Nucleotide sequence and expression of clcD, a plasmid-borne dienelactone hydrolase gene from Pseudomonas sp. strain B13.

The clcD structural gene encodes dienelactone hydrolase (EC 3.1.1.45), an enzyme that catalyzes the conversion of dienelactones to maleylacetate. The gene is part of the clc gene cluster involved in the utilization of chlorocatechol and is carried on a 4.3-kilobase-pair BglII fragment subcloned from the Pseudomonas degradative plasmid pAC27. A 1.9-kilobase-pair PstI-EcoRI segment subcloned from the BglII fragment was shown to carry the clcD gene, which was expressed inducibly under the tac promoter at levels similar to those found in 3-chlorobenzoate-grown Pseudomonas cells carrying the plasmid pAC27. In this study, we present the complete nucleotide sequence of the clcD gene and the amino acid sequence of dienelactone hydrolase deduced from the DNA sequence. The NH2-terminal amino acid sequence encoded by the clcD gene from plasmid pAC27 corresponds to a 33-residue sequence established for dienelactone hydrolase encoded by the Pseudomonas sp. strain B13 plasmid pWR1. A possible relationship between the clcD gene and pcaD, a Pseudomonas putida chromosomal gene encoding enol-lactone hydrolase (EC 3.1.1.24) is suggested by the fact that the gene products contain an apparently conserved pentapeptide neighboring a cysteinyl side chain that presumably lies at or near the active sites; the cysteinyl residue occupies position 60 in the predicted amino acid sequence of dienelactone hydrolase.     

Cloning and characterization of the genes for two distinct cephalosporin acylases from a Pseudomonas strain.

Pseudomonas sp. strain SE83 converts cephalosporin C and 7 beta-(4-carboxybutanamido)cephalosporanic acid (GL-7ACA) to 7-aminocephalosporanic acid (7ACA). A DNA library of this strain was constructed in Escherichia coli and screened for the ability to deacylate GL-7ACA to 7ACA. Apparently, two distinct genes, designated acyI and acyII, were cloned on 4.8- and 6.0-kilobase-pair BglII fragments, respectively. The enzymes encoded by the two genes showed different substrate specificities, and the acyII-encoded enzyme was found to yield 7ACA from cephalosporin C by direct deacylation. Expression of the two genes in E. coli was strongly dependent on a promoter of the vector. The coding regions for acyI and acyII were localized on the 2.5- and 2.8-kilobase-pair fragments, respectively, by subcloning experiments, and high expression of both genes was obtained by placing them under the control of the lacUV5 promoter. The acyII-encoded enzyme was purified and shown to be composed of two nonidentical subunits with molecular weights of 26,000 and 57,000. Maxicell analysis revealed three acyII-specific polypeptides, two of which corresponded to the above subunits. The third polypeptide with a molecular weight of 83,000 was suggested to be the precursor of both subunits.     

Isolation, hyperexpression, and sequencing of the aceA gene encoding isocitrate lyase in Escherichia coli.

A structural gene for isocitrate lyase was isolated from a cosmid containing an ace locus of the Escherichia coli chromosome. Cloning and expression under control of the tac promoter in a multicopy plasmid showed that a 1.7-kilobase-pair DNA segment was sufficient for complementation of an aceA deletion mutation and overproduction of isocitrate lyase. DNA sequence analysis of the cloned gene and N-terminal protein sequencing of the cloned and wild-type enzymes revealed an entire aceA gene which encodes a 429-amino-acid residue polypeptide whose C-terminus is histidine. The deduced amino acid sequence for the 47.2-kilodalton subunit of E. coli isocitrate lyase could be aligned with that for the 64.8-kilodalton subunit of the castor bean enzyme with 39% identity except for limited N- and C-terminal regions and a 103-residue stretch that was unique for the plant enzyme and started approximately in the middle of that peptide.     

Structure and regulation of the anthranilate synthase genes in Pseudomonas aeruginosa: I. Sequence of trpG encoding the glutamine amidotransferase subunit

We have determined the DNA sequence of the distal 148 codons of trpE and all of trpG in Pseudomonas aeruginosa. These genes encode, respectively, the large and small (glutamine amidotransferase) subunits of anthranilate synthase, the first enzyme in the tryptophan synthetic pathway. The sequenced region of trpE is homologous with the distal portion of E. coli and Bacillus subtilis trpE, whereas the trpG sequence is homologous to the glutamine amidotransferase subunit genes of a number of bacterial and fungal anthranilate synthases. The two coding sequences overlap by 23 bp. Codon usage in these Pseudomonas genes shows a marked preference for codons ending in G or C, thereby resembling that of trpB, trpA, and several other chromosomal loci from this species and others with a high G + C content in their DNA. The deduced amino acid sequence for the P. aeruginosa trpG gene product differs to a surprising extent from the directly determined amino acid sequence of the glutamine amidotransferase subunit of P. putida anthranilate synthase (Kawamura et al. 1978). This suggests that these two proteins are encoded by loci that duplicated much earlier in the phylogeny of these organisms but have recently assumed the same function. We have also determined 490 bp of DNA sequence distal to trpG but have not ascertained the function of this segment, though it is rich in dyad symmetries.      

