. The thermodynamically stable globin module of FHP, obtained after cleaving off the oxidoreductase module, also contains the phospholipid and can therefore be considered as a phospholipid-binding protein. Single amino acid exchanges designed to decrease the lipid-binding site revealed both the possibility of blocking incorporation of the phospholipid and its capability to evade steric barriers. Conformational changes in the phospholipid can also be induced by binding heme-ligating compounds. Phospholipid binding is not a general feature of flavohemoproteins, because the Escherichia coli and the yeast protein exhibit less and no lipid affinity, respectively.     

Comparison of the membrane-bound hydrogenases from Alcaligenes eutrophus H16 and Alcaligenes eutrophus type strain

Whereas the membrane-bound hydrogenase from Alcaligenes eutrophus H16 is an integral membrane protein and can only be solubilized by detergent treatment, the membrane-bound hydrogenase of Alcaligenes eutrophus type strain was found to be present in a soluble form after cell disruption. For the enzyme of A. eutrophus H16 a new, highly effective purification procedure was developed including phase separation with Triton X-114 and triazine dye chromatography on Procion Blue H-ERD-Sepharose. The purification led to an homogeneous hydrogenase preparation with a specific activity of 269 U/mg protein (methylene blue reduction) and a yield of 45%. During purification and storage the enzyme was optimally stabilized by the presence of 0.2 mM MnCl2. The hydrogenase of A. eutrophus type strain was purified from the soluble extract by a similar procedure, however, with less specific activity and activity yield. Comparison of the two purified enzymes revealed no significant differences: They have the same molecular weight, both consist of two different subunits (Mr = 62,000, 31,000) and both have an isoelectric point near pH 7.0. They have the same electron acceptor specificity reacting with similar high rates and similar Km values. The acceptors reduced include viologen dyes, flavins, quinones, cytochrome c, methylene blue, 2,6-dichlorophenolindophenol, phenazine methosulfate and ferricyanide. Ubiquinones and NAD were not reduced. The two hydrogenases were shown to be immunologically identical and both have identical electrophoretic mobility. For the membrane-bound hydrogenase of A. eutrophus H16 it was demonstrated that this type of hydrogenase in its solubilized, purified state is able to catalyze also the reverse reaction, the H2 evolution from reduced methyl viologen.     

An oxygen-binding flavohemoprotein from Alcaligenes eutrophus.

A procedure is described for the purification of a soluble flavohemoprotein from the hydrogen bacterium Alcaligenes eutrophus. The isolated protein exists as a monomer with a molecular weight of approx. 43,000. The molecule contains two prosthetic groups, 1 mol each of noncovalently bound FAD and protoheme per monomer. The absorption spectra of the protein in its ferric, ferrous-deoxy and ferrous-carboxy forms are similar to those of hemoglobins, with the exception of the flavin contribution (absorption maxima--ferric form: 395, 456, 483, 645 nm; ferrous-deoxy form: 436, 560 nm; ferrour-CO form: 423, 539, 569 nm). The flavohemoprotein when reduced by NADH in aerobic solution is capable of binding oxygen reversibly. The stable oxygenated complex exhibits absorption maxima at 414, 541, and 576 nm. The protein catalyzes the reduction of various dyes and cytochrome c by NADH.     

Nickel transport in Alcaligenes eutrophus

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.     

DNA topoisomerase I from Alcaligenes eutrophus H16

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     

An immobilized hydrogenase from Alcaligenes eutrophus H-16

Alcaligenes Eutrophus H-16 was grown in continuous culture under conditions which induced hydrogenase production. The hydrogenase enzyme was extracted, partially purified and immobilized on porous glass. This enzyme was then studied both in solution and in immobilized form as a possible candidate for a number of industrial applications. It proved to have a stability (storage and operational) which was highly temperature dependent. Temperatures near freezing caused the enzyme to retain its activity for long periods of time. Although its kinetics were more favorable at elevated temperatures of up to 40 degrees C, the loss of stability outweighed this gain substantially. The effects of buffer type and pH on enzyme activity were also studied. This enzyme has only a modest sensitivity to destruction by oxygen during storage, in contrast to hydrogenases produced by several other microorganisms.     

