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PubMed1996

Overproduction of D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 in Escherichia coli and its purification.

We constructed the high-expression plasmid for D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6. The appropriate Shine-Dalgarno sequence (AAGGAG) was introduced to the eight bases upstream of start codon (ATG) of D-aminoacylase structural gene by site-directed mutagenesis, and then the 1.75-kb DNA fragment including the open reading frame was inserted into the downstream of the tac promoter of plasmid vector pKK223-3. The resultant plasmid, which was named pKNSD2, showed a high D-aminoacylase activity in Escherichia coli JM109 cells transformed with it. The enzyme was purified to homogeneity in only two steps with a final yield of 24% (sp act, 2023 U/mg).

Alcaligenes↗
PubMedDate not supplied

Cloning and nucleotide sequence of a D,L-haloalkanoic acid dehalogenase encoding gene from Alcaligenes xylosoxidans ssp. denitrificans ABIV.

We have cloned DNA fragments of plasmid pFL40 from Alcaligenes xylosoxidans ssp. denitrificans ABIV encoding a D,L-2-haloalkanoic acid halidohydrolase (DhlIV). A 6.5-kb EcoRI/SalI-fragment with inducible expression of the halidohydrolase was cloned in Pseudomonas fluorescens and Escherichia coli. A 1.9-kb HindII-fragment demonstrated expression of the dehalogenase only due to the presence of the promoter from the pUC vector in Escherichia coli. The nucleotide sequence of this DNA-fragment was determined. It had an open reading frame coding for 296 amino acid residues (molecular weight of 32783 D). The dhlIV gene showed sequence homology to a short segment of a D-specific dehalogenase (hadD) from Pseudomonas putida AJ1, but not to any other known DNA sequences. Restriction enzyme patterns indicated similarity between dhlIV and the D,L- isomer specific dehl dehalogenase gene from Pseudomonas putida PP3. There are some indications from restriction enzyme patterns and initial sequencing data, that a gene encoding a sigma 54-dependent activator protein, similar to the dehRI regulatory gene from Pseudomonas putida PP3 is located upstream of dhlIV. In contrast to DehI, dehalogenation of D- or L-chloropropionic acid by the DhlIV-protein leads to lactic acid of inverted configuration.

Alcaligenes↗
PubMed1990

Initial steps in the degradation of benzene sulfonic acid, 4-toluene sulfonic acids, and orthanilic acid in Alcaligenes sp. strain O-1.

Alcaligenes sp. strain O-1 grew with benzene sulfonate (BS) as sole carbon source for growth with either NH4+ or NH4+ plus orthanilate (2-aminobenzene sulfonate, OS) as the source(s) of nitrogen. The intracellular desulfonative enzyme did not degrade 3- or 4-aminobenzene sulfonates in the medium, although the enzyme in cell extracts degraded these compounds. We deduce the presence of a selective permeability barrier to sulfonates and conclude that the first step in sulfonate metabolism is transport into the cell. Cell-free desulfonation of BS in standard reaction mixtures required 2 mol of O2 per mol. One mol of O2 was required for a catechol 2,3-dioxygenase. When meta ring cleavage was inhibited with 3-chlorocatechol in desalted extracts, about 1 mol each of O2 and of NAD(P)H per mol of BS were required for the reaction, and SO3(2-) and catechol were recovered in high yield. Catechol was shown to be formed by dioxygenation in an experiment involving 18O2. 4-Toluene sulfonate was subject to NAD(P)H-dependent dioxygenation to yield SO3(2-) and 4-methylcatechol, which was subject to meta cleavage. OS also required 2 mol of O2 per mol and NAD(P)H for degradation, and SO3(2-) and NH4+ were recovered quantitatively. Inhibition of ring cleavage with 3-chlorocatechol reduced the oxygen requirement to 1 mol per mol of OS SO3(2-) (1 mol) and an unidentified organic intermediate, but no NH4+, were observed.

Alcaligenes↗
PubMed1995

Purification and characterization of N-carbamoyl-L-amino acid amidohydrolase with broad substrate specificity from Alcaligenes xylosoxidans.