Cloning and nucleotide sequence of the maltopentaose-forming amylase gene from Pseudomonas sp. KO-8940.

The gene coding for the maltopentaose-(G5)-forming amylase of Pseudomonas sp. KO-8940 was cloned into Escherichia coli and its nucleotides were sequenced. It was expected that a long open reading frame composed of 1,842-bp that encoded 614 amino acid residues for secretory precursor polypeptide including the typical signal sequence with an NH2-terminal was the gene. An extract of Escherichia coli carrying the cloned G5-forming amylase gene had amylolytic activity with which produced only G5 from starch, the same as that of the donor strain enzyme. In the deduced primary structure of this enzyme, the four conserved regions of many alpha-amylases were found, and the COOH-terminal portion of this enzyme showed high homology with other raw starch digesting amylases.     

Characterization and derivation of the gene coding for mitochondrial carbamyl phosphate synthetase I of rat

The nucleotide sequence of rat carbamyl phosphate synthetase I mRNA has been determined from the complementary DNA. The mRNA comprises minimally 5,645 nucleotides and codes for a polypeptide of 164,564 Da corresponding to the precursor form of the rat liver enzyme. The primary sequence of mature rat carbamyl phosphate synthetase I indicates that the precursor is cleaved at one of two leucines at residues 38 or 39. The derived amino acid sequence of carbamyl phosphate synthetase I is homologous to the sequences of carbamyl phosphate synthetase of Escherichia coli and yeast. The sequence homology extends along the entire length of the rat polypeptide and encompasses the entire sequences of both the small and large subunits of the E. coli and yeast enzymes. The protein sequence data provide strong evidence that the carbamyl phosphate synthetase I gene of rat, the carAB gene of E. coli, and the CPA1 and CPA2 genes of yeast were derived from common ancestral genes. Part of the rat carbamyl phosphate synthetase I gene has been characterized with two nonoverlapping phage clones spanning 28.7 kilobases of rat chromosomal DNA. This region contains 13 exons ranging in size from 68 to 195 base pairs and encodes the 453 carboxyl-terminal amino acids of the rat protein. Southern hybridization analysis of rat genomic DNA indicates the carbamyl phosphate synthetase I gene to be present in single copy.     

Isolation, sequence, and expression in Escherichia coli of the Pseudomonas sp. strain ACP gene encoding 1-aminocyclopropane-1-carboxylate deaminase.

Pseudomonas sp. strain ACP is capable of growth on 1-aminocyclopropane-1-carboxylate (ACC) as a nitrogen source owing to induction of the enzyme ACC deaminase and the subsequent conversion of ACC to alpha-ketobutyrate and ammonia (M. Honma, Agric. Biol. Chem. 49:567-571, 1985). The complete amino acid sequence of purified ACC deaminase was determined, and the sequence information was used to clone the ACC deaminase gene from a 6-kb EcoRI fragment of Pseudomonas sp. strain ACP DNA. DNA sequence analysis of an EcoRI-PstI subclone demonstrated an open reading frame (ORF) encoding a polypeptide with a deduced amino acid sequence identical to the protein sequence determined chemically and a predicted molecular mass of 36,674 Da. The ORF also contained an additional 72 bp of upstream sequence not predicted by the amino acid sequence. Escherichia coli minicells containing the 6-kb clone expressed a major polypeptide of the size expected for ACC deaminase which was reactive with ACC deaminase antiserum. Furthermore, a lacZ fusion with the ACC deaminase ORF resulted in the expression of active enzyme in E. coli. ACC is a key intermediate in the biosynthesis of ethylene in plants, and the use of the ACC deaminase gene to manipulate this pathway is discussed.     

