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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]

We previously identified a novel protein elicitor, PebC1, from Botrytis cinerea and described its enhancement of plant growth, drought tolerance and disease resistance in tomato. Here, we have investigated the defense-associated molecular responses in Arabidopsis thaliana after treatment with recombinant PebC1. PebC1 was expressed in Escherichia coli. Recombinant protein treatments improved plant resistance to Botrytis infection and maintained plant defenses for more than 21 days. The purified protein at 10 μg ml activated extracellular medium alkalization (pH) and reactive oxygen species and nitric oxide generation and also induced defense gene expression. Arabidopsis mutants that are insensitive to salicylic acid had increased resistance to Botrytis infection after PebC1 treatment but PebC1 did not affect the resistance of mutants with jasmonic acid and ethylene transduction pathways. The results suggest that PebC1 can function as an activator of plant disease resistance and can promote disease resistance to Botrytis in A. thaliana through the ethylene signal transduction pathway. [ABSTRACT FROM AUTHOR]

Genetic manipulation was undertaken in order to understand the mechanism involved in the heterologous synthesis of lycopene in Escherichia coli. Knockout of the central carbon metabolic gene zwf (glucose-6-phosphate dehydrogenase) resulted in the enhancement of lycopene production (above 130 % relative to control). The amplification and overexpression of rate-limiting steps encoded by idi (isopentenyl diphosphate isomerase), dxs (1-deoxyxylulose-5-phosphate synthase) and ispDF (4-diphosphocytidyl-2C-methyl- d-erythritol synthase and 2C-methyl- d-erythritol 2,4-cyclodiphosphate synthase) genes improved lycopene synthesis from 0.89 to 5.39 mg g DCW. The combination of central metabolic genes knockout with the amplification of MEP pathway genes yielded best amounts of lycopene (6.85-7.55 mg g DCW). Transcript profiling revealed that idi and dxs were up-regulated in the zwf knock-out strain, providing a plausible explanation for the increase in lycopene yield observed in this strain. An increase in precursor availability might also have contributed to the improved lycopene production. [ABSTRACT FROM AUTHOR]

The cya gene encoding adenylate cyclase was cloned from Arthrobacter sp. CGMCC 3584 by thermal asymmetric interlaced PCR for the first time. It exhibited an open reading frame containing 1,125 bp and encoding 374 amino acids. Amino acid sequence analysis showed that this enzyme was a class III adenylate cyclase. Expression of the cya gene was carried out in Escherichia coli Rosetta, and purification was performed via Ni-NTA agarose gel column. SDS-PAGE indicated that the molecular mass of the recombinant adenylate cyclase was 45 kDa. The V and K were determined to be 5.06 μmol/min/mg and 7.56 mM, respectively. The optimum pH and temperature were 8.0 and 35 °C. Several divalent metal ions were found to activate the enzyme to different extents, and the maximal specific activity reached 3.04 μmol/min/mg when 50 mM Mg was added. This was the first report of the cloning of an adenylate cyclase gene from Arthrobacter sp. [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]