Molecular cloning and expression of a proteinase gene from Lactococcus lactis subsp. cremoris H2 and construction of a new lactococcal vector pFX1

The 6.5 kb HindIII DNA fragment of the Lactococcus lactis subsp. cremoris H2 plasmid pDI21 was cloned into Escherichia coli POP13 with NM1149, and also directly into Lactococcus lactis subsp. lactis 4125 using a newly-constructed broad host-range vector pFX1. Proteinase was experessed in both transformed organisms. The proteinase resembles a PI type since it preferentially degraded -casein. The restriction map of the 6.5 kb proteinase gene fragment has minor differences from those of published plamid proteinase genes. High-efficiency electroporation with pFX1 provides a direct approach for gene cloning in lactococci.Abbreviations cfu colony forming units - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid] Dedicated to Prof. Dr. G. Drews on the occasion of his 65th birthday     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

A novel site for streptomycin resistance in the “530 loop” of chloroplast 16S ribosomal RNA

The chloroplast gene for 16S rRNA was cloned from two maternally inherited streptomycin-resistant mutants ofNicotiana differing in degree of resistance at the whole plant and isolated chloroplast level. A single-nucleotide change in the 16S rRNA gene was detected for each mutant: a C to T transition at nucleotide 860 (Escherichia coli coordinate C₉₁₂) which is an often mutated site, and a novel transition of C to T at nucleotide 472 (E. coli coordinate C₅₂₅). The novel mutation is located in the phylogenetically conserved 530 loop.     

Molecular cloning of Bacillus subtilis genes involved in DNA metabolism

Summary Different clones carrying a chromosomal DNA fragment able to transform Bacillus subtilis mutants dnaA13, dnaB19, dnaG5, recG40 and polA42 to a wild-type phenotype were isolated from a library constructed in plasmid pJH101. A recombinant clone carrying a chromosomal fragment able to transform dnaC mutants was obtained from a Charon 4A library. A restriction map of the cloned DNA fragments was constructed. The 11.3 kb cloned DNA fragment of plasmid pMP60-13 containing the wild-type sequence of dnaG5 was shown to transform a recF33 mutant as well.     

Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum

The gene for the catalytic subunit of cellulose synthase from Acetobacter xylinum has been cloned by using an oligonucleotide probe designed from the N-terminal amino acid sequence of the catalytic subunit (an 83 kDa polypeptide) of the cellulose synthase purified from trypsin-treated membranes of A. xylinum. The gene was located on a 9.5 kb HindIII fragment of A. xylinum DNA that was cloned in the plasmid pUC18. DNA sequencing of approximately 3 kb of the HindIII fragment led to the identification of an open reading frame of 2169 base pairs coding for a polypeptide of 80 kDa. Fifteen amino acids in the N-terminal region (positions 6 to 20) of the amino acid sequence, deduced from the DNA sequence, match with the N-terminal amino acid sequence obtained for the 83 kDa polypeptide, confirming that the DNA sequence cloned codes for the catalytic subunit of cellulose synthase which transfers glucose from UDP-glucose to the growing glucan chain. Trypsin treatment of membranes during purification of the 83 kDa polypeptide cleaved the first 5 amino acids at the N-terminal end of this polypeptide as observed from the deduced amino acid sequence, and also from sequencing of the 83 kDa polypeptide purified from membranes that were not treated with trypsin. Sequence analysis suggests that the cellulose synthase catalytic subunit is an integral membrane protein with 6 transmembrane segments. There is no signal sequence and it is postulated that the protein is anchored in the membrane at the N-terminal end by a single hydrophobic helix. Two potential N-glycosylation sites are predicted from the sequence analysis, and this is in agreement with the earlier observations that the 83 kDa polypeptide is a glycoprotein [13]. The cloned gene is conserved among a number of A. xylinum strains, as determined by Southern hybridization.     

