Molecular cloning and nucleotide sequence of the factor IIIlac gene of Lactobacillus casei

The lactose-specific factor III (FIIIlac of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was isolated from Lactobacillus casei and purified to homogeneity by conventional protein purification methods. Its apparent native Mr, estimated from steric exclusion chromatography (approx. 39 kDa), and subunit Mr, estimated from sodium dodecyl sulfate-polyacrylamide gels, indicated that it exists as a trimer of identical subunits of 13 kDa. The gene for FIII L. casei lac was cloned into Escherichia coli using the vector pUC18. The coding sequences were contained on an 860-bp BglII-HindIII DNA fragment of the L. casei lactose plasmid, pLZ64. A protein identical in properties to FIII L. casei lac was isolated from clones of E. coli carrying this DNA insert. The nucleotide sequence of the FIII L. casei lac gene was determined by the dideoxy chain-termination technique. The 336-bp open reading frame for FIII L. casei lac was followed by a stem-loop structure, analogous to a Rho-independent terminator. We concluded that the FIII L. casei lac was the terminal gene in what appears to be an operon comprised of the lactose-PTS-P-beta Gal-coding genes. Comparison of the deduced amino acid sequence of FIII L. caseilac with the amino acid sequence of FIII S. aureus lac (derived from peptide sequencing) demonstrated a high degree of homology (49 identical residues and 21 conservative exchanges out of 103 total aa residues). The FIII L. casei lac lacked his82, previously identified as the phosphorylation site of FIII S. aureus. lac His80 was proposed to be the site of histidyl phosphorylation of FIII L. casei lac.     

Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein.

The sequence of a 1,693-base-pair plasmid DNA fragment from Flavobacterium sp. strain ATCC 27551 containing the parathion hydrolase gene (opd) was determined. Within this sequence, there is only one open reading frame large enough to encode the 35,000-dalton membrane-associated hydrolase protein purified from Flavobacterium extracts. Amino-terminal sequence analysis of the purified Flavobacterium hydrolase demonstrated that serine is the amino-terminal residue of the hydrolase protein. The amino-terminal serine corresponds to a TCG codon located 87 base pairs downstream of the presumptive ATG initiation codon in the nucleotide sequence. The amino acid composition of the purified protein agrees well with that predicted from the nucleotide sequence, using serine as the amino-terminal residue. These data suggest that the parathion hydrolase protein is processed at its amino terminus in Flavobacterium sp. Construction in Escherichia coli of a lacZ-opd gene fusion in which the first 33 amino-terminal residues of opd were replaced by the first 5 residues of lacZ resulted in the production of an active hydrolase identical in molecular mass to the hydrolase isolated from Flavobacterium sp. E. coli cells containing the lacZ-opd fusion showed higher levels of hydrolase activity than did cells containing the parent plasmid.     

Folylpoly-gamma-glutamate synthetase-dihydrofolate synthetase. Cloning and high expression of the Escherichia coli folC gene and purification and properties of the gene product

The Escherichia coli gene for folylpolyglutamate synthetase-dihydrofolate synthetase was localized to plasmids pLC22-45, 24-31, and 28-44 of the Clarke-Carbon E. coli colony bank (Clarke, L., and Carbon, J. (1976) Cell 9, 91-99) by screening the bank by replica mating with an E. coli folC mutant. The folC gene was subcloned from pLC22-45 and inserted into a high copy number plasmid containing the lambda replication control region under the control of the temperature-sensitive cI857 repressor and into a high expression plasmid containing the lambda PL promoter and the cI857 repressor. The folC structural gene was located on a 1.52-kilobase PvuI fragment, sufficient to code for a protein of maximum Mr 55,000. E. coli transformants containing the recombinant plasmids, when induced by culturing at 42 degrees C, had folylpolyglutamate synthetase and dihydrofolate synthetase levels that were 100- to 400-fold higher than in wild type strains and which represented up to 4% of the soluble cell protein. The E. coli folylpolyglutamate synthetase-dihydrofolate synthetase has been purified to homogeneity from the transformants. Both activities are catalyzed by a single protein of Mr 47,000. Some kinetic properties of the enzymes and a new spectrophotometric method for assaying dihydrofolate synthetase activity are described.     

Replacement of the folC gene, encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli, with genes mutagenized in vitro.

