Conjugative Transfer of a New Broad Host Range Expression Vector To

Heinze et al. BMC Microbiology (2018) 18:56 https://doi.org/10.1186/s12866-018-1198-4

APPLIEDMICROBIOLOGY Open Access Transmating: conjugative transfer of a new broad host range expression vector to various Bacillus species using a single protocol Simon Heinze1, Petra Kornberger1* , Christian Grätz1, Wolfgang H. Schwarz1, Vladimir V. Zverlov1,2 and Wolfgang Liebl1

Abstract Background: The genus Bacillus includes a great variety of species with potential applications in biotechnology. While species such as B. subtilis or B. licheniformis are well-known and used to provide various products at industrial scale, other Bacillus species are less characterized and are not yet used in commercial processes. One reason for this is the fact that genetic manipulation of new isolates is usually complicated with conventional techniques which have to be adapted to each new strain. Even in well-established strains, the available transformation protocols often suffer from low efficiencies. Results: In this paper, we provide a new broad host range E. coli/Bacillus shuttle vector, named pBACOV (Bacillus conjugation vector), that can be efficiently transferred to various Bacillus species using a single protocol. A variant of pBACOV carrying the sfGFP gene was successfully transferred to eight different species from the genus Bacillus and to one Paenibacillus species using triparental conjugation (“transmating”). This was achieved using a single protocol and worked for nine out of eleven tested acceptor species. The transmating procedure was used to test expression of the heterologous reporter gene sfGFP under control of the PaprE-promoter from B. subtilis in several Bacillus species in parallel. Expression of sfGFP was found in eight out of nine transmates. For several of the tested species, this is the first report of a method for genetic modification and heterologous gene expression. The expression level, analyzed by measuring the relative sfGFP-fluorescence normalized to the cell density of the cultures, was highest in B. mojavensis. Conclusions: The new shuttle vector pBACOV can be transferred to many different Bacillus and Paenibacillus species using a simple and efficient transmating protocol. It is a versatile tool facilitating the application of recombinant DNA technology in new as well as established strains, or selection of an ideal host for heterologous gene expression from a multitude of strains. This paves the way for the genetic modification and biotechnological exploitation of the broad diversity of species of Bacillus and related genera as well as different strains from these species. Keywords: Triparental conjugation, Broad host range, Shuttle vector, sfGFP, Bacillus, Paenibacillus,Heterologous expression, Genetic modification, Plasmid transfer, Transmating

* Correspondence: [email protected] 1Department of Microbiology, Technical University of Munich, Emil-Ramann-Str. 4, D-85354 Freising-Weihenstephan, Germany Full list of author information is available at the end of the article

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/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://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Heinze et al. BMC Microbiology (2018) 18:56 Page 2 of 10

