Multicopy Vector Research Articles

A K-252a-resistance gene, sksl +, encodes a protein similar to the caenorhabditis elegans F37A4.5 gene product and confers multidrug resistance in schizosaccharomyces pombe

A gene named sksl +was cloned as a suppressor of the K-252a-sensitivity phenotype of Schizosaccharomyces pombe (Sp) from a gene library of the parental Sp chromosomal DNA constructed with a multicopy vector pDB248'. The gene encoded a 308-amino-acid (aa) protein similar to the Caenorhabditis elegans F37A4.5 gene product and to the mouse and Drosophila Mov34 gene products. The sksl + null mutants obtained by gene disruption were non-viable, indicating that sksl + is essential for vegetative growth. The parental Sp strain carrying multiple copies of sksl + showed distinct cross-resistance to staurosporine, thiabendazole and vanadate in addition to K-252a, although Sksl has no similarity in aa sequence to those of ATP-binding cassette (ABC)-type transporters. The multicopy plasmid containing sksl + conferred multidrug resistance (MDR), even in a mutant cell defective in pmdl + encoding an ABC-type transporter. It is therefore unlikely that the function of pmdl + is involved in MDR conferred by sksl + . These results suggest that sksl + is a functionally novel MDR gene.

Functional expression in Saccharomyces cerevisiae of the Lactococcus lactis mleS gene encoding the malolactic enzyme

Malolactic fermentation, a crucial step in winemaking, results mostly in degradation by lactic acid bacteria of L-malic acid into l-lactic acid. This direct decarboxylation is catalysed by the malolactic enzyme. Recently we, and others, have cloned the mleS gene of Lactococcus lactis encoding malolactic enzyme. Heterologous expression of mleS in Saccha-romyces cerevisiae was tested to perform simultaneously alcoholic and malolactic fermentations by yeast. mleS gene was cloned in a yeast multicopy vector under a strong promoter. Malolactic activity was present in crude extracts of recombinant yeasts. Malic acid degradation was tested during alcoholic fermentation in synthetic media and must. Yeasts expressing the mleS gene actually produced l-lactate from l-malate; nevertheless malate degradation was far from complete.

Cloning and sequencing the degS-degU operon from an alkalophilic Bacillus brevis

The sacU region from an alkalophilic Bacillus brevis was cloned and sequenced. The two open reading frames of the degS-degU operon encode polypeptides that gave calculated molecular masses of 43.8 kDa and 27.0 kDa, respectively. Sequence comparisons at the amino acid level to the B. subtilis degS-degU genes showed 74% and 84% similarity, respectively. On a multicopy vector the B. brevis degS-degU genes were found to cause hypersecretion of several extracellular enzymes in a B. subtilis rec- strain as well as in a B. subtilis sacU(HY) strain.

Negative control of plasmid DNA replication by iterons. Correlation with initiator binding affinity.

Initiation of DNA replication and negative control of initiation frequency in many bacterial plasmids are mediated by multiple binding sites (iterons) for the replicon-encoded initiator protein. Here we show that a single iteron of plasmid P1, when cloned in a multicopy vector, pUC19, can control replication of a miniP1 plasmid effectively. Using this assay system, several iterons with single point mutations were analyzed. The degree of control was directly correlated with the binding affinity of the iterons for the initiator protein in vitro. The control was also unaffected whether or not a wild type iteron was flanked with strong transcription terminators. We conclude that initiator binding is the only activity of P1 iteron sequences relevant to replication control.

Construction and application of streptomycete promoter probe vectors which employ the Streptomyces glaucescens tyrosinase-encoding gene as reporter

We have constructed multicopy and integrative streptomycete promoter probe vectors that employ the promoterless tyrosinase ( melC) operon of Streptomyces glaucescens as the chromogenic transcriptional reporter. Each vector contains the reporter cassette, RC3, which comprises part of the melC operon flanked by transcription terminators; RC3 may be easily inserted into any vector. We demonstrate the use of the pIJ101-based mel vector, pMT3010, for the isolation of mutations which alter expression of the S. coelicolor glycerol operon. The S. glaucescens mel reporter system is well-suited for the visual, non-selective identification of regulatory mutants and can be used efficiently for screening several thousand clones at high colony density.

Cloning and expression of the penicillinase from a borderline methicillin-susceptible Staphylococcus aureus strain in Escherichia coli.

