Degradation of Aromatics and Chloroaromatics by Pseudomonas sp. Strain B13: Purification and Characterization of 3-Oxoadipate:Succinyl-Coenzyme A (CoA) Transferase and 3-Oxoadipyl-CoA Thiolase

The degradation of 3-oxoadipate in Pseudomonas sp. strain B13 was investigated and was shown to proceed through 3-oxoadipyl-coenzyme A (CoA) to give acetyl-CoA and succinyl-CoA. 3-Oxoadipate:succinyl-CoA transferase of strain B13 was purified by heat treatment and chromatography on phenyl-Sepharose, Mono-Q, and Superose 6 gels. Estimation of the native molecular mass gave a value of 115,000 +/- 5,000 Da with a Superose 12 column. Polyacrylamide gel electrophoresis under denaturing conditions resulted in two distinct bands of equal intensities. The subunit A and B values were 32,900 and 27,000 Da. Therefore it can be assumed that the enzyme is a heterotetramer of the type A2B2 with a molecular mass of 120,000 Da. The N-terminal amino acid sequences of both subunits are as follows: subunit A, AELLTLREAVERFVNDGTVALEGFTHLIPT; subunit B, SAYSTNEMMTVAAARRLKNGAVVFV. The pH optimum was 8.4. Km values were 0.4 and 0.2 mM for 3-oxoadipate and succinyl-CoA, respectively. Reversibility of the reaction with succinate was shown. The transferase of strain B13 failed to convert 2-chloro- and 2-methyl-3-oxoadipate. Some activity was observed with 4-methyl-3-oxoadipate. Even 2-oxoadipate and 3-oxoglutarate were shown to function as poor substrates of the transferase. 3-oxoadipyl-CoA thiolase was purified by chromatography on DEAE-Sepharose, blue 3GA, and reactive brown-agarose. Estimation of the native molecular mass gave 162,000 +/- 5,000 Da with a Superose 6 column. The molecular mass of the subunit of the denatured protein, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 42 kDa. On the basis of these results, 3-oxoadipyl-CoA thiolase should be a tetramer of the type A4. The N-terminal amino acid sequence of 3-oxoadipyl-CoA thiolase was determined to be SREVYI-DAVRTPIGRFG. The pH optimum was 7.8. Km values were 0.15 and 0.01 mM for 3-oxoadipyl-CoA and CoA, respectively. Sequence analysis of the thiolase terminus revealed high percentages of identity (70 to 85%) with thiolases of different functions. The N termini of the transferase subunits showed about 30 to 35% identical amino acids with the glutaconate-CoA transferase of an anaerobic bacterium but only an identity of 25% with the respective transferases of aromatic compound-degrading organisms was found.     

Pea early browning virus RNA1 encodes four polypeptides including a putative zinc-finger protein.

We have determined the complete nucleotide sequence of RNA1 of the tobravirus pea early browning virus [PEBV] from an overlapping series of cDNA clones. The 7073 nucleotide sequence contains four open reading frames [ORFs]. The 5' proximal ORF encodes a 141K polypeptide, and readthrough of the opal [UGA] termination codon of this ORF would lead to the synthesis of a second, 201K polypeptide. Both of these polypeptides have extensive amino acid homology with the putative replicase proteins of tobacco rattle virus [TRV] and tobacco mosaic virus [TMV]. The third ORF encodes a 30K polypeptide which has homology with the TRV 29K and TMV 30K putative cell-to-cell spread proteins. The fourth, 3' proximal ORF encodes a 12K polypeptide which has extensive homology with the TRV 16K protein whose function is unknown. Examination of the amino acid sequences of the 12K and 16K gene products reveals in each the presence of two multiple-cysteine/histidine motifs, a finding which suggests that these proteins might have zinc and/or nucleic acid-binding properties.     

Cloning and sequencing of a cluster of genes encoding branched-chain alpha-keto acid dehydrogenase from Streptomyces avermitilis and the production of a functional E1 [alpha beta] component in Escherichia coli.