Background In addition, it can be troublesome to transfer previously Bacillus species are among the most extensively exam- established genetic transformation methods (such as ined bacteria and are used for a variety of applications. chemical/heat transformation, protoplast transformation Most prominent among them is Bacillus subtilis, which or electroporation) even between strains of the same was the first Bacillus strain successfully transformed species, due to differences e.g. in special medium re- with purified DNA [1]. Today, B. subtilis and other quirements (for example regeneration media after proto- members of the genus are major workhorses in indus- plast transformation) or differences in their restriction/ trial microbiology, mainly due to their ability to secrete modification systems. large quantities of extracellular enzymes [2]. While spe- Methods for the efficient introduction of genes into cies such as B. subtilis and B. licheniformis have already multiple species and strains of bacilli without the need been isolated in the nineteenth century and are therefore to adapt the protocol to each strain of interest are cur- well-known and widely applied, new species and strains rently not available, but would be of high value, for ex- continue to be isolated. Some of these new isolates have in- ample to enable fast examination which strain or species teresting properties for potential applications, but are not is best suited for expression of a given gene. yet widely used. Additional File 1 gives a non-exhaustive overview of Bacillus strains which are already applied for Results the production of enzymes and metabolites, as well as ra- To enable fast evaluation of the suitability of multiple ther “exotic” strains which are not yet applied but show bacterial species for heterologous gene expression and to promising characteristics. The listed species were also used assess their genetic accessibility, we established a new in this study. broad-host range shuttle vector in combination with a Recombinant DNA technology can greatly improve simple procedure for plasmid transfer by triparental con- the performance of microorganisms for selected applica- jugation. The method, named “transmating”, should be tions. Although Bacillus species exhibit promising char- suited for a broad variety of strains without the need of acteristics, currently the Gram-negative Escherichia coli protocol adaptation for the individual strains. A sche- is the most widely used host for heterologous gene ex- matic overview of the method is given in Fig. 1. pression, especially in the context of metabolic engineering and production of small molecules. This is due to Design of pBACOV the fact that efficient genetic manipulation techniques The newly developed plasmid pBACOV (Bacillus conjuga- and system-level strategies exist for this microorganism tion vector) includes replication origins and selection [3]. While efficient transformation protocols and markers for E. coli and Bacillus as well as the expression methods for manipulation of the chromosome are avail- cassette from pBE-S and the RK2 conjugation origin (oriT/ able for B. subtilis, it is more challenging to perform traJ) from plasmid pKVM4, a mobilizable plasmid used to such experiments with sufficient efficiency in other Ba- generate markerless gene deletions in B. licheniformis [8]. cillus hosts, especially in wild-type strains which are dif- The expression cassette contains the B. subtilis promoter ficult to modify genetically [2, 4]. One reason for this is PaprE,theaprE signal peptide for secretory protein expres- that transformation of bacteria with plasmid DNA gen- sion, a multiple cloning site (MCS) and the coding se- erally requires specified protocols and adaptation of the quence for a C-terminal hexahistidine tag (His6-tag) for procedures for each strain. protein purification (see Materials and Methods). Common methods to introduce recombinant DNA are To generate pBACOV-sfGFP (Fig. 2), the sfGFP gene the use of natural competence in case of B. subtilis, coding for super-folder green fluorescent protein [9] was protoplast transformation (often used for B. megater- inserted into pBACOV, while removing the aprE signal ium), electroporation, and mobilization of plasmids from peptide. Thus, sfGFP is expressed intracellularly, allowing E. coli to Gram-positive recipients by conjugation. In to compare promoter strength between different strains most cases, the elaborate preparation of naturally or containing pBACOV-sfGFP without having to be con- otherwise competent cells is required and usually, differ- cerned about additional influences that secretion might ent methods are needed for different strains. In addition have on the level of sfGFP produced. The expected plas- to the fact that the available methods are usually specific mid sequences of pBACOV and pBACOV-sfGFP were for one strain, they also have other drawbacks: proto- confirmed by sequencing. plasts are difficult to handle and do not survive freezing; natural competence is observed in B. subtilis but not in Establishment of the minimal inhibitory concentrations of most other Bacillus species and the efficiency is rather kanamycin and polymyxin B for all tested strains low; electroporation is an efficient method, but requires The selection agar contained kanamycin (Kan) for the individual determination of ideal parameters such as selection of colonies carrying the plasmid and polymyxin medium composition and electric field strength [5–7]. B (Pol) for counter-selection against the E. coli donor Heinze et al. BMC Microbiology (2018) 18:56 Page 3 of 10

Fig. 2 Schematic map of the shuttle vector pBACOV with the inserted target gene sfGFP. ColE1 ori: origin of replication for E. coli; AmpR: Ampicillin resistance gene for selection in E. coli; oriT/traJ: origin of transfer for conjugative plasmid transfer; KanR: Kanamycin resistance gene for selection in Bacillus; pUB ori: origin of replication