Evidence for novel mechanisms of polychlorinated biphenyl metabolism in Alcaligenes eutrophus H850

Previous studies indicated that Alcaligenes eutrophus H850 attacks a different spectrum of polychlorinated biphenyl (PCB) congeners than do most PCB-degrading bacteria and that novel mechanisms of PCB degradation might be involved. To delineate this, we have investigated the differences in congener selectivity and metabolite production between H850 and Corynebacterium sp. strain MB1, an organism that apparently degrades PCBs via a 2,3-dioxygenase. H850 exhibited a superior ability to degrade congeners via attack on 2-, 2,4-, 2,5-, or 2,4,5-chlorophenyl rings in PCBs but an inferior ability to degrade congeners via attack on a 4-chlorophenyl ring. Reactivity preferences were also reflected in the products formed from unsymmetrical PCBs; thus MB1 attacked the 2,3-chlorophenyl ring of 2,3,2',5'-tetrachlorobiphenyl to yield 2,5-dichlorobenzoic acid, while H850 attacked the 2,5-chlorophenyl ring to yield 2,3-dichlorobenzoic acid and a novel metabolite, 2',3'-dichloroacetophenone. Furthermore, H850 oxidized 2,4,5,2',4',5'-hexachlorobiphenyl, a congener with no adjacent unsubstituted carbons, to 2',4',5'-trichloroacetophenone. The atypical congener selectivity pattern and novel metabolites produced suggest that A. eutrophus H850 may degrade certain PCB congeners by a new route beginning with attack by some enzyme other than the usual 2,3-dioxygenase.     

Pleotropic mutants from Alcaligenes eutrophus defective in the metabolism of hydrogen,nitrate, urea,and fumarate

Chromosomal mutants of Alcaligenes eutrophus unable to grow with molecular hydrogen as the energy source also failed to grow with nitrate as the terminal electron acceptor or as a nitrogen source. The mutants (Hno^–) (i) formed neither soluble nor particulate hydrogenase antigens, (ii) expressed only about 50% the wild type level of ribulosebisphosphate carboxylase activity, and (iii) transported nickel, an essential constituent of active hydrogenase, at a significantly lower rate than wild type cells. Moreover, the mutants grew very slowly with urea as nitrogen source and did not express urease. Growth on formamide was also affected and formamidase activity was induced to only a very low level. Growth of the Hno^– mutants on succinate, glutamate, fumarate, and malate was significantly slower than wild type, and a reduced rate of succinate incorporation into the mutant cells was demonstrated. The highly pleiotropic phenotype of Hno^– mutants is indicative of a chromosomal gene with a considerable physiological importance. It affected the expression of both chromosomal and megaplasmid encoded systems of energy, carbon, and nitrogen metabolism. Thus, the hno mutation restricts the metabolic versatility but does not affect the basic metabolic functions of the organism.     

Three nitrate reductase activities in Alcaligenes eutrophus

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.     

Redox-dependent inactivation of the NAD-dependent hydrogenase from Alcaligenes eutrophus Z1

A novel inactivation mechanism of the NAD-dependent hydrogenase from Alcaligenes eutrophus Z1 comprising redox-dependent steps is described. The model of the hydrogenase inactivation process is proposed which implies that the enzyme may exist in several forms which differ in their stability and spectral properties. One of these forms, existing within a limited (approximately -200 +/- 30 mV) potential range, undergoes a rapid and irreversible inactivation. The dissociation of the FMN prosthetic group from the apohydrogenase appears to be the main reason for the enzyme inactivation. The rationale for the enzyme stabilization under real operational conditions based on the chemical modification of the hydrogenase molecule is suggested.     

The lysis of Gram-negative Alcaligenes eutrophus by enzymes from Cytophaga

Summary The use of Cytophaga lysing enzymes was investigated for the liberation of poly--hydroxybutyrate (PHB) granules from the Gram-negative bacterium Alcaligenes eutrophus. Complete cell lysis was approached within a 60 minute period. Contrary to previous findings for the lysis of Gram-negative bacteria, prior removal of the outer membrane was not essential for enzymic lysis. The destabilisation of the outer membrane by the removal of divalent cations resulted in no significant improvement in the disruption process.     

The iron-sulphur centres of soluble hydrogenase from Alcaligenes eutrophus.