Cloning, sequencing, and expression of the gene for NADH-sensitive citrate synthase of Pseudomonas aeruginosa

The structural gene for the allosteric citrate synthase of Pseudomonas aeruginosa has been cloned from a genomic library by using the Escherichia coli citrate synthase gene as a hybridization probe under conditions of reduced stringency. Subcloning of portions of the original 10-kilobase-pair (kbp) clone led to isolation of the structural gene, with its promoter, within a 2,083-bp length of DNA flanked by sites for KpnI and BamHI. The nucleotide sequence of this fragment is presented; the inferred amino acid sequence was 70 and 76% identical, respectively, with the citrate synthase sequences from E. coli and Acinetobacter anitratum, two other gram-negative bacteria. DEAE-cellulose chromatography of P. aeruginosa citrate synthase from an E. coli host harboring the cloned P. aeruginosa gene gave three peaks of activity. All three enzyme peaks had subunit molecular weights of 48,000; the proteins were identical by immunological criteria and very similar in kinetics of substrate saturation and NADH inhibition. Because the cloned gene contained only one open reading frame large enough to encode a polypeptide of such a size, the three peaks must represent different forms of the same protein. A portion of the cloned P. aeruginosa gene was used as a hybridization probe under stringent conditions to identify highly homologous sequences in genomic DNA of a second strain classified as P. aeruginosa and isolates of P. putida, P. stutzeri, and P. alcaligenes. When crude extracts of each of these four isolates were mixed with antiserum raised against purified P. aeruginosa citrate synthase, however, only the P. alcaligenes extract cross-reacted.     

Organization of genes expressing the blood-group-M-specific hemagglutinin of Escherichia coli: identification and nucleotide sequence of the M-agglutinin subunit gene

The organization of genes encoding the blood group M-specific hemagglutinin (M-agglutinin) of Escherichia coli strain IH11165 was studied with a cloned 6.5-kb DNA segment. This DNA segment contains at least five genes which code for the polypeptides of 12.5, 30, 80, 18.5 and 21 kDa. The 30-, 80- and 21-kDa polypeptides are synthesized as precursors that are approximately 2 kDa larger. The 21-kDa polypeptide was identified as the M-agglutinin subunit by its reactivity with anti-M-agglutinin serum. Nucleotide sequence analysis of the corresponding gene showed that the M-agglutinin precursor had a 24-amino acid (aa) signal sequence, while the mature protein is 146 aa residues long. Although the organization of the M-agglutinin gene cluster resembles those of other E. coli adhesins, there is no significant sequence homology between the M-agglutinin subunit and the subunits of the other potentially related proteins in E. coli.     

Molecular cloning, sequencing, and expression of the gene encoding alkaline ceramidase from Pseudomonas aeruginosa. Cloning of a ceramidase homologue from Mycobacterium tuberculosis

We previously reported the purification and characterization of a novel type of alkaline ceramidase from Pseudomonas aeruginosa strain AN17 (Okino, N., Tani, M., Imayama, S., and Ito, M. (1998) J. Biol. Chem. 273, 14368-14373). Here, we report the molecular cloning, sequencing, and expression of the gene encoding the ceramidase of this strain. Specific oligonucleotide primers were synthesized using the peptide sequences of the purified ceramidase obtained by digestion with lysylendopeptidase and used for polymerase chain reaction. DNA fragments thus amplified were used as probes to clone the gene encoding the ceramidase from a genomic library of strain AN17. The open reading frame of 2,010 nucleotides encoded a polypeptide of 670 amino acids including a signal sequence of 24 residues, 64 residues of which matched the amino acid sequence determined for the purified enzyme. The molecular weight of the mature enzyme was estimated to be 70,767 from the deduced amino acid sequence. Expression of the ceramidase gene in Escherichia coli, resulted in production of a soluble enzyme with the identical N-terminal amino acid sequence. Recombinant ceramidase was purified to homogeneity from the lysate of E. coli cells and confirmed to be identical to the Pseudomonas enzyme in its specificity and other enzymatic properties. No significant sequence similarities were found in other known functional proteins including human acid ceramidase. However, we found a sequence homologous to the ceramidase in hypothetical proteins encoded in Mycobacterium tuberculosis, Dictyostelium discoideum, and Arabidopsis thaliana. The homologue of the ceramidase gene was thus cloned from an M. tuberculosis cosmid and expressed in E. coli, and the gene was demonstrated to encode an alkaline ceramidase. This is the first report for the cloning of an alkaline ceramidase.     

Cloning and nucleotide sequencing of a novel 7 beta-(4-carboxybutanamido)cephalosporanic acid acylase gene of Bacillus laterosporus and its expression in Escherichia coli and Bacillus subtilis.