for Bacillus;PaprE: aprE promoter from B. subtilis; sfGFP-His6: target gene sfGFP fused with a sequence encoding a C-terminal His6-tag b selection agar was 10 μg/ml for Kan and 40 μg/ml for Pol. These concentrations were found to be suited for B. licheniformis DSM13, B. pumilus DSM27, B. sonorensis DSM13779 and Paenibacillus polymyxa DSM356. Other Bacillus species tested showed resistance to 10 μg/ml Kan or sensitivity to 40 μg/ml Pol. In these cases, the minimal inhibitory concentrations of Kan or Pol had to be established by serial dilution tests before transmating with pBACOV-sfGFP could be attempted. The results of the serial dilution tests are listed in Additional File 2. Based on these results, seven additional strains were selected for Fig. 1 Steps for transfer of pBACOV-sfGFP from E. coli to Bacillus species transmating experiments with adapted concentrations of by transmating. Triparental conjugation (transmating) requires an acceptor strain (strain of a Bacillus species of choice), a donor strain (e. g. the selection antibiotics: B. mycoides DSM2048,B.mega- E. coli TOP10 carrying pBACOV-sfGFP) and a helper strain (E. coli HB101 terium DSM32, B. pseudomycoides DSM12442 (concen- pRK2013). After conjugation on agar plates without selection, the cells trations used: Pol: 40 μg/ml, Kan: 50 μg/ml), B. subtilis are transferred to appropriate antibiotic-containing plates (Pol RIK1285,B.mojavensisDSM9205,B.vallismortis concentration < MIC of acceptor, Kan concentration > MIC of acceptor) DSM11031 (Pol: 10 μg/ml, Kan: 10 μg/ml) and B. olero- for selection of transmates (a). Plasmid content and the identity of the μ μ host strain were confirmed by colony PCR and 16S rRNA gene nius WS8036 (Pol: 2.5 g/ml, Kan: 10 g/ml). sequencing (b) before further characterization of the transmates (c). MIC: minimal inhibitory concentration, Kan: kanamycin, Pol: polymyxin B Transmating pBACOV-sfGFP to different Bacillus species Plasmid pBACOV-sfGFP, which was constructed as a derivative of vector pBACOV (see above and Fig. 2), was and helper strains. The antibiotic concentration in the se- used to study (i) transfer to a large number of different lection agar must be adjusted such that the sensitivity to- Bacillus species by a single routine transmating proced- ward Kan was high enough to enable selection for colonies ure and (ii) the expression of the heterologous reporter carrying the plasmid, while sufficient resistance against Pol gene sfGFP in all transmates. was necessary to ensure effective counter-selection against The transfer of pBACOV-sfGFP from E. coli TOP10 to E. coli. The standard concentration of antibiotics in the various Bacillus acceptor strains was achieved by Heinze et al. BMC Microbiology (2018) 18:56 Page 4 of 10

transmating using E. coli HB101 pRK2013 as helper polymyxa DSM356 (total number of colonies: 1750) and strain [10, 11] as described in Material & Methods. Plas- B. subtilis RIK1285 (several thousand colonies, partially mid pRK2013 is a self-transmissible plasmid (size: 48 confluent growth on selection plates). kbp) containing the broad host range transfer system of RK2 [10, 12]. It is efficiently transferred between Measurement of sfGFP fluorescence Gram-negative bacteria and can promote the conjugal Production of sfGFP by selected transmates was studied in transfer of unrelated plasmids [10, 12]. The mechanism order to demonstrate the usefulness of pBACOV as broad for triparental plasmid transfer is thought to involve two host range expression plasmid. Non-transmated cultures of steps: first, the helper plasmid pRK2013 is transferred to the same species served as negative controls. SM-Cas the donor strain which carries the mobilizable, but not medium was used, because autofluorescence was not de- self-transmissible, expression plasmid pBACOV. Now, tectable with this medium. After 24 h of incubation, the the genes necessary for conjugation can be expressed in the cultures were diluted to an OD600 of about 1 and the fluor- donor strain and thus the expression plasmid pBACOV can escence was measured (excitation: 470 nm, emission: be transferred to the acceptor strain. For each acceptor 520 nm). Table 2 summarizes the relative fluorescence nor- strain, three transmating batches were made. These con- malized to the OD600 of the cultures (fluorescence inten- tained either cells from the overnight pre-cultures, cells sity/OD600). In eight out of nine cultures of strains carrying from the mid-exponential growth phase (OD600 between pBACOV-sfGFP, sfGFP-fluorescence was detectable, which 0.6 and 0.9) or cells from the late exponential phase shows that the PaprE-promoter is functional in these bac- (OD600 ≥ 1.2). After incubation of the selection plates, col- teria. B. sonorensis was the only transmated species which onies were counted and correct transmates were identified did not show detectable sfGFP fluorescence. Hence, PaprE by analytical PCR using plasmid specific primers (pBA- can be used for the expression of heterologous genes in al- COV-seq_4894fw and pBACOV-Seq-rv). The 16S rRNA most 90% (eight of nine) of the tested strains. The strongest gene sequence of selected transmates was determined to relative fluorescence was observed with B. mojavensis. The verify that the clones obtained after selection/counter-selec- sfGFP-fluorescence was also visualized by fluorescence mi- tion belonged to the correct acceptor species. This was es- croscopy. Images of B.licheniformis,B.mojavensisand P. pecially important for B. mojavensis, B. vallismortis and B. polymyxa with or without pBACOV-sfGFP are shown in oleronius, i. e. the strains grown at reduced Pol concentra- Fig. 3. tions, in order to eliminate the possibility of growth of E. coli under these milder counter-selection conditions. Discussion Additionally, special attention was paid to the colony Conjugal transfer of plasmid DNA from Gram-negative morphology: to determine the transmating efficiency, only E. coli to Gram-positive bacteria has been reported colonies with the morphology typical for the respective acceptor strains were considered. The Pol concentration of Table 1 Yield of colonies obtained in representative 10 μg/ml used for the matings with B. mojavensis and B. transmating experiments with each acceptor strain vallismortis could not inhibit growth of E. coli completely, Species number of colonies sincesomeofthetestedcolonieswereE. coli,whileothers with typical morphology were Bacillus transmates. Here, the colony morphology B. subtilis > 5000 * was helpful for the identification of correct clones as a pre- P. polymyxa 1750 screening before 16S rRNA gene sequencing. In the case of B. vallismortis 849 B. oleronius all colonies chosen from the selection plates for B. pumilus 655 16S rRNA gene sequencing turned out to be E. coli. This indicates that although the minimal inhibitory Pol concen- B. mojavensis 574 tration for E. coli in liquid cultures was below 2.5 μg/ml, ef- B. sonorensis 178 fective counter-selection against E. coli was not possible on B. licheniformis 37 agar plates at this concentration. Thus, transfer of B. mycoides n. q. ** pBACOV-sfGFP to B. oleronius wasregardedasnot B. pseudomycoides n. q. ** successful. B. oleronius 0 Transfer of pBACOV-sfGFP to all other selected acceptor strains was successful with the only exception of B. megaterium 0 B. megaterium. The total number of colonies on selec- Only colonies with the typical morphology of the respective acceptor strain were counted. On selection plates with polymyxin B concentrations ≤10 μg/ tion agar varied between the different species as well as ml, additional growth of E. coli was observed. *: for B. subtilis, several thousand the three transmating batches for each strain, but was distinct colonies and partially confluent growth were observed. **: for B. mycoides and B. pseudomycoides, the exact number of colonies could not be never below 25 (for examples of yielded colony numbers, quantified (n.q.), due to confluent growth of large, mycelium-like see Table 1). The most colonies were obtained with P. colonies (diameter > 10 mm) Heinze et al. BMC Microbiology (2018) 18:56 Page 5 of 10