Cloning and expression in Escherichia coli K-12 of the biosynthetic dehydroquinase function of the arom cluster gene from the eucaryote, Aspergillus nidulans

Summary A 1.35 Md DNA HindIII fragment containing part of the arom gene cluster or cluster gene of Aspergillus nidulans encoding biosynthetic dehydroquinase (5-dehydroquinate hydrolyase) has been cloned in plasmid pBR322 on the basis of functional expression in Escherichia coli. The fungal fragment on pBR322, designated pHK29, complements a corresponding E. coli dehydroquinase structural gene (aroD) mutation. pHK29 contains one BamHI, HpaII, PstI, SmaI, XhoI and surprisingly, one HindIII site since pHK29 hybrid Aspergillus DNA is a HindIII fragment itself. The biosynthetic dehydroquinase activity extracted from E. coli strains, containing pHK29, had properties similar to those of the enzyme activity from Aspergillus. The protein specified by pHK29 appears to be 80 Kd. No increase of dehydroquinase activity was found in polynucleotide phosphorylase deficient strains (pnp) of E. coli.Standard Abbreviations Used SSC Standard saline citrate (3 M Sodium Chloride, 0.15 M Sodium citrate) - EDTA Ethylenediaminetetra acetic acid - DTT Dithiothreitol - PMSF Phenyl methyl sulphonyl fluoride - TEMED N N N N, Tetramethylethylenediamine - Md Megadaltons - Kd Kilodaltons     

The bacteriophage T4 dexA gene: sequence and analysis of a gene conditionally required for DNA replication

Summary We have cloned and sequenced a bacteriophage T4 EcoRI fragment that complements T4 del (39-56) infections of an optA defective Escherichia coli strain. Bacteria containing this recombinant plasmid synthesize two new proteins with molecular weights of 9 and 26 kilodaltons. We have identified the gene encoding the 26 kilodalton protein as essential for T4 infections of optA defective E. coli. Genetic and biochemical results are consistent with the identification of this protein as the product of the dexA gene, which encodes a 3 to 5 exonuclease.     

Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae

Summary A gene conferring high-level resistance to tylosin in Streptomyces lividans and Streptomyces griseofuscus was cloned from a tylosin-producing strain of Streptomyces fradiae. The tylosin-resistance (Tyl^r) gene (tlrA) was isolated on five overlapping DNA fragments which contained a common 2.6 Kb KpnI fragment. The KpnI fragment contained all of the information required for the expression of the Tyl^r phenotype in S. lividans and S. griseofuscus. Southern hybridization indicated that the sequence conferring tylosin resistance was present on the same 5 kb SalI fragment in genomic DNA from S. fradiae and several tylosin-sensitive (Tyl^s) mutants. The cloned tlrA gene failed to restore tylosin resistance in two Tyl^s mutants derived by protoplast formation and regeneration, and it restored partial resistance in a Tyl^s mutant obtained by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The tlrA gene conferred resistance to tylosin, carbomycin, niddamycin, vernamycin-B and, to some degree, lincomycin in S. griseofuscus, but it had no effect on sensitivity to streptomycin or spectinomycin, suggesting that the cloned gene is an MLS (macrolide, lincosamide, streptogramin-B)-resistance gene. Twenty-eight kb of S. fradiae DNA surrounding the tlrA gene was isolated from a genomic library in bacteriophage Charon 4. Introduction of these DNA sequence into S. fradiae mutants blocked at different steps in tylosin biosynthesis failed to restore tylosin production, suggesting that the cloned Tyl^r gene is not closely linked to tylosin biosynthetic genes.     