The blaZ gene contained in a single 17.2-kb beta-lactamase plasmid from a borderline methicillin-susceptible Staphylococcus aureus strain (a53) has been cloned in Escherichia coli. A Bluescript II derivative in which the ampicillin resistance gene has been replaced with the chloramphenicol resistance gene was used as a multi-copy vector. One ampicillin-resistant colony was detected among 31 chloramphenicol-resistant transformants selected. This E. coli clone harbored a recombinant plasmid (pAH12) containing two different staphylococcal HindIII inserts (7.0 and 5.3 kb), of which only the former hybridized with a blaZ probe. The clone showed an ampicillin MIC of > 1024 micrograms ml-1, independently of the inoculum size used, and produced large amounts of beta-lactamase, which hydrolyzed nitrocefin and penicillin G but not methicillin of the beta-lactamase substrate, padac. In contrast, S. aureus a53 hydrolyzed all four substrates. The fact that high levels of staphylococcal penicillinase are unable to cause methicillin hydrolysis confirms that penicillinase hyperproduction is unlikely to be the true mechanism responsible for the borderline phenotype. These results also suggest that the two different beta-lactamases (penicillinase and methicillinase) associated with borderline S. aureus strains have a different genetic origin.

Development of a newBacillus carboxyl esterase for use in the resolution of chiral drugs

We have screened a new enzyme for the resolution of R, S-naproxen enantiomers. The enzyme is free of lipase activity, and possesses a very high sterospecificity on S-naproxen [2-(6-methoxy-2-naphthyl)-propionic acid] esters and esters of related drugs. The primary structure of the enzyme, determined from the nucleotide sequence, shows limited homology with the catalytic site of lipases. The gene coding for the steroselective carboxylesterase has been cloned and expressed in Bacillus subtilis. Using a multicopy vector and an additional strong promoter an efficient production process was developed. The enzyme was shown to be sensitive to very high concentrations of the products formed during the reaction it catalyses. To increase the resistance of the enzyme, lysine residues thought to be responsible for this phenomnon were replaced through site-directed mutagenesis. Enzymes with improved stability were obtained. An explanation is given in terms of a model in which a reaction of the acid moiety of naproxen with free lysine NH2 groups is a major cause of inactivation.

Dominance of Lactobacillus plantarum strains in grass silage as demonstrated by a novel competition assay

An assay was developed for assessing the competitive ability of potential Lactobacillus plantarum silage inoculants. This assay was based on the ability of the test inoculant to outcompete a standard strain (Lact. plantarum DCU101) co‐inoculated at the same rate of 5 × 105 colony forming units g‐1 of grass. Total populations of Lact. plantarum were enumerated with a selective medium and Lact. plantarum DCU101 was identified with a strain‐specific DNA probe. The DNA probe was based on a small (2.2 kb), cryptic, indigenous plasmid which was cloned into pAT153, a multicopy cloning vector. Seven Lact. plantarum strains, six of which were isolated from well‐preserved grass silages, were used to inoculate laboratory scale silos, and variation in strain dominance was monitored over the 14 d ensilage period.

Cloning of a galacturonic acid uptake gene fromErwinia chrysanthemiEC16

A 3.4 kb fragment of Erwinia chrysanthemi EC16 DNA capable of complementing galacturonic acid uptake mutants (exuT) was identified and cloned into a multicopy vector. In E. chrysanthemi B374 exuT mutants, the cloned DNA provided for growth of the mutant strains on galacturonic acid by complementing the galacturonic acid uptake defect. Alkaline phosphatase (phoA) gene fusions with the cloned DNA suggested that most of the cloned DNA was necessary for complementation of exuT mutant strains. Using anti-alkaline phosphatase antibody, a hybrid ExuT-PhoA protein was localized to the membrane fraction of the bacterium.

Cloning and characterization of a yeast cytochrome b5-encoding gene which suppresses ketoconazole hypersensitivity in a NADPH- P-450 reductase-deficient strain

Cytochrome P-450 (Cyp) 51 or lanosterol-C14-demethylase is the main target for antifungal compounds of the triazole family like ketoconazole (Kz). Disruption of the associated NADPH- P-450 reductase-encoding gene ( YRED) is not lethal, but decreases by about 20-fold the Kz resistance (Kz R of wild-type (wt) Saccharomyces cerevisiae. Transformation of a YRED-disrupted strain by a yeast genomic library based on a multicopy vector allowed us to identify a suppressor of Kz hypersensitivity. Deletion analysis of the 5-kb cloned fragment indicated that yeast cytochrome b 5-encoding gene ( CYB5), which encodes a 120-amino-acid (aa) protein, is required and sufficient for the suppressor effect. The encoded polypeptide shares about 30% aa identity with mammalian cytochromes b 5 (Cyb5). CYB5 disruption and tetrad analysis demonstrate that yeast Cyb5 is not required for growth in a Yred + strain. Determination of the microsomal content of b-type cytochromes by differential spectra indicated the presence of a strongly decreased or null Cyb5 level in the disrupted strain. This confirms that we have cloned the gene encoding the major microsomal form of Cyb5 which appears not to be essential. Minor Cyb5 isoforms could also be present in yeast or other redox proteins could substitute for the pleiotropic roles of Cyb5 in the sterol and lipid biosynthesis pathways.