The folylpolyglutamate synthetase-dihydrofolate synthetase gene (folC) in Escherichia coli was deleted from the bacterial chromosome and replaced by a selectable Kmr marker. The deletion strain required a complementing gene expressing folylpolyglutamate synthetase encoded on a plasmid for viability, indicating that folC is an essential gene in E. coli. The complementing folC gene was cloned into the vector pPM103 (pSC101, temperature sensitive for replication), which segregated spontaneously at 42 degrees C in the absence of selection. This complementing plasmid was replaced in the folC deletion strain by compatible pUC plasmids containing folC genes with mutations generated in vitro, producing strains which express only mutant folylpolyglutamate synthetase. Mutant folC genes expressing insufficient enzyme activity could not complement the chromosomal deletion, resulting in retention of the pPM103 plasmid. Some mutant genes expressing low levels of enzyme activity replaced the complementing plasmid, but the strains produced were auxotrophic for products of folate-dependent pathways. The folylpolyglutamate synthetase gene from Lactobacillus casei, which may lack dihydrofolate synthetase activity, replaced the complementing plasmid, but the strain was auxotrophic for all folate end products.     

UTP: alpha-D-glucose-1-phosphate uridylyltransferase of Escherichia coli: isolation and DNA sequence of the galU gene and purification of the enzyme.

The galU gene of Escherichia coli, thought to encode the enzyme UTP:alpha-D-glucose-1-phosphate uridylyltransferase, had previously been mapped to the 27-min region of the chromosome (J. A. Shapiro, J. Bacteriol. 92:518-520, 1966). By complementation of the membrane-derived oligosaccharide biosynthetic defect of strains with a galU mutation, we have now identified a plasmid containing the galU gene and have determined the nucleotide sequence of this gene. The galU gene is located immediately downstream of the hns gene, and its open reading frame would be transcribed in the direction opposite that of the hns gene (i.e., clockwise on the E. coli chromosome). The nucleotide sequences of five galU mutations were also determined. The enzyme UTP:alpha-D-glucose-1-phosphate uridylyltransferase was purified from a strain containing the galU gene on a multicopy plasmid. The amino-terminal amino acid sequence (10 residues) of the purified enzyme was identical to the predicted amino acid sequence (after the initiating methionine) of the galU-encoded open reading frame. The functional enzyme appears to be a tetramer of the galU gene product.     

Cloning and characterization of the gene coding for cytoplasmic seryl-tRNA synthetase from Saccharomyces cerevisiae.

We have screened a Saccharomyces cerevisiae expression library with antibodies against seryl-tRNA synthetase (SerRS) from baker's yeast. In this way we obtained clones which contain serS, the structural gene for seryl-tRNA synthetase. Genomic Southern blots show that the serS gene resides on a 5.0 kb SalI fragment. Nucleotide sequence analysis of the genes revealed a single open reading frame from which we deduced the amino acid sequence of the enzyme consistent with that of two peptides isolated from SerRS. The enzyme is comprised of 462 amino acids consistent with earlier determinations of its molecular weight. The codon usage of serS is typical of abundant yeast proteins. Nuclease S1 analysis of serS mRNA defined the RNA initiation site 20-40 bases downstream from an AT rich sequence containing the TATA box and 21-39 nucleotides upstream of the translation initiation codon. Yeast strains transformed with the cloned gene overproduce seryl-tRNA synthetase in vivo.     

Nucleotide sequence of the gene ereA encoding the erythromycin esterase in Escherichia coli

We have cloned and determined the nucleotide sequence of the gene ereA of plasmid pIP1100 which confers high-level resistance to erythromycin (Em) in Escherichia coli. The gene was defined by initiation and termination codons and by in vitro insertion-inactivation into an open reading frame (ORF) of 1032 bp corresponding to a product with an Mr of 37 765. However, the enzyme, an Em esterase, displayed an apparent Mr of 43 000 upon electrophoresis of a minicell extract on the SDS-polyacrylamide gels. The G + C content (50.5%) of the gene ereA and the preferential codon usage in its ORF suggest that this resistance determinant should be indigenous to E. coli.     