Table 2 sfGFP fluorescence of different bacterial species in this work, is a new broad host range vector which can carrying pBACOV-sfGFP relative to B. subtilis RIK1285 pBACOV- easily be transferred to an acceptor strain of choice using sfGFP triparental conjugation (“transmating”) and can be used Species Relative fluorescence [%] for heterologous gene expression in various Bacillales pBACOV-sfGFP wild-type species. pBACOV-sfGFP, containing sfGFP as reporter B. mojavensis 383 ± 5.1 n. d. gene, was successfully transferred to strains of widely B. vallismortis 239 ± 5.9 0.9 ± 0.1 used and well-studied species such as B. licheniformis or B. subtilis, but also to more “exotic” species. To our B. licheniformis 160 ± 8.8 n. d. knowledge, this is the first report of recombinant DNA B. subtilis 100 ± 0.6 n. d. technology applied in B. sonorensis, B. mycoides, B. pseu- B. pumilus 56.1 ± 0.9 n. d. * domycoides, B. vallismortis and B. mojavensis, thus mak- B. pseudomycoides 41.0 ± 1.5 n. d. ing these species accessible for genetic engineering. P. polymyxa 16.7 ± 0.5 3.4 ± 0.2 Since Bacillus species in general have high potential for B. mycoides 14.9 ± 0.1 3.1 ± 0.0 biotechnological applications, there is a great interest in expanding the number of genetically accessible strains. B. sonorensis n. d. 1.1 ± 0.1 Usage of the pBACOV vector in combination with the Strains were grown for 24 h at 37 °C, 180 rpm (B. mycoides: 72 h at 30 °C, 180 rpm) in SM-Cas medium with supplements, if appropriate. For details, see transmating protocol described herein can pave the way Material & Methods. Fluorescence was detected with cultures diluted to to future applications. OD600 = 1 and the fluorescence intensity (excitation: 470 nm, emission In this study, we strived to test the applicability of 520 nm) and the OD600 were determined. The relative fluorescence (fluorescence intensity/OD600) was calculated by normalizing the measured pBACOV and our transmating method in as many spe- fluorescence intensity by the OD600. The relative fluorescence of B. subtilis cies as possible from the genus Bacillus and closely re- RIK1285 pBACOV-sfGFP was defined as 100%. The given values are mean values ± standard deviation of triplicate measurements. The asterisk * lated genera. While the majority of the tested strains indicates that the B. pumilus wild-type culture did not reach an OD600 of 1 belonged to the genus Bacillus,pBACOValsoworked within the 24 h of incubation and therefore was not diluted for fluorescence measurements. n. d.: not detectable (defined as relative fluorescence < 0.7%) in the genus Paenibacillus,aswasdemonstratedwithP. polymyxa DSM356. The pUB origin of replication before [13, 14], including B. licheniformis and thermo- stems from Staphylococcus aureus and is known to be philic Bacillus strains [4, 14, 15], but no comprehensive active in a wide range of low GC Gram-positive study exists showing the versatility of this gene transfer bacteria (Firmicutes), including anaerobic Clostridia method to introduce plasmids into various different spe- [16, 17]. Therefore it is likely that pBACOV can be cies of mesophilic bacilli. Plasmid pBACOV, introduced transferred to even more genera.

