Electron microscopic localization of hydrogenase genes on the megaplasmid pHG 1 in Alcaligenes eutrophus

Summary Plasmids carrying hydrogenase genes in Alcaligenes eutrophus wild type H 16 and in two transposon Tn5 —induced mutants have been investigated by electron microscopy. Besides the pHG1 megaplasmid (458±27 kb) carrying genes coding for structural and regulatory properties of hydrogenases, small plasmids of unknown significance have been detected. The sizes of EcoRI fragments obtained from pHG1 were measured from electron micrographs. They were significantly different from sizes determined previously by agarose gel electrophoresis.Plasmid pHG1 isolated from the wild type H 16 was shown to contain two inverted repeats (IR 16-1 and IR 16-2) with sizes similar to known transposons.From electron microscopic hybridization studies, it was deduced that the sites of insertion of Tn5 into a regulation gene on pHG1 for both soluble and membrane-bound hydrogenase, and of Tn5-Mob into the gene coding for structural properties of the soluble hydrogenase, are about 67.2 kb apart. One of the inverted repeats (IR 16-1) was localized in between these sites.     

Analysis of a pleiotropic gene region involved in formation of catalytically active hydrogenases in Alcaligenes eutrophus H16

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.     

Involvement of megaplasmids in heterotrophic derepression of the carbon-dioxide assimilating enzyme system in Alcaligenes spp.

The facultatively chemolithoautotrophic hydrogen-oxidizing bacteria Alcaligenes eutrophus and Alcaligenes hydrogenophilus partially derepressed the formation of phosphoribulokinase and ribulosebisphosphate carboxylase during heterotrophic growth on fructose or gluconate. We examined whether the indigenous magaplasmids in these bacteria that encode the ability to oxidize hydrogen affected this derepression. The results suggest an involvement of the plasmids in the derepression for the following reasons: (i) wild-type strains, except A. eutrophus TF93, exhibited the derepressible phenotype; (ii) plasmid-cured mutants formed the enzymes with formate as autotrophic growth substrate but did not derepress their formation during heterotrophic growth; (iii) the phenotype of the wild type was restored by transfer of the plasmids into plasmid-cured mutants. Plasmid pHG2 from strain TF93 differed from the other wild-type plasmids by conferring a non-derepressible phenotype onto the harboring strain. Mutants of A. eutrophus H16 carrying deletions in plasmid pHG1 showed a similar phenotype as that of the plasmid-cured mutants. We concluded that the plasmids from the various strains studied encode a regulatory ability to derepress phosphoribulokinase and ribulosebisphosphate carboxylase under heterotrophic growth conditions.Abbreviations PRK phosphoribulokinase - RuBPC ribulosebisphosphate carboxylase - Hox ability to oxidize hydrogen - Cfx ability to fix carbon dioxide autotrophically Dedicated to Prof. Dr. H. G. Schlegel on the occasion of his 60th birthday     

Isolation of hydrogenase regulatory mutants of hydrogen-oxidizing bacteria by a colony-screening method

Mutants derepressible for hydrogenases (Hox d) have been isolated from the wild type of Alcaligenes hydrogenophilus which is inducible for hydrogenases (Hox i). The mutants are able to form the hydrogenases during growth on gluconate under air while the wild type requires molecular hydrogen for hydrogenase systhesis.Mutant selection involved alternating growth under autotrophic and heterotrophic conditions. Mutants derepressed for hydrogenases after growth on gluconate were recognized by a new colony-screening method allowing differentiation between colonies of hydrogenase-containing and hydrogenase-free cells of aerobic hydrogen-oxidizing bacteria. The method is based on the ability of the colonies to reduce triphenyltetrazolium chloride in the presence of monoiodoacetate and gaseous hydrogen to its water-insoluble purple formazan. Endogenous dye reduction (under nitrogen) and the function of the cytoplasmic NAD-reducing hydrogenase were completely inhibited by monoiodoacetate. The applicability of the method has been demonstrated for wild type strains and mutants of various hydrogen-oxidizing bacteria. When mutants of A. hydrogenophilus and A. eutrophus H16 lacking the Hox-encoding plasmids pHG21-a and pHG1, respectively, were used as recipients and Hox d mutant M 201 of A. hydrogenophilus as a donor transconjugants appeared which had received the Hox d character and the megaplasmid pHG21-a.Abbreviations MIAc monoiodoacetate - TTC 2,3,5-triphenyl-2-tetrazolium chloride - Hox ability to oxidize hydrogen Dedicated to Gerhard Drews on the occasion of his 60th birthday, remembering the education and inspiration we received from our teacher Johannes Buder at the Martin-Luther University of Halle     