Cloning and characterization of an amidase gene from Rhodococcus species N-774 and its expression in Escherichia coli

For investigation of an unknown open reading frame which is present upstream of the nitrile hydratase (NHase) gene from Rhodococcus sp. N-774, a longer DNA fragment covering the entire gene was cloned in Escherichia coli. Nucleotide sequencing and detailed subcloning experiments predicted a single open reading frame consisting of 521 amino acid residues of Mr 54,671. The amino acid sequence, especially its NH2-terminal portion, showed significant homology with those of indoleacetamide hydrolases from Pseudomonas savastanoi and Agrobacterium tumefaciens, and acetamidase from Aspergillus nidulans. The 521-amino acid coding region was therefore expressed by use of the E. coli lac promoter in E. coli, and was found to direct a considerable amidase activity. This amidase hydrolyzed propionamide efficiently, and also hydrolyzed, at a lower efficiency, acetamide, acrylamide and indoleacetamide. These data clearly show that the unknown open reading frame present upstream of the NHase coding region encodes an amidase. Because the TAG translational stop codon of the amidase is located only 75 base pairs apart from the ATG start codon of the alpha-subunit of NHase, these genes are probably translated in a polycistronic manner.     

Nucleotide sequences and genomic constitution of five tryptophan genes of Lactobacillus casei

Five trp genes, trpD, trpC, trpF, trpB, and trpA, of Lactobacillus casei were cloned by transformation of tryptophan auxotrophic mutants of the respective trp genes in Escherichia coli. These trp genes appear to constitute an operon and are located in the above order in a segment of DNA of 6,468 base pairs. The entire nucleotide sequence of this DNA segment was determined. Five contiguous open reading frames in this segment can encode proteins consisting of 341, 260, 199, 406, and 266 amino acids, respectively, in the same direction. The amino acid sequences of these proteins exhibit 25.5-50.2% homology with the amino acid sequences of the corresponding trp enzymes of E. coli. Two trp genes, trpC and trpF, from L. casei can complement mutant alleles of the corresponding genes of E. coli. However, neither the trpA gene nor the trpB gene of L. casei can complement mutations in the E. coli trpA gene and the trpB gene, respectively, suggesting that the protein products of the L. casei and E. coli trpA and trpB genes, respectively, cannot form heterodimers of tryptophan synthetase with activity. Other features of the coding and flanking regions of the trp genes are also described.     

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 nucleotide sequence determination of the isopenicillin N synthetase gene from Streptomyces clavuligerus

The isopenicillin N synthetase (IPNS) gene from Streptomyces clavuligerus was isolated from an Escherichia coli plasmid library of S. clavuligerus genomic DNA fragments using a 44-mer mixed oligodeoxynucleotide probe. The nucleotide sequence of a 3-kb region of the cloned fragment from the plasmid, pBL1, was determined and analysis of the sequence showed an open reading frame that could encode a protein of 329 amino acids with an Mr of 36,917. When the S. clavuligerus DNA from pBL1 was introduced into an IPNS-deficient mutant of S. clavuligerus on the Streptomyces vector pIJ941, the recombinant plasmid was able to complement the mutation and restore IPNS activity. The protein coding region of the S. clavuligerus IPNS gene shows about 63% and 62% similarity to the Cephalosporium acremonium and Penicillium chrysogenum IPNS nucleotide sequences, respectively, and the predicted amino acid sequence of the encoded protein showed about 56% similarity to both fungal sequences.     

Amplification of the bacA gene confers bacitracin resistance to Escherichia coli.

An Escherichia coli genomic library was constructed in order to facilitate selection for genes which confer bacitracin resistance through amplification. One of the plasmids from the library, plasmid pXV62, provided a high level of bacitracin resistance for E. coli. Deletion and nucleotide sequence analyses of bacitracin resistance plasmid pXV62 revealed that a single open reading frame, designated the bacA gene, was sufficient for antibiotic resistance. The bacA gene mapped to approximately 67 min on the E. coli chromosome by proximity to a previously mapped locus. The deduced amino acid sequence of the bacA-encoded protein suggests an extremely hydrophobic protein of 151 amino acids, approximately 65% of which were nonpolar amino acids. E. coli cells containing plasmid pXV62 have increased isoprenol kinase activity. The physical characteristics of the deduced protein and enhanced lipid kinase activity suggest that the bacA gene may confer resistance to bacitracin by phosphorylation of undecaprenol.     

