Identification and molecular characterization of the acetyl coenzyme A synthetase gene (acoE) of Alcaligenes eutrophus.

The gene locus acoE, which is involved in the utilization of acetoin in Alcaligenes eutrophus, was identified as the structural gene of an acetyl coenzyme A synthetase (acetate:coenzyme A ligase [AMP forming]; EC 6.2.1.1). This gene was localized on a 3.8-kbp SmaI-EcoRI subfragment of an 8.1-kbp EcoRI restriction fragment (fragment E) that was cloned recently (C. Fründ, H. Priefert, A. Steinbüchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989). The 1,983 bp acoE gene encoded a protein with a relative molecular weight of 72,519, and it was preceded by a putative Shine-Dalgarno sequence. A comparison analysis of the amino acid sequence deduced from acoE revealed a high degree of homology to primary structures of acetyl coenzyme A synthetases from other sources (amounting to up to 50.5% identical amino acids). Tn5 insertions in two transposon-induced mutants of A. eutrophus, that were impaired in the catabolism of acetoin were mapped 481 and 1,159 bp downstream from the translational start codon of acoE. The expression of acoE in Escherichia coli led to the formation of an acyl coenzyme A synthetase that accepted acetate as the preferred substrate (100% relative activity) but also reacted with propionate (46%) and hydroxypropionate (87%); fatty acids consisting of four or more carbon atoms were not accepted. In addition, evidence for the presence of a second acyl coenzyme A synthetase was obtained; this enzyme exhibited a different substrate specificity. The latter enzyme is obviously required for the activation of propionate, e.g., during the formation of the storage compound poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) when propionate is provided as the sole carbon source. An analysis of mutants provided evidence that the expression of the uptake protein for propionate depends on the presence of alternate sigma factor sigma 54.     

Cloning of the Alcaligenes eutrophus alcohol dehydrogenase gene

Mutants of Alcaligenes eutrophus which are altered with respect to the utilization of 2,3-butanediol and acetoin were isolated after transposon mutagenesis. The suicide vehicle pSUP5011 was used to introduce the drug resistance transposable element Tn5 into A. eutrophus. Kanamycin-resistant transconjugants of the 2,3-butanediol-utilizing parent strains CF10141 and AS141 were screened for mutants impaired in the utilization of 2,3-butanediol or acetoin. Eleven mutants were negative for 2,3-butanediol but positive for acetoin; they were unable to synthesize active fermentative alcohol dehydrogenase protein (class 1). Forty mutants were negative for 2,3-butanediol and for acetoin (class 2). Tn5-mob was also introduced into a Smr derivative of the 2,3-butanediol-nonutilizing parent strain H16. Of about 35,000 transconjugants, 2 were able to grow on 2,3-butanediol. Both mutants synthesized the fermentative alcohol dehydrogenase constitutively (class 3). The Tn5-labeled EcoRI fragments of genomic DNA of four class 1 and two class 3 mutants were cloned from a cosmid library. They were biotinylated and used as probes for the detection of the corresponding wild-type fragments in a lambda L47 and a cosmid gene bank. The gene which encodes the fermentative alcohol dehydrogenase in A. eutrophus was cloned and localized to a 2.5-kilobase (kb) SalI fragment which is located within a 11.5-kb EcoRI-fragment. The gene was heterologously expressed in A. eutrophus JMP222 and in Pseudomonas oxalaticus. The insertion of Tn5-mob in class 3 mutants mapped near the structural gene for alcohol dehydrogenase on the same 2.5-kb SalI fragment.     

Poly-beta-hydroxybutyrate (PHB) biosynthesis in Alcaligenes eutrophus H16. Identification and characterization of the PHB polymerase gene (phbC)

The phbC gene encoding the third enzyme of the poly-beta-hydroxybutyrate biosynthetic pathway, poly-beta-hydroxybutyrate polymerase, in Alcaligenes eutrophus H16 has been identified by the complementation of poly-beta-hydroxybutyrate negative mutants of A. eutrophus H16. These results demonstrate that the three enzymes of the poly-beta-hydroxybutyrate biosynthetic pathway are organized phbC-phbA-phbB. Expression of all three genes in Escherichia coli results in a significant level (50% dry cell weight) of poly-beta-hydroxybutyrate production. phbC encodes a polypeptide of Mr = 63,900 which has a hydropathy profile distinct from typical membrane proteins indicating that poly-beta-hydroxybutyrate biosynthesis probably does not involve a membrane complex.     

