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PubMed1991

Purification and characterization of haloalcohol dehalogenase from Arthrobacter sp. strain AD2.

An enzyme capable of dehalogenating vicinal haloalcohols to their corresponding epoxides was purified from the 3-chloro-1,2-propanediol-utilizing bacterium Arthrobacter sp. strain AD2. The inducible haloalcohol dehalogenase converted 1,3-dichloro-2-propanol, 3-chloro-1,2-propanediol, 1-chloro-2-propanol, and their brominated analogs, 2-bromoethanol, as well as chloroacetone and 1,3-dichloroacetone. The enzyme possessed no activity for epichlorohydrin (3-chloro-1,2-epoxypropane) or 2,3-dichloro-1-propanol. The dehalogenase had a broad pH optimum at about 8.5 and a temperature optimum of 50 degrees C. The enzyme followed Michaelis-Menten kinetics, and the Km values for 1,3-dichloro-2-propanol and 3-chloro-1,2-propanediol were 8.5 and 48 mM, respectively. Chloroacetic acid was a competitive inhibitor, with a Ki of 0.50 mM. A subunit molecular mass of 29 kDa was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With gel filtration, a molecular mass of 69 kDa was found, indicating that the native protein is a dimer. The amino acid composition and N-terminal amino acid sequence are given.

Amino Acid Sequence↗
PubMed1991

Choline oxidase, a catabolic enzyme in Arthrobacter pascens, facilitates adaptation to osmotic stress in Escherichia coli.

Choline oxidase (EC 1.1.3.17) is a bifunctional enzyme that is capable of catalyzing glycine betaine biosynthesis from choline via betaine aldehyde. A gene (cox) encoding this enzyme in the gram-positive soil bacterium Arthrobacter pascens was isolated and characterized. This gene is contained within a 1.9-kb fragment that encodes a polypeptide of approximately 66 kDa. Transfer of this gene to an Escherichia coli mutant that is defective in betaine biosynthesis resulted in an osmotolerant phenotype. This phenotype was associated with the ability of the host to synthesize and assemble an enzymatically active choline oxidase that could catalyze biosynthesis of glycine betaine from an exogenous supply of choline. Although glycine betaine functions as an osmolyte in several different organisms, it was not found to have this role in A. pascens. Instead, both choline and glycine betaine were utilized as carbon sources. In A. pascens synthesis and activity of choline oxidase were modulated by carbon sources and were susceptible to catabolite repression. Thus, cox, a gene concerned with carbon utilization in A. pascens, was found to play a role in adaptation to an environmental stress in a heterologous organism. In addition to providing a possible means of manipulating osmotolerance in other organisms, the cox gene offers a model system for the study of choline oxidation, an important metabolic process in both procaryotes and eucaryotes.

Alcohol Oxidoreductases↗
PubMed1991

Mannopine and mannopinic acid as substrates for Arthrobacter sp. strain MBA209 and Pseudomonas putida NA513.

The characteristics of mannopine and mannopinic acid utilization by Agrobacterium tumefaciens B6S3, Arthrobacter sp. strain MBA209, and Pseudomonas putida NA513 were studied. Strain B6S3 utilized the four mannityl opines, mannopine, mannopinic acid, agropine, and agropinic acid. It also utilized several mannityl opine analogs, which were modified in either the sugar or the amino acid moiety. It utilized mannopine more rapidly after preincubation on mannopine, mannopinic acid, or glutamine than after pregrowth on glucose, mannose, or mannitol. Strains MBA209 and NA513 utilized mannopine and mannopinic acid, but not the other two mannityl opines. They utilized few mannityl opine analogs, sometimes because of failure to utilize the products of initial cleavage of the analog. Utilization of mannopine and mannopinic acid by strain NA513 was strictly dependent on prior growth on these substrates. A spontaneous regulatory variant of strain NA513 remained unable to utilize most of the mannityl opine analogs. Glutamine, mannose, and several analogs had no inhibitory effect on [14C]mannopine utilization by strain NA513.