N-Carbamoyl-L-amino acid amidohydrolase was purified to homogeneity for the first time from Alcaligenes xylosoxidans. The enzyme showed high affinity toward N-carbamoyl-L-amino acids with long-chain aliphatic or aromatic substituents, and hydrolyzed those with short-chain substituents quite well. The enzyme hydrolyzed N-formyl- and N-acetylamino acids quickly and very slowly, respectively. The enzyme did not hydrolyze beta-ureidopropionate and ureidosuccinate. The relative molecular mass of the native enzyme was about 135,000 and the enzyme consisted of two identical polypeptide chains. The enzyme activity was significantly inhibited by sulfhydryl reagents and required the following divalent metal ions: Mn2+, Ni2+ and Co2+.

Alcaligenes↗
PubMed1991

Biodegradation of 4-chlorophenol by adsorptive immobilized Alcaligenes sp. A 7-2 in soil.

Alcaligenes sp. A 7-2 immobilized on granular clay has been applied in a percolator to degrade 4-chlorophenol in sandy soil. Good adsorption rates on granular clay were achieved using cell suspensions with high titres and media at pH 8.0. The influence of various parameters such as aeration rate, pH, temperature, concentration of 4-chlorophenol and size of inoculum on the degradation rate were investigated. During fed-batch fermentations under optimal culture conditions, concentrations of 4-chlorophenol up to 160 mg.l-1 could be degraded. Semicontinuous culture experiments demonstrated that the degradation potential in soil could be well established and enhanced by the addition of immobilized bacteria. Continuous fermentation was performed with varying 4-chlorophenol concentrations in the feed and different input levels. The maximum degradation rate was 1.64 g.l-1.day-1.

Alcaligenes↗
PubMed1992

Cloning and heterologous expression of a novel arylmalonate decarboxylase gene from Alcaligenes bronchisepticus KU 1201.

We have cloned and sequenced a DNA fragment that encodes the arylmalonate decarboxylase (AM-Dase) gene from Alcaligenes bronchisepticus KU 1201. The AMDase gene consists of an open reading frame of 720 nucleotides, which specifies a 240-amino-acid protein of relative molecular mass (M(r)) 24734. The M(r) deduced from the AMDase gene is in good agreement with that of the AMDase isolated from A. bronchisepticus. No TATA or TTGA sequence was observed within the cloned DNA fragment, but the fragment was expressed in Escherichia coli by the lac promoter of pUC19. The enzyme produced in E. coli has the same M(r) and the same enzyme activity as that purified from A. bronchisepticus. Comparison of the DNA sequence and the deduced amino acid sequence of AMDase with available DNA and amino acid sequence data bases revealed that there are no significant sequence homologies.

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↗
PubMed1990

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.

Alcaligenes↗
PubMed1993

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.

Alcaligenes↗
PubMed1993

Metabolism of 2-chloro-4-methylphenoxyacetate by Alcaligenes eutrophus JMP 134.

2-Chloro-4-methylphenoxyacetate is not a growth substrate for Alcaligenes eutrophus JMP 134 and JMP 134-1. It is, however, being transformed by enzymes of 2,4-dichlorophenoxyacetic acid metabolism to 2-chloro-4-methyl-cis,cis-muconate, which is converted by enzymatic 1,4-cycloisomerization to 4-carboxymethyl-2-chloro-4-methylmuconolactone as a dead end metabolite. Chemically, only 3,6-cycloisomerization occurs, giving rise to both diastereomers of 4-carboxychloromethyl-3-methylbut-2-en-4-olide. Those lactones harboring a chlorosubstituent on the 4-carboxymethyl side chain were surprisingly stable under physiological as well as acidic conditions.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1990

On the operon structure of the cfx gene clusters in Alcaligenes eutrophus.

Three transposon Tn5-induced mutants deficient in autotrophic CO2 fixation were isolated from a megaplasmid pHG1-cured strain of Alcaligenes eutrophus H16. Their phenotypes were initially characterized by their ability to form both key enzymes of the Calvin cycle, ribulose-1,5-bisphosphate carboxylase (Rubisco) and phosphoribulokinase (PRK). Since the transposon insertions were at different sites within the chromosomal cluster of cfx genes encoding Calvin cycle enzymes, the individual mutants showed different inactivation patterns for Rubisco and PRK synthesis. These data together with already known sequence data and the arrangement of cfx genes suggested that the Rubisco, fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase and PRK genes are constituents of the same operon. This was further confirmed by trans complementation analyses which indicated that the very similarly organized pHG1-encoded cfx genes additionally present in wild-type strain H16 are functional and also form a common operon. Each operon may also include a glyceraldehyde-3-phosphate dehydrogenase gene. Thus, the duplicated cfx operons of A. eutrophus H16 are large transcriptional units comprising at least about 8 kilobase pairs (kb) and possibly as much as 11 kb.

