NORMAL GENOMIC

• Vitreschak AG, Mironov AA, Lyubetsky VA, Gelfand MS
• Comparative genomic analysis of T-box regulatory systems in bacteria.
• RNA. 2008; 14: 717-35
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• T-box antitermination is one of the main mechanisms of regulation of genesinvolved in amino acid metabolism in Gram-positive bacteria. T-boxregulatory sites consist of conserved sequence and RNA secondary structureelements. Using a set of known T-box sites, we constructed the commonpattern and used it to scan available bacterial genomes. New T-boxes werefound in various Gram-positive bacteria, some Gram-negative bacteria(delta-proteobacteria), and some other bacterial groups(Deinococcales/Thermales, Chloroflexi, Dictyoglomi). The majority ofT-box-regulated genes encode aminoacyl-tRNA synthetases. Two other groupsof T-box-regulated genes are amino acid biosynthetic genes andtransporters, as well as genes with unknown function. Analysis ofcandidate T-box sites resulted in new functional annotations. We assignedthe amino acid specificity to a large number of candidate amino acidtransporters and a possible function to amino acid biosynthesis genes. Wethen studied the evolution of the T-boxes. Analysis of the constructedphylogenetic trees demonstrated that in addition to the normal evolutionconsistent with the evolution of regulated genes, T-boxes may beduplicated, transferred to other genes, and change specificity. Weobserved several cases of recent T-box regulon expansion following theloss of a previously existing regulatory system, in particular, arginineregulon in Clostridium difficile and methionine regulon inLactobacillaceae. Finally, we described a new structural class of T-boxescontaining duplicated terminator-antiterminator elements and unusualreduced T-boxes regulating initiation of translation in theActinobacteria.

• Rodionov DA, Vitreschak AG, Mironov AA, Gelfand MS
• Comparative genomics of the methionine metabolism in Gram-positivebacteria: a variety of regulatory systems.
• Nucleic Acids Res. 2004; 32: 3340-53
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• Regulation of the methionine biosynthesis and transport genes in bacteriais rather diverse and involves two RNA-level regulatory systems and atleast three DNA-level systems. In particular, the methionine metabolism inGram-positive bacteria was known to be controlled by the S-box and T-boxmechanisms, both acting on the level of premature termination oftranscription. Using comparative analysis of genes, operons and regulatoryelements, we described the methionine metabolic pathway and the methionineregulons in available genomes of Gram-positive bacteria. A large number ofmethionine-specific RNA elements were identified. S-boxes were shown to bewidely distributed in Bacillales and Clostridia, whereasmethionine-specific T-boxes occurred mostly in Lactobacillales. Acandidate binding signal (MET-box) for a hypothetical methionineregulator, possibly MtaR, was identified in Streptococcaceae, the onlyfamily in the Bacillus/Clostridium group of Gram-positive bacteria havingneither S-boxes, nor methionine-specific T-boxes. Positional analysis ofmethionine-specific regulatory sites complemented by genome contextanalysis lead to identification of new members of the methionine regulon,both enzymes and transporters, and reconstruction of the methioninemetabolism in various bacterial genomes. In particular, we found candidatetransporters for methionine (MetT) and methylthioribose (MtnABC), as wellas new enzymes forming the S-adenosylmethionine recycling pathway.Methionine biosynthetic enzymes in various bacterial species are quitevariable. In particular, Oceanobacillus iheyensis possibly uses a homologof the betaine-homocysteine methyltransferase bhmT gene from vertebratesto substitute missing bacterial-type methionine synthases.