Cloning and characterization of the glycogen branching enzyme gene existing in tandem with the glycogen debranching enzyme from Pectobacterium chrysanthemi PY35

The glycogen branching enzyme gene (glgB) from Pectobacterium chrysanthemi PY35 was cloned, sequenced, and expressed in Escherichia coli. The glgB gene consisted of an open reading frame of 2196bp encoding a protein of 731 amino acids (calculated molecular weight of 83,859Da). The glgB gene is upstream of glgX and the ORF starts the ATG initiation codon and ends with the TGA stop codon at 2bp upstream of glgX. The enzyme was 43-69% sequence identical with other glycogen branching enzymes. The enzyme is the most similar to GlgB of E. coli and contained the four regions conserved among the alpha-amylase family. The glycogen branching enzyme (GlgB) was purified and the molecular weight of the enzyme was estimated to be 84kDa by SDS-PAGE. The glycogen branching enzyme was optimally active at pH 7 and 30 degrees C.     

Cloning and sequencing of the gene encoding the large subunit of glutathione synthetase of Schizosaccharomyces pombe

The gene for the large subunit of glutathione synthetase (EC 6.3.2.3) of Schizosaccharomyces pombe was cloned from a S. pombe genomic DNA library by complementation of cadmium hypersensitivity of a glutathione synthetase deficient mutant of S. pombe. A long open reading frame was found in the cloned DNA sequence. Amino acid sequence predicted from the long open reading frame coincided with amino acid sequences of peptides obtained by V8 protease digestion of the large subunit of the purified glutathione synthetase. The glutathione synthetase deficient mutant which harbored plasmids containing the glutathione synthetase large subunit gene exhibited glutathione synthetase activity higher than the activity in the wild type strain, though the plasmid did not contain the gene for the small subunit of the enzyme.     

Mutagenesis of the folC gene encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli

The folC gene of Escherichia coli, cloned in a pUC19 vector, was mutagenized by progressive deletions from both the 5' and the 3' ends and by TAB linker insertion. A number of 5'-deleted genes, which had the initiator ATG codon removed, produced a truncated gene product, in reduced amounts, from a secondary initiation site. The most likely position of this site at a GTG codon located 35 codons downstream of the normal start site. This product could complement the folC mutation in E. coli strain SF4 as well as a strain deleted in the folC gene. The specific activity of extracts of the mutant enzyme are 4-16% that of the wild type enzyme for the folylpolyglutamate synthetase activity and 6-19% for the dihydrofolate synthetase activity. The relative amount of protein expressed by the mutant, compared to the wild type, in maxicells was comparable to the relative specific activity, suggesting that the kcat of the mutant enzyme is similar to that of the wild type. Mutants with up to 14 amino acids deleted from the carboxy terminal could still complement the folC deletion mutant. Seven out of ten linker insertions dispersed through the coding region of the gene showed complementation of the folC mutation in strain SF4 but none of these insertion mutants were able to complement the strain containing a deleted folC gene. None of the carboxy terminal or linker insertion mutants had a specific activity greater than 0.5% that of the wild type enzyme. The dihydrofolate synthetase and folylpolyglutamate synthetase activities behaved similarly in all mutants, both retaining a large fraction of the wild type activity in the amino terminal deletions and both being very low in the carboxy terminal deletions and linker insertion mutants. These studies are consistent with a single catalytic site for the two activities catalyzed by this enzyme.     

Biosynthesis of riboflavin: cloning, sequencing, mapping, and expression of the gene coding for GTP cyclohydrolase II in Escherichia coli.

GTP cyclohydrolase II catalyzes the first committed step in the biosynthesis of riboflavin. The gene coding for this enzyme in Escherichia coli has been cloned by marker rescue. Sequencing indicated an open reading frame of 588 bp coding for a 21.8-kDa peptide of 196 amino acids. The gene was mapped to a position at 28.2 min on the E. coli chromosome and is identical with ribA. GTP cyclohydrolase II was overexpressed in a recombinant strain carrying a plasmid with the cloned gene. The enzyme was purified to homogeneity from the recombinant strain. The N-terminal sequence determined by Edman degradation was identical to the predicted sequence. The sequence is homologous to the 3' part of the central open reading frame in the riboflavin operon of Bacillus subtilis.