Genetic transfer of lithoautotrophy mediated by a plasmid-cointegrate from Pseudomonas facilis

Pseudomonas facilis (DSM 620) is host of two plasmids one of which (pHG22-a) has been shown to be involved in lithoautotrophic metabolism. The lithoautotrophic marker was transferred via conjugation to mutants of two wild type strains of P. facilis and to the heterotrophic bacterium Pseudomonas delafieldii. The transfer required mobilization by the IncP1 plasmid RP4. Transconjugants contained a plasmid which neither correlated in size with RP4 nor with pHG22-a. This newly formed plasmid, pHG22-c, was shown to be a cointegrate consisting of RP4 DNA and a 50-kb insert derived from the native plasmid pHG22-a. DNA-DNA hybridization using lithoautotrophic genes of Alcaligenes eutrophus as DNA probes, revealed the presence of hydrogenase structural and regulatory genes in addition to genes of autotrophic carbon dioxide fixation on the cointegrate pHG22-c.     

Transfer and Expression of Lithoautotrophy and Denitrification in a Host Lacking These Metabolic Activities

The conjugative 450-kilobase-pair megaplasmid pHG1 from Alcaligenes eutrophus H16 was transferred to the herbicide-degrading soil bacterium A. eutrophus JMP134. This transfer was achieved by means of RP4 mobilization and a Tn5-Mob insertion provided in trans on the megaplasmid replicon. Although kanamycin-resistant transconjugants also occurred with other gram-negative species such as Rhizobium, Agrobacterium, and thiobacteria, A. eutrophus JMP134 was the only recipient which stably maintained the megaplasmid. pHG1-containing transconjugants derived from JMP134 expressed all metabolic functions associated with the plasmid: the ability to oxidize hydrogen through catalysis of two hydrogenases, to assimilate carbon dioxide via the Calvin cycle pathway, and to grow with nitrate anaerobically. All of these metabolic activities were absent in the original strain JMP134.     

Structural aspects of the soluble NAD-dependent hydrogenase isolated from Alcaligenes eutrophus H16 and from Nocardia opaca 1b

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.     

Location, catalytic activity, and subunit composition of NAD-reducing hydrogenases of some Alcaligenes strains and Rhodococcus opacus MR22

Six new strains of Alcaligenes enriched for and isolated as nickel-resistant bacteria resemble Alcaligenes eutrophus H16 and contain both an NAD-reducing, tetrameric soluble hydrogenase and a membrane-bound hydrogenase. None of the soluble hydrogenases share with the Rhodococcus opacus MR11 enzyme tetramer the property of being cleaved easily into two dimeric moieties [a hydrogenase (βδ) and an NADH:acceptor oxidoreductase (αγ)], in the absence of nickel or at low ionic strength. The soluble hydrogenase of the newly isolated strain MR22 of R. opacus equalled that of strain MR11. The absence of a membrane-bound hydrogenase in Alcaligenes denitrificans strain 4a-2 and in Alcaligenes ruhlandii was confirmed. Received: 14 May 1996 / Accepted: 7 November 1996     

Analysis of the cbbF genes from Alcaligenes eutrophus that encode fructose-1,6-/sedoheptulose-1,7-bisphosphatase