Overproduction of a stable acetaldehyde dehydrogenase with high affinity to acetaldehyde by Escherichia coli expressing the ACOD gene of Alcaligenes eutrophus

Summary The acoD gene of Alcaligenes eutrophus, which encodes a very stable NAD dependent aldehyde dehydrogenase with high affinity toward acetaldehyde (K _m = 4M), was overexpressed in Escherichia coli. Plasmid pDel087, a deletion derivative of a plasmid constructed recently (Priefert et al., 1992), conferred acetaldehyde dehydrogenase activity of 2.5 U/mg of protein to E. coli, which was about 8-fold higher than the activity in ethanol-grown cells of A. eutrophus.     

Cloning and characterization of the Alcaligenes eutrophus 2-oxoglutarate dehydrogenase complex

Abstract Nucleotide sequence analysis of a 3.3-kb genomic Eco RI fragment and of relevant subfragments of a genomic 13.2-kb Sma I fragment of Alcaligenes eutrophus , which were identified by using a dihydrolipoamide dehydrogenase-specific DNA probe, revealed the structural genes of the 2-oxoglutarate dehydrogenase complex in a 7.5-kb genomic region. The genes odhA (2850 bp), odhB (1248 bp), and odhL (1422 bp), encoding 2-oxoglutarate dehydrogenase (El), dihydrolipoamide succinyltransferase (E2), and dihydrolipoamide dehydrogenase (E3), respectively, occur co-linearly in one gene cluster downstream of a putative −35 / −10 promoter in the order odhA, odhB , and odhL . In comparison to other bacteria, the occurrence of genes for two E3 components for the pyruvate as well as for the 2-oxoglutarate dehydrogenase complexes is unique. Heterologous expression of the A. eutrophus odh genes in E. coli XL1-Blue and in the kgdA mutant Pseudomonas putida JS347 was demonstrated by the occurrence of protein bands in electropherograms, by spectrometric detection of enzyme activities, and by phenotypic complementation, respectively.     

Physiological and biochemical characterization of the soluble formate dehydrogenase, a molybdoenzyme from Alcaligenes eutrophus.

Organoautotrophic growth of Alcaligenes eutrophus on formate was dependent on the presence of molybdate in the medium. Supplementation of the medium with tungstate lead to growth cessation. Corresponding effects of these anions were observed for the activity of the soluble, NAD(+)-linked formate dehydrogenase (S-FDH; EC 1.2.1.2) of the organism. Lack of molybdate or presence of tungstate resulted in an almost complete loss of S-FDH activity. S-FDH was purified to near homogeneity in the presence of nitrate as a stabilizing agent. The native enzyme exhibited an M(r) of 197,000 and a heterotetrameric quaternary structure with nonidentical subunits of M(r) 110,000 (alpha), 57,000 (beta), 19,400 (gamma), and 11,600 (delta). It contained 0.64 g-atom of molybdenum, 25 g-atom of nonheme iron, 20 g-atom of acid-labile sulfur, and 0.9 mol of flavin mononucleotide per mol. The fluorescence spectrum of iodine-oxidized S-FDH was nearly identical to the form A spectrum of milk xanthine oxidase, proving the presence of a pterin cofactor. The molybdenum-complexing cofactor was identified as molybdopterin guanine dinucleotide in an amount of 0.71 mol/mol of S-FDH. Apparent Km values of 3.3 mM for formate and 0.09 mM for NAD+ were determined. The enzyme coupled the oxidation of formate to a number of artificial electron acceptors and was strongly inactivated by formate in the absence of NAD+. It was inhibited by cyanide, azide, nitrate, and Hg2+ ions. Thus, the enzyme belongs to a new group of complex molybdo-flavo Fe-S FDH that so far has been detected in only one other aerobic bacterium.     

Identification of a lipoamide dehydrogenase gene as second locus affected in poly(3-hydroxybutyric acid)-leaky mutants of Alcaligenes eutrophus

Ribosomal rRNA gene fragments (rDNA) encompassing the 16S rDNA, the 16S-23S rDNA spacer region and part of the 23S rDNA of 95 strains belonging to 13 well-described taxa of the eubacterial family Comamonadaceae (beta subclass of the Proteobacteria or rRNA superfamily III) were enzymatically amplified using conserved primers. The fragments of approximately 2400 base pairs were subjected to restriction analysis. Restriction fragment length patterns obtained with HinfI enabled us to distinguish 9 of the 13 taxa studied. Restriction with CfoI was necessary to differentiate Acidovorax delafieldii from A. temperans and Hydrogenophaga flava from H. pseudoflava. The results indicate that amplified rDNA restriction analysis is a simple and reliable tool for the identification of bacterial species.     

