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Origin of replication (ori in theta-replicating plasmids or dso in rolling circle replicating plasmids) initiates plasmid replication in a broad range of bacteria. These two kinds of plasmids were both identified in Streptococcus, a genus composed of both human commensal bacteria and pathogens with the ability to cause severe community-acquired infections, including meningitides, septicemia, and respiratory tract diseases. Given the important roles of Streptococcus in the exchange of genetic elements with other symbiotic microbes, the genotypes and phenotypes of both Streptococcus spp. and other symbiotic species could be changed during colonization of the host. Therefore, an improved plasmid system is required to study the functional, complicated, and changeable genomes of Streptococcus. In this study, a dual-replicon shuttle vector system named pDRE was constructed to achieve heterologous gene expression. The vector system contained theta replicon for Escherichia coli. The origin of rolling circle replicon was synthesized according to pMV158 in Gram-positive bacteria. By measuring the products of inserted genes at multiple cloning sites, the ability of this vector system in the replication and expression of heterologous genes was assessed in four Streptococcus and three other Gram-positive bacteria: Bacillus subtilis, Lactococcus lactis, and Staphylococcus aureus. The results showed that the newly constructed vector could simultaneously replicate and express heterologous genes in a broad range of Gram-positive and Gram-negative bacteria, thus providing a potentially powerful genetic tool for further functional analysis. [ABSTRACT FROM AUTHOR]

In this study, the cellulase gene celD from Clostridium thermocellum was cloned into expression vectors pET-20b(+) and pHsh. While high expression can be achieved by means of both these expression systems, only the pHsh expression system gives soluble proteins. By weakening the mRNA secondary structure and replacing the rare codons for the N-terminal amino acids of the target protein, the expression level of CelD was increased from 4.1 ± 0.3 to 6.4 ± 0.4 U ml in LB medium. Recombinant CelD was purified by heat treatment followed by Ni-NTA affinity. The purified CelD exhibited the highest activity at pH 5.4 and 60°C, and retained more than 50% activity after incubation at 70°C for 1 h. The cellulase activity of CelD was significantly enhanced by Ca but inhibited by EDTA. The favorable properties of CelD offer the potential for genetic modification of strains for biomass degradation. Presently, one of the major bottlenecks for industrial cellulase users is the high cost of enzyme production. The high level expression of soluble enzymes from the pHsh expression system offers a novel approach for the production of cellulases to be used in various agro-industrial processes such as chemical, food and textile. [ABSTRACT FROM AUTHOR]