The cbbF genes of the facultative chemoautotroph Alcaligenes eutrophus H16 are part of two highly homologous cbb operons. Both the chromosomal and the megaplasmid pHG1-borne copy of cbbF were cloned and sequenced. Subsequent analyses including comparison with known sequences from other organisms and heterologous expression in Escherichia coli revealed that each of the genes encodes fructose-1,6-bisphosphatase (FBPase). A closely related activity likewise operating in the Calvin carbon reduction cycle, sedoheptulose-1,7-bisphosphatase, was also catalyzed by the two isoenzymes which were purified from autotrophically grown cells of A. eutrophus. Two-dimensional gel electrophoresis allowed the separation of the cbbF gene products. Preliminary physical evidence by Southern hybridization with a heterologous gene probe was obtained for the existence of a third FBPase gene, fbp, on the chromosome of the organism. Its product is probably involved in the heterotrophic carbon metabolism.     

Genetic analysis of the parB+ locus of plasmid R1

Summary Megaplasmid DNA from mutants has been analysed physically for deletions and insertions in order to identify the location of hydrogenase (hox) genes in Alcaligenes eutrophus. Four classes of mutants have been examined: mutants defective in genes coding for soluble NAD-dependent hydrogenase (hoxS), mutants impaired in the membrane-bound hydrogenase (hoxP), mutants altered in the regulation of hox gene expression (hoxC) and mutants with lesions in the carbon dioxide fixing enzyme system (cfx). A comparison of the restriction patterns with EcoRI, BamHI and HindIII, complementation studies with cloned DNA and DNA - DNA hybridization experiments showed that genes coding for hox and cfx are clustered on a 100-kb region of the 450-kb plasmid pHG1.     

Transfer and expression of PCB-degradative genes into heavy metal resistantAlcaligenes eutrophus strains

Sites polluted with organic compounds frequently contain inorganic pollutants such as heavy metals. The latter might inhibit the biodegradation of the organics and impair bioremediation. Chromosomally located polychlorinated biphenyl (PCB) catabolic genes ofAlcaligenes eutrophus A5,Achromobacter sp. LBS1C1 andAlcaligenes denitrificans JB1 were introduced into the heavy metal resistantAlcaligenes eutrophus strain CH34 and related strains by means of natural conjugation. Mobile elements containing the PCB catabolic genes were transferred fromA. eutrophus A5 andAchromobacter sp. LB51C1 intoA. eutrophus CH34 after transposition onto their endogenous IncP plasmids pSS50 and pSS60, respectively. The PCB catabolic genes ofA. denitrificans JB1 were transferred intoA. eutrophus CH34 by means of RP4::Mu3A mediated prime plasmid formation. TheA. eutrophus CH34 transconjugant strains expressed both catabolic and metal resistance markers. Such constructs may be useful for the decontamination of sites polluted by both organics and heavy metals.     

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.     

Nickel resistance of Alcaligenes denitrificans strain 4a-2 is chromosomally coded

A newly isolated aerobic hydrogen-oxidizing bacterium, Alcaligenes denitrificans strain 4a-2, differs from related autotrophic bacteria by containing only a single cytoplasmic, NAD-reducing hydrogenase, and by its high resistance to nickel ions, i.e. tolerance to 20 mM NiCl₂. Strain 4a-2 harbors a single plasmid of about 250 kb. On helper-assisted mating of 4a-2 with Alcaligenes eutrophus strains H16,G29, and M85 nickelresistant transconjugants were selected; these did not contain the donor plasmid, however. All three transconjugants tolerated 3 to 10 mM NiCl₂. The resistance was constitutively expressed. DNA/DNA hybridization showed homology with EcoRI-digested DNA of the wild type 4a-2 and transconjugants using a DNA probe containing nickel resistance genes of pMOL28. This indicated that the 4a-2 nickel resistance genes are located on the chromosome.     

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.     

