Involvement of two plasmids in the degradation of carbaryl by Arthrobacter sp. strain RC100.
A bacterium capable of utilizing carbaryl (1-naphthyl N-methylcarbamate) as the sole carbon source was isolated from carbaryl-treated soil. This bacterium was characterized taxonomically as Arthrobacter and was designated strain RC100. RC100 hydrolyzes the N-methylcarbamate linkage to 1-naphthol, which was further metabolized via salicylate and gentisate. Strain RC100 harbored three plasmids (designated pRC1, pRC2, and pRC3). Mutants unable to degrade carbaryl arose at a high frequency after treating the culture with mitomycin C. All carbaryl-hydrolysis-deficient mutants (Cah-) lacked pRC1, and all 1-naphthol-utilization-deficient mutants (Nat-) lacked pRC2. The plasmid-free strain RC107 grew on gentisate as a carbon source. These two plasmids could be transferred to Cah- mutants or Nat- mutants by conjugation, resulting in the restoration of the Cah and Nah phenotypes. (+info
Conversion of (+/-)-synephrine into p-hydroxyphenylacetaldehyde by Arthrobacter synephrinum. A novel enzymic reaction.
A partically purified enzyme from Arthrobacter synephrinum was found to catalyse the conversion of (+/-)-synphrine into p-hydroxyphrenylacetaldehyde and methylamine. The enzyme is highly specific for synephrine and is distinctly different from monoamine oxidase. (+info
Action of chondroitinases. II. Numerical calculation of the degree of multiple attack.
Further investigation was carried out on the action patterns of two chondroitinase-AC [EC 126.96.36.199.] preparations obtained from Arthrobacter aurescens and Flavobacterium heparinum. To infer the action patterns of the chondroitinases, we proposed a new method for the calculation of the degree of multiple attack, based on the concept established by Robyt and French ((1967) Arch. Biochem. Biophys. 122, 8-16). It was shown that the degree of multiple attack (DM) is represented by the ratio of the initial velocity of number-average degree of scission to that of viscosity-average degree of scission. By this method, DM for A-Chase was estimated to be 3.03 and for F-chase, 1.31. (+info
Construction and characterization of two recombinant bacteria that grow on ortho- and para-substituted chlorobiphenyls.
Cloning and expression of the aromatic ring dehalogenation genes in biphenyl-growing, polychlorinated biphenyl (PCB)-cometabolizing Comamonas testosteroni VP44 resulted in recombinant pathways allowing growth on ortho- and para-chlorobiphenyls (CBs) as a sole carbon source. The recombinant variants were constructed by transformation of strain VP44 with plasmids carrying specific genes for dehalogenation of chlorobenzoates (CBAs). Plasmid pE43 carries the Pseudomonas aeruginosa 142 ohb genes coding for the terminal oxygenase (ISPOHB) of the ortho-halobenzoate 1,2-dioxygenase, whereas plasmid pPC3 contains the Arthrobacter globiformis KZT1 fcb genes, which catalyze the hydrolytic para-dechlorination of 4-CBA. The parental strain, VP44, grew only on low concentrations of 2- and 4-CB by using the products from the fission of the nonchlorinated ring of the CBs (pentadiene) and accumulated stoichiometric amounts of the corresponding CBAs. The recombinant strains VP44(pPC3) and VP44(pE43) grew on, and completely dechlorinated high concentrations (up to 10 mM), of 4-CBA and 4-CB and 2-CBA and 2-CB, respectively. Cell protein yield corresponded to complete oxidation of both biphenyl rings, thus confirming mineralization of the CBs. Hence, the use of CBA dehalogenase genes appears to be an effective strategy for construction of organisms that will grow on at least some congeners important for remediation of PCBs. (+info
Arthrobacter rhombi sp. nov., isolated from Greenland halibut (Reinhardtius hippoglossoides).