Arthrobacter↗
PubMed1992

Purification and properties of an Arthrobacter oxydans P52 carbamate hydrolase specific for the herbicide phenmedipham and nucleotide sequence of the corresponding gene.

Arthrobacter oxydans P52 isolated from soil samples was found to degrade the phenylcarbamate herbicides phenmedipham and desmedipham cometabolically by hydrolyzing their central carbamate linkages. The phenylcarbamate hydrolase (phenmedipham hydrolase) responsible for the degradative reaction was purified to homogeneity. The enzyme was shown to be a monomer with a molecular weight of 55,000. A 41-kb wild-type plasmid (pHP52) was identified in A. oxydans P52, but not in a derivative of this strain that had spontaneously lost the ability to hydrolyze phenylcarbamates, indicating that the gene for phenylcarbamate degradation (pcd) is plasmid encoded. Determination of two partial amino acid sequences allowed the localization of the coding sequence of the pcd gene on a 3.3-kb PstI restriction fragment within pHP52 DNA by hybridization with synthetic oligonucleotides. The phenylcarbamate hydrolase was functionally expressed in Escherichia coli under control of the lacZ promoter after the 3.3-kb PstI fragment was subcloned into the vector pUC19. A stretch of 1,864 bases within the cloned Pst fragment was sequenced. Sequence analysis revealed an open reading frame of 1,479 bases containing the amino acid partial sequences determined for the purified enzyme. Sequence comparisons revealed significant homology between the pcd gene product and the amino acid sequences of esterases of eukaryotic origin. Subsequently, it was demonstrated that the esterase substrate p-nitrophenylbutyrate is hydrolyzed by phenmedipham hydrolase.

Amino Acid Sequence↗
PubMed1993

A new lipopeptide biosurfactant produced by Arthrobacter sp. strain MIS38.

A biosurfactant termed arthrofactin produced by Arthrobacter species strain MIS38 was purified and chemically characterized as 3-hydroxydecanoyl-D-leucyl-D-asparagyl-D-threonyl-D- leucyl-D-leucyl-D-seryl-L-leucyl-D-seryl-L-isoleucyl-L-isoleucyl-L-as paragyl lactone. Surface activity of arthrofactin was examined, with surfactin as a control. Critical micelle concentration values of arthrofactin and surfactin were around 1.0 x 10(-5) M and 7.0 x 10(-5) M at 25 degrees C, respectively. Arthrofactin was found to be five to seven times more effective than surfactin. The minimum surface tension value of arthrofactin was 24 mN/m at a concentration higher than the critical micelle concentration. According to the oil displacement assay, arthrofactin was a better oil remover than synthetic surfactants, such as Triton X-100 and sodium dodecyl sulfate. Arthrofactin is one of the most effective lipopeptide biosurfactants.

Amino Acid Sequence↗
PubMed1994

Molecular cloning and expression of an isomalto-dextranase gene from Arthrobacter globiformis T6.

The gene encoding an extracellular isomalto-dextranase, designated imd, was isolated from the chromosomal DNA of Arthrobacter globiformis T6 and cloned and expressed in Escherichia coli. A single open reading frame consisting of 1,926 base pairs that encoded a polypeptide composed of a signal peptide of 39 amino acids and a mature protein of 602 amino acids (M(r), 65,900) was found. The primary structure had no significant homology with the structures of any other reported carbohydrases, including two other dextranases. Transformed E. coli cells carrying the 2.3-kb fragment overproduced isomalto-dextranase into the periplasmic space under control of the promoter of the imd gene itself.

Amino Acid Sequence↗
PubMed1995

A manganese-dependent dioxygenase from Arthrobacter globiformis CM-2 belongs to the major extradiol dioxygenase family.

