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.     

A novel gamma-N-methylaminobutyrate demethylating oxidase involved in catabolism of the tobacco alkaloid nicotine by Arthrobacter nicotinovorans pAO1.

Nicotine catabolism, linked in Arthrobacter nicotinovorans to the presence of the megaplasmid pAO1, leads to the formation of gamma-N-methylaminobutyrate from the pyrrolidine ring of the alkaloid. Until now the metabolic fate of gamma-N-methylaminobutyrate has been unknown. pAO1 carries a cluster of ORFs with similarity to sarcosine and dimethylglycine dehydrogenases and oxidases, to the bifunctional enzyme methylenetetrahydrofolate dehydrogenase/cyclohydrolase and to formyltetrahydrofolate deformylase. We cloned and expressed the gene carrying the sarcosine dehydrogenase-like ORF and showed, by enzyme activity, spectrophotometric methods and identification of the reaction product as gamma-aminobutyrate, that the predicted 89 395 Da flavoprotein is a demethylating gamma-N-methylaminobutyrate oxidase. Site-directed mutagenesis identified His67 as the site of covalent attachment of FAD and confirmed Trp66 as essential for FAD binding, for enzyme activity and for the spectral properties of the wild-type enzyme. A Km of 140 microm and a kcat of 800 s(-1) was determined when gamma-N-methylaminobutyrate was used as the substrate. Sarcosine was also turned over by the enzyme, but at a rate 200-fold slower than gamma-N-methylaminobutyrate. This novel enzyme activity revealed that the first step in channelling the gamma-N-methylaminobutyrate generated from nicotine into the cell metabolism proceeds by its oxidative demethylation.     

pAO1 of Arthrobacter nicotinovorans and the Spread of Catabolic Traits by Horizontal Gene Transfer in Gram-Positive Soil Bacteria

The 165-kb megaplasmid pAO1 of Arthrobacter nicotinovorans carries two large gene clusters, one involved in nicotine catabolism (nic-gene cluster) and one in carbohydrate utilization (ch-gene cluster). Here, we propose that both gene clusters were acquired by A. nicotinovorans by horizontal gene transfer mediated by pAO1. Protein–protein blast search showed that none of the published Arthrobacter genomes contains nic-genes, but Rhodococcus opacus carries on its chromosome a nic-gene cluster highly similar to that of pAO1. Analysis of the nic-genes in the two species suggested a recombination event between their nic-gene clusters. Apparently, there was a gene exchange between pAO1, or a precursor plasmid, and a nic-gene cluster of an as yet unidentified Arthrobacter specie or other soil bacterium, possibly related to Rhodococcus, leading to the transfer by pAO1 of this catabolic trait to A. nicotinovorans. Analysis of the pAO1 ch-gene cluster revealed a virtually identical counterpart on the chromosome of Arthrobacter phenanthrenivorans. Moreover, the sequence analysis of the genes flanking the ch-gene cluster suggested that it was acquired by pAO1 by Xer-related site directed recombination and transferred via the plasmid to A. nicotinovorans. The G+C content, the level of sequence identity, gene co-linearity of nic- and ch-gene clusters as well as the signs of recombination events clearly supports the notion of pAO1 and its precursor plasmids as vehicles in HGT among Gram + soil bacteria.     

A pAO1-encoded molybdopterin cofactor gene (moaA) ofArthrobacter nicotinovorans: characterization and site-directed mutagenesis of the encoded protein

