Cloning and characterization of genes responsible for metabolism of nitrile compounds from Pseudomonas chlororaphis B23.

The nitrile hydratase (NHase) of Pseudomonas chlororaphis B23, which is composed of two subunits, alpha and beta, catalyzes the hydration of nitrile compounds to the corresponding amides. The NHase gene of strain B23 was cloned into Escherichia coli by the DNA-probing method with the NHase gene of Rhodococcus sp. strain N-774 as the hybridization probe. Nucleotide sequencing revealed that an amidase showing significant similarity to the amidase of Rhodococcus sp. strain N-774 was also coded by the region just upstream of the subunit alpha-coding sequence. In addition to these three proteins, two open reading frames, P47K and OrfE, were found just downstream of the coding region of subunit beta. The direction and close locations to each other of these open reading frames encoding five proteins (amidase, subunits alpha and beta, P47K, and OrfE, in that order) suggested that these genes were cotranscribed by a single mRNA. Plasmid pPCN4, in which a 6.2-kb sequence covering the region coding for these proteins is placed under control of the lac promoter, directed overproduction of enzymatically active NHase and amidase in response to addition of isopropyl-beta-D-thiogalactopyranoside. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cell extract showed that the amount of subunits alpha and beta of NHase was about 10% of the total cellular proteins and that an additional 38-kDa protein probably encoded by the region upstream of the amidase gene was also produced in a large amount. The 38-kDa protein, as well as P47K and OrfE, appeared to be important for efficient expression of NHase activity in E. coli cells, because plasmids containing the NHase and amidase genes but lacking the region coding for the 38-kDa protein or the region coding for P47K and OrfE failed to express efficient NHase activity.     

DNA sequences of the cysB regions of Salmonella typhimurium and Escherichia coli

Nucleotide sequences of the cysB region of Salmonella typhimurium and Escherichia coli have been determined and compared. A total of 1759 nucleotides were sequenced in S. typhimurium and 1840 in E. coli. Both contain a 972-nucleotide open reading frame identified as the coding region for the cysB regulatory protein on the basis of sequence homology and by comparison of the deduced amino acid sequences with known physicochemical properties of this protein. The DNA sequence identity for the cysB coding region in the two species is 80.5%. The deduced amino acid sequences are 95% identical. The predicted cysB polypeptide molecular weights are 36,013 for S. typhimurium and 36,150 for E. coli. For both proteins a helix-turn-helix region similar to that found in other DNA-binding proteins is predicted from the deduced amino acid sequence. Sequences upstream to cysB contain open reading frames which represent the carboxyl-terminal end of the topA gene product, DNA topoisomerase I. A pattern of highly conserved nucleotide sequences in the 151 nucleotides immediately preceding the cysB initiator codon in both species suggests that this region may contain multiple signals for the regulation of cysB expression.     

Cloning, nucleotide sequence and expression in Escherichia coli of two cobalt-containing nitrile hydratase genes from Rhodococcus rhodochrous J1.

Rhodococcus rhodochrous J1 produces two kinds of cobalt-containing nitrile hydratases (NHases); one is a high molecular mass-NHase (H-NHase) and the other is a low molecular mass-NHase (L-NHase). Both NHases are composed of two subunits of different sizes (alpha and beta subunits). The H- and L-NHase genes were cloned into Escherichia coli by a DNA-probing method using the NHase gene of Rhodococcus sp. N-774, a ferric ion-containing NHase producing strain, as the hybridization probe and their nucleotide sequences were determined. In each of the H- and L-NHase genes, the open reading frame for the beta subunit was located just upstream of that for the alpha subunit, which probably belongs to the same operon. The amino acid sequences of each subunit of the H- and L-NHases from R. rhodochrous J1 showed generally significant similarities to those from Rhodococcus sp. N-774, but the arrangement of the coding sequences for two subunits is reverse 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 control of lac promoter, only when they were cultured in the medium supplemented with CoCl2.     

Cloning and expression of the nitrile hydratase and amidase genes from Bacillus sp. BR449 into Escherichia coli

A moderate thermophile, Bacillus sp. BR449 was previously shown to exhibit a high level of nitrile hydratase (NHase) activity when growing on high levels of acrylonitrile at 55 degrees C. In this report, we describe the cloning of a 6.1 kb SalI DNA fragment encoding the NHase gene cluster of BR449 into Escherichia coli. Nucleotide sequencing revealed six ORFs encoding (in order), two unidentified putative proteins, amidase, NHase beta- and alpha-subunits and a small putative protein of 101 amino acids designated P12K. Spacings and orientation of the coding regions as well as their gene expression in E. coli suggest that the beta-subunit, alpha-subunit, and P12K genes are co-transcribed. Analysis of deduced amino acid sequences indicate that the amidase (348 aa, MW 38.6 kDa) belongs to the nitrilase-related aliphatic amidase family, and that the NHase beta- (229 aa, MW 26.5 kDa) and alpha- (214 aa, MW 24.5 kDa) subunits comprise a cobalt-containing member of the NHase family, which includes Rhodococcus rhodochrous J1 and Pseudomonas putida 5B NHases. The amidase/NHase gene cluster differs both in arrangement and composition from those described for other NHase-producing strains. When expressed in Escherichia coli DH5alpha, the subcloned NHase genes produced significant levels of active NHase enzyme when cobalt ion was added either to the culture medium or cell extracts. Presence of the P12K gene and addition of amide compounds as inducers were not required for this expression.     