Almost all bacterial ring cleavage dioxygenases contain iron as the catalytic metal center. We report here the first available sequence for a manganese-dependent 3,4-dihydroxyphenylacetate (3,4-DHPA) 2,3-dioxygenase and its further characterization. This manganese-dependent extradiol dioxygenase from Arthrobacter globiformis CM-2, unlike iron-dependent extradiol dioxygenases, is not inactivated by hydrogen peroxide. Also, ferrous ions, which activate iron extradiol dioxygenases, inhibit 3,4-DHPA 2,3-dioxygenase. The gene encoding 3,4-DHPA 2,3-dioxygenase, mndD, was identified from an A. globiformis CM-2 cosmid library. mndD was subcloned as a 2.0-kb SmaI fragment in pUC18, from which manganese-dependent extradiol dioxygenase activity was expressed at high levels in Escherichia coli. The mndD open reading frame was identified by comparison with the known N-terminal amino acid sequence of purified manganese-dependent 3,4-DHPA 2,3-dioxygenase. Fourteen of 18 amino acids conserved in members of the iron-dependent extradiol dioxygenase family are also conserved in the manganese-dependent 3,4-DHPA 2,3-dioxygenase (MndD). Thus, MndD belongs to the extradiol family of dioxygenases and may share a common ancestry with the iron-dependent extradiol dioxygenases. We propose the revised consensus primary sequence (G,T,N,R)X(H,A)XXXXXXX(L,I,V,M,F)YXX(D,E,T,N,A)PX(G,P) X(2,3)E for this family. (Numbers in brackets indicate a gap of two or three residues at this point in the sequence.) The suggested common ancestry is also supported by sequence obtained from genes flanking mndD, which share significant sequence identity with xylJ and xylG from Pseudomonas putida.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1995

Analysis of a novel gene and beta-galactosidase isozyme from a psychrotrophic Arthrobacter isolate.

We have characterized a new psychrotrophic Arthrobacter isolate which produces beta-galactosidase isozymes. When DNA from this isolate was transformed into an Escherichia coli host, we obtained three different fragments, designated 12, 14, and 15, each encoding a different beta-galactosidase isozyme. The beta-galactosidase produced from fragment 12 was of special interest because the protein subunit was smaller (about 71 versus 116 kDa) than those typically encoded by the lacZ family. The isozyme encoded by fragment 12 was purified, and its activity and thermostability were examined. Although the enzyme is highly specific towards beta-D-galactoside substrates, its levels in the isolate do not increase in cells grown with lactose. Nucleotide sequence determination showed that the gene encoding isozyme 12 is not similar to the other members of the lacZ family but has regions similar to beta-galactosidase isozymes from Bacillus stearothermophilus and B. circulans. Addition of the isozyme 12 sequence to the database made it possible to examine these enzymes as possible members of a new, separate family. Our analysis of this new family showed some conserved amino acids corresponding to the lacZ acid-base catalytic region but no homology with the nucleophilic region. On the basis of these comparisons, we designated this a new lacG family.

Amino Acid Sequence↗
PubMed1996

Cold shock and cold acclimation proteins in the psychrotrophic bacterium Arthrobacter globiformis SI55.

The psychrotrophic bacterium Arthrobacter globiformis SI55 was grown at 4 and 25 degrees C, and the cell protein contents were analyzed by two-dimensional electrophoresis. Cells subjected to cold shocks of increasing magnitude were also analyzed. Correspondence analysis of protein appearance distinguished four groups of physiological significance. Group I contained cold shock proteins (Csps) overexpressed only after a large temperature downshift. Group II contained Csps with optimal expression after mild shocks. Group III contained proteins overexpressed after all cold shocks. These last proteins were also overexpressed in cells growing at 4 degrees C and were considered to be early cold acclimation proteins (Caps). Group IV contained proteins which were present at high concentrations only in 4 degrees C steady-state cells and appeared to be late Caps. A portion of a gene very similar to the Escherichia coli cspA gene (encoding protein CS7.4) was identified. A synthetic peptide was used to produce an antibody which detected a CS7.4-like protein (A9) by immunoblotting two-dimensional electrophoresis gels of A. globiformis SI55 total proteins. Unlike mesophilic microorganisms, this CS7.4-like protein was still produced during prolonged growth at low temperature, and it might have a particular adaptive function needed for balanced growth under harsh conditions. However, A9 was induced at high temperature by chloramphenicol, suggesting that CS7.4-like proteins have a more general role than their sole implication in cold acclimation processes.