Nucleotide sequence of the celA gene encoding a cellodextrinase of Ruminococcus flavefaciens FD-1

Summary The nucleotide sequence of a 3.6 kb DNA fragment containing a cellodextrinase gene (celA) fromRuminococcus flavefaciens FD-1 was determined. The gene was expressed from its own regulatory region inEscherichia coli and a putative consensus promoter sequence was identified upstream of a ribosome binding site and a TTG start codon. The complete amino acid sequence of the CeIA enzyme (352 residues) was deduced and showed no significant homology to cellulases from other oganisms. Two lysozymetype active sites were found in the amino-terminal third of the enzyme. InE. coli the cloned CeIA protein was translocated into the periplasm. The lack of a typical signal sequence, and the results of transposonphoA mutagenesis experiments indicated that CeIA is secreted by a mechanism other than a leader peptide.Abbreviations CMCase carboxymethylcellulase - celA gene coding for CeIA - CelA cellodextrinase - ORF open reading frame - phoA gene encoding alkaline phosphatase - pNPC p-nitrophenyl--d-cellobioside     

Cloning and characterization of the bgxA gene from Erwinia chrysanthemi D1 which encodes a β-glucosidase/xylosidase enzyme

A -glucosidase/xylosidase gene from Erwinia chrysanthemi strain D1 was cloned and sequenced. This gene, named bgxA, encodes a ca. 71 kDa protein product which, following removal of the leader peptide, resulted in a ca. 69 kDa mature protein that accumulated in the periplasmic space of E. chrysanthemi strain D1 and Escherichia coli cells expressing the cloned gene. The protein exhibited both -glucosidase and -xylosidase activities but gave no detectable activity on xylan or carboxymethyl cellulose. The enzyme was classified as a type 3 glycosyl hydrolase, but was unusual in having a truncated B region at the carboxyl-terminus. Several E. chrysanthemi strains isolated from corn produced the glucosidase/xylosidase activity but not those isolated from dicot plants. However, bgxA marker exchange mutants of strain D1 were not detectably altered in virulence on corn leaves.     

Molecular cloning of the uvrD gene of Escherichia coli that controls ultraviolet sensitivity and spontaneous mutation frequency

Summary The uvrD gene of Escherichia coli that control UV sensitivity and spontaneous mutation frequency has been cloned with phage as vector. The increased sensitivity to ultraviolet light (UV) of uvrD3, uvrE502, recL152, and pdeB41 mutants, high mutability of uvrD3 and pdeB41 mutants, and conditional lethality of strain TS41 that carried pdeB41, polA1, and sup126 mutations were all suppressed by lysogenization of the mutant cells with uvrD ⁺. These results were consistent with the idea that the uvrD, uvrE, recL, and pdeB mutations are alleles of the uvrD gene. In addition to the uvD gene, uvrD ⁺ carried the corA gene that controls transport of Mg⁺⁺, Mn⁺⁺, and Co⁺⁺ through the cell membrane. Hybrid plasmids carrying both uvrD and corA genes were also constructed by using pKY2289 as a cloning vehicle. Orientational isomers that carried the same 12.0 kb fragment in the opposite direction were equally efficient in complementing the UvrD^- as well as CorA^- defects of the transformed host cells, suggesting that the DNA insert contains all the genetic signals needed to express the two gene products. Insertion of the sequence into recombinant plasmids was performed to generate appropriate restriction endonuclease target sites in the cloned DNA fragments.     

Cloning and expression in Escherichia coli, Bacillus subtilis, and Streptococcus sanguis of a gene for staphylokinase--a bacterial plasminogen activator

Summary The gene coding for the bacterial plasminogen activator staphylokinase was cloned from the Staphylococcus aureus phage 42D, a serogroup F phage used for lysotyping, onto the standard Escherichia coli plasmid vector pACYC184. The coding and flanking sequences of the sak42D gene were largely identical to those of a sak gene cloned from the serologically different S. aureus phage SøC (Sako and Tsuchida 1983). Subcloning of a 2.5 kb phage 42D DNA fragment onto plasmid pGB3631 allowed the sak42D gene to be introduced into the gram-positive hosts Bacillus subtilis and Streptococcus sanguis. The sak42D gene was expressed and secreted most efficiently by B. subtilis cells (25 g/ml of culture supernatant) reduced in exoprotease production. In this host expression and secretion of Sak was initiated at the early growth phase and continued through the logarithmic phase. Formation of Sak was, however, also observed with the other cloning hosts. The Sak elaborated by the heterologous hosts was serologically identical with authentic Sak derived from S. aureus.     