Cloning and expression of the Mycobacterium bovis BCG gene for extracellular alpha antigen.

The gene for the extracellular alpha antigen of Mycobacterium bovis BCG was cloned by using a single probe restricted to G or C in the third position. This technique should have great potential for the isolation of mycobacterial antigen genes. The gene analysis revealed that the alpha antigen gene encoded 323 amino acid residues, including 40 amino acids for signal peptide followed by 283 amino acids for mature protein. This is the first report on the structure of the mycobacterial signal peptide. The promoter-like sequence and ribosome-binding site were observed upstream of the open reading frame. In the coding region, the third position of the codon showed high G + C content (86%). The gene was expressed as an unfused protein in Escherichia coli by using an E. coli expression vector. This protein, which reacted with polyclonal antibody raised against alpha antigen from Mycobacterium tuberculosis, would be applicable to the immunodiagnosis of tuberculosis.     

Tetanus toxin: primary structure, expression in E. coli, and homology with botulinum toxins.

A pool of synthetic oligonucleotides was used to identify the gene encoding tetanus toxin on a 75-kbp plasmid from a toxigenic non-sporulating strain of Clostridium tetani. The nucleotide sequence contained a single open reading frame coding for 1315 amino acids corresponding to a polypeptide with a mol. wt of 150,700. In the mature toxin molecule, proline (2) and serine (458) formed the N termini of the 52,288 mol. wt light chain and the 98,300 mol. wt heavy chain, respectively. Cysteine (467) was involved in the disulfide linkage between the two subchains. The amino acid sequences of the tetanus toxin revealed striking homologies with the partial amino acid sequences of botulinum toxins A, B, and E, indicating that the neurotoxins from C. tetani and C. botulinum are derived from a common ancestral gene. Overlapping peptides together covering the entire tetanus toxin molecule were synthesized in Escherichia coli and identified by monoclonal antibodies. The promoter of the toxin gene was localized in a region extending 322 bp upstream from the ATG codon and was shown to be functional in E. coli.     

Nitrile pathway involving acyl-CoA synthetase: overall metabolic gene organization and purification and characterization of the enzyme

Two open reading frames (nhpS and acsA) were identified immediately downstream of the previously described Pseudomonas chlororaphis B23 nitrile hydratase (NHase) gene cluster (encoding aldoxime dehydratase, amidase, the two NHase subunits, and an uncharacterized protein). The amino acid sequence deduced from acsA shows similarity to that of acyl-CoA synthetase (AcsA). The acsA gene product expressed in Escherichia coli showed acyl-CoA synthetase activity toward butyric acid and CoA as substrates, with butyryl-CoA being synthesized. From the E. coli transformant, AcsA was purified to homogeneity and characterized. The quality of the recombinant protein was verified by the NH2-terminal amino acid sequence and the results of matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The apparent Km values for butyric acid, CoA, and ATP were 0.32 +/- 0.04, 0.37 +/- 0.02, and 0.22 +/- 0.02 mm, respectively. AcsA was shown to be a short-chain acyl-CoA synthetase, according to the catalytic efficiencies (kcat/Km) for various acids. The substrate specificity of AcsA was similar to those of aldoxime dehydratase, NHase, and amidase, the genes of which coexist in the same orientation in the gene cluster. P. chlororaphis B23 grew when cultured in a medium containing butyraldoxime as the sole carbon and nitrogen source. The activities of aldoxime dehydratase, NHase, and amidase were detected together with that of acyl-CoA synthetase under the culture conditions used. Moreover, on culture in a medium containing butyric acid as the sole carbon source, acyl-CoA synthetase activity was also detected. Together with the adjacent locations of the aldoxime dehydratase, NHase, amidase, and acyl-CoA synthetase genes, these findings suggest that the four enzymes are sequentially correlated with one another in vivo to utilize butyraldoxime as a carbon and nitrogen source. This is the first report of an overall "nitrile pathway" (aldoxime-->nitrile-->amide-->acid-->acyl-CoA) comprising these enzymes.     

Nucleotide sequence of the gene ompA coding the outer membrane protein II of Escherichia coli K-12.

A nucleotide sequence of 2271 basepairs has been determined from cloned E. coli DNA which contains ompA. Withing that sequence, starting at nucleotide 1037, an open translational reading frame encodes a protein of 367 amino acids which starting with amino acid 22 agrees with the primary structure of protein II. The preceeding 21 amino acids constitute a typical signal sequence. There is a non-translated region of 360 nucleotides in front of the translational start. The insertion point of an IS1 element 110 nucleotides upstream from the start codon and an amber codon at the position of amino acid residue 28 have been localized in the DNA from two ompA mutants.     