A strain of Bacillus species which produced an enzyme named glutaryl 7-ACA acylase which converts 7 beta-(4-carboxybutanamido)cephalosporanic acid (glutaryl 7-ACA) to 7-amino cephalosporanic acid (7-ACA) was isolated from soil. The gene for the glutaryl 7-ACA acylase was cloned with pHSG298 in Escherichia coli JM109, and the nucleotide sequence was determined by the M13 dideoxy chain termination method. The DNA sequence revealed only one large open reading frame composed of 1,902 bp corresponding to 634 amino acid residues. The deduced amino acid sequence contained a potential signal sequence in its amino-terminal region. Expression of the gene for glutaryl 7-ACA acylase was performed in both E. coli and Bacillus subtilis. The enzyme preparations purified from either recombinant strain of E. coli or B. subtilis were shown to be identical with each other as regards the profile of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were composed of a single peptide with the molecular size of 70 kDa. Determination of the amino-terminal sequence of the two enzyme preparations revealed that both amino-terminal sequences (the first nine amino acids) were identical and completely coincided with residues 28 to 36 of the open reading frame. Extracellular excretion of the enzyme was observed in a recombinant strain of B. subtilis.     

Primary structure requirements for the maturation in vivo of penicillin acylase from Escherichia coli ATCC 11105

The two constituent subunits of the enzyme penicillin acylase from Escherichia coli strain ATCC 11105 are derived from a single precursor polypeptide by post-translational processing. Mutant penicillin acylase precursors were constructed carrying insertions and deletions in various domains and they were analysed for their processing behaviour. It was found that an endopeptide region of appropriate size and an intact C-terminus were absolutely necessary for the maturation process. Internal deletions within the beta-subunit domain also prevented post-translational cleavage. Processing competence, therefore, was not merely determined by the amino acid sequence in the vicinity of the processing sites but relied on a correct overall conformation of the protein. The processing pathway in vivo proceeds via an intermediate comprising the alpha subunits plus endopeptide and is thus identical to the pathway which has been determined previously by in vitro analysis. The post-translational modification of the precursor is probably not carried out by a specific processing enzyme(s) as the heterologous expression of the penicillin acylase (pac) structural gene yielded processed and active enzyme in different enterobacteria and in a Pseudomonas species.     

Cloning and nucleotide sequence of the aspartase gene of Pseudomonas fluorescens

The aspartase gene (aspA) of Pseudomonas fluorescens was cloned and the nucleotide sequence of the 2,066-base-pair DNA fragment containing the aspA gene was determined. The amino acid sequence of the protein deduced from the nucleotide sequence was confirmed by N- and C-terminal sequence analysis of the purified enzyme protein. The deduced amino acid composition also fitted the previous amino acid analysis results well (Takagi et al. (1984) J. Biochem. 96, 545-552). These results indicate that aspartase of P. fluorescens consists of four identical subunits with a molecular weight of 50,859, composed of 472 amino acid residues. The coding sequence of the gene was preceded by a potential Shine-Dalgarno sequence and by a few promoter-like structures. Following the stop codon there was a structure which is reminiscent of the Escherichia coli rho-independent terminator. The G + C content of the coding sequence was found to be 62.3%. Inspection of the codon usage for the aspA gene revealed as high as 80.0% preference for G or C at the third codon position. The deduced amino acid sequence was 56.3% homologous with that of the enzyme of E. coli W (Takagi et al. (1985) Nucl. Acids Res. 13, 2063-2074). Cys-140 and Cys-430 of the E. coli enzyme, which had been assigned as functionally essential (Ida & Tokushige (1985) J. Biochem. 98, 793-797), were substituted by Ala-140 and Ala-431, respectively, in the P. fluorescens enzyme.     

Unorthodox expression of an enzyme: evidence for an untranslated region within carA from Pseudomonas aeruginosa.

The genes encoding carbamoylphosphate synthetase from Pseudomonas aeruginosa PAO1 were cloned in Escherichia coli. Deletion and transposition analysis determined the locations of carA, encoding the small subunit, and carB, encoding the large subunit, on the chromosomal insert. The nucleotide sequence of carA and the flanking regions was determined. The derived amino acid sequence for the small subunit of carbamoylphosphate synthetase from P. aeruginosa exhibited 68% homology with its counterparts in E. coli and Salmonella typhimurium. The derived sequences in the three organisms were essentially identical in the three polypeptide segments that are conserved in glutamine amidotransferases but showed low homology at the amino- and carboxy-terminal regions. The amino-terminal amino acid sequences were determined for the large and small subunits. The first 15 amino acids of the large subunit were identical to those derived from the carB sequence. However, comparison of the derived sequence for carA with the amino-terminal amino acid sequence for the small subunit suggested that codons 5 to 8 are not translated. The DNA sequence for the region encompassing these four codons was confirmed by direct sequencing of chromosomal DNA after amplification by the polymerase chain reaction. The mRNA sequence was also deduced by in vitro synthesis of cDNA, enzymatic amplification, and sequencing, confirming that 12 nucleotides in the 5' terminal of carA are transcribed but are not translated.     