• Nahvi A, Barrick JE, Breaker RR
• Coenzyme B12 riboswitches are widespread genetic control elements inprokaryotes.
• Nucleic Acids Res. 2004; 32: 143-50
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• Recent studies have begun to reveal that numerous fundamental metabolicpathways in bacteria are regulated by riboswitches residing within certainmessenger RNAs. These riboswitches selectively bind metabolites andmodulate gene expression in response to changing ligand concentrations.Previously, we provided evidence that the btuB mRNAs of Escherichia coliand Salmonella typhimurium each carry a coenzyme B12-dependent riboswitchthat causes repressed translation of the encoded cobalamin-transportprotein at elevated coenzyme concentrations. Herein, we use a phylogeneticanalysis to define a consensus sequence and secondary structure model forthe ligand- binding domain of this riboswitch class. RNA structures thatconform to this model are widespread in both Gram-positive andGram-negative organisms. In addition, we find that the 5'-untranslatedregion (5'-UTR) of the cobalamin biosynthesis (cob) operon ofS.typhimurium carries an RNA motif that matches this consensus sequence.Biochemical and genetic characterization of this motif confirms that theRNA directly binds coenzyme B12, and that it likely serves as a geneticcontrol element for regulating expression of the 25-gene operon forcobalamin production in this pathogen.

• Vitreschak AG, Rodionov DA, Mironov AA, Gelfand MS
• Regulation of the vitamin B12 metabolism and transport in bacteria by aconserved RNA structural element.
• RNA. 2003; 9: 1084-97
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• Cobalamin in the form of adenosylcobalamin (Ado-CBL) is known to repressexpression of genes for vitamin B(12) biosynthesis and be transported by aposttranscriptional regulatory mechanism, which involves direct binding ofAdo-CBL to 5'untranslated gene regions (5'UTR). Using comparative analysisof genes and regulatory regions, we identified a highly conserved RNAstructure, the B12-element, which is widely distributed in 5'UTRs ofvitamin B(12)-related genes in eubacteria. Multiple alignment ofapproximately 200 B12-elements from 66 bacterial genomes reveals theircommon secondary structure and several extended regions of sequenceconservation, including the previously known B12-box motif. In analogy tothe model of regulation of the riboflavin and thiamin biosynthesis, wesuggest Ado-CBL-mediated regulation based on formation of alternative RNAstructures including the B12-element. In Gram-negative proteobacteria, aswell as in cyanobacteria, actinobacteria, and the CFB group, the cobalaminbiosynthesis and vitamin B(12) transport genes are predicted to beregulated by inhibition of translation initiation, whereas in theBacillus/Clostridium group of Gram-positive bacteria, these genes seem tobe regulated by transcriptional antitermination. Phylogenetic analysis ofthe B12-elements reveals a large number of likely duplications ofB12-elements in several bacterial genomes. These lineage-specificduplications of RNA regulatory elements seem to be a major evolutionarymechanism for expansion of the vitamin B(12) regulon.

• Rodionov DA, Vitreschak AG, Mironov AA, Gelfand MS
• Regulation of lysine biosynthesis and transport genes in bacteria: yetanother RNA riboswitch?
• Nucleic Acids Res. 2003; 31: 6748-57
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• Comparative analysis of genes, operons and regulatory elements was appliedto the lysine biosynthetic pathway in available bacterial genomes. Wereport identification of a lysine-specific RNA element, named the LYSelement, in the regulatory regions of bacterial genes involved inbiosynthesis and transport of lysine. Similarly to the previouslydescribed RNA regulatory elements for three vitamins (riboflavin, thiaminand cobalamin), purine and methionine regulons, this regulatory RNAstructure is highly conserved on the sequence and structural levels. TheLYS element includes regions of lysine-constitutive mutations previouslyidentified in Escherichia coli and Bacillus subtilis. A possible mechanismof the lysine-specific riboswitch is similar to the previously definedmechanisms for the other metabolite-specific riboswitches and involveseither transcriptional or translational attenuation in various groups ofbacteria. Identification of LYS elements in Gram-negativegamma-proteobacteria, Gram-positive bacteria from the Bacillus/Clostridiumgroup, and Thermotogales resulted in description of the previouslyuncharacterized lysine regulon in these bacterial species. Positionalanalysis of LYS elements led to identification of a number of newcandidate lysine transporters, namely LysW, YvsH and LysXY. Finally, themost likely candidates for genes of lysine biosynthesis missing in Gram-positive bacteria were identified using the genome context analysis.