The Alcaligenes eutrophus ldh structural gene encodes a novel type of lactate dehydrogenase

Abstract The lactate dehydrogenase gene, ldh , of Alcaligenes eutrophus H16 was identified on a 14-kbp Eco RI restriction fragment of a genomic library in the cosmid pHC79 by hybridization with a 50-mer synthetic oligonucleotide which was derived from the N-terminal amino acid sequence of the purified enzyme. Recombinant strains of Escherichia coli JM83, which harboured a 2.0-kbp Pst I subfragment in pUC9-1, expressed LDH at a high level, if ldh was downstream from and colinear to the E. coli lac promoter. The nucleotide sequence of a region of 4245 bp revealed several open reading frames which might represent coding regions. One represented the ldh gene. The amino acid sequence deduced from ldh exhibited 29% and 36% identity to the L-malate dehydrogenase of Methanothermus fervidus and to the putative translation product of an E. coli sequence of unknown function, respectively. The ldh was separated by short intergenic regions from two other open reading frames: ORF5 was located downstream of and colinear to ldh , and its putative translational product revealed 38 to 56% amino acid identity to penicillin-binding proteins. ORF3 was located upstream of and colinear to ldh , and its putative gene translational product represented a hydrophobic protein. A sequence, which resembled the A. eutrophus alcohol dehydrogenase promoter, was detected upstream of ORF3, which most probably represents the first transcribed gene of an operon consisting of ORF3, ldh and ORF5.     

Construction and characterization of heavy metal-resistant haloaromatic-degrading Alcaligenes eutrophus strains.

Alcaligenes eutrophus strains exhibiting both plasmid-borne heavy metal resistance and haloaromatic-degrading functions were obtained by intraspecific conjugation. The strains which we constructed expressed catabolic and resistance markers together. Degradation of various polychlorinated biphenyl isomers and 2,4-D (2,4-dichlorophenoxyacetic acid) was observed in the presence of 1 mM nickel or 2 mM zinc, provided that the metal resistance determinant was present in the catabolizing strain. Such strains may be useful for decontamination of sites that are polluted with both organic compounds and heavy metals.     

Molecular and genetic characterization of an Alcaligenes eutrophus insertion element.

An insertion element, ISAE1, was discovered during the molecular analysis of mutants defective in the autotrophic growth (Aut-) of Alcaligenes eutrophus H1-4, a mitomycin C-generated derivative of strain H1. ISAE1 is 1,313 bp long, has 12-bp nearly perfect inverted terminal repeats, and contains an open reading frame that has a coding capacity of 408 amino acids. Direct repeats of 8 bp were generated by insertion of ISAE1 into chromosomes or plasmids. Most insertion were found in the AT-rich target sites. The distribution of ISAE1 is limited to A. eutrophus H1 (ATCC 17698) and H16 (ATCC 17699). Variants with newly transposed copies of ISAE1 could be isolated at an elevated frequency by changing the growth conditions.     

Structural gene (nirS) for the cytochrome cd1 nitrite reductase of Alcaligenes eutrophus H16.

Denitrification by Alcaligenes eutrophus H16 is genetically linked to megaplasmid pHG1. Unexpectedly, the gene encoding the nitrite reductase (nirS) was identified on chromosomal DNA. The nirS product showed extensive homology with periplasmic nitrite reductases of the heme cd1-type. Disruption of nirS abolished nitrite-reducing ability, indicating that NirS is the enzyme essential for denitrification in A.eutrophus.     

