Overproduction of d-xylose isomerase in Escherichia coli by cloning the d-xylose isomerase gene

The Escherichia coli d-xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) gene, xylA, has been cloned on various E. coli plasmids. However, it has been found that high levels of overproduction of the d-xylose isomerase, the protein product of the xylA gene, cannot be accomplished by cloning the intact gene on high copy-number plasmids alone. This is believed to be due to the fact that the expression of the gene through its natural promoter is highly regulated in E. coli. In order to overcome this, the xylA structural gene has been fused with other strong promoters such as tac and lac, resulting in the construction of a number of fused genes. Analysis of the E. coli transformants containing the fused genes, cloned on high copy-number plasmids, indicated that a 20-fold overproduction of the enzyme can now be obtained. It is expected that overproduction of the enzyme in E. coli can still be substantially improved through additional manipulation with recombinant DNA techniques.     

Plasmid composition and the chpG gene determine the virulence level of Clavibacter capsici natural isolates in pepper

The gram-positive bacterial species Clavibacter capsici causes necrosis and canker in pepper plants. Genomic and functional analyses of C. capsici type strain PF008 have shown that multiple virulence genes exist in its two plasmids. We aimed to identify the key determinants that control the virulence of C. capsici. Pepper leaves inoculated with 54 natural isolates exhibited significant variation in the necrosis. Six isolates showed very low virulence, but their population titres in plants were not significantly different from those of the highly virulent isolates. All six isolates lacked the pCM1_Cc plasmid that carries chpG, which has been shown to be required for virulence and encodes a putative serine protease, but two of them, isolates 1,106 and 1,207, had the intact chpG elsewhere in the genome. Genomic analysis of these two isolates revealed that chpG was located in the pCM2_Cc plasmid, and two highly homologous regions were present next to the chpG locus. The chpG expression in isolate 1,106 was not induced in plants. Introduction of chpG of the PF008 strain into the six low-virulence isolates restored their virulence to that of PF008. Our findings indicate that there are at least three different variant groups of C. capsici and that the plasmid composition and the chpG gene are critical for determining the virulence level. Moreover, our findings also indicate that the virulence level of C. capsici does not directly correlate with bacterial titres in plants.     

Reverse genetics system for human rotaviruses

A reverse genetics technology is an incredibly useful technique both for a proper understanding of different aspects of virus biology and for the generation of complementary DNA (cDNA)-derived infectious viruses, which can act as safe and effective vaccines and viral vectors. Rotaviruses (RVAs), especially human RVAs (HuRVAs), had been very refractory to this technology until very recently. Here, we describe the historical background of the development of a long-awaited HuRVA reverse genetics system, culminating in the generation of replicative HuRVAs entirely from cloned cDNAs.     

Mutational Analysis of the Multicopy hao Gene Coding for Hydroxylamine Oxidoreductase in Nitrosomonas sp. Strain ENI-11

The ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11 contains three copies of the hao gene (hao ₁, hao ₂, and hao ₃) coding for hydroxylamine oxidoreductase (HAO). Three single mutants (hao ₁::kan, hao ₂::kan, or hao ₃::kan) had 68 to 75% of the wild-type growth rate and 58 to 89% of the wild-type HAO activity when grown under the same conditions. A double mutant (hao ₁::kan and hao ₃::amp) also had 68% of the wild-type growth and 37% of the wild-type HAO activity.     

A novel vector element providing multicopy vector integration in Arxula adeninivorans

An Arxula adeninivorans vector element has been identified that provides multicopy integration in an atrp1 host strain. The element consists of the ATRP1 selection marker fused to a newly generated truncated ALEU2 promoter of 53 bp. In the described example eight copies of an amyA expression vector encoding heterologous alpha-amylase from Bacillus amyloliquefaciens are integrated in the genome of the recombinant strain instead of a single copy observed when using the ATRP1 element with the complete promoter. The high copy number results in strains of superior productivity for a secreted recombinant alpha-amylase. The vector design enables the integration of a small vector fragment that consists of yeast DNA only providing high transformation frequencies and a high mitotic stability.     