• Rodionov DA, Vitreschak AG, Mironov AA, Gelfand MS
• Comparative genomics of thiamin biosynthesis in procaryotes. New genes andregulatory mechanisms.
• J Biol Chem. 2002; 277: 48949-59
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• Vitamin B(1) in its active form thiamin pyrophosphate is an essentialcoenzyme that is synthesized by coupling of pyrimidine(hydroxymethylpyrimidine; HMP) and thiazole (hydroxyethylthiazole)moieties in bacteria. Using comparative analysis of genes, operons, andregulatory elements, we describe the thiamin biosynthetic pathway inavailable bacterial genomes. The previously detected thiamin-regulatoryelement, thi box (Miranda-Rios, J., Navarro, M., and Soberon, M. (2001)Proc. Natl. Acad. Sci. U. S. A. 98, 9736-9741), was extended, resulting ina new, highly conserved RNA secondary structure, the THI element, which iswidely distributed in eubacteria and also occurs in some archaea. Searchfor THI elements and analysis of operon structures identified a largenumber of new candidate thiamin-regulated genes, mostly transporters, invarious prokaryotic organisms. In particular, we assign the thiamintransporter function to yuaJ in the Bacillus/Clostridium group and the HMPtransporter function to an ABC transporter thiXYZ in some proteobacteriaand firmicutes. By analogy to the model of regulation of the riboflavinbiosynthesis, we suggest thiamin-mediated regulation based on formation ofalternative RNA structures involving the THI element. Eithertranscriptional or translational attenuation mechanism may operate indifferent taxonomic groups, dependent on the existence of putativehairpins that either act as transcriptional terminators or sequestertranslation initiation sites. Based on analysis of co-occurrence of thethiamin biosynthetic genes in complete genomes, we predict thateubacteria, archaea, and eukaryota have different pathways for the HMP andhydroxyethylthiazole biosynthesis.

• Roessner CA, Huang KX, Warren MJ, Raux E, Scott AI
• Isolation and characterization of 14 additional genes specifying theanaerobic biosynthesis of cobalamin (vitamin B12) in Propionibacteriumfreudenreichii (P. shermanii).
• Microbiology. 2002; 148: 1845-53
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• A search for genes encoding enzymes involved in cobalamin (vitamin B12)production in the commercially important organism Propionibacteriumfreudenreichii (P. shermanii) has resulted in the isolation of anadditional 14 genes encoding enzymes responsible for 17 steps of theanaerobic B12 pathway in this organism. All of the genes believed to benecessary for the biosynthesis of adenosylcobinamide from uroporphyrinogenIII have now been isolated except two (cbiA and an as yet unidentifiedgene encoding cobalt reductase). Most of the genes are contained in twodivergent operons, one of which, in turn, is closely linked to the operonencoding the B12-dependent enzyme methylmalonyl-CoA mutase. The closelinkage of the three genes encoding the subunits of transcarboxylase tothe hemYHBXRL gene cluster is reported. The functions of the P.freudenreichii B12 pathway genes are discussed, and a mechanism for theregulation of cobalamin and propionic acid production by oxygen in thisorganism is proposed.

• Raux E, Schubert HL, Warren MJ
• Biosynthesis of cobalamin (vitamin B12): a bacterial conundrum.
• Cell Mol Life Sci. 2000; 57: 1880-93
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• The biosynthesis of cobalamin (vitamin B12) is described, revealing howthe concerted action of around 30 enzyme-mediated steps results in thesynthesis of one of Nature's most structurally complex 'small molecules'.The plethora of genome sequences has meant that bacteria capable ofcobalamin synthesis can be easily identified and their biosynthetic genescompared. Whereas only a few years ago cobalamin synthesis was thought tooccur by one of two routes, there are apparently a number of variations onthese two pathways, where the major differences seem to be concerned withthe process of ring contraction. A comparison of what is currently knownabout these pathways is presented. Finally, the process of cobaltchelation is discussed and the structure/function of the cobalt chelataseassociated with the oxygen-independent pathway (CbiK) is described.