Fig. 3 Visualization of sfGFP expression by fluorescence microscopy. Top row from left to right: Transmates of B. licheniformis, B. mojavensis and P. polymyxa carrying pBACOV-sfGFP. Bottom row from left to right: B. licheniformis, B. mojavensis and P. polymyxa wild type strains. The scale bar corresponds to 5 μm. Images were recorded using a Zeiss Axio Imager.M1 fluorescence microscope equipped with a Zeiss AxioCam MRm camera Heinze et al. BMC Microbiology (2018) 18:56 Page 6 of 10

In this study we did not aim to optimize the frequency Pol concentration of 2.5 μg/ml, a substantial number of E. of transfer between the E. coli donor and the acceptor coli colonies was detected on the selection agar. In some in- bacilli but rather wanted to develop a simple, versatile stances, colonies of E. coli even grew at 10 μg/ml, resulting method that can be applied to many strains without ex- in false-positive clones which had to be eliminated via tensive adaptation to the different acceptor strains. In rigorous discriminating tests. In this work we used as- our hands, transmates were obtained for nine of the sessment of the characteristic colony morphology and eleven Bacillus and Paenibacillus species tested. Trans- 16S rRNA gene sequencing for the unequivocal identifi- mating failed in only two of the cases tested, i.e. for B. cation of the B. mojavensis and B. vallismortis trans- megaterium DSM32 and B. oleronius WS8036. Regard- mates, but other methods, including Gram-staining or ing B. megaterium, the reason for the failed transmating differentiating physiological tests may also be useful to is not known, but the cell envelope composition [18]or discriminate between real acceptor strain transmates other special features may play a role. and false-positive E. coli clones. Alternative options for To express heterologous target genes, pBACOV fea- effective counter-selection against E. coli could include tures the PaprE promoter from B. subtilis [19–22]. We the use of spontaneous antibiotic-resistant mutants of demonstrate that PaprE is functional in seven additional the acceptor strain or culture media which inhibit Bacillus species using sfGFP as a reporter gene and re- growth of E. coli, such as MRS agar [23]. port for the first time heterologous gene expression in four new Bacillus species (B. mojavensis, B. mycoides, B. Conclusions pseudomycoides and B. vallismortis). The observed sfGFP This study introduces a universal, simple and efficient expression levels ranged between about 0.15- and method for the transfer of plasmid DNA into a large num- 3.8-fold of the value measured in B. subtilis. The reasons ber of different Bacillus and Paenibacillus species. We for the up to 25-fold differences in the apparent sfGFP stress that the same procedure worked for most tested expression levels found between different host strains strains without the need for individual optimization. The are currently unknown, but various differences between use of pBACOV enabled for the first time the demonstra- the hosts’ gene expression machineries (differences in tion of heterologous gene expression in B. mojavensis, B. promoter PaprE recognition, mRNA stability, sfGFP fold- mycoides, B. pseudomycoides, and B. vallismortis. Its ex- ing efficiency etc.) and also differences in the plasmid’s pression promoter PaprE from B. subtilis was functional in copy number may contribute to this observation. eight out of nine transmated species. It is possible to obtain large numbers of colonies with The new plasmid pBACOV in combination with the some strains (e.g. P. polymyxa DSM356, B. subtilis transmating protocol facilitates application of recombin- RIK1285). This makes the transmating system suitable ant DNA technology in new as well as established Bacil- for the screening of libraries, e. g. signal peptide librar- lus and Paenibacillus strains. It can aid in the selection ies, in some of the new host strains from this work for of the best-suited host for heterologous gene expression optimizing the expression of a secretory protein of inter- from a large number of candidate strains, thereby enab- est. This holds true even for species where fewer col- ling the research community to tap into the abundant onies were obtained, as the procedure can be easily source of hitherto unused Bacillus species with potential scaled up. This is a big advantage over transformation for application in biotechnology. procedures using purified plasmid DNA, where purification of large quantities of plasmid DNA is needed. Methods One limitation of our system is that sensitivity to Kan Bacterial strains and growth conditions: and resistance to Pol are prerequisites for the acceptor The bacterial strains used in this study are listed in strain. Of the 24 bacteria included in this study, only 10 Additional File 2.AllBacillus and Paenibacillus strains (approx. 42%) showed the necessary characteristics. The were obtained from DSMZ (Leibniz-Institut DSMZ-Deutsche selection for transmates relies on Kan resistance con- Sammlung von Mikroorganismen und Zellkulturen ferred by the plasmid. By replacing the Kan resistance GmbH, Braunschweig, Germany), except B. bataviensis cassette of pBACOV with a different selection marker WS4576, B. ginsengihumi WS8095, B. horikoshii WS2157, (such as chloramphenicol or tetracycline resistance), or B. niabensis WS9147, B. niacini WS4575, B. oleronius by equipping the vector with two or even three selection WS8036, Fictibacillus arsenicus WS4538 (obtained from markers, it should be possible to increase the versatility of the strain collection of the Chair for Microbial Ecology, pBACOV. For counter-selection against the E. coli donor Department of Biosciences, Technical University of Mun- and helper strains, the minimal inhibitory concentration of ich, Freising, Germany) and B. subtilis RIK1285 (Takara-- Pol is critical. In our experience, a concentration of at least Bio Inc., Kusatsu, Japan). E. coli TOP10 (Life technologies, 10 μg/ml Pol is required for sufficient counter-selection. At Carlsbad, USA) was used for cloning and as donor strain the attempt to transfer pBACOV-sfGFP to B. oleronius at a in transmating experiments. E. coli HB101 carrying the Heinze et al. BMC Microbiology (2018) 18:56 Page 7 of 10