A cluster of genes encoding the E1 alpha, E1 beta, and E2 subunits of branched-chain alpha-keto acid dehydrogenase (BCDH) of Streptomyces avermitilis has been cloned and sequenced. Open reading frame 1 (ORF1) (E1 alpha), 1,146 nucleotides long, would encode a polypeptide of 40,969 Da (381 amino acids). ORF2 (E1 beta), 1,005 nucleotides long, would encode a polypeptide of 35,577 Da (334 amino acids). The intergenic distance between ORF1 and ORF2 is 73 bp. The putative ATG start codon of the incomplete ORF3 (E2) overlaps the stop codon of ORF2. Computer-aided searches showed that the deduced products of ORF1 and ORF2 resembled the corresponding E1 subunit (alpha or beta) of several prokaryotic and eukaryotic BCDH complexes. When these ORFs were overexpressed in Escherichia coli, proteins of about 41 and 34 kDa, which are the approximate masses of the predicted S. avermitilis ORF1 and ORF2 products, respectively, were detected. In addition, specific E1 [alpha beta] BCDH activity was detected in E. coli cells carrying the S. avermitilis ORF1 (E1 alpha) and ORF2 (E1 beta) coexpressed under the control of the T7 promoter.     

Sequence analysis and expression of the aspartokinase and aspartate semialdehyde dehydrogenase operon from rifamycin SV-producing amycolatopsis mediterranei.

A approximately 4.8 kb KpnI fragment, from the upstream region of the methylmalonyl-CoA mutase gene (mutAB) of rifamycin SV-producing Amycolatopsis mediterranei, was cloned and partially sequenced. Codon preference analysis showed three complete ORFs. ORF2 is internal to ORF1, and encodes a polypeptide corresponding to 172 amino acids, whereas ORF1 encodes a polypeptide of 421 amino acids. They were identified as the encoding genes of aspartokinase alpha- and beta-subunits by comparing the amino acid sequences with those in the database. The downstream ORF3, whose start codon was overlapped with the stop codon of both ORF1 and ORF2 by 1 bp, was identified as the aspartate semialdehyde dehydrogenase gene (asd), encoding a polypeptide of 346 amino acids. Subclones containing either the ask gene or the asd gene were constructed, in which the genes could be expressed under Lac promoters. Two subclones could transform E. coli CGSC 5074 (ask-) and E. coli X6118 (asd-) to prototrophy, supporting the functional assignments. Southern hybridisation indicated that the approximately 4.8 kb sequenced region represented a continuous segment in the A. mediterranei chromosome. It is concluded that ask and asd genes are present in an operon in A. mediterranei, and therefore that organisation of these two genes is the same as in most gram-positive bacteria, such as Mycobacteria, Corynebacterium glutamicum and Bacillus subtilis, but is different from Streptomyces akiyoshiensis.     

Cloning and sequencing of the bovine adenovirus type 2 early region 4

Bovine adenovirus type 2 (BAV2) is a medium size double-stranded DNA virus which infects both bovine and ovine species, resulting in mild respiratory and gastrointestinal disorders. To better understand the virus and its growth characterisitics in Madin-Darby bovine kidney (MDBK) cells, we have cloned and sequenced the extreme right-end segment of the BAV2 genome (90.5–100 map units). Analysis of the nucleotide sequence revealed 40 potential open reading frames (ORFs) with coding capacity for polypeptides that are 25 or more amino acid (aa) residues long. Six of these ORFs encode polypeptides that show homology to well-characterized early region 4 (E4) proteins of human adenovirus type 2 (Ad2) and Ad12. ORF1 has the potential to encode a 114 aa long polypeptide that is 54% homologous to the E4 14 kDa protein of Ad2. ORF2 encodes a 78 aa long polypeptide that exhibits 40% homology to the E4 13 kDa protein of Ad2. ORFs 3–6 encode polypeptides that have homology to the E4 34 kDa protein encoded by ORF6 of Ad2 and Ad12. ORFs 3, 4 and 5 encode 128, 96 and 31 aa long polypeptides, respectively. The 128-aa polypeptide exhibits 59% homology, while the 96 and 31 aa long polypeptides exhibit 61% and 70% homology to the E4 34 kDa protein, respectively. ORF6 has the potential to encode a 57 aa long polypeptide that has 67% homology to the E4 34 kDa protein of Ad2 and 50% homology to the E4 34 kDa protein of Ad12.     