• Nojiri M et al.
• Functional expression of nitrile hydratase in Escherichia coli:requirement of a nitrile hydratase activator and post-translationalmodification of a ligand cysteine.
• J Biochem. 1999; 125: 696-704
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• The nitrile hydratase (NHase) from Rhodococcus sp. N-771 is aphotoreactive enzyme that is inactivated on nitrosylation of the non-hemeiron center and activated on photo-dissociation of nitric oxide (NO). Thenitrile hydratase operon consists of six genes encoding NHase regulator 2,NHase regulator 1, amidase, NHase alpha subunit, NHase beta subunit andNHase activator. We overproduced the NHase in Escherichia coli using a T7expression system. The NHase was functionally expressed in E. coli onlywhen the NHase activator encoded downstream of the beta subunit gene wasco-expressed and the transformant was grown at 30 degrees C or less. Aligand cysteine, alphaCys112, of the recombinant NHase was alsopost-translationally modified to a cysteine-sulfinic acid similar to forthe native NHase. Although another modification of alphaCys114 could notbe identified because of the instability under acidic conditions, therecombinant NHase could be reversibly inactivated by nitric oxide.

• Mizunashi W, Nishiyama M, Horinouchi S, Beppu T
• Overexpression of high-molecular-mass nitrile hydratase from Rhodococcusrhodochrous J1 in recombinant Rhodococcus cells.
• Appl Microbiol Biotechnol. 1998; 49: 568-72
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• High-molecular-mass nitrile hydratase (H-NHase, 530 kDa) is acobalt-containing enzyme produced by Rhodococcus rhodochrous J1. Forefficient production of H-NHase in R. rhodochrous ATCC12674, severalplasmids were constructed. The enzyme was produced in the recombinantRhodococcus cells only in the presence of an upstream region(approximately 4 kb) of the H-NHase gene under the control of the promoterfor the amidase-NHase gene cluster from Rhodococcus sp. N-774. AlthoughH-NHase was produced as a soluble protein in the cells, the protein didnot show NHase activity. However, when the recombinant R. rhodochrousATCC12674 cells were cultured in the presence of amide compounds, such ascrotonamide and methacrylamide, markedly high NHase activity was detected,Gel-filtration chromatography revealed that the NHases produced by thecells grown in the presence and absence of the amide compounds had amolecular mass of more than 500 kDa and 50-80 kDa respectively. Theseresults suggest that the amide compounds are essential for subunitassembly to form an enzymatically active multimer. By the use of therecombinant expression system, NHase activity 1.7 times higher than thatof the original strain, R. rhodochrous J1, was achieved.

• Komeda H, Kobayashi M, Shimizu S
• A novel gene cluster including the Rhodococcus rhodochrous J1 nhlBA genesencoding a low molecular mass nitrile hydratase (L-NHase) induced by itsreaction product.
• J Biol Chem. 1996; 271: 15796-802
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• The 3.5 kilobases (kb) of the 5'-upstream region from nhlBA encoding acobalt-containing low molecular mass nitrile hydratase (L-NHase) fromRhodococcus rhodochrous J1 was found to be required for theamide-dependent expression of nhlBA in experiments using a Rhodococcustransformation system. Sequence analysis of the 3.5-kb fragment revealedthe presence of two open reading frames (nhlD and nhlC) in this fragment.NhlD has similarity to regulators MerR, CadC, and ArsR. NhlC hassimilarity to the regulators AmiC, for the expression of an aliphaticamidase from Pseudomonas aeruginosa, and NhhC, for the expression of ahigh molecular mass nitrile hydratase from R. rhodochrous J1. Assays ofNHase activity of transformants carrying nhlD deletion or nhlC deletionmutations suggest a negative regulatory role for nhlD and a positiveregulatory role for nhlC in the process of the L-NHase formation. Assaysof NHase and amidase activities and Western blot analyses of eachRhodococcus transformant carrying various deletion plasmids, have shownthat nhlBA and amdA encoding an amidase, which is located 1.9 kbdownstream of nhlBA, were regulated in the same manner. These findingspresent the genetic evidence for a novel gene cluster controlling theexpression of L-NHase, which is induced by the reaction product (amide) inthe "practical microorganism" R. rhodochrous J1.