A novel R-stereoselective amidase from Pseudomonas sp. MCI3434 acting on piperazine-2-tert-butylcarboxamide.

A novel amidase acting on (R,S)-piperazine-2-tert-butylcarboxamide was purified from Pseudomonas sp. MCI3434 and characterized. The enzyme acted R-stereoselectively on (R,S)-piperazine-2-tert-butylcarboxamide to yield (R)-piperazine-2-carboxylic acid, and was tentatively named R-amidase. The N-terminal amino acid sequence of the enzyme showed high sequence identity with that deduced from a gene named PA3598 encoding a hypothetical hydrolase in Pseudomonas aeruginosa PAO1. The gene encoding R-amidase was cloned from the genomic DNA of Pseudomonas sp. MCI3434 and sequenced. Analysis of 1332 bp of the genomic DNA revealed the presence of one open reading frame (ramA) which encodes the R-amidase. This enzyme, RamA, is composed of 274 amino acid residues (molecular mass, 30 128 Da), and the deduced amino acid sequence exhibits homology to a carbon-nitrogen hydrolase protein (PP3846) from Pseudomonas putida strain KT2440 (72.6% identity) and PA3598 protein from P. aeruginosa strain PAO1 (65.6% identity) and may be classified into a new subfamily in the carbon-nitrogen hydrolase family consisting of aliphatic amidase, beta-ureidopropionase, carbamylase, nitrilase, and so on. The amount of R-amidase in the supernatant of the sonicated cell-free extract of an Escherichia coli transformant overexpressing the ramA gene was about 30 000 times higher than that of Pseudomonas sp. MCI3434. The intact cells of the E. coli transformant could be used for the R-stereoselective hydrolysis of racemic piperazine-2-tert-butylcarboxamide. The recombinant enzyme was purified to electrophoretic homogeneity from cell-free extract of the E. coli transformant overexpressing the ramA gene. On gel-filtration chromatography, the enzyme appeared to be a monomer. It had maximal activity at 45 degrees C and pH 8.0, and was completely inactivated in the presence of p-chloromercuribenzoate, N-ethylmaleimide, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Ag+, Cd2+, Hg2+, or Pb2+. RamA had hydrolyzing activity toward the carboxamide compounds, in which amino or imino group is connected to beta- or gamma-carbon, such as beta-alaninamide, (R)-piperazine-2-carboxamide (R)-piperidine-3-carboxamide, D-glutaminamide and (R)-piperazine-2-tert-butylcarboxamide. The enzyme, however, did not act on the other amide substrates for the aliphatic amidase despite its sequence similarity to RamA.     

Cloning and overproduction of biodegradative threonine deaminase from Escherichia coli W strain.

We have cloned the structural gene (tdcB) of biodegradative threonine deaminase from Escherichia coli W strain by utilizing the polymerase chain reaction. The JM109/pUCTDA strain, which was obtained by transforming E. coli JM109 with a vector plasmid (pUCTDA) containing the cloned tdcB gene, produced a large amount of the enzyme corresponding to more than 5% of the total soluble protein. Amino acid sequence analysis of this recombinant enzyme showed that the amino acid sequence is identical to the nucleotide-deduced sequence of biodegradative threonine deaminase from E. coli K-12.     

Genetic organization and sequence of the Pseudomonas cepacia genes for the alpha and beta subunits of protocatechuate 3,4-dioxygenase.

The locations of the genes for the alpha and beta subunits of protocatechuate 3,4-dioxygenase (EC 1.13.11.3) on a 9.5-kilobase-pair PstI fragment cloned from the Pseudomonas cepacia DBO1 chromosome were determined. This was accomplished through the construction of several subclones into the broad-host-range cloning vectors pRO2317, pRO2320, and pRO2321. The ability of each subclone to complement mutations in protocatechuate 3,4-dioxygenase (pcaA) was tested in mutant strains derived from P. cepacia, Pseudomonas aeruginosa, and Pseudomonas putida. These complementation studies also showed that the two subunits were expressed from the same promoter. The nucleotide sequence of the region encoding for protocatechuate 3,4-dioxygenase was determined. The deduced amino acid sequence matched that determined by N-terminal analysis of regions of the isolated enzyme. Although over 400 nucleotides were sequenced before the start of the genes, no homology to known promoters was found. However, a terminator stem-loop structure was found immediately after the genes. The deduced amino acid sequence showed extensive homology with the previously determined amino acid sequence of protocatechuate 3,4-dioxygenase from another Pseudomonas species.