Acclimatization↗
PubMed1996

Genetic characterization of site-specific integration functions of phi AAU2 infecting "Arthrobacter aureus" C70.

All the essential genetic determinants for site-specific integration of corynephage phi AAU2 are contained within a 1,756-bp DNA fragment, carried on the integrative plasmid p5510, and are shown to be functional in Escherichia coli. One open reading frame, ORF4, encoding a protein of 266 amino acids was shown to represent the phi AAU2 integrase. The nucleotide sequence of the phi AAU2 attachment site, attP, and the attB, attL, and attR sequences in the host "Arthrobacter aureus" C70 were determined. Identical nucleotide sequences were shown to be responsible for the integration of p5510 in the chromosomes of Corynebacterium glutamicum, Brevibacterium divaricatum, and B. lactofermentum, and a sequence almost identical to attB was found to be present in these three strains. In contrast to other phage site-specific recombination systems, a plasmid encompassing only int-attP failed to integrate into the host chromosome. This led to the identification of an 800-bp noncoding region, immediately upstream of int, absolutely required for site-specific integration of p5510.

Amino Acid Sequence↗
PubMed1997

capA, a cspA-like gene that encodes a cold acclimation protein in the psychrotrophic bacterium Arthrobacter globiformis SI55.

By use of Arthrobacter globiformis SI55, a psychrotrophic bacterium capable of growth between -5 and +32 degrees C, we cloned and sequenced capA, a gene homologous to cspA encoding the major cold shock protein in Escherichia coli. The deduced protein sequence has a high level of identity with the sequences of other CspA-related proteins from various sources, and no particular residue or domain that could be specific to cold-adapted microorganisms emerged. We show that CapA was produced very rapidly following cold shock, but unlike its mesophilic counterparts, it was still expressed during prolonged growth at low temperature. Its synthesis is regulated at the translational level, and we showed that growth resumption following a temperature downshift correlated with CapA expression. Transient inhibitions in protein synthesis during the first stages of the cold shock response severely impaired the subsequent acclimation of A. globiformis SI55 to low temperature and delayed CapA expression. The cold shock response in A. globiformis SI55 is an adaptative process in which CapA may play a crucial role. We suggest that low-temperature acclimation is conditioned mainly by the ability of cells to restore an active translational machinery after cold shock in a process that may be different from that present in mesophiles.

Arthrobacter↗
PubMed1997

A novel Arthrobacter beta-galactosidase with homology to eucaryotic beta-galactosidases.

An Arthrobacter beta-galactosidase has homology with the lysosomal acid beta-galactosidases from humans and mice and with a Xanthomonas manihotis enzyme. Phylogenetic analysis of the deduced amino acid sequence showed an unusual pattern, with this procaryotic enzyme clustering within the animal clade. The gene encodes a subunit of 52 kDa, and the enzyme appears to be active as a dimer. The enzyme hydrolyzed substrates with either a beta-1,4 or a beta-1,3 linkage.

Amino Acid Sequence↗
PubMed2001

Plasmid-encoded phthalate catabolic pathway in Arthrobacter keyseri 12B.