Nucleotide sequence of a novel kanamycin resistance gene, aphA-7, from Campylobacter jejuni and comparison to other kanamycin phosphotransferase genes

A novel kanamycin phosphotransferase gene, aphA-7, was cloned from a 14-kb plasmid obtained from a strain of Campylobacter jejuni and the nucleotide sequence of the gene was determined. The presumed open reading frame of the aphA-7 structural gene was 753 bp in length and encoded a protein of 251 amino acids with a calculated weight of 29,691 Da. A 29-kDa protein was demonstrated in Escherichia coli maxicells containing the cloned aphA-7 gene. A ribosomal binding site corresponding to 5 of 8 bases of the 3' end of the E. coli 16S rRNA was 8 bp upstream of the start codon. Sequences corresponding to the -35 and -10 regions of the consensus promoter sequences of E. coli were upstream of the presumed initiation codon of the gene. The DNA sequence was most closely related to the aphA-3 gene from Streptococcus faecalis, showing 55.4% sequence similarity. There was 45.6% identity at the amino acid level between the aphA-3 and the aphA-7 proteins. Of the three conserved regions noted previously in phosphotransferase genes, the aphA-7 amino acid sequence was identical to the six conserved amino acids in motif 3, but differed in one of the five conserved amino acids in motif 1 (if gaps are permitted) and 3 of the 10 conserved residues in motif 2. The 32.8% G + C ratio in the open reading frame of the aphA-7 kanamycin resistance gene, which is similar to that of the C. jejuni chromosome, suggests that the aphA-7 may be indigenous to Campylobacters.     

Cloning and characterization of the hemA region of the Bacillus subtilis chromosome.

A 3.8-kilobase DNA fragment from Bacillus subtilis containing the hemA gene has been cloned and sequenced. Four open reading frames were identified. The first is hemA, encoding a protein of 50.8 kilodaltons. The primary defect of a B. subtilis 5-aminolevulinic acid-requiring mutant was identified as a cysteine-to-tyrosine substitution in the HemA protein. The predicted amino acid sequence of the B. subtilis HemA protein showed 34% identity with the Escherichia coli HemA protein, which is known to code for the NAD(P)H:glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid synthesis. The B. subtilis HemA protein also complements the defect of an E. coli hemA mutant. The second open reading frame in the cloned fragment, called ORF2, codes for a protein of about 30 kilodaltons with unknown function. It is not the proposed hemB gene product porphobilinogen synthase. The third open reading frame is hemC, coding for porphobilinogen deaminase. The fourth open reading frame extends past the sequenced fragment and may be identical to hemD, coding for uroporphyrinogen III cosynthase. Analysis of deletion mutants of the hemA region suggests that (at least) hemA, ORF2, and hemC may be part of an operon.     

Purification, cloning, and primary structure of an enantiomer-selective amidase from Brevibacterium sp. strain R312: structural evidence for genetic coupling with nitrile hydratase.

An enantiomer-selective amidase active on several 2-aryl and 2-aryloxy propionamides was identified and purified from Brevibacterium sp. strain R312. Oligonucleotide probes were designed from limited peptide sequence information and were used to clone the corresponding gene, named amdA. Highly significant homologies were found at the amino acid level between the deduced sequence of the enantiomer-selective amidase and the sequences of known amidases such as indoleacetamide hydrolases from Pseudomonas syringae and Agrobacterium tumefaciens and acetamidase from Aspergillus nidulans. Moreover, amdA is found in the same orientation and only 73 bp upstream from the gene coding for nitrile hydratase, strongly suggesting that both genes are part of the same operon. Our results also showed that Rhodococcus sp. strain N-774 and Brevibacterium sp. strain R312 are probably identical, or at least very similar, microorganisms. The characterized amidase is an apparent homodimer of Mr 2 x 54,671 which exhibited under our conditions a specific activity of about 13 to 17 mumol of 2-(4-hydroxyphenoxy)propionic R acid formed per min per mg of enzyme from the racemic amide. Large amounts of an active recombinant enzyme could be produced in Escherichia coli at 30 degrees C under the control of an E. coli promoter and ribosome-binding site.     

Ribosomal protein S14 transcripts are edited in Oenothera mitochondria

The gene encoding ribosomal protein S14 (rps14) in Oenothera mitochondria is located upstream of the cytochrome b gene (cob). Sequence analysis of independently derived cDNA clones covering the entire rps14 coding region shows two nucleotides edited from the genomic DNA to the mRNA derived sequences by C to U modifications. A third editing event occurs four nucleotides upstream of the AUG initiation codon and improves a potential ribosome binding site. A CGG codon specifying arginine in a position conserved in evolution between chloroplasts and E. coli as a UGG tryptophan codon is not edited in any of the cDNAs analysed. An inverted repeat 3' of an unidentified open reading frame is located upstream of the rps14 gene. The inverted repeat sequence is highly conserved at analogous regions in other Oenothera mitochondrial loci.