• Komeda H, Kobayashi M, Shimizu S
• Characterization of the gene cluster of high-molecular-mass nitrilehydratase (H-NHase) induced by its reaction product in Rhodococcusrhodochrous J1.
• Proc Natl Acad Sci U S A. 1996; 93: 4267-72
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• The 4.6-kb region 5'-upstream from the gene encoding a cobalt-containingand amide-induced high molecular mass-nitrile hydratase (H-NHase) fromRhodococcus rhodochrous J1 was found to be required for the expression ofthe H-NHase gene with a host-vector system in a Rhodococcus strain.Sequence analysis has revealed that there are at least five open readingframes (H-ORF1 approximately 5) in addition to H-NHase alpha- andbeta-subunit genes. Deletion of H-ORF1 and H-ORF2 resulted in decrease ofNHase activity, suggesting a positive regulatory role of both ORFs in theexpression of the H-NHase gene. H-ORF1 showed significant similarity to aregulatory protein, AmiC, which is involved in regulation of amidaseexpression by binding an inducer amide in Pseudomonas aeruginosa. H-ORF4,which has been found to be uninvolved in regulation of H-NHase expressionby enzyme assay for its deletion transformant and Northern blot analysisfor R. rhodochrous J1, showed high similarity to transposases frominsertion sequences of several bacteria. Determination of H-NHase activityand H-NHase mRNA levels in R. rhodochrous J1 has indicated that theexpression of the H-NHase gene is regulated by an amide at thetranscriptional level. These findings suggest the participation of H-ORF4(IS1164) in the organization of the H-NHase gene cluster and theinvolvement of H-ORF1 in unusual induction mechanism, in which H-NHase isformed by amides (the products in the NHase reaction), but not by nitriles(the substrates).

• Roth JR, Lawrence JG, Rubenfield M, Kieffer-Higgins S, Church GM
• Characterization of the cobalamin (vitamin B12) biosynthetic genes ofSalmonella typhimurium.
• J Bacteriol. 1993; 175: 3303-16
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• Salmonella typhimurium synthesizes cobalamin (vitamin B12) de novo underanaerobic conditions. Of the 30 cobalamin synthetic genes, 25 areclustered in one operon, cob, and are arranged in three groups, each groupencoding enzymes for a biochemically distinct portion of the biosyntheticpathway. We have determined the DNA sequence for the promoter region andthe proximal 17.1 kb of the cob operon. This sequence includes 20translationally coupled genes that encode the enzymes involved in parts Iand III of the cobalamin biosynthetic pathway. A comparison of these geneswith the cobalamin synthetic genes from Pseudomonas denitrificans allowsassignment of likely functions to 12 of the 20 sequenced Salmonella genes.Three additional Salmonella genes encode proteins likely to be involved inthe transport of cobalt, a component of vitamin B12. However, not allSalmonella and Pseudomonas cobalamin synthetic genes have apparenthomologs in the other species. These differences suggest that thecobalamin biosynthetic pathways differ between the two organisms. Theevolution of these genes and their chromosomal positions is discussed.