Localization and stability of hydrogenases from aerobic hydrogen bacteria

Alcaligenes eutrophus strains H 16, B 19, G 27 and N9A contained two different hydrogenases. One enzyme catalyzed the reduction of NAD by hydrogen and was strictly localized in the soluble cell fraction, while the second enzyme was found to be particulate and unable to react with NAD.All other tested strains, Alcaligenes paradoxus SA 29, Pseudomonas facilis, P. palleronii RH 2, Pseudomonas sp. strain GA 3, Paracoccus denitrificans, Aquaspirillum autotrophicum SA 32, and Corynebacterium autotrophicum 14g and 7C contained only a single enzyme exclusively bound to membranes. This was established using fractional centrifugation, indicator enzyme systems, gentle methods of cell disintegration and discontinuous sucrose density gradient centrifugation. In cell-free extracts obtained by rough disruption (sonication) of cells, hydrogenase was associated to particles of different size and sedimentation velocity. A partial solubilization of hydrogenase caused by sonication was observed with P. facilis.Without exception, the particulate hydrogenases were found (1) to be unable to reduce pyridine nucleotides, and (2) to reduce methylene blue at an extremely high activity. The eminent reaction rate of 34 moles H₂ oxidized per min and mg protein has been determined in particle suspensions of Pseudomonas sp. strain GA 3. All hydrogenases were stable during storage under hydrogen atmosphere, except the soluble enzyme from A. eutrophus H 16 which was shown to be more stable under aerobic conditions.     

A cytochrome cd 1-type nitrite reductase mediates the first step of denitrification in Alcaligenes eutrophus

Respiratory nitrite reductase (NIR) has been purified from the soluble extract of denitrifying cells of Alcaligenes eutrophus strain H16 to apparent electrophoretic homogeneity. The enzyme was induced under anoxic conditions in the presence of nitrite. Purified NIR showed typical features of a cytochrome cd ₁-type nitrite reductase. It appeared to be a dimer of 60 kDa subunits, its activity was only weakly inhibited by the copper chelator diethyldithiocarbamate, and spectral analysis revealed absorption maxima which were characteristic for the presence of heme c and heme d ₁. The isoelectric point of 8.6 was considerably higher than the pI determined for cd ₁ nitrite reductases from pseudomonads. Eighteen amino acids at the N-terminus of the A. eutrophus NIR, obtained by protein sequencing, showed no significant homology to the N-terminal region of nitrite reductases from Pseudomonas stutzeri and Pseudomonas aeruginosa.     

Isolation and characterization of megaplasmid DNA from lithoautotrophic bacteria

A method is described for the preparative isolation of megaplasmids ranging in size from 340 to 700 kb. These plamids were isolated from chemolithoautotrophic bacteria including the species Alcaligenes, Pseudomonas, and Paracoccus. The procedure was based on alkaline sodium dodecyl sulfate lysis of the cells, followed by heat treatment, salt precipitation, several phenol extractions, dialysis steps, and proteinase and RNase treatment. The various parameters were evaluated and controlled. Hydrogen-oxidizing-ability (Hox) encoding plasmids were compared by EcoRI restriction enzyme analysis. pHG plasmids from Alcaligenes eutrophus wild-type strains appeared to be closely related; plasmids derived from the type strain TF93 and from A. hydrogenophilus exhibited major differences in restriction sites. Two cryptic plasmids harbored by Pseudomonas facilis and Paracoccus denitrificans showed scarcely detectable similarity to the plasmid species of Alcaligenes.     

Translational system of the hydrogen-oxidizing bacterium Alcaligenes eutrophus

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.     

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     

Chromosome mapping in Alealigenes eutrophus CH34

Mutants and mobilizing plasmids were developed as genetic tools in Alcaligenes eutrophus CH34. In order to map the chromosome, spontaneous and ethyl methane sulphonate (EMS)-induced mutants (mostly auxotrophs) were isolated. Another source of mutants was provided by the phenomenon of temperature-induced mortality and mutagenesis that is observed at 37° C and is characteristic of many metallotolerant strains of A. eutrophus. Plasmid pULB113 (RP4::miniMu) was used to map the available mutations. Twenty-five loci were ordered in a circular map. pMOL50, a rearranged derivative of plasmid pMOL28, which was obtained in a survivor at 37° C and displayed chromosome mobilizing activity (Cma+), was also used to mobilize chromosomal markers: resulting linkages were stronger than with pULB113, allowing confirmation of the circularity of the A. eutrophus CH34 chromosome with a small number of crosses.