Escherichia coli phenylalanyl-tRNA synthetase operon: characterization of mutations isolated on multicopy plasmids

Plasmid pB1 carries the genes for threonyl-tRNA synthetase, phenylalanyl-tRNA synthetase, and translation initiation factor IF3. Strains carrying this plasmid overproduce phenylalanyl-tRNA synthetase about 100-fold. Spontaneous mutant plasmids were obtained which no longer caused the overproduction of the enzyme. Three classes of mutations were found. (i) Deletion mutations were found, some of which had the interesting property of fusing different genes together, e.g., putting phenylalanyl-tRNA synthetase under the control of the threonyl-tRNA synthetase promoter. (ii) Insertion mutations were found; one insertion in particular was studied. This insertion is located in front of the structural gene for phenylalanyl-tRNA synthetase and is shown to interrupt a cis-acting regulatory region. (iii) Mutations that showed no major change in DNA structure were found. One of these mutations is apparently purely structural, as it produces a small subunit of phenylalanyl-tRNA synthetase with a reduced molecular weight. This protein is less stable than the wild-type enzyme. These mutations represent useful tools to investigate how the phenylalanyl-tRNA synthetase operon is regulated.     

Escherichia coli phenylalanyl-tRNA synthetase operon: transcription studies of wild-type and mutated operons on multicopy plasmids

The construction of three lambda bacteriophages containing parts of the structural gene for threonyl-tRNA synthetase, thrS, and those for the two subunits of phenylalanyl-tRNA synthetases, pheS and pheT, is described. These phages were used as hybridization probes to measure the in vivo levels of mRNA specific to these three genes. Plasmid pB1 carries the three genes thrS, pheS, and pheT, and strains carrying the plasmid show enhanced levels of mRNA corresponding to these genes. Although the steady-state levels of threonyl-tRNA synthetase and phenylalanyl-tRNA synthetase produced by the presence of the plasmid differed by a factor of 10, their pulse-labeled mRNA levels were about the same. Mutant derivatives of pB1 were also analyzed. Firstly, a cis-acting insertion located before the structural genes for phenylalanyl-tRNA synthetase caused a major decrease in both pheS and pheT mRNA. Secondly, mutations affecting either structural gene pheS or pheT caused a reduction in the mRNA levels for both pheS and pheT. This observation suggests that autoregulation plays a role in the expression of phenylalanyl-tRNA synthetase.     

Mutationally altered ribonucleotide reductase from Escherichia coli: characterization of mutations isolated on multicopy plasmids.

The Escherichia coli ribonucleotide reductase genes (nrd genes) were mutagenized at random. Point mutations were introduced in vitro into a recombinant nrd plasmid. Transformants were initially screened for altered tolerance toward the drug hydroxyurea and further characterized by enzymatic and immunological methods. The screening procedure could pick out defects in either of the two subunits of ribonucleotide reductase. Cells carrying the nrd plasmid pPS2 were earlier shown to have levels of ribonucleotide reductase molecules that were 10 to 20 times higher than those in wild-type cells. We now demonstrate that the enzymatic activity in gently lysed pPS2-containing cells on cellophane disks is six times higher than in wild-type cells. Supplementation of the pPS2-containing lysates with a purified thioredoxin system results in a further 4.5-fold stimulation of the enzymatic activity, which implies a functional shortage of the electron donor system(s) for ribonucleotide reduction in pPS2-containing cells.     

pBR322-derived multicopy plasmids harboring large inserts are often dimers in Escherichia coli K-12

pBR322-related plasmids that are 2.3 to 5.1 kb were found predominantly as monomers, while plasmids that are 7.7 to 15.2 kb were found predominantly as dimers in rec+ cells of Escherichia coli K-12.     

Production of restriction endonucleases using multicopy Hsd plasmids occurring naturally in pathogenic Escherichia coli and Shigella boydii

A convenient procedure has been devised for detection of restriction endonucleases in the Escherichia coli-Shigella group. With this procedure, two restriction endonucleases, designated Sbo 13 and Eco T22, were found and later were identified as isoschizomers of NruI and AvaIII, respectively. These endonucleases were shown to be produced from small multicopy plasmids. They were isolated from nonpathogenic E. coli into which the plasmids had been introduced by transformation, and purified from contaminating nuclease activity. The yield was high, 1,000 units/g of wet cells for Sbo 13 and 500 units/g for Eco T22. Sbo 13 and Eco T22 should be preferable to NruI and AvaIII because of the high yield and ease in handling the producer cells.     

Hyperproduction of aspartase by a catabolite repression-resistant mutant of Escherichia coli B harboring multicopy aspA and par recombinant plasmids

Recombinant plasmids bearing the Escherichia coli K-12 aspartase gene (aspA) and the plasmid partition locus (par) were introduced into a catabolite repression-resistant strain of E. coli B, AT202, constructed by mutational and transductional methods. Plasmid pNK101(pBR322-aspA-par) was stably maintained in cells of AT202 even after 30 cell generations, while pYT471(pBR322-aspA), which bore no par locus, was lost at high frequencies from the host cells. Strain AT202 harboring pNK101 produced 3-fold and 80-fold more aspartase than the wild-type E. coli B harboring pNK101 and the wild-type E. coli B strain, respectively. The maximum amount of aspA product (aspartase) was 40–45% of the total cellular protein.     

Cloning of genes that suppress an Escherichia coli K-12 alanine auxotroph when present in multicopy plasmids.