• Duran R, Nishiyama M, Horinouchi S, Beppu T
• Characterization of nitrile hydratase genes cloned by DNA screening fromRhodococcus erythropolis.
• Biosci Biotechnol Biochem. 1993; 57: 1323-8
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• Southern hybridization analysis using the genes encoding the alpha- andbeta-subunits of nitrile hydratase (NHase) from Rhodococcus sp. N-774 asprobe suggested that two R. erythropolis strains, JCM6823 and JCM2892,among 31 strains mainly from Japan Culture of Microorganisms (JCM) haveNHase genes. Restriction analysis of DNA fragments showing positivehybridization showed that each fragment carried a nucleotide sequence verysimilar to that of the NHase genes from Rhodococcus sp. N-774. Nucleotidesequence analysis of the DNA fragment cloned from R. erythropolis JCM6823showed the presence of the genes encoding the alpha- and beta-subunits ofNHase, which show 94.7% and 96.2% identity in amino acid sequence to thoseof Rhodococcus sp. N-774, respectively, as well as a C-terminal portion ofthe amidase gene upstream from these genes. Despite the extremely highamino acid sequence similarity in both NHases and amidases from R.erythropolis JCM6823 and Rhodococcus sp. N-774, the NHases and amidasesfrom R. erythropolis strains showed broader substrate specificity whencompared to those from Rhodococcus sp. N-774. This suggests that a verylimited number of amino acid residues are responsible for the differencein substrate specificity. Although the NHase of Rhodococcus sp. N-774 areconstitutively produced, the NHases of both R. erythropolis strains wereinducibly produced by addition of epsilon-caprolactam as an inducer.

• Kobayashi M et al.
• Amidase coupled with low-molecular-mass nitrile hydratase from Rhodococcusrhodochrous J1. Sequencing and expression of the gene and purification andcharacterization of the gene product.
• Eur J Biochem. 1993; 217: 327-36
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• The cloned 9.4-kb insert of plasmid pNHJ20L containing low-molecular-massnitrile hydratase (L-NHase) gene from Rhodococcus rhodochrous J1[Kobayashi, M. et al. (1991) Biochim. Biophys. Acta 1129, 23-33] wasdigested with various restriction enzymes, and the trimmed fragments wereinserted into pUC18 or pUC19. A 1.96-kb EcoRI-SphI region located 1.9-kbdownstream of the L-NHase gene was found to be essential for theexpression of amidase activity in Escherichia coli; the gene arrangementof the amidase and the NHase in R. rhodochrous J1 differed from those inRhodococcus species including N-774 and Pseudomonas chlororaphis B23. Thenucleotide-determined sequence indicated that the amidase consists of 515amino acids (54626 Da) and the deduced amino acid sequence of the amidasehad high similarity to those of amidases from Rhodococcus speciesincluding N-774 and P. chlororaphis B23 and to indole-3-acetamidehydrolase from Pseudomonas savastanoi. The amidase gene modified in thenucleotide sequence upstream from its start codon expressed 8% of thetotal soluble protein in E. coli under the control of lac promoter. Thelevel of amidase activity in cell-free extracts of E. coli was 0.468unit/mg using benzamide as a substrate. This amidase was purified tohomogeneity from extracts of the E. coli transformant with 30.4% overallrecovery. The molecular mass of the enzyme estimated by HPLC was about 110kDa and the enzyme consists of two subunits identical in molecular mass(55 kDa). The enzyme acted upon aliphatic amides such as propionamide andalso upon aromatic amides such as benzamide. The apparent Km values forpropionamide and benzamide were 0.48 mM and 0.15 mM, respectively. Thisamidase was highly specific for the S-enantiomer of 2-phenylpropionamide,but could not recognize the configuration of 2-chloropropionamide. It alsocatalyzed the transfer of an acyl group from an amide to hydroxylamine toproduce the corresponding hydroxamate.

• Kobayashi M, Nishiyama M, Nagasawa T, Horinouchi S, Beppu T, Yamada H
• Cloning, nucleotide sequence and expression in Escherichia coli of twocobalt-containing nitrile hydratase genes from Rhodococcus rhodochrous J1.
• Biochim Biophys Acta. 1991; 1129: 23-33
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• Rhodococcus rhodochrous J1 produces two kinds of cobalt-containing nitrilehydratases (NHases); one is a high molecular mass-NHase (H-NHase) and theother is a low molecular mass-NHase (L-NHase). Both NHases are composed oftwo subunits of different sizes (alpha and beta subunits). The H- andL-NHase genes were cloned into Escherichia coli by a DNA-probing methodusing the NHase gene of Rhodococcus sp. N-774, a ferric ion-containingNHase producing strain, as the hybridization probe and their nucleotidesequences were determined. In each of the H- and L-NHase genes, the openreading frame for the beta subunit was located just upstream of that forthe alpha subunit, which probably belongs to the same operon. The aminoacid sequences of each subunit of the H- and L-NHases from R. rhodochrousJ1 showed generally significant similarities to those from Rhodococcus sp.N-774, but the arrangement of the coding sequences for two subunits isreverse of the order found in the NHase gene of Rhodococcus sp. N-774.Each of the NHase genes was expressed in E. coli cells under the controlof lac promoter, only when they were cultured in the medium supplementedwith CoCl2.