地中海拟无枝菌酸菌U32中amrC／amkC基因的序列分析及在大肠杆 …

从力复霉素ＳＶ产生菌－－地中海拟无枝菌酸菌（Ａｍｙｃｏｌａｔｏｐｓｉｓ ｍｅｄｉｔｅｒｒａｎｅｉ）Ｕ３２的硝酸盐同化基因簇的上游克隆了一个２．６ｋｂ的ＥｃｏＲＩ－ＸｈｏＩ ＤＮＡ片段并测定其序列。序列分析表明,该ＤＮＡ片段编码两个完整的开放阅读框架（ＯＲＦ）,ＯＲＦ２的起始密码子ＧＴＧ与ＯＲＦ１的终止密码子ＴＧＡ在ＴＧ处重叠。ＯＲＦ１编码一个含２２４个氨基酸的多肽,它同放线菌中典型的应答调节蛋白包     

Conservation of a long open reading frame in two Neurospora mitochondrial plasmids

The nucleotide sequence of a specific region of the mitochondrial plasmid from the Neurospora intermedia Varkud-lc strain was determined. Analysis of the sequence revealed the presence of a long (up to 710 amino acids) ORF. This ORF is almost identical to a previously characterized ORF in the mitochondrial plasmid from the Neurospora crassa Mauriceville-lc strain. When the ORFs from the two plasmids are compared over their entire length of 2,133 bp, only 34 nucleotide substitutions are found (greater than 98% identity). These substitutions result in only nine amino acid replacements in the protein sequences predicted from the two ORFs. Though no function can be assigned to the putative products of these ORFs, their high conservation of nucleotide and deduced amino acid sequence suggest that they are under selective pressure, presumably to preserve the function of some protein.      

Characterization of the genes encoding beta-ketoadipate: succinyl-coenzyme A transferase in Pseudomonas putida.

beta-Ketoadipate:succinyl-coenzyme A transferase (beta-ketoadipate:succinyl-CoA transferase) (EC 2.8.3.6) carries out the penultimate step in the conversion of benzoate and 4-hydroxybenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the beta-ketoadipate pathway. This report describes the characterization of a DNA fragment from Pseudomonas putida that encodes this enzyme. The fragment complemented mutants defective in the synthesis of the CoA transferase, and two proteins of sizes appropriate to encode the two nonidentical subunits of the enzyme were produced in Escherichia coli when the fragment was placed under the control of a phage T7 promoter. DNA sequence analysis revealed two open reading frames, designated pcaI and pcaJ, that were separated by 8 bp, suggesting that they may comprise an operon. A comparison of the deduced amino acid sequence of the P. putida CoA transferase genes with the sequences of two other bacterial CoA transferases and that of succinyl-CoA:3-ketoacid CoA transferase from pig heart suggests that the homodimeric structure of the mammalian enzyme may have resulted from a gene fusion of the bacterial alpha and beta subunit genes during evolution. Conserved functional groups important to the catalytic activity of CoA transferases were also identified.     

The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus.