A gene homologous tomoaA, the gene responsible for the expression of a protein involved in an early step in the synthesis of the molybdopterin cofactor ofEscherichia coli, was found to be located 2.7-kb upstream of the nicotine dehydrogenase (ndh) operon on the catabolic plasmid pAO1 ofArthrobacter nicotinovorans. The MoaA protein, containing 354 amino acids, migrated on an SDS-polyacrylamide gel with an apparent molecular weight of 40,000, in good agreement with the predicted molecular weight of 38,880. The pAO1-encodedmoaA gene fromA. nicotinovorans was expressed inE. coli as an active protein that functionally complementedmoaA mutants. Its reduced amino acid sequence shows 43% identity to theE. coli MoaA, 44% to the NarAB gene product fromBacillus subtilis, and 42% to the gene product of two contiguous ORFs fromMethanobacterium formicicum. N-terminal sequences, including the motif CxxxCxYC, are conserved among the MoaA and NarAB proteins. This motif is also present in proteins involved in PQQ cofactor synthesis in almost all the NifB proteins reported so far and in thefixZ gene product fromRhizobium leguminosarum. Mutagenesis of any of these three conserved cysteine residues to serine abolished the biological activity of MoaA, while substitution of the tyrosine by either serine, phenylalanine, or alanine did not alter the capacity of the protein to complement themoaA mutation inE. coli. A second Cys-rich domain with the motif FCxxC(13x)C is found close to the C-terminus of MoaA and NarAB proteins. These two Cys-rich sequences may be involved in the coordination of a metal ions. The pAO1 copy ofmoaA may not be unique in theA. nicotinovorans genome since the molybdopterin cofactor oxidation products were detected in cell extracts from a plasmidless strain.     

Interaction of the regulatory protein NicR1 with the promoter region of the pAO1-encoded 6-hydroxy-D-nicotine oxidase gene of Arthrobacter oxidans

The D,L-nicotine catabolism of the Gram-positive soil bacterium Arthrobacter oxidans is linked to the presence within the cells of the 160 kb catabolic plasmid pAO1. pAO1-cured cells lost the catabolic enzymes and reintroduction of pAO1 by electroporation into cured cells reestablished the nic+ phenotype. DNA band shift assays with extracts from cured and pAO1+ cells suggested that pAO1 encodes the regulatory protein NicR1. Footprint analysis revealed that two homologous palindromes (IR1 and IR2), present in the 5'-regulatory region of the 6-HDNO gene, were protected from DNase I digestion. Binding of NicR1 to the palindromes is symmetrical, co-operative, and stronger to IR1 containing the 6-HDNO gene promoter than to IR2. Site-directed mutagenesis revealed that steric constraints and sequence requirements for NicR1-binding are located exclusively in the palindromic sequences. Deletions and insertions in the interpalindromic region and in the 6-HDNO promoter -10 sequence had no effect on the binding characteristics of NicR1 to the 6-HDNO regulatory region. Acting as a repressor, NicR1 prevents binding of the E. coli RNA-polymerase to the consensus sigma 70 promoter in vitro. However, the interaction of NicR1 with the 6-HDNO promoter region in extracts of nicotine-induced cells from various growth stages did not differ from that observed with extracts of nicotine-uninduced cells.     

Plasmids for nicotine-dependent and -independent gene expression in Arthrobacter nicotinovorans and other arthrobacter species

The first inducible Arthrobacter overexpression system, based on the promoter/operator and the repressor of the 6-D-hydroxynicotine oxidase gene of Arthrobacter nicotinovorans, is described here. Nicotine-dependent overproduction and affinity purification of recombinant proteins are presented. The system will allow the production of complex enzymes and genetic complementation studies in Arthrobacter species.     

Plasmid pAO1 of Arthrobacter oxidans encodes 6-hydroxy-D-nicotine oxidase: cloning and expression of the gene in Escherichia coli

Summary The 160 kb plasmid pAO1 from Arthrobacter oxidans (Brandsch and Decker 1984) was subcloned in Escherichia coli with the aid of the plasmid vectors pUR222 and pBR322. Screening of the recombinant clones for enzyme activity revealed that the flavoenzyme 6-hydroxy-d-nicotine oxidase (6-HDNO), one of the enzymes of the nicotine-degradative pathway in A. oxidans, is encoded on pAO1. Immunoprecipitation of ³⁵S-methionine-labelled E. coli cells with 6-HDNO-specific antiserum and expression of recombinant plasmid DNA in E. coli maxicells revealed that 6-HDNO is made as a 52,000 dalton protein, approximately 4,500 daltons larger than 6-HDNO from A. oxidans. The 6-HDNO activity was constitutively expressed in E. coli cells, possibly from an A. oxidans promoter, as shown by subcloning of the 6-HDNO gene in pBR322, using the expression vector pKK223-3 and the promoter probe vector pCB192.     

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.     