The soluble hydrogenase (hydrogen:NAD+ oxidoreductase (EC 1.12.1.2) from Alcaligenes eutrophus has been purified to homogeneity by an improved procedure, which includes preparative electrophoresis as final step. The specific activity of 57 mumol H2 oxidized/min per mg protein was achieved and the yield of pure enzyme from 200 g cells (wet weight) was about 16 mg/purification. After removal of non-functional iron, analysis of iron and acid-labile sulphur yielded average values of 11.5 and 12.9 atoms/molecule of enzyme, respectively. p-Chloromercuribenzoate was a strong inhibitor of hydrogenase and apparently competed with NAD not with H2. Chelating agents, CO and O2 failed to inhibit enzyme activity. The oxidized hydrogenase showed an EPR spectrum with a small signal at g = 2.02. On reduction the appearance of a high temperature (50--77 K) signal at g = 2.04, 1.95 and a more complex low temperature (less than 30 K) spectrum at g = 2.04, 2.0, 1.95, 1.93, 1.86 was observed. The pronounced temperature dependence and characteristic lineshape of the signals obtained with hydrogenase in 80--85% dimethylsulphoxide demonstrated that iron-sulphur centres of both the [2Fe-2S] and [4Fe-4S] types are present in the enzyme. Quantitation of the EPR signals indicated the existence of two identical centres each of the [4Fe-4S] and of the [2Fe-2S] type. The midpoint redox potentials of the [4Fe-4S] and the [2Fe-2S] centres were determined to be -445 mV and -325 mV, respectively. Spin coupling between two centres, indicated by the split feature of the low temperature spectrum of the native hydrogenase around g = 1.95, 1.93, has been established by power saturation studies. On reduction of the [Fe-4S] centres, the electron spin relaxation rate of the [2Fe-2S] centres was considerably increased. Treatment of hydrogenase with CO caused no change in EPR spectra.     

Formation of Dimethylmuconolactones from Dimethylphenols by Alcaligenes eutrophus JMP 134

2,3-, 2,4-, 2,5-, 3,4-, and 3,5-dimethylphenols were cometabolized by 2,4-dichlorophenoxyacetate-grown Alcaligenes eutrophus JMP 134 or the constitutive derivative JMP 134-1 via the ortho pathway into dimethylmuconolactones as dead-end products. Formation of two distinct lactones from 3,4-dimethylphenol is indicative of 2- as well as 6-hydroxylation. Induction of the meta-cleavage pathway by 2,3- and 3,4-dimethylphenols resulted in growth and no accumulation of products. In contrast, 3,5-dimethylphenol is not metabolized by the meta-cleavage pathway.     

Evidence for novel mechanisms of polychlorinated biphenyl metabolism in Alcaligenes eutrophus H850.

Previous studies indicated that Alcaligenes eutrophus H850 attacks a different spectrum of polychlorinated biphenyl (PCB) congeners than do most PCB-degrading bacteria and that novel mechanisms of PCB degradation might be involved. To delineate this, we have investigated the differences in congener selectivity and metabolite production between H850 and Corynebacterium sp. strain MB1, an organism that apparently degrades PCBs via a 2,3-dioxygenase. H850 exhibited a superior ability to degrade congeners via attack on 2-, 2,4-, 2,5-, or 2,4,5-chlorophenyl rings in PCBs but an inferior ability to degrade congeners via attack on a 4-chlorophenyl ring. Reactivity preferences were also reflected in the products formed from unsymmetrical PCBs; thus MB1 attacked the 2,3-chlorophenyl ring of 2,3,2',5'-tetrachlorobiphenyl to yield 2,5-dichlorobenzoic acid, while H850 attacked the 2,5-chlorophenyl ring to yield 2,3-dichlorobenzoic acid and a novel metabolite, 2',3'-dichloroacetophenone. Furthermore, H850 oxidized 2,4,5,2',4',5'-hexachlorobiphenyl, a congener with no adjacent unsubstituted carbons, to 2',4',5'-trichloroacetophenone. The atypical congener selectivity pattern and novel metabolites produced suggest that A. eutrophus H850 may degrade certain PCB congeners by a new route beginning with attack by some enzyme other than the usual 2,3-dioxygenase.