Several 2-substituted benzoates (including 2-trifluoromethyl-, 2-chloro-, 2-bromo-, 2-iodo-, 2-nitro-, 2-methoxy-, and 2-acetyl-benzoates) were converted by phthalate-grown Arthrobacter keyseri (formerly Micrococcus sp.) 12B to the corresponding 2-substituted 3,4-dihydroxybenzoates (protocatechuates). Because these products lack a carboxyl group at the 2 position, they were not substrates for the next enzyme of the phthalate catabolic pathway, 3,4-dihydroxyphthalate 2-decarboxylase, and accumulated. When these incubations were carried out in iron-containing minimal medium, the products formed colored chelates. This chromogenic response was subsequently used to identify recombinant Escherichia coli strains carrying genes encoding the responsible enzymes, phthalate 3,4-dioxygenase and 3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase, from the 130-kbp plasmid pRE1 of strain 12B. Beginning with the initially cloned 8.14-kbp PstI fragment of pRE824 as a probe to identify recombinant plasmids carrying overlapping fragments, a DNA segment of 33.5 kbp was cloned from pRE1 on several plasmids and mapped using restriction endonucleases. From these plasmids, the sequence of 26,274 contiguous bp was determined. Sequenced DNA included several genetic units: tnpR, pcm operon, ptr genes, pehA, norA fragment, and pht operon, encoding a transposon resolvase, catabolism of protocatechuate (3,4-dihydroxybenzoate), a putative ATP-binding cassette transporter, a possible phthalate ester hydrolase, a fragment of a norfloxacin resistance-like transporter, and the conversion of phthalate to protocatechuate, respectively. Activities of the eight enzymes involved in the catabolism of phthalate through protocatechuate to pyruvate and oxaloacetate were demonstrated in cells or cell extracts of recombinant E. coli strains.

Amino Acid Sequence↗
PubMed2001

Gene cluster on pAO1 of Arthrobacter nicotinovorans involved in degradation of the plant alkaloid nicotine: cloning, purification, and characterization of 2,6-dihydroxypyridine 3-hydroxylase.

A 27,690-bp gene cluster involved in the degradation of the plant alkaloid nicotine was characterized from the plasmid pAO1 of Arthrobacter nicotinovorans. The genes of the heterotrimeric, molybdopterin cofactor (MoCo)-, flavin adenine dinucleotide (FAD)-, and [Fe-S] cluster-dependent 6-hydroxypseudooxynicotine (ketone) dehydrogenase (KDH) were identified within this cluster. The gene of the large MoCo subunit of KDH was located 4,266 bp from the FAD and [Fe-S] cluster subunit genes. Deduced functions of proteins encoded by open reading frames (ORFs) of the cluster were correlated to individual steps in nicotine degradation. The gene for 2,6-dihydroxypyridine 3-hydroxylase was cloned and expressed in Escherichia coli. The purified homodimeric enzyme of 90 kDa contained 2 mol of tightly bound FAD per mol of dimer. Enzyme activity was strictly NADH-dependent and specific for 2,6-dihydroxypyridine. 2,3-Dihydroxypyridine and 2,6-dimethoxypyridine acted as irreversible inhibitors. Additional ORFs were shown to encode hypothetical proteins presumably required for holoenzyme assembly, interaction with the cell membrane, and transcriptional regulation, including a MobA homologue predicted to be specific for the synthesis of the molybdopterin cytidine dinucleotide cofactor.

Amino Acid Sequence↗
PubMed2003

Biochemical characterization of a beta-galactosidase with a low temperature optimum obtained from an Antarctic arthrobacter isolate.

A psychrophilic gram-positive isolate was obtained from Antarctic Dry Valley soil. It utilized lactose, had a rod-coccus cycle, and contained lysine as the diamino acid in its cell wall. Consistent with these physiological traits, the 16S ribosomal DNA sequence showed that it was phylogenetically related to other Arthrobacter species. A gene (bgaS) encoding a family 2 beta-galactosidase was cloned from this organism into an Escherichia coli host. Preliminary results showed that the enzyme was cold active (optimal activity at 18 degrees C and 50% activity remaining at 0 degrees C) and heat labile (inactivated within 10 min at 37 degrees C). To enable rapid purification, vectors were constructed adding histidine residues to the BgaS enzyme and its E. coli LacZ counterpart, which was purified for comparison. The His tag additions reduced the specific activities of both beta-galactosidases but did not alter the other characteristics of the enzymes. Kinetic studies using o-nitrophenyl-beta-D-galactopyranoside showed that BgaS with and without a His tag had greater catalytic activity at and below 20 degrees C than the comparable LacZ beta-galactosidases. The BgaS heat lability was investigated by ultracentrifugation, where the active enzyme was a homotetramer at 4 degrees C but dissociated into inactive monomers at 25 degrees C. Comparisons of family 2 beta-galactosidase amino acid compositions and modeling studies with the LacZ structure did not mimic suggested trends for conferring enzyme flexibility at low temperatures, consistent with the changes affecting thermal adaptation being localized and subtle. Mutation studies of the BgaS enzyme should aid our understanding of such specific, localized changes affecting enzyme thermal properties.