Isolation of a CDC25 family gene, MSI2/LTE1, as a multicopy suppressor of ira1.

We have identified MSI2 as a gene of Saccharomyces cerevisiae which, when on a multicopy vector, suppresses the heat shock sensitivity caused by the loss of the IRA1 product, a negative regulator of the RAS protein. The multicopy MSI2 also suppresses the heat shock sensitivity of cells with the RAS2val19 mutation but not those with the bcy1 mutation, suggesting that the MSI2 protein may interfere with the activity of the RAS protein. The sequence analysis of MSI2 reveals that it is identical to LTE1 belonging to the CDC25 family: CDC25, SCD25 and BUD5, each of which encodes a guanine nucleotide exchange factor for the ras superfamily gene products. Deletion of the entire MSI2 coding region reveals that MSI2 is not essential but the disruptant shows a cold-sensitive phenotype. Under the non-permissive conditions, more than 70% of the msi2 disruptants arrested at telophase as large budded cells with two nuclei divided completely and elongated spindles, indicating that the msi2 deletion is a cell division cycle mutation. These results suggest that MSI2 is involved in the termination of M phase and that this process is regulated by a ras superfamily gene product.

Use of β‐lactamase as a secreted reporter of promoter function in yeast

K1 preprotoxin is the 316 residue precursor of the K1 killer toxin secreted by the yeast Saccharomyces cerevisiae. The SP beta la reporter consists of the mature, secreted form of beta-lactamase (beta la) fused to S and P, two fragments of preprotoxin. S is the N-terminal 34 residues, including the secretion signal. P, a 67 residue 'processing' segment with three sites for N-glycosylation, terminates in a Lys Arg site for cleavage by the Kex2 protease. Expression of SP beta 1a in yeast results in efficient secretion, processing by signal peptidase and glycosylation in the endoplasmic reticulum, producing pro beta la. Kex2 cleavage of pro beta la in the lumen of a late Golgi compartment releases beta la, which accumulates stably in culture media buffered at pH 5.8-7. The half-life of secretion is 11 min at 30 degrees C; 10-12% of the total activity in exponential-phase cells is intracellular, mostly in the form of pro beta la, indicating that transit from the endoplasmic reticulum to the Golgi is rate limiting. We have used SP beta la expression in single- and multi-copy vectors to compare the PGK, GAL1, GAL10, PHO5 and CUP1 promoters under varying nutritional conditions. In exponential-phase cells, secretion of beta la over a 40-fold range and up to several micrograms/ml was proportional to transcript level, demonstrating that SP beta la can be employed as a convenient secreted reporter of promoter function in yeast.

Leptomycin B targets a regulatory cascade of crm1, a fission yeast nuclear protein, involved in control of higher order chromosome structure and gene expression.

The molecular action of leptomycin B (LMB), an agent inducing arrest of the eukaryotic cell cycle at G1 and G2 phases, was investigated by analyzing an LMB resistance gene of Schizosaccharomyces pombe. A genomic library of an LMB-resistant mutant was screened for LMB resistance, and a DNA fragment containing an open reading frame (ORF) of 1078 amino acids was cloned on a multicopy vector. The plasmid was found to confer drug resistance specifically to LMB. Nucleotide sequencing revealed that the ORF was a mutant gene for the essential nuclear protein crm1, which had been reported to complement a cold-sensitive mutation causing deformed nuclear morphology. The gene product named crm1-N1 had two amino acid replacements (Gly-503 to Asp and Met-546 to Ile). Two allelic mutants of crm1 (crm1-809 and crm1-119) were found to be hypersensitive and resistant, respectively, to LMB. Nuclear morphology of the cold-sensitive crm1-809 mutant at the restrictive temperature was almost the same as that of the wild-type cells treated with LMB. Furthermore, a low concentration of LMB induced the intracellular accumulation of a 25-kDa protein in the wild-type cells, which was immunologically identical to the protein accumulating in the crm1-809 mutant cells. These results strongly suggest that LMB primarily inhibits the function of the crm1 gene which is required for maintaining higher order chromosome structures, correct gene expression, and cell growth in the fission yeast.

The archaebacterial membrane protein bacterio-opsin is expressed and N-terminally processed in the yeast Saccharomyces cerevisiae.

