A stable plasmid vector and control of its copy number in Bacillus brevis 47, a protein-producing bacterium

A low-copy-number plasmid vector, pHY481, was constructed by combining a macrolide resistance gene of a Staphylococcus aureus plasmid with a cryptic plasmid found in a Bacillus brevis strain isolated from soil. The plasmid introduced into B. brevis 47, an extensively investigated protein-producing bacterium, was maintained very stably in the absence of selective antibiotics. A Bacillus megaterium alpha-amylase gene subcloned into pHY481 was retained much more stably in B. brevis 47 than one subcloned into a plasmid of S. aureus origin. B. brevis 47 mutants were also isolated in which the copy number of pHY481 was amplified about 10-fold. The copy number of pHY481 with the inserted amylase gene also increased in the mutants. As a result, a severalfold-higher amount of the enzyme was produced in the mutants compared with that produced in wild-type B. brevis 47. Thus, the plasmid vector constructed here and the copy-number mutants of B. brevis 47 are useful for cloning foreign genes and performing genetic engineering in the protein-producing bacterium.     

A stable plasmid vector and control of its copy number in Bacillus brevis 47, a protein-producing bacterium.

A low-copy-number plasmid vector, pHY481, was constructed by combining a macrolide resistance gene of a Staphylococcus aureus plasmid with a cryptic plasmid found in a Bacillus brevis strain isolated from soil. The plasmid introduced into B. brevis 47, an extensively investigated protein-producing bacterium, was maintained very stably in the absence of selective antibiotics. A Bacillus megaterium alpha-amylase gene subcloned into pHY481 was retained much more stably in B. brevis 47 than one subcloned into a plasmid of S. aureus origin. B. brevis 47 mutants were also isolated in which the copy number of pHY481 was amplified about 10-fold. The copy number of pHY481 with the inserted amylase gene also increased in the mutants. As a result, a severalfold-higher amount of the enzyme was produced in the mutants compared with that produced in wild-type B. brevis 47. Thus, the plasmid vector constructed here and the copy-number mutants of B. brevis 47 are useful for cloning foreign genes and performing genetic engineering in the protein-producing bacterium.     

The yeast 2-micron plasmid Rep2 protein has Rep1-independent partitioning function

Equal partitioning of the multi-copy 2-micron plasmid of the budding yeast Saccharomyces cerevisiae requires association of the plasmid Rep1 and Rep2 proteins with the plasmid STB partitioning locus. Determining how the Rep proteins contribute has been complicated by interactions between the components. Here, each Rep protein was expressed fused to the DNA-binding domain of the bacterial repressor protein LexA in yeast harboring a replication-competent plasmid that had LexA-binding sites but lacked STB. Plasmid transmission to daughter cells was increased only by Rep2 fusion expression. Neither Rep1 nor a functional RSC2 complex (a chromatin remodeler required for 2-micron plasmid partitioning) were needed for the improvement. Deletion analysis showed the carboxy-terminal 65 residues of Rep2 were required and sufficient for this Rep1-independent inheritance. Mutation of a conserved basic motif in this domain impaired Rep1-independent and Rep protein/STB-dependent plasmid partitioning. Our findings suggest Rep2, which requires Rep1 and the RSC2 complex for functional association with STB, directly participates in 2-micron plasmid partitioning by linking the plasmid to a host component that is efficiently partitioned during cell division. Further investigation is needed to reveal the host factor targeted by Rep2 that contributes to the survival of these plasmids in their budding yeast hosts.     

The FLP protein of the 2-micron plasmid of yeast. Inter- and intramolecular reactions

We have studied the mechanism of reaction of the FLP protein of the yeast 2-micron plasmid on linear substrates. The products of the reaction are dependent upon the concentration of FLP protein. At low concentrations of FLP, products resulting from intramolecular recombination between two FLP target sites accumulate. At higher concentrations of FLP, intermolecular recombination results in the accumulation of products which are larger than the starting substrate. At higher concentrations still, FLP-promoted recombination is inhibited. Potassium chloride (0.15 M) inhibits the intermolecular reaction and also prevents the inhibition of FLP-mediated recombination caused by high concentrations of FLP protein. We present a model that explains these findings.     

The par region of pSC101 affects plasmid copy number as well as stability

The par locus is a segment of pSC101 that has been identified as a cis-acting determinant of plasmid stability. We show that par also determines copy number and must, therefore, play a role in plasmid replication. The segregation defect, but not the copy-number reduction, of par- replication origins is completely suppressed by a short sequence from the bacteriophage lambda gene O which is present in plasmid pKO-4. Thus, replication and segregation functions are separable from each other.     

High frequency of yeast transformation by plasmids carrying part or entire 2-micron yeast plasmid.