Biosynthesis and characterization of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in Alcaligenes eutrophus

Copolyesters of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) were produced by Alcaligenes eutrophus at 30 degrees C in nitrogen-free culture solutions containing gamma-butyrolactone alone or with fructose or butyric acid as the carbon sources. When gamma-butyrolactone was used as the sole carbon source, the 4HB fraction in copolyester increased from 9 to 21 mol% as the concentration of gamma-butyrolactone in the culture solution increased from 10 to 25 g/l. The addition of fructose to the culture solution of gamma-butyrolactone resulted in a decrease in the 4HB fraction in copolyester. The copolyesters produced from gamma-butyrolactone and fructose by A. eutrophus were shown to have random sequence distribution of 3HB and 4HB units by analysis of the 125 MHz 13C n.m.r. spectra. In contrast, a mixture of random copolyesters with two different 4HB fractions was produced by A. eutrophus when gamma-butyrolactone and butyric acid were used as the carbon sources. These results are discussed on the basis of a proposed biosynthetic pathway of P(3HB-co-4HB). The copolyester films became soft with an increase in the 4HB fraction, and the elongation to break at 23 degrees C increased from 5 to 444% as the 4HB fraction increased from 0 to 16 mol%. The P(3HB-co-10% 4HB) film was shown to be biodegradable in an activated sludge.     

Plasmids required for utilization of molecular hydrogen by Alcaligenes eutrophus

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     

Purification and characterization of dichloromuconate cycloisomerase from Alcaligenes eutrophus JMP 134.

Dichloromuconate cycloisomerase from Alcaligenes eutrophus JMP 134 was purified to homogeneity. The enzyme has an Mr of about 270,000 as determined by gel filtration and consists of six to eight subunits of identical Mr 40,000 as determined by SDS/PAGE. Mn2+ ions as well as thiol groups are required for activity. A high Km value of about 4 mM for cis,cis-muconate explains the reported low activity with this compound. Relatively high Km values were also calculated for monochloro-substituted cis,cis-muconates (300-500 microM), in contrast with the low Km value of 20 microM for 2,4-dichloro-cis,cis-muconate. The catalytic constant of the pure enzyme was 3820 min-1 when measured with 2,4-dichloro-cis,cis-muconate.     

hoxX (hypX) is a functional member of the Alcaligenes eutrophus hyp gene cluster

The role of HoxX in hydrogenase biosynthesis of Alcaligenes eutrophus H16 was re-examined. The previously characterized hoxX deletion mutant HF344 and a newly constructed second hoxX mutant carrying a smaller in-frame deletion were studied. The second mutant was impaired in the activity of both the soluble and the membrane-bound hydrogenase. The two hydrogenase activities were reduced by approximately 50% due to delayed processing of the active-site-containing large subunits, while hydrogenase gene expression was not affected. We conclude that the mutation in mutant HF344 causes polarity resulting in the observed regulatory phenotype of this mutant. The data presented in this report point to an enhancing function of HoxX in the conversion of the soluble hydrogenase and of the membrane-bound hydrogenase large-subunit precursor. Thus, hoxX encodes a member of the Hyp proteins that are required for the formation of active hydrogenase and was accordingly renamed hypX. Received: 15 June 1998 / Accepted: 5 August 1998     

Purification and properties of chorismate mutase-prephenate dehydratase and prephenate dehydrogenase from Alcaligenes eutrophus.

Chorismate mutase and prephenate dehydratase from Alcaligenes autophus H16 were purified 470-fold with a yield of 24%. During the course of purification, including chromatography on diethylaminoethyl (DEAE)-cellulose, phenylalanine-substituted Sepharose, Sephadex G-200 and hydrogyapatite, both enzymes appeared in association. The ratio of their specific activities remained almost constant. The molecular weight of chorismate mutase-prephenast dehydratase varied from 144,000 to 187,000 due to the three different determination methods used. Treatment of electrophoretically homogeneous mutase-dehydratase with sodium dodecyl sulfate dissociated the enzyme into a single component of molecular weight 47,000, indicating a tetramer of identical subunits. The isoelectric point of the bifunctional enzyme was 5.8. Prephenate dehydrogenase was not associated with other enzyme activities; it was separated from mutasedehydratase by DEAE-cellulose chromatgraphy. Chromatography on DEAE Sephadex, Sephadex G-200, and hydroxyapatite resulted in a 740-fold purification with a yield of 10%. The molecular weight of the enzyme was 55,000 as determined by sucrose gradient centrifugation and 65,000 as determined by gel filtration or electrophoresis. Its isoelectric point was pH 6.6. In the overall conversion of chorismate to phenylpyruvate, free prephenate was formed which accumulated in the reaction mixture. The dissociation of prephenate allowed prephenate dehydrogenase to compete with prephenate dehydratase for the substrate.