The nucleotide sequence of the lipo-penicillinase gene of alkalophilic Bacillus sp. strain 170

The lipo-penicillinase (LIPEN) gene from an alkalophilic Bacillus sp. strain 170 was cloned in Escherichia coli using the vector pHSG399. A plasmid, pFAP121, was isolated from an ampicillin resistant transformant and the cloned LIPEN gene was found to be in a 2.2 kb DNA fragment. The nucleotide sequence of a 1.9 kb segment encoding the LIPEN was determined. This segment showed an open reading frame which would encode a polypeptide of 310 amino acids. The amino acid sequence of this LIPEN gene product has strong homology with those of the Bacillus cereus -lactamase III and Bacillus licheniformis penicillinase.     

The sequence and function of the recA gene and its protein in Pseudomonas aeruginosa PAO

Summary The recA gene of Pseudomonas aeruginosa has been isolated and its nucleotide sequence has been determined. The coding region of the recA gene has 1038 bp specifying 346 amino acids. The recA protein of P. aeruginosa showed a striking homology with that of Escherichia coli except for the carboxy-terminal region both at the nucleotide and amino acid level. The recA ⁺-carrying plasmids restored the UV sensitivity and recombination ability of several rec mutants of P. aeruginosa. The precise location of the recA gene on the chromosome was deduced from the analysis of R plasmids.     

Isolation of a new cellulase gene from a thermophilic anaerobe and its expression inEscherichia coli

Summary A new cellulase gene was cloned and expressed inEscherichia coli from a thermophilic anaerobe, strain NA10. A 7.4 kbEcoRI fragment of NA10 DNA encoded the cellulase which hydrolyzed carboxymethyl cellulose, lichenan, andp-nitrophenyl--d-cellobioside, but could not digest laminarin andp-nitrophenyl--d-glucoside. The cloned enzyme could digest cellooligosaccharides and release cellobiose as a main product from cellotetraose but could not digest cellobiose. It was distinct from the endoglucanase which was cloned by us previously from NA10 strain in terms ofp-nitrophenyl--d-cellobioside degradation activity and the location of restriction enzyme sites. The enzyme produced byE. coli transformant was extremely heat-stable and the optimum temperature for the enzymatic reaction was 80°C. Fifty three percent of the cloned enzyme was detected in the periplasm and the remaining activity existed in the cellular fraction in theE. coli transformant.     

Cloning and expression of the structural gene for beta-glucosidase of Kluyveromyces fragilis in Escherichia coli and Saccharomyces cerevisiae

Summary Cellobiose, the last product in cellulose degradation, is converted into two molecules of glucose by a -glucosidase. S. cerevisiae does posses the structural gene for a -glucosidase, but it is very poorly expressed; we thus decided to isolate and characterize that of Kluyveromyces fragilis.We constructed in E. coli HB101 strain a genomic library of the Kluyveromyces fragilis Y610 strain (ATCC 12424), a yeast able to grow on cellobiose and which constitutively produces the -glucosidase. The structural gene for -glucosidase was identified by its expression in E. coli. The initial isolated cosmid KF1 contained an insert of 35 Kb and by successive subcloning the insert size was reduced to 3.5 Kb (KF4).This cloned -glucosidase gene introduced in S. cerevisiae by transformation is expressed at a level of about 500 times that of K. fragilis. We checked by Southern hybridization that the high expression level was not due to a rearrangement of K. fragilis DNA during the cloning experiments. Nevertheless to obtain yeast transformants able to grow on cellobiose a yeast strain whose permeability to sugar is increased must be used and this last point is discussed.     