Alcaligenes↗
PubMed1993

Conversion of 2-chloromaleylacetate in Alcaligenes eutrophus JMP134.

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 funneled 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.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1993

Chromosome mapping in Alcaligenes 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 degrees 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 degrees 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.

Alcaligenes↗
PubMed1992

Characterization of an Azospirillum brasilense Sp7 gene homologous to Alcaligenes eutrophus phbB and to Rhizobium meliloti nodG.

A 4 kb SalI fragment from Azospirillum brasilense Sp7 that shares homology with a 6.8 kb EcoRI fragment carrying nodGEFH and part of nodP of Rhizobium meliloti 41 was cloned in pUC18 to yield pAB503. The nucleotide sequence of a 2 kb SalI-SmaI fragment of the pAB503 insert revealed an open reading frame, named ORF3, encoding a polypeptide sharing 40% identity with R. meliloti NodG. The deduced polypeptide also shared 60% identity with the Alcaligenes eutrophus NADPH-dependent acetoacetyl-CoA (AA-CoA) reductase, encoded by the phbB gene and involved in poly-beta-hydroxybutyrate (PHB) synthesis. Northern blot analysis and promoter extension mapping indicated that ORF3 is expressed as a monocistronic operon from a promoter that resembles the Escherichia coli sigma 70 consensus promoter. An ORF3-lacZ translational fusion was constructed and was very poorly expressed in E. coli, but was functional and constitutively expressed in Azospirillum. Tn5-Mob insertions in ORF3 did not affect growth, nitrogen fixation, PHB synthesis or NAD(P)H-linked AA-CoA reductase activity. An ORF3 DNA sequence was used to probe total DNA of several Azospirillum strains. No ORF3 homologues were found in A. irakense, A. amazonense, A. halopraeferens or in several A. lipoferum strains.

Alcaligenes↗
PubMed1995

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.

Alcaligenes↗
PubMed1994

A cytochrome cd1-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 cd1-type nitrite reductase. It appeared to be a dimer of 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 d1. The isoelectric point of 8.6 was considerably higher than the pI determined for cd1 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.

Alcaligenes↗
PubMed1995

Analysis of the genes forming the distal parts of the two cbb CO2 fixation operons from Alcaligenes eutrophus.

In the facultative chemoautotroph Alcaligenes eutrophus H16, most of the genes (cbb genes) encoding enzymes of the Calvin carbon reduction cycle are organized within two highly homologous cbb operons, one located on the chromosome and the other on the megaplasmid pHG1. Nucleotide sequencing of the promoter-distal part of the operons revealed three open reading frames, designated cbbG, cbbK, and cbbA. Similarity searches in databases and heterologous expressions of the subcloned genes in Escherichia coli identified them as genes encoding the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and a class II fructose-1,6-bisphosphate aldolase, respectively. The aldolase could be grouped together with the enzymes from Rhodobacter sphaeroides and Bacillus subtilis as a new subtype of class II aldolases. A phenotypic complementation analysis with a cbb operon mutant of A. eutrophus showed that the cbbG product is essential for autotrophic growth of the organism, whereas the products of cbbK and cbbA can apparently be substituted by isoenzymes encoded elsewhere on the chromosome. No or only low constitutive promoter activity was associated with cbbK and cbbA, respectively, confirming the two genes as parts of the cbb operon. Downstream of cbbA, the very high overall nucleotide sequence identity (about 94%) prevailing throughout the two cbb operons discontinues, suggesting that cbbA is the most promoter-distal gene of the operon.

Alcaligenes↗
PubMed1978

Mutants of Alcaligenes eutrophus defective in autotrophic metabolism.

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.

Alcaligenes↗