The bop gene codes for the membrane protein bacterio-opsin (BO), which on binding all-trans-retinal, constitutes the light-driven proton pump bacteriorhodopsin (BR) in the archaebacterium Halobacterium salinarium. This gene was cloned in a yeast multi-copy vector and expressed in Saccharomyces cerevisiae under the control of the constitutive ADH1 promoter. Both the authentic gene and a modified form lacking the precursor sequence were expressed in yeast. Both proteins are incorporated into the membrane in S. cerevisiae. The presequence is thus not required for membrane targeting and insertion of the archaebacterial protein in budding yeast, or in the fission yeast Schizosaccharomyces pombe, as has been shown previously. However, in contrast to S. pombe transformants, which take on a reddish colour when all-trans-retinal is added to the culture medium as a result of the in vivo regeneration of the pigment, S. cerevisiae cells expressing BO do not take on a red colour. The precursor of BO is processed to a protein identical in size to the mature BO found in the purple membrane of Halobacterium. The efficiency of processing in S. cerevisiae is dependent on growth phase, as well as on the composition of the medium and on the strain used. The efficiency of processing of BR is reduced in S. pombe and in a retinal-deficient strain of H. salinarium, when retinal is present in the medium.

InhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis.

Isoniazid (isonicotinic acid hydrazide, INH) is one of the most widely used antituberculosis drugs, yet its precise target of action on Mycobacterium tuberculosis is unknown. A missense mutation within the mycobacterial inhA gene was shown to confer resistance to both INH and ethionamide (ETH) in M. smegmatis and in M. bovis. The wild-type inhA gene also conferred INH and ETH resistance when transferred on a multicopy plasmid vector to M. smegmatis and M. bovis BCG. The InhA protein shows significant sequence conservation with the Escherichia coli enzyme EnvM, and cell-free assays indicate that it may be involved in mycolic acid biosynthesis. These results suggest that InhA is likely a primary target of action for INH and ETH.

Preparation and ligand screening of a lambda gt11 lysogen library.

The λgt11 system has been designed to allow expression of cDNAs either in phage plaques or in bacteria lysogenized with recombinant λgtll phages (, ). cDNAs are cloned into a unique EcoRl restriction site near the 3′ end of a truncated E. coli lactose operon (lacP/O-lacZ), allowing inducible, high-level transcription of lacZ-cDNA-encoded fusion mRNAs that can be translated into fusion proteins if the cDNA coding strand is properly oriented with respect to lac transcription, and in phase with the p-galactosidase reading frame. A host strain is chosen that carries a lacl q allele on a high-copy number plasmid (pMC9), assuring efficient repression of lac transcription in the absence of an inducer. Since λgtll encodes a temperature-sensitive cl repressor (c/857), it can be lysogenized and stably maintained as a lysogen (i.e., as a “single copy vector”) at the permissive temperature, and subsequently made to switch to a “multi copy vector” on inducing lytic growth of the prophage by a temperature shift. An amber mutation in the S gene (SlOO&mb) renders the phage lysis-defective in a nonsuppressor host, allowing the accumulation of phage-encoded products inside the bacterial host cells. To maximize stability of lacZ- cDNA-encoded fusion protein, a host strain is used that carries a deletion in the gene for the Ion protease. λgtl 1 can also form plaques if grown on a host that carries an amber suppressor mutation (for a recent review on the λgtll system, see ref. )

Suppression of the serT42 mutation with modified tRNA(1Ser) and tRNA(5Ser) genes.

Serine tRNA gene derivatives with altered anticodons were introduced to the temperature-sensitive serT42 mutant, whose tRNA(1Ser) shows a base substitution of A10 for wild type G10. When a low copy number vector-system was used, the growth and beta-galactosidase synthetic activity of the serT42 mutant were restored by complementation with the tRNA(5Ser) (T34) gene or the tRNA(1Ser) (G34) gene as well as the tRNA(1Ser) (wt) gene, but not with tRNA(5Ser) (wt), tRNA(1Ser) (A34) or tRNA(1Ser) (C34) genes at 42 degrees C. When multicopy vectors were used, the transformation even with tRNA(1Ser) (A10) gene restored the growth and beta-galactosidase synthetic activity at 42 degrees C. The tRNA(1Ser) (A10) showed no thermosensitivity in serine acceptor activity by in vitro assay. At 42 degrees C, the amount of tRNA(1Ser) (A10) in the serT42 mutant was almost the same as those in the wild type. The nucleotides in the tRNA(1Ser) (A10) were found to be fully modified like those in the wild type tRNA(1Ser). Both of the tRNAs transcribed from tRNA(5Ser) (T34) and tRNA(1Ser) (G34) genes showed serine acceptor activity. Modified nucleosides of these tRNAs were also analyzed.

