Abstract
BackgroundBacterial strains of the genus Geobacillus grow at high temperatures of 50–75 °C and could thus be useful for biotechnological applications. However, genetic manipulation of these species is difficult because the current techniques for transforming Geobacillus species are not efficient. In this study, we developed an easy and efficient method for transforming Geobacillus kaustophilus using the conjugative plasmid pLS20cat.ResultsWe constructed a transformation system comprising (i) a mobilizable Bacillus subtilis–G. kaustophilus shuttle plasmid named pGK1 that carries the elements for selection and replication in Geobacillus, and (ii) a pLS20cat-harboring B. subtilis donor strain expressing the dam methylase gene of Escherichia coli and the conjugation-stimulating rapLS20 gene of pLS20cat. This system can be used to efficiently introduce pGK1 into G. kaustophilus by mobilization in a pLS20cat-dependent way. Whereas the thermostable kanamycin marker and Geobacillus replication origin of pGK1 as well as expression of dam methylase in the donor were indispensable for mobilization, ectopic expression of rapLS20 increased its efficiency. In addition, the conditions of the recipient influenced mobilization efficiency: the highest mobilization efficiencies were obtained using recipient cells that were in the exponential growth phase. Furthermore, elimination of the origin of transfer from pLS20cat enhanced the mobilization.ConclusionsWe describe a novel method of plasmid mobilization into G. kaustophilus recipient from B. subtilis donor depending on the helper function of pLS20cat, which enables simple, rapid, and easy transformation of the thermophilic Gram-positive bacterium.
Highlights
Bacterial strains of the genus Geobacillus grow at high temperatures of 50–75 °C and could be useful for biotechnological applications
A conjugation assay between B. subtilis donor strain STM1 [(Table 1); trpC2 epr::(PrpsO-dam, ble) pLS20cat] and G. kaustophilus recipient strain MK244 (ΔpyrFR, ΔGK1378–GK1390, ΔGK0343–GK0346) [17] failed to form colonies on chloramphenicol-containing plates after incubation at 65 °C in our repeated experiments, indicating that pLS20cat did not transfer itself to G. kaustophilus
At least three possible reasons may explain this failure: (i) pLS20cat could not transfer itself to G. kaustophilus, (ii) pLS20cat could transfer itself to G. kaustophilus but is unable to replicate in G. kaustophilus, and/or (iii) pLS20cat could transfer itself to G. kaustophilus but the gene for chloramphenicol acetyl transferase did not function in G. kaustophilus at 65 °C
Summary
Bacterial strains of the genus Geobacillus grow at high temperatures of 50–75 °C and could be useful for biotechnological applications. Bacteria reproduce asexually and their genetic traits are inherited vertically from mother to daughter cells. They can acquire different traits from other species via horizontal gene transfer (HGT), a mechanism that contributes importantly to the genetic diversity of bacteria [1,2,3,4]. Elongation of the DNA at the generated hydroxyl group at the 3′-end of the nic site causes displacement of the strand at which the relaxase is attached. Mobilizable plasmids can be divided into two groups Members of both groups transfer their ssDNA strand through the connecting pore generated by the conjugative plasmid. Plasmids of the other group do not encode a relaxase gene; they merely contain a copy of the oriT that is present on the conjugative plasmid
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