• Hashimoto Y, Nishiyama M, Ikehata O, Horinouchi S, Beppu T
• Cloning and characterization of an amidase gene from Rhodococcus speciesN-774 and its expression in Escherichia coli.
• Biochim Biophys Acta. 1991; 1088: 225-33
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• For investigation of an unknown open reading frame which is presentupstream of the nitrile hydratase (NHase) gene from Rhodococcus sp. N-774,a longer DNA fragment covering the entire gene was cloned in Escherichiacoli. Nucleotide sequencing and detailed subcloning experiments predicteda single open reading frame consisting of 521 amino acid residues of Mr54,671. The amino acid sequence, especially its NH2-terminal portion,showed significant homology with those of indoleacetamide hydrolases fromPseudomonas savastanoi and Agrobacterium tumefaciens, and acetamidase fromAspergillus nidulans. The 521-amino acid coding region was thereforeexpressed by use of the E. coli lac promoter in E. coli, and was found todirect a considerable amidase activity. This amidase hydrolyzedpropionamide efficiently, and also hydrolyzed, at a lower efficiency,acetamide, acrylamide and indoleacetamide. These data clearly show thatthe unknown open reading frame present upstream of the NHase coding regionencodes an amidase. Because the TAG translational stop codon of theamidase is located only 75 base pairs apart from the ATG start codon ofthe alpha-subunit of NHase, these genes are probably translated in apolycistronic manner.

• Nishiyama M, Horinouchi S, Kobayashi M, Nagasawa T, Yamada H, Beppu T
• Cloning and characterization of genes responsible for metabolism ofnitrile compounds from Pseudomonas chlororaphis B23.
• J Bacteriol. 1991; 173: 2465-72
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• The nitrile hydratase (NHase) of Pseudomonas chlororaphis B23, which iscomposed of two subunits, alpha and beta, catalyzes the hydration ofnitrile compounds to the corresponding amides. The NHase gene of strainB23 was cloned into Escherichia coli by the DNA-probing method with theNHase gene of Rhodococcus sp. strain N-774 as the hybridization probe.Nucleotide sequencing revealed that an amidase showing significantsimilarity to the amidase of Rhodococcus sp. strain N-774 was also codedby the region just upstream of the subunit alpha-coding sequence. Inaddition to these three proteins, two open reading frames, P47K and OrfE,were found just downstream of the coding region of subunit beta. Thedirection and close locations to each other of these open reading framesencoding five proteins (amidase, subunits alpha and beta, P47K, and OrfE,in that order) suggested that these genes were cotranscribed by a singlemRNA. Plasmid pPCN4, in which a 6.2-kb sequence covering the region codingfor these proteins is placed under control of the lac promoter, directedoverproduction of enzymatically active NHase and amidase in response toaddition of isopropyl-beta-D-thiogalactopyranoside. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of the cell extract showed thatthe amount of subunits alpha and beta of NHase was about 10% of the totalcellular proteins and that an additional 38-kDa protein probably encodedby the region upstream of the amidase gene was also produced in a largeamount. The 38-kDa protein, as well as P47K and OrfE, appeared to beimportant for efficient expression of NHase activity in E. coli cells,because plasmids containing the NHase and amidase genes but lacking theregion coding for the 38-kDa protein or the region coding for P47K andOrfE failed to express efficient NHase activity.