Antarctic Regions↗
PubMed2003

Sequence of the 165-kilobase catabolic plasmid pAO1 from Arthrobacter nicotinovorans and identification of a pAO1-dependent nicotine uptake system.

The 165-kb catabolic plasmid pAO1 enables the gram-positive soil bacterium Arthrobacter nicotinovorans to grow on the tobacco alkaloid L-nicotine. The 165,137-nucleotide sequence, with an overall G+C content of 59.7%, revealed, besides genes and open reading frames (ORFs) for nicotine degradation, a complete set of ORFs for enzymes essential for the biosynthesis of the molybdenum dinucleotide cofactor, as well as ORFs related to uptake and utilization of carbohydrates, sarcosine, and amino acids. Of the 165 ORFs, approximately 50% were related to metabolic functions. pAO1 conferred to A. nicotinovorans the ability to take up L-[(14)C]nicotine from the medium, with an K(m) of 5.6 +/- 2.2 micro M. ORFs of putative nicotine transporters formed a cluster with the gene of the D-nicotine-specific 6-hydroxy-D-nicotine oxidase. ORFs related to replication, chromosome partitioning, and natural transformation functions (dprA) were identified on pAO1. Few ORFs showed similarity to known conjugation-promoting proteins, but pAO1 could be transferred by conjugation to a pAO1-negative strain at a rate of 10(-2) to 10(-3) per donor. ORFs with no known function represented approximately 35% of the pAO1 sequence. The positions of insertion sequence elements and composite transposons, corroborated by the G+C content of the pAO1 sequence, suggest a modular composition of the plasmid.

Arthrobacter↗
PubMed2005

An alpha/beta-fold C--C bond hydrolase is involved in a central step of nicotine catabolism by Arthrobacter nicotinovorans.

The enzyme catalyzing the hydrolytic cleavage of 2,6-dihydroxypseudooxynicotine to 2,6-dihydroxypyridine and gamma-N-methylaminobutyrate was found to be encoded on pAO1 of Arthrobacter nicotinovorans. The new enzyme answers an old question about nicotine catabolism and may be the first C--C bond hydrolase that is involved in the biodegradation of a heterocyclic compound.

Alkaloids↗
PubMed2005

Characterization of PmfR, the transcriptional activator of the pAO1-borne purU-mabO-folD operon of Arthrobacter nicotinovorans.

Nicotine catabolism by Arthrobacter nicotinovorans is linked to the presence of the megaplasmid pAO1. Genes involved in this catabolic pathway are arranged on the plasmid into gene modules according to function. During nicotine degradation gamma-N-methylaminobutyrate is formed from the pyrrolidine ring of nicotine. Analysis of the pAO1 open reading frames (ORF) resulted in identification of the gene encoding a demethylating gamma-N-methylaminobutyrate oxidase (mabO). This gene was shown to form an operon with purU- and folD-like genes. Only in bacteria grown in the presence of nicotine could transcripts of the purU-mabO-folD operon be detected, demonstrating that this operon constitutes part of the pAO1 nicotine regulon. Its transcriptional start site was determined by primer extension analysis. Transcription of the operon was shown to be controlled by a new transcriptional regulator, PmfR, the product of a gene that is transcribed divergently from the purU, mabO, and folD genes. PmfR was purified, and electromobility shift assays and DNase I-nuclease digestion experiments were used to determine that its DNA binding site is located between -48 and -88 nucleotides upstream of the transcriptional start site of the operon. Disruption of pmfR by homologous recombination with a chloramphenicol resistance cassette demonstrated that PmfR acts in vivo as a transcriptional activator. Mutagenesis of the PmfR target DNA suggested that the sequence GTTT-14 bp-AAAC is the core binding site of the regulator upstream of the -35 promoter region of the purU-mabO-folD operon.

Amino Acid Sequence↗