Transport of aromatic amino acids and its influence on overproduction of the amino acids in Corynebacterium glutamicum

Abstract The uptake of phenylalanine, tyrosine and tryptophan by wild-type Corynebacterium glutamicum ATCC 13032 was demonstrated to occur mainly via a common transport system by competition experiments using these amino acids which were either 14C-labeled or unlabeled. Strain HCA1 defective in the common transport system was derived from a phenylalanine auxotroph, C. glutamicum KY9182, by selecting for resistance to 5-fluorotryptophan and subsequent screening for requirement for a high concentration of phenylalanine. The wild-type gene responsible for the common transport system was cloned into a multicopy vector, based on its ability to make strain HCA1 less dependent on phenylalanine and sensitive to 5-fluorotrytophan. The resulting plasmid pCA5 conferred on strain HCA1 a fewfold increase in the uptake activities of all three aromatic amino acids relative to the wild-type levels. Although strains KY9182 and HCA1 acquired the productivity of aromatic amino acids by introducing a plasmid coexpressing a set of feedback-resistant enzymes involved in phenylalanine or trytophan biosynthesis, the production levels were higher in the HCA1 strain background than in the KY9182 background. In contrast, introduction of pCA5 into trytophan-producing C. glutamicum KY9225 resulted in a drastic decrease in the trytophan production. However, when another compatible plasmid specifying feedback-resistant tryptophan-biosynthetic enzymes coexisted in the transformant, the tryptophan productivity was restored to an appreciable level. These results indicated that prevention of amino acid transport, as well as deregulation of biosynthetic pathways, was one of the crucial factors in amino acid overproduction.

Fpg protein protects Escherichia coli K-12 from mutation induction by the carcinogen 4-nitroquinoline 1-oxide.

This paper investigates the role of the fpg gene product in protecting Escherichia coli cells against the lethal and mutagenic effects of 4-nitroquinoline 1-oxide (4NQO). To this end, the araD81 mutation which make the cells sensitive to L-arabinose was combined with an fpg-1::Knr allele, in either an uvrA+ or uvrA-, umuC+ or umuC-, genetic background. Mutation induction was monitored by selecting forward mutations to L-arabinose resistance (Arar). The formamidopyrimidine-DNA glycosylase (Fpg protein) protected bacteria from 4NQO-induced mutagenesis since Fpg- defective cells showed greater Arar mutation induction than fpg+ bacteria did. This was confirmed since the increased sensitivity of the fpg- cells to mutagenesis by 4NQO was suppressed when the Fpg protein was overproduced by placing the fpg gene in a multicopy plasmid vector. The fpg- mutation had no detectable influence on 4NQO mutagenesis in a uvrA- genetic background, but its effect was magnified in umuC- cells. No influence on cell survival was observed after 4NQO treatment. Our data suggest that 8-hydroxyguanine, a non-lethal, non-bulky and directly miscoding lesion, might be responsible for the detected influence of Fpg protein expression on mutation induction by 4NQO. This is in agreement with the reported in vivo formation of 8-hydroxyguanine in cellular DNA after 4NQO exposure. The increased 4NQO-induction of GC to TA transversions on fpg- bacteria further support such a possibility. This work reinforces the role of Fpg protein in the bacterial defense against the mutagenicity by genotoxic agents.

Genetics of pentose-phosphate pathway enzymes in Saccharomyces cerevisiae

Deletion mutants of transketolase and transaldolase were examined for their growth on xylulose medium. Xylulose can be phosphorylated and degraded via the pentose-phosphate pathway in yeast. Deletion mutants of transaldolase ( TAL1) can still grow very slowly with xylulose as the carbon source. In-vitro mutagenesis of TAL1 showed that lysine 144 is essential for the catalytic activity of transaldolase. Two genes code for transketolase in S. cerevisiae. Mutants in the gene TKL2 are unaffected by their growth on xylulose medium, while deletion of TKL1 leads to mutants with only very slow growth when xylulose is used as the carbon source. Double mutants for TKL1 and TKL2 could not use xylulose as the carbon source at all, indicating a complete block of the transketolase reaction. Transformants overexpressing transaldolase, transketolase or both enzymes were constructed by placing their genes on the multicopy vector GS42. The activity of transaldolase or transkatolase, or a combination of both enzymes, could be increased by more than 100 fold compared to the wild-type strain measured in parallel. Growth of the transformants on agar plates with xylulose as the carbon source was not accelerated compared to the wild-type strain.