Miyano et al. Microb Cell Fact (2018) 17:127 https://doi.org/10.1186/s12934-018-0969-9 Microbial Cell Factories RESEARCH Open Access A novel method for transforming the thermophilic bacterium Geobacillus kaustophilus Megumi Miyano1, Kosei Tanaka1, Shu Ishikawa1, Kotaro Mori1, Andrés Miguel‑Arribas2, Wilfried J. J. Meijer2* and Ken‑ichi Yoshida1* Abstract Background: Bacterial 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 difcult because the cur‑ rent techniques for transforming Geobacillus species are not efcient. In this study, we developed an easy and efcient method for transforming Geobacillus kaustophilus using the conjugative plasmid pLS20cat. Results: We 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-har‑ boring 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 efciently 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 efciency. In addition, the conditions of the recipient infuenced mobilization efciency: the highest mobilization efciencies were obtained using recipient cells that were in the exponential growth phase. Furthermore, elimination of the origin of transfer from pLS20cat enhanced the mobilization. Conclusions: We 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 thermo‑ philic Gram-positive bacterium. Keywords: Geobacillus kaustophilus, Bacillus subtilis, Plasmid, Conjugation, Mobilization, Transformation Background transduction, and conjugation. Transformation involves In general, bacteria reproduce asexually and their genetic the acquisition of naked DNA from the extracellu- traits are inherited vertically from mother to daugh- lar environment [5], transduction involves transfer of ter cells. However, they can also acquire diferent traits genetic information through bacteriophage infection [6], from other species via horizontal gene transfer (HGT), a and conjugation involves physical cell-to-cell contact for mechanism that contributes importantly to the genetic DNA transfer, mediated by conjugative elements that can diversity of bacteria [1–4]. Tree diferent mecha- be embedded in the bacterial genome (named Integra- nisms are mainly responsible for HGT: transformation, tive Conjugative Element, ICE) or present on plasmids (named conjugative plasmids) [7]. A conjugative element *Correspondence: [email protected]; kenyoshi@kobe‑u.ac.jp renders a complete set of genes required for DNA trans- 1 Department of Science, Technology and Innovation, Kobe University, fer. A cell harboring a conjugative plasmid can act as a 1‑1 Rokkodai, Nada, Kobe 657 8501, Japan donor to transfer the plasmid to a recipient cell lacking 2 Centro de Biología Molecular ‘Severo Ochoa’ (CSIC‑UAM), Instituto de Biología Molecular ‘Eladio Viñuela’ (CSIC), Universidad Autónoma, Canto the plasmid. Conjugation has important environmen- Blanco, 28049 Madrid, Spain tal and medical implications. In addition, conjugation is © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Miyano et al. Microb Cell Fact (2018) 17:127 Page 2 of 13 exploited as a tool for genetic modifcation of bacteria into two groups. Members of both groups transfer their thereby serving research and industrial purposes. Plas- ssDNA strand through the connecting pore generated mids can be classifed into two categories: conjugative by the conjugative plasmid. Plasmids belonging to one and non-conjugative; of which only the former can trans- of these groups encode their proper relaxase that acts on fer themselves to recipient cells (Fig. 1; in most cases, the cognate oriT present on the plasmid; the relaxase and only one DNA strand of the conjugative plasmid is trans- oriT of the plasmids are unrelated to those present on the ferred into the recipient cell through a pore that connects conjugative plasmid. Plasmids of the other group do not the donor and recipient cells). One of the initial steps of encode a relaxase gene; they merely contain a copy of the the conjugation route involves so-called relaxosome pro- oriT that is present on the conjugative plasmid. teins processing the DNA to generate the single-stranded Te genus Geobacillus was frst described in 2001 [8]. DNA, which is transferred to the recipient cell. Te key It comprises thermophilic bacteria that were previously enzyme of the relaxosome complex is a relaxase that rec- included in the genus Bacillus. Geobacilli are Gram- ognizes and binds to specifc sequences in a region on positive, endospore-forming, aerobic or facultative the plasmid named the origin of transfer (oriT). After anaerobic thermophiles and are isolated from various binding, the relaxase cleaves the DNA in a strand- and environments, such as soil, hot springs, oilfelds, and hay site-specifc manner at a specifc position, named the nic compost [9–12]. Geobacilli grow optimally at 50–75 °C. site, and remains covalently attached to the 5′-end of the Tis feature makes them benefcial for biotechnological nicked strand. Elongation of the DNA at the generated applications. For instance, the high temperature prevents hydroxyl group at the 3′-end of the nic site causes dis- the growth of possible contaminant mesophilic bacteria, placement of the strand at which the relaxase is attached. and also saves energy and costs to remove fermentation Te relaxase and its attached DNA is delivered to the heat. In addition, it can facilitate the recovery of volatile pore and subsequently transferred into the recipient cell. products from culture media. Successful use of bacte- As may be expected, many plasmids lacking the conju- rial species for biotechnological applications requires gation genes cannot be transferred. However, despite efcient ways to modify it genetics in order to engineer lacking the conjugation genes, a rather large group of strains with desired and/or optimized features. A few plasmids can be transferred when they are co-resident techniques to modify Geobacillus species are available with a conjugative plasmid via a process named mobi- [13]. For example, a protoplast method for Geobacillus lization (Fig. 1). Mobilizable plasmids can be divided stearothermophilus NUB36 [14] and an electroporation a Chromosome Conjugative plasmid Donor Recipient Conjugation Transconjugant b Chromosome Conjugative plasmid Mobilizable plasmid Donor Recipient Mobilization Transconjugant Fig. 1 Schematic presentation of conjugation and mobilization. Conjugation refers to transfer of a conjugative plasmid from donor to recipient (a), whereas mobilization refers to mobilization of a mobilizable plasmid from donor to recipient mediated by the helper function of a co‑resident conjugative plasmid (b). Large light circle, conjugative plasmid; small bold circle, mobilizable plasmid; light rounded rectangle, donor cell; and bold rounded rectangle, recipient cell Miyano et al. Microb Cell Fact (2018) 17:127 Page 3 of 13 method for Geobacillus thermoglucosidasius DL44 [15] requiring at least 6 h of incubation on solid media for have been reported. However, besides that these tech- mating. niques are not efcient, transformation by the protoplast pLS20, a conjugative plasmid isolated from B. subtilis technique is laborious and the electroporation method natto [19], can transfer itself to various B. subtilis-related requires the optimized conditions for each strain. Here, Gram-positive bacteria, including Bacillus anthracis, we describe a novel method to modify Geobacillus Bacillus cereus, Bacillus licheniformis, Bacillus megate- kaustophilus strain HTA426 that is based on interspe- rium, Bacillus pumilus, and Bacillus thuringiensis [20]. cies mobilization. Te technique is simple, rapid and pLS20cat, a derivative of pLS20 carrying a chloram- reproducible. phenicol resistance gene possesses the outstanding abil- Geobacillus kaustophilus HTA426 can grow in lysogeny ity of rapid transfer: after simply mixing liquid cultures of broth (LB) medium at high temperatures ranging from 42 donor and recipient cells the plasmid is efciently trans- to 74 °C under aerobic conditions as rapidly as Escheri- ferred within 15 min [21–26]. In addition, pLS20cat can chia coli at 37 °C. Growth has low nutrient demands and mobilize a co-resident plasmid if it contains a functional various carbon sources can be used, which include glyc- copy of the