Transduction and transfection of difficult-to-transfect cells: Systematic attempts for the transfection of protozoa Leishmania

Cell-penetrating peptides (CPPs) are used to internalize different cargoes, including DNA, into live mammalian and plant cells. Despite many cells being easily transfected with this approach, other cells are rather “difficult” or “hard to transfect,” including protist cells of the genus Leishmania. Based on our previous results in successfully internalizing proteins into Leishmania tarentolae cells, we used single CPPs and three different DNA-binding proteins to form protein-like complexes with plasmids covered with CPPs. We attempted magnetofection, electroporation, and transfection using a number of commercially available detergents. While complex formation with negatively charged DNA required substantially higher amounts of CPPs than those necessary for mostly neutral proteins, the cytotoxicity of the required amounts of CPPs and auxiliaries was thoroughly studied. We found that Leishmania cells were indeed susceptible to high concentrations of some CPPs and auxiliaries, although in a different manner compared with that for mammalian cells. The lack of successful transfections implies the necessity to accept certain general limitations regarding DNA internalization into difficult-to-transfect cells. Only electroporation allowed reproducible internalization of large and rigid plasmid DNA molecules through electrically disturbed extended membrane areas, known as permeable membrane macrodomains.     

Occurrence and diversity of plasmids in populations of streptomycetes in soil

Studies were made of naturally occurring plasmids hosted in Streptomyces strains isolated from two different terrestrial ecosystems: an agricultural field and a protected forest area. Six out of the 147 screened isolates contained plasmids. The strains containing these plasmids were all isolated from the agricultural soil. Plasmids were not found among the strains isolated from the forest area. Cross hybridization of the six newly isolated plasmids revealed very high similarities between four of them. However, no similarities were found between the six newly isolated plasmids and well studied streptomycete plasmids such as pIJ101 and SCP2^*. The host strains of the four similar plasmids belonged to three different species S. anulatus, S. rochei and S. diastaticus. This implies a possible conjugative transfer of these plasmids within the streptomycete population in the agricultural area. The reason for the absence of streptomycete plasmids from the populations derived from the forest area is discussed.     

Determination of the copy number of the nuclear rDNA and beta-tubulin genes of Pneumocystis carinii f. sp. hominis using PCR multicompetitors

Multiple copies of a gene may lead to difficulty in the interpretation of typing results because polymorphism of the copies may wrongly lead to the conclusion that different types are present in a specimen. To determine the copy number per genome of the nuclear rDNA and beta-tubulin genes analyzed for the typing of Pneumocystis carinii f. sp. hominis, we developed a strategy based on the use of the same multicompetitor molecule in two different quantitative-competitive PCRs, one for the gene under study and the other for a reference single copy gene, allowing direct comparison of the results of both PCRs. Control experiments showed that the strategy was sensitive enough to detect duplication of a gene. The copy number of the nuclear rDNA operon was determined by amplification of the intron of the 26S rDNA gene and that of the beta-tubulin by amplification of the region surrounding the intron no. 6. The method was first tested on P. c. carinii, the special form commonly infecting rats. Pneumocystis c. carinii was found to contain a single copy of the rDNA operon. The method was then applied to P. c. hominis. The results confirmed that P. c. hominis genome contains a single copy of the nuclear rDNA and beta-tubulin genes.     

Detection and distribution of six linear mitochondrial plasmids in the shiitake mushroom,Lentinula edodes

The presence of plasmids was surveyed in 90 wild isolates ofLentinula edodes collected from geographically different world regions. DNA plasmids of different sizes were found in about 80% of the isolates. The plasmids detected were of six kinds, designated as pLE1 (9.0 kb), pLE2 (11.1 kb,=pLLE1 described by other authors), pLE3A (9.8 kb), pLE3B (10.8 kb), pLE3C (12.1 kb), and pLE3D (12.3 kb). Hybridization analysis suggested that pLE1 and pLE2 were distinct plasmid types of different homology groups to each other, and the four other plasmids were variant types belonging to a third homology group. These plasmids had no homology with their host's and non-host's nuclear and mitochondrial genome DNAs. Restriction analysis and electron microscopy indicated that the plasmids are linear in form. Since all six plasmids were transmitted uniparentally in sexual crosses and were consistently associated with the DNA preparations from mitochondria fractionated from mycelia of representative isolates, they were suggested to be located in mitochondria, similar to many other known fungal DNA plasmids. Geographically, pLE1 and pLE2 were widely distributed in natural populations ofL. edodes, while the remaining four plasmids were uniquely present in delimited natural populations. Contribution No. 322 from the Tottori Mycological Institute.