To facilitate molecular analyses of a previously uncharacterized gene involved in alanine synthesis, attempts were made to clone the wild-type allele of this gene, alaA, with a mini-Mu plasmid element used for in vivo cloning. Seventy-six independent Ala+ plasmids were isolated and characterized. Physiological, enzymological, and restriction endonuclease analyses indicated that three different genes, none of them alaA, were cloned. These genes were avtA+, which encodes the alanine-valine transaminase (transaminase C); tyrB+, which encodes the tyrosine-repressible transaminase (transaminase D); and a previously undescribed gene, called alaB, which encodes an alanine-glutamate transaminase.     

Effects of multicopy LeuO on the expression of the acid-inducible lysine decarboxylase gene in Escherichia coli.

We previously reported that mutations in hns, the structural gene for the histone-like protein H-NS, cause derepressed expression of cadA, which encodes the acid-inducible lysine decarboxylase at noninducing pH (pH 8.0). This study reports the characterization of a plasmid isolated from an Escherichia coli library that suppresses the effect of an hns mutation on cadA expression. A previously sequenced open reading frame, leuO, proves to be the gene that causes the hns-complementing phenotype. The mechanism for this phenotype appears to be overexpression of leuO from a multicopy plasmid, which drastically reduces production of CadC, the essential activator for cadA induction. These results show an in vivo regulatory phenotype for leuO, consistent with its proposed protein sequence.     

Construction of hybrid plasmids containing the lysA gene of Escherichia coli: studies of expression in Escherichia coli and Saccharomyces cerevisiae

Summary The lysA gene of Escherichia coli has been cloned from a transducing phage on various plasmids, present in different copy numbers in bacterial cells. Synthesis of the product of this gene, diaminopimelate (DAP)-decarboxylase, and its regulation have been studied. Expression does not follow a simple gene dosage effect, maximal expression already being obtained with a six-copy plasmid. This result suggests that either a positive or an autogenous regulatory mechanism is involved. We also used one of the hybrid plasmids to look for expression of the bacterial lysA gene in Saccharomyces cerevisiae. The results indicate that the product of the E. coli gene is not actively translated in yeast.     

Discriminant analysis of promoter regions in Escherichia coli sequences

We have previously developed a general method based on the statisticaltechnique of discriminant analysis to predict splice junctionsin eukaryotic mRNA sequences [Nakata, K., Kanehisa, M. and DeLisi,C. (1985) Nucleic Acids Res., 13, 5327–5340]. In orderto evaluate further applicability of this method, we now analyzethe promoter region of Escherichia coli sequences. The attributesused for discrimination include the accuracy of consensus sequencepatterns measured by the perceptron algorithm, the thermal stabilitymap, the base composition and the Calladine-Dickerson rulesfor helical twist angle, roll angle, torsion angle and propellertwist angle. When applied to selected E. coli sequences in theGenBank database, the method correctly identifies 75 % of thetrue promoter regions. Received on May 15, 1987; accepted on April 17, 1988     

Protein isoaspartate methyltransferase is a multicopy suppressor of protein aggregation in Escherichia coli

We used preS2-S'-beta-galactosidase, a three-domain fusion protein that aggregates extensively at 43 degrees C in the cytoplasm of Escherichia coli, to search for multicopy suppressors of protein aggregation and inclusion body formation and took advantage of the known differential solubility of preS2-S'-beta-galactosidase at 37 and 43 degrees C to develop a selection procedure for the gene products that would prevent its aggregation in vivo at 43 degrees C. First, we demonstrate that the differential solubility of preS2-S'-beta-galactosidase results in a lactose-positive phenotype at 37 degrees C as opposed to a lactose-negative phenotype at 43 degrees C. We searched for multicopy suppressors of preS2-S'-beta-galactosidase aggregation by selecting pink lactose-positive colonies on a background of white lactose-negative colonies at 43 degrees C after transformation of bacteria with an E. coli gene bank. We discovered that protein isoaspartate methyltransferase (PIMT) is a multicopy suppressor of preS2-S'-beta-galactosidase aggregation at 43 degrees C. Overexpression of PIMT reduces the amount of preS2-S'-beta-galactosidase found in inclusion bodies at 43 degrees C and increases its amount in soluble fractions. It reduces the level of isoaspartate formation in preS2-S'-beta-galactosidase and increases its thermal stability in E. coli crude extracts without increasing the thermostability of a control protein, citrate synthase, in the same extracts. We could not detect any induction of the heat shock response resulting from PIMT overexpression, as judged from amounts of DnaK and GroEL, which were similar in the PIMT-overproducing and control strains. These results suggest that PIMT might be overburdened in some physiological conditions and that its overproduction may be beneficial in conditions in which protein aggregation occurs, for example, during biotechnological protein overproduction or in protein aggregation diseases.     

Studies on transformation of Escherichia coli with plasmids

Factors that affect the probability of genetic transformation of Escherichia coli by plasmids have been evaluated. A set of conditions is described under which about one in every 400 plasmid molecules produces a transformed cell. These conditions include cell growth in medium containing elevated levels of Mg2+, and incubation of the cells at 0 degrees C in a solution of Mn2+, Ca2+, Rb+ or K+, dimethyl sulfoxide, dithiothreitol, and hexamine cobalt (III). Transformation efficiency declines linearly with increasing plasmid size. Relaxed and supercoiled plasmids transform with similar probabilities. Non-transforming DNAs compete consistent with mass. No significant variation is observed between competing DNAs of different source, complexity, length or form. Competition with both transforming and non-transforming plasmids indicates that each cell is capable of taking up many DNA molecules, and that the establishment of a transformation event is neither helped nor hindered significantly by the presence of multiple plasmids.