plasmid pRK2013 (DSMZ 5599) [10, 11] was used as oriT/traJ region necessary for conjugative plasmid transfer helper strain for transmating experiments. All strains were was inserted between the antibiotic resistance genes. routinely grown in LB liquid medium [24]oragarplates Fragment 1 for Gibson Assembly (i. e. the pUB110-derived at 37 °C (liquid cultures were shaken at 180 rpm), except region and expression cassette, bp 3453–557 of pBE-S) for B. mycoides, which was grown at 30 °C. E. coli HB101 was created using the primers pUB110_backbone_fw and pRK2013 was grown with 50 μg/ml kanamycin (Appli- pUB110_backbone_rv with pBE-S as template. Fragment 2 Chem GmbH, Darmstadt, Germany) and E. coli TOP10 (the pUC-derived portion of pBE-S, bp 1642–3452) was carrying pBACOV or pBACOV-sfGFP was grown with amplified using the primers Fw_pUC_for_Gibson and 100 μg/ml carbenicillin (Carl Roth GmbH, Karlsruhe, Rv_pUC_from_pBE-S and pBE-S as template. Fragment 3 Germany). Bacillus transmates were streaked on LB selec- (oriT/traJ)wasamplifiedbyusingtheprimersoriT_- tion agar containing polymyxin B (Carl Roth GmbH) and traJ_fw and oriT_traJ_rv and the plasmid pKVM4 as Kan (for the applied concentrations see below, section template [8]. To maintain compatibility of pBACOV “plasmid transfer by transmating”) or grown in liquid LB with the signal peptide library from the B. subtilis medium with Kan (same concentrations as the selection Secretory Protein Expression System Kit (Takara-Bio agar). Inc.), the EagI restriction site within the traJ gene was removed by two-step QuikChange mutagenesis Construction of pBACOV and pBACOV-sfGFP [26] using primers QC_g2015t_1 and QC_g2015t_2. The To construct pBACOV, Gibson Assembly of three over- incorporated mutation (g2015t) is silent, exchanging the lapping DNA fragments was applied [25], using the com- original alanine-codon GCC to GCA while removing the mercially available pBE-S plasmid (Takara-Bio Inc., size: EagI restriction site (CGGCCG is mutated to CTGCCG). 5938 bp) as chassis. pBE-S is composed of a fragment of For construction of pBACOV-sfGFP, the sfGFP-gene was the pUC plasmid, comprising the ColE1 origin of replica- amplified using the primers sfGFP-fw and sfGFP-rv and tion and ampicillin resistance gene (bp 1645–3556 of the plasmid pET28A-sfGFP (kindly provided by Mark pBE-S) for propagation in E. coli, and a piece of plasmid Teese) as template. The sfGFP coding sequence corre- pUB110, including the pUB origin of replication and the sponds to the sequences published in [27]. pBACOV was kanamycin resistance gene for Bacillus (bp 3557–5936 of linearized using the restriction endonucleases MluIand pBE-S). Furthermore, an expression cassette is present XbaI, thereby removing the coding sequence of the aprE after the pUB ori (bp 5937–517 of pBE-S). The expression signal peptide. sfGFP was inserted into pBACOV using cassette consists of the B. subtilis promoter PaprE,theaprE Gibson Assembly. All primers used are listed in Table 3. signal peptide for secretory protein expression, a multiple The vector sequences of pBACOV (accession number: cloning site (MCS) and the coding sequence for a MG599120.1) and pBACOV-sfGFP (accession num- C-terminal hexahistidine tag (His6-tag) for protein purifi- ber:MG599121.1) were deposited at GenBank. cation. A stem-loop was predicted from bp 486 to 519, followed by a stretch of five T nucleotides, constituting a Determination of minimal inhibitory concentrations of potential rho-independent transcriptional terminator. For kanamycin and polymyxin B construction of pBACOV, (i) an un-annotated segment of A single colony of the respective strain was carefully re- pBE-S (bp 580–1642 of pBE-S) was removed and (ii) a suspended in 20 ml LB medium. Four glass test tubes

Table 3 Primers used in this study Primer Name Sequence 5′–3′ pUB110_backbone_fw GTCTAAGAAACCATTATTATCATGAC pUB110_backbone_rv CATAATAGTTATGCAGTTTGTAGAATGC Fw_pUC_for_Gibson CAAACTGCATAACTATTATGTACGAGCAAAAGGCCAGC Rv_pUC_from_pBE-S GTCAGGTGGCACTTTTCG oriT_traJ_fw TCCCCGAAAAGTGCCACCTGACTTAGTGCTTTACGGCACCTCG oriT_traJ_rv CATGATAATAATGGTTTCTTAGACGCTTCGGGGTCATTATAGCG sfGFP-fw TAAGCAAAAGGAGAGGGACGCGTATGAGCAAAGGTGAAGAACTG sfGFP-rv CATTAGTGGTGATGATGGTGATGTCTAGATTTATACAGTTCATCCATACCATG pBACOV-seq_4894fw CGAGTCTCTACGGAAATAGC pBACOV-Seq-rv CGATGAGCGCATTGTTAG QC_g2015t_1 GCGGCGGCGGCAGGCATGAGCCTG QC_g2015t_2 CAGGCTCATGCCTGCCGCCGCCGC Heinze et al. BMC Microbiology (2018) 18:56 Page 8 of 10