Reclassification of two strains of Arthrobacter oxydans and proposal of Arthrobacter nicotinovorans sp. nov.

Arthrobacter oxydans DSM 419 and DSM 420 have chemical and microbiological properties that are consistent with assignment to the genus Arthrobacter. Both organisms have the lysine-alanine-threonine-alanine peptidoglycan type. DNA-DNA pairing studies indicated that A. oxydans DSM 419 should be reclassified as Arthrobacter ureafaciens and that A. oxydans DSM 420T forms the nucleus of a distinct genomic species. We propose that A. oxydans DSM 420 should be reclassified as Arthrobacter nicotinovorans sp. nov. The type strain is strain DSM 420.     

A functional mobA gene for molybdopterin cytosine dinucleotide cofactor biosynthesis is required for activity and holoenzyme assembly of the heterotrimeric nicotine dehydrogenases of Arthrobacter nicotinovorans

Two Arthrobacter nicotinovorans molybdenum enzymes hydroxylate the pyridine ring of nicotine. Molybdopterin cytosine dinucleotide (MCD) was determined to be a cofactor of these enzymes. A mobA gene responsible for the formation of MCD could be identified and its function shown to be required for assembly of the heterotrimeric molybdenum enzymes.     

Functional analysis through site-directed mutations and phylogeny of the Candida albicans LYS1-encoded saccharopine dehydrogenase

Candida albicans LYS1-encoded saccharopine dehydrogenase (CaLys1p, SDH) catalyzes the final biosynthetic step (saccharopine to lysine + α-ketoglutarate) of the novel α-aminoadipate pathway for lysine synthesis in fungi. The reverse reaction catalyzed by lysine-α-ketoglutarate reductase (LKR) is used exclusively in animals and plants for the catabolism of excess lysine. The 1,146 bp C. albicans LYS1 ORF encodes a 382 amino acid SDH. In the present investigation, we have used E. coli-expressed recombinant C. albicans Lys1p for the determination of both forward and reverse SDH activities in vitro, compared the sequence identity of C. albicans Lys1p with other known SDHs and LKRs, performed extensive site-directed mutational analyses of conserved amino acid residues and analyzed the phylogenetic relationship of C. albicans Lys1p to other known SDHs and LKRs. We have identified 14 of the 68 amino acid substitutions as essential for C. albicans Lys1p SDH activity, including two highly conserved functional motifs, H93XXF96XH98 and G138XXXG142XXG145. These results provided new insight into the functional and phylogenetic characteristics of the distinct biosynthetic SDH in fungi and catabolic LKR in higher eukaryotes.     

Cloning and expression of the Arthrobacter globiformis fcb genes in Bacillus subtilis]

The fcb genes of Arthrobacter globiformis KZT1 coding for the dehalogenase (4-chlorobenzoate-4-hydroxylase) activity have been cloned. The characteristics of fcb genes expression have been studied. The recombinant strains of Bacillus subtilis 6JM15 (pCBS 311) and 6JM15 (pCBS1) have shown the decreased level of substrate dehalogenation as compared with the one in the parent strain KZT1 and the recombinant strains of Escherichia coli and Pseudomonas putida.     

Purification and characterization of aminopropionaldehyde dehydrogenase from Arthrobacter sp. TMP-1

Aminopropionaldehyde dehydrogenase was purified to apparent homogeneity from 1,3-diaminopropane-grown cells of Arthrobacter sp. TMP-1. The native molecular mass and the subunit molecular mass of the enzyme were approximately 20,5000 and 52,000, respectively, suggesting that the enzyme is a tetramer of identical subunits. The apparent Michaelis constant (K(m)) for 1,3-diaminopropane was approximately 3 microM. The enzyme equally used both NAD(+) and NADP(+) as coenzymes. The apparent K(m) values for NAD(+) and NADP(+) were 255 microM and 108 microM, respectively. The maximum reaction rates (V(max)) for NAD(+) and NADP(+) were 102 and 83.3 micromol min(-1) mg(-1), respectively. Some tested aliphatic aldehydes and aromatic aldehydes were inert as substrates. The optimum pH was 8.0-8.5. The enzyme was sensitive to sulfhydryl group-modifying reagents.