Two strains of a hitherto undescribed Gram-positive coryneform bacterium isolated from Greenland halibut (Reinhardtius hippoglossoides) were characterized by phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing studies demonstrated that the unknown strains constitute a new line within the genus Arthrobacter. The nearest relatives of the bacterium from fish were members of the Arthrobacter nicotianael Arthrobacter sulfureus group. The unknown bacterium was readily distinguished from these species by phenotypic methods. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as Arthrobacter rhombi sp. nov. The type strain of Arthrobacter rhombi is CCUG 38813T. (+info
Bacterial 2,4-dioxygenases: new members of the alpha/beta hydrolase-fold superfamily of enzymes functionally related to serine hydrolases.
1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (Qdo) from Pseudomonas putida 33/1 and 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) from Arthrobacter ilicis Ru61a catalyze an N-heterocyclic-ring cleavage reaction, generating N-formylanthranilate and N-acetylanthranilate, respectively, and carbon monoxide. Amino acid sequence comparisons between Qdo, Hod, and a number of proteins belonging to the alpha/beta hydrolase-fold superfamily of enzymes and analysis of the similarity between the predicted secondary structures of the 2,4-dioxygenases and the known secondary structure of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 strongly suggested that Qdo and Hod are structurally related to the alpha/beta hydrolase-fold enzymes. The residues S95 and H244 of Qdo were found to be arranged like the catalytic nucleophilic residue and the catalytic histidine, respectively, of the alpha/beta hydrolase-fold enzymes. Investigation of the potential functional significance of these and other residues of Qdo through site-directed mutagenesis supported the hypothesis that Qdo is structurally as well as functionally related to serine hydrolases, with S95 being a possible catalytic nucleophile and H244 being a possible catalytic base. A hypothetical reaction mechanism for Qdo-catalyzed 2,4-dioxygenolysis, involving formation of an ester bond between the catalytic serine residue and the carbonyl carbon of the substrate and subsequent dioxygenolysis of the covalently bound anionic intermediate, is discussed. (+info
Cloning and sequence analysis of the gene for glucodextranase from Arthrobacter globiformis T-3044 and expression in Escherichia coli cells.
The gld gene for glucodextranase from Arthrobacter globiformis T-3044 was cloned by using a combination of gene walking and probe methods and expressed on the recombinant plasmid pGD8, which was constructed with pUC118, in Escherichia coli cells. The enzyme gene consisted of a unique open reading frame of 3,153 bp. The comparison of the DNA sequence data with the N-terminal and 6 internal amino acid sequences of the purified enzyme secreted from A. globiformis T-3044 suggested the enzyme was translated from mRNA as a secretory precursor with a signal peptide of 28 amino acids residues. The deduced amino acids sequence of the mature enzyme contained 1,023 residues, resulting in a polypeptide with a molecular mass of 107,475 daltons. The deduced sequence showed about 38% identity to that of the glucoamylase from Clostridium sp. G0005. The glucodextranase activity of transformant harboring pGD8 was about 40 mU/ml at 30 degrees C for a 16-h culture. Although the GDase that was produced from the transformant was shorter than authentic GDase by 2 amino acid residues at the N-terminal end side, its enzymatic properties were almost same as the authentic one. Two kinds of genes, dex1 and dex2, for endo-dextranases from A. globiformis T-3044 were also cloned into Escherichia coli cells. The N-terminal of the purified endo-dextranase from A. globiformis T-3044 agreed with the deduced amino acid sequence, after the 33rd alanine residue, of only the dex1 gene for edo-dextranase. This result suggests that the endo-dextranase is translated from mRNA as a secretory precursor with a signal peptide of 32 amino acids residues. The deduced sequence of endo-dextranase 1 and endo-dextranase 2 showed about 93% and 65% identity with that of known endo-dextranase from Arthrobacter sp. CB-8, respectively. (+info
Cloning and nucleotide sequence of a gene encoding a glycogen debranching enzyme in the trehalose operon from Arthrobacter sp. Q36.
A gene located just upstream of the treYZ operon was isolated from Arthrobacter sp. strain Q36. The gene, designated treX, encoded an 823-amino acid protein. The amino acid sequence of the protein had 50% identity with the TreX protein (isoamylase) from Sulfolobus acidocaldarius ATCC 33909 which has a treZXY operon on the genome. We suggest that Arthrobacter treX is an isoamylase gene, and that it is a component of a treXYZ operon. (+info