The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus (BSMV) has been determined. The sequence is 3289 nucleotides in length and contains four open reading frames (ORFs) which code for proteins of Mr 22,147 (ORF1), Mr 58,098 (ORF2), Mr 17,378 (ORF3), and Mr 14,119 (ORF4). The predicted N-terminal amino acid sequence of the polypeptide encoded by the ORF nearest the 5'-end of the RNA (ORF1) is identical (after the initiator methionine) to the published N-terminal amino acid sequence of BSMV coat protein for 29 of the first 30 amino acids. ORF2 occupies the central portion of the coding region of RNA beta and ORF3 is located at the 3'-end. The ORF4 sequence overlaps the 3'-region of ORF2 and the 5'-region of ORF3 and differs in codon usage from the other three RNA beta ORFs. The coding region of RNA beta is followed by a poly(A) tract and a 238 nucleotide tRNA-like structure which are common to all three BSMV genomic RNAs.     

A Functional 4-Hydroxysalicylate/Hydroxyquinol Degradative Pathway Gene Cluster Is Linked to the Initial Dibenzo-p-Dioxin Pathway Genes in Sphingomonas sp. Strain RW1

The bacterium Sphingomonas sp. strain RW1 is able to use dibenzo-p-dioxin, dibenzofuran, and several hydroxylated derivatives as sole sources of carbon and energy. We have determined and analyzed the nucleic acid sequence of a 9,997-bp HindIII fragment downstream of cistrons dxnA1A2, which encode the dioxygenase component of the initial dioxygenase system of the corresponding catabolic pathways. This fragment contains 10 colinear open reading frames (ORFs), apparently organized in one compact operon. The enzymatic activities of some proteins encoded by these genes were analyzed in the strain RW1 and, after hyperexpression, in Escherichia coli. The first three ORFs of the locus, designated dxnC, ORF2, and fdx3, specify a protein with a low homology to bacterial siderophore receptors, a polypeptide representing no significant homology to known proteins, and a putative ferredoxin, respectively. dxnD encodes a 69-kDa phenol monooxygenase-like protein with activity for the turnover of 4-hydroxysalicylate, and dxnE codes for a 37-kDa protein whose sequence and activity are similar to those of known maleylacetate reductases. The following gene, dxnF, encodes a 33-kDa intradiol dioxygenase which efficiently cleaves hydroxyquinol, yielding maleylacetate, the ketoform of 3-hydroxy-cis,cis-muconate. The heteromeric protein encoded by dxnGH is a 3-oxoadipate succinyl coenzyme A (succinyl-CoA) transferase, whereas dxnI specifies a protein exhibiting marked homology to acetyl-CoA acetyltransferases (thiolases). The last ORF of the sequenced fragment codes for a putative transposase. DxnD, DxnF, DxnE, DxnGH, and DxnI (the activities of most of them have also been detected in strain RW1) thus form a complete 4-hydroxysalicylate/hydroxyquinol degradative pathway. A route for the mineralization of the growth substrates 3-hydroxydibenzofuran and 2-hydroxydibenzo-p-dioxin in Sphingomonas sp. strain RW1 thus suggests itself.     

Isolation and characterization of kikA, a region on IncN group plasmids that determines killing of Klebsiella oxytoca.

Transfer of the IncN group plasmid pCU1 from Escherichia coli to Klebsiella oxytoca by conjugation kills a large proportion (90 to 95%) of the recipients of plasmid DNA, whereas transfer to E. coli or even to the closely related Enterobacter aerogenes does not. Two regions, kikA and kikB, have been identified on pCU1 that contribute to the Kik (killing in klebsiellas) phenotype. We have localized the kikA region to 500 bp by deletion analysis and show by DNA-DNA hybridization that kikA is highly conserved among the plasmids of incompatibility group N. The expression in K. oxytoca of kikA under the control of the strong inducible E. coli tac promoter results in loss of cell viability. The nucleotide sequence showed two overlapping open reading frames (ORFs) within the kikA region. The first ORF codes for a putative polypeptide of 104 amino acids (ORF104). The second ORF codes for a 70-amino-acid polypeptide (ORF70). The properties of the putative protein encoded by ORF104 and gene fusions of kikA to alkaline phosphatase by using TnphoA suggest that killing may involve an association with the bacterial membrane; however, we could not rule out the possibility that ORF70 plays a role in the Kik phenotype.     