Isolation of Hem3 mutants from Candida albicans by sequential gene disruption

Summary Molecular methods for directed mutagenesis in Candida albicans have relied on a combination of gene disruption by transformation to inactivate one allele and UV-induced mitotic recombination or point mutation to produce lesions in the second allele. An alternate method which uses two sequential gene disruptions was developed and used to construct a C. albicans mutant defective in a gene essential for synthesizing tetrapyrrole (uroporphyrinogen I synthase). The Candida gene was cloned from a random library by complementation of the hem3 mutation in Saccharomyces cerevisiae. The complementing region was limited to a 2.0 kb fragment by subcloning and a BglII site was determined to be within an essential region. Linear fragments containing either the Candida URA3 or LEU2 gene inserted into the BglII site were used to disrupt both alleles of a leu2, ura3 mutant by sequential transformation. Ura⁺, Leu⁺ heme-requiring strains were recovered and identified as hem3 mutants by Southern hybridization, transformation to heme independence by the cloned gene, and enzyme assays.     

Enzymes of N-methylputrescine biosynthesis in relation to hyoscyamine formation in transformed root cultures of Datura stramonium and Atropa belladonna

The activities of enzymes related to the biosynthesis of N-methylputrescine, a precursor of the alkaloid hyoscyamine, have been measured in root cultures of Datura stramonium L. and Atropa belladonna L. transformed with Agrobacterium rhizogenes. Ornithine -Nmethyltransferase and -N-methylornithine decafboxylase were undetectable, indicating that -N-methylornithine is an unlikely intermediate in the formation of N-methylputrescine. The activity of putrescine-N-methyltransferase (EC 2.1.1.53) was comparable to, or greater than, that of arginine decarboxylase (EC 4.1.1.19) or ornithine decarboxylase (EC 4.1.1.17). Radiolabel from dl-[5-¹⁴C]ornithine, l-[U-¹⁴C]arginine, [U-¹⁴C]agmaine and [1,4-¹⁴C]putrescine was incorporated into hyosyamine by Datura cultures. Hyoscyamine production by Datura cultures was substantially inhibited by the arginine-decarboxylase inhibitor, dl--difluoromethylarginine, but not by the corresponding ornithine-decarboxylase inhibitor, dl--difluoromethylornithine. Together with the demonstration that label was incorporated from [U-¹⁴C]agmatine, this indicates clearly that arginine is metabolised to hyoscyamine at least in part via decarboxylation to agmatine, even though a high activity of arginase (EC 3.5.3.1) was measurable under optimal conditions. The effect of unlabelled putrescine in diminishing the incorporation into hyoscyamine of label from dl-[ 5-¹⁴C] ornithine and l-[U-¹⁴C] arginine does not lend support to the theory that ornithine is metabolised via a bound, asymmetric putrescine intermediate.Abbreviations DFMA dl--difluoromethylarginine - DFMO dl--difluoromethylornithine We thank Miss E. Bent for valuable technical assistance and J. Eagles, K. Parsley and Dr. F. Mellon for mass-spectrometric analysis. We are grateful to Dr. A.J. Parr and Dr. M.J.C. Rhodes for helpful discussions. We are indebted to the Merrell Dow Research Institute, Cincinnati, Ohio, USA for supplying DFMA and DFMO.     

The cya gene region of Erwinia chrysanthemi B374: organisation and gene products

Summary A DNA fragment containing the cya gene region of Erwinia chrysanthemi, B374 was cloned in vivo and transferred into cells of E. coli using a plasmid pULB113 derived from RP4 followed by subcloning in vitro into the vector pBR322. The cya gene encodes a 95 kDal protein that complemented E. coli cya mutants. Apparently, cya genes truncated at the 3 end could still produce proteins complementing cya-defective strains, thus showing that adenylate cyclase truncated at its carboxy-terminal end could synthesise cAMP. A protein of unknown function (40 kDal) is encoded by a gene that is transcribed divergently from the control region of the adenylate cyclase gene.