By using two chimeric plasmids containing yeast ura3 gene and 2-micron yeast DNA linked to the bacterial plasmid pCR1, yeast transformation of a high frequency has been achieved. The first plasmid is such that the 2-micron DNA part, in which the ura3 gene is incorporated, can be removed in one step and thus the 2-micron-ura3 sequence can be considered as a "transposable" block. In contrast, the second one bears the entire 2-micron plasmid and the ura3 gene is inserted in the bacterial plasmid part. As shown through hybridization experiments and genetic studies, the ura3 gene was maintained as a cytoplasmic element. Plasmids recovered from the yeast transformants were used to transform Escherichia coli. Their analysis by EcoRI showed that in many cases the vector had recombined with the endogenous 2-micron DNA of the recipient strain. The specific activity of orotidine 5'-monophosphate decarboxylase (coded by ura3) in yeast transformants was 10- to 30-fold higher than in the wild type.     

FLP site-specific recombinase of yeast 2-micron plasmid. Topological features of the reaction

The 2-micron plasmid of the yeast Saccharomyces cerevisiae encodes a site-specific recombinase (FLP) that promotes inversion across a unique site contained in each of the 599-base-pair inverted repeats of the plasmid. We have studied the topological changes generated in supercoiled substrates after exposure to the purified FLP protein in vitro. When a supercoiled substrate bearing two FLP target sequences in inverse orientation is treated with FLP, the products are multiply knotted structures that arise as a result of random entrapment of interdomainal supercoils. Likewise, a supercoiled substrate bearing two target sequences in direct orientation yields multiply interlocked catenanes as the product. Both types of substrate seem to be able to undergo repeated rounds of recombination that result in products of further complexity. The FLP protein also acts as a site-specific topoisomerase during the recombination reaction.     

Some stochastic models for plasmid copy number

Some stochastic models for the copy number of plasmids in a cell line are studied. When considering the behavior of copy number in the whole cell line, the theory of multitype branching processes is appropriate. Attention is paid to the cure rate in the cell line, and the asymptotic fractions of cells containing a given number of plasmids. These quantities are used to compare the models numerically.     

Isolation of recipient yeast strains for the stable reproduction of 2-micron DNA vectors

Most Leu- clones of yeast transformants (cir0, pJDB219) can stabilize the replication of 2 micron-vectors with REP3, the stability obtained being comparable to the one for the standard cir0 strain. One of the Leu- clones was used to isolate a plasmid with Rep 1.2 functions ("Rep-helper plasmid"). The plasmid was shown to carry a partially active LEU2 gene by transforming both E. coli and S. cerevisiae to Leu+ phenotype. A restriction analysis performed demonstrated that the Rep-helper plasmid has lost approximately 1.9 kb compared to the parent pJDB219, deletion and rearrangement having taken place at the bacterial and 2 mem components boundary. The Rep-helper plasmid carrying host strains allows to quantify the REP3 function on different 2 microns vectors. Some but not all cir+ stabilized vectors show greater stability in Rep-helper strains compared to the standard cir0 ones. Manipulating the Rep-helper plasmid level, by selecting for Leu+ phenotype, stabilized REP3 +/- plasmid p3030, but mostly destabilizes REP3+ plasmid YEp13HIS3.     

Salinity-dependent copy number increase of a marine cyanobacterial endogenous plasmid

Listeria monocytogenes possessed glucose oxidase and NADH oxidase activities in whole cells and lysed protoplasts respectively. The NADH oxidase activity sedimented with the membrane fraction and was inhibited by the respiratory inhibitors rotenone, 2-heptyl-4-hydroxy-quinoline-N-oxide and cyanide, suggesting the presence of a membrane associated respiratory chain.     

The expression of integrated plasmid DNA depends on copy number

The effect of copy number, integration site, and enhancers on the expression of stably integrated exogenous DNA was examined in Chinese hamster cells. Three similar plasmids were constructed with the mouse beta maj-globin promoter fused to the galK gene either with no enhancer or with the SV40 or Harvey sarcoma virus (HaSV) enhancer. Eighteen stable cell lines were obtained and characterized with respect to plasmid copy number and galactokinase activity. At copy numbers of four or less, the enhancers showed detectable activity and a DNase I hypersensitive site was present. Above four copies, gene activity decreased as the copy number increased, the enhancer sequences were apparently inactive, and the DNase I hypersensitive site disappeared. These data suggest that, at least in this model system, when exogenous DNA is integrated as multiple head-to-tail copies, the entire multigene unit expresses poorly and inappropriately. When the same exogenous DNA integrates as a single (or low number) copy, expression appears to be relatively normal as judged by enhancer stimulation and DNase I hypersensitivity.