The DNA [adenine-N6]methyltransferase (Dam) of bacteriophage T4

A functional bacteriophage T4 dam+ gene, which specifies a DNA [adenine-N6]methyltransferase (Dam), was cloned on a 1.8-kb HindIII fragment [Schlagman and Hattman, Gene 22 (1983) 139-156]. Sequence analysis [Macdonald and Mosig, EMBO J. 3 (1984) 2863-2871] revealed two overlapping in-phase open reading frames (ORFs). The 5' proximal ORF initiates translation at an AUG and encodes a 30-kDa polypeptide, whereas the downstream ORF initiates translation at a GUG and encodes a 26-kDa polypeptide. Analysis of BAL 31 deletions in our original dam+ clone has verified that at least one of these overlapping ORFs, in fact, encodes T4 Dam. To investigate where T4 Dam translation is initiated, we have constructed plasmids in which a tac or lambda PL promoter is placed 5' to either the longer ORF or just the shorter ORF. Only clones which contain a promoter in front of the longer ORF produce active T4 Dam. This indicates that the 26-kDa polypeptide alone cannot be T4 Dam. Additional experiments suggest that only the 30-kDa polypeptide is required for enzyme activity and that the shorter ORF is not translated in plasmid-carrying cells. We also present evidence that T4 Dam is capable of methylating 5'-GATC-3', GATm5C, and GAThmC sequences; non-canonical sites (e.g., GACC) are also methylated, but much less efficiently.     

Gene cloning, structural gene and promoter identification, and active assay of the phosphatidylcholine synthase of Pseudomonas sp. strain 593

Pseudomonas sp. strain 593, a soil bacterium, is able to use exogenous choline to synthesize phosphatidylcholine via phosphatidylcholine synthase (Pcs). A 2020 bp DNA fragment that hybridized to a Pcs probe was cloned. This fragment contained a large open reading frame (ORF) with two potential ATG start sites that would encode for 293 and 231 amino acid proteins. Fragments containing the two ORFs encoded Pcs when they were inserted into the expression vector pET23a and expressed under the control of the T7 promoter in Escherichia coli BL21(DE3) pLysS. However, when the two ORFs were inserted into the cloning vector pMD18-T and expressed without control of the plasmid promoter in E. coli DH5α, only the larger clone exhibited Pcs activity. This suggested that the larger fragment contained a native promoter driving expression of the smaller ORF. A promoter activity assay, in which DNA fragments were inserted into the promoter-probe plasmid pCB182 and β-galactosidase activity of E. coli transformants was tested, demonstrated that a promoter is indeed present in the DNA region. All results together indicate that the 696 bp ORF, not the larger 897 bp ORF, encodes the Pcs in Pseudomonas sp. strain 593 and carries a promoter in front of its 5' terminus.     

cDNA-derived amino acid sequence of rat mitochondrial 3-oxoacyl-CoA thiolase with no transient presequence: structural relationship with peroxisomal isozyme.

The sorting of homologous proteins between two separate intracellular organelles is a major unsolved problem. 3-Oxoacyl-CoA thiolase is localized in mitochondria and peroxisomes, and provides a good system for the study on the problem. Unlike most mitochondrial matrix proteins, mitochondrial 3-oxoacyl-CoA thiolase in rats is synthesized with no transient presequence and possess information for mitochondrial targeting and import in the mature protein. Two overlapping cDNA clones contained an open reading frame encoding a polypeptide of 397 amino acid residues (predicted Mr = 41,868), a 5' untranslated sequence of 164 bp, a 3' untranslated sequence of 264 bp and a poly(A) tract. The amino acid sequence of the mitochondrial thiolase is 37% identical with that of the mature portion of rat peroxisomal 3-oxoacyl-CoA thiolase precursor. These results suggest that the two thiolases have a common origin and obtained information for targeting to respective organelles during evolution. Two portions in the mitochondrial thiolase that may serve as a mitochondrial targeting signal are presented.