were each filled with 2.5 ml of this suspension while a Eppendorf reaction tubes (Eppendorf AG, Hamburg, fifth glass test tube was filled with 5 ml. The 5 ml cul- Germany). The cells were harvested from the filters by ture was supplemented with Kan (final concentration: adding 1 ml of LB medium and vortexing. These cell 200 μg/ml) or Pol (final concentration: 40 μg/ml). Then, suspensions were then plated onto selection agar plates a dilution series was made by transferring 2.5 ml from one (LB agar containing Pol and Kan) and incubated over- tube to the next, which resulted in a 2-fold dilution of the night at 37 °C or for up to three days at 30 °C until col- antibiotic per step (from 200 μg/ml to 12.5 μg/ml Kan; onies appeared. Routinely, the selection agar contained from 40 μg/ml to 2.5 μg/ml Pol). The tubes were incu- 10 μg/ml Kan and 40 μg/ml Pol. For Bacillus strains bated overnight at 180 rpm at the growth temperature of sensitive toward 40 μg/ml Pol or resistant against the respective test strain (see above). The minimal inhibi- 10 μg/ml Kan, the respective antibiotic concentration tory concentration (MIC) was defined as the lowest con- was adapted based on the determined MIC as follows: centration at which no growth was detectable. 40 μg/ml Pol and 50 μg/ml Kan for B. mycoides, B. megaterium and B. pseudomycoides;10μg/ml Pol and Plasmid transfer by transmating 10 μg/ml Kan for B.subtilis,B.mojavensisand B. val- To prepare pre-cultures for transmating experiments, lismortis, and 2.5 μg/ml Pol and 10 μg/ml Kan for B. 100 ml Erlenmeyer flasks containing 20 ml LB medium oleronius. Correct transmates were identified by analyt- (supplemented with antibiotics if appropriate) were inocu- ical PCR using two plasmid specific primers and by se- lated with single colonies of the Bacillus acceptor strain(s), quencing of the 16S rRNA gene using the primers the donor strain (E. coli TOP10 pBACOV-sfGFP) and the 616 V and 630R [28]. helper strain (E. coli HB101 pRK2013), respectively. The pre-cultures were incubated overnight as indicated above. The next day, the OD600 of all cultures was determined Expression of sfGFP and determination of relative and the cultures were used to inoculate fresh flasks with fluorescence 20 ml LB (supplemented with antibiotics if appropriate) to SM-Cas medium, based on Spizizen’s Minimal Medium an OD600 of 0.2. The overnight pre-cultures were stored [1, 29], was used for sfGFP-expression experiments. The on ice for later use. The OD600 of the freshly inoculated medium corresponds to the SM medium published online flasks was checked regularly, starting 90 min after the be- at SubtiWiki [30], with the addition of 1 g/l casamino ginning of incubation. Once the OD600 was between 0.6 acids but without tryptophane. The final composition of and 0.9, 2 ml samples of each culture were taken and SM-Cas medium was 17.5 g/l K2HPO4, 7.5 g/l KH2PO4, stored on ice. Incubation of the main cultures was contin- 5 g/l glucose, 4 g/l L-glutamate, 2 g/l (NH4)2SO4,1.26g/l ued until the OD600 was at least 1.2, at which point an- trisodium citrate dihydrate, 1 g/l casamino acids, 250 mg/l other round of 2 ml samples was collected and stored on MgSO4,5.5mg/lCaCl2, 1 mg/l MnCl2∙4H2O, 1.7 mg/l ice. The collected donor, acceptor and helper samples of ZnCl2,0.33mg/lCuCl2∙2H2O, 0.6 mg/l CoCl2∙6H2O, the same growth phase (overnight, OD600 between 0.6 and 0.6 mg/l Na2MoO4∙2H2O, 1.35 mg/l FeCl3∙6H2O. For P. 0.9 or OD600 ≥ 1.2) were then mixed as follows: 2 ml of polymyxa DSM356, biotin was added to a final concentra- the donor cells were pelleted by centrifugation and the tion of 150 μg/l and for the tryptophan auxotrophic B. supernatant was discarded. All centrifugation steps were subtilis RIK1285 tryptophan was added to a final concen- carried out at 1900 x g, 4 °C, for 5 min. The donor cell tration of 50 mg/l. 20 ml of SM-Cas (with Kan at appropri- pellet was resuspended in 2 ml of acceptor culture and ate concentrations) were inoculated with a single colony of centrifuged again. The pellet was resuspended in 2 ml of the Bacillus transmates carrying pBACOV-sfGFP or the the helper culture and centrifuged. The resulting pellet respective wild-type strains as negative controls and in- containing all three strains was washed twice with 1 ml of cubatedat37°C,180rpm(B. mycoides:30°C).After LB to remove traces of antibiotics. The cell pellet was re- 24 h (B. mycoides: 72 h), the OD600 of the cultures was suspended in a small volume of remaining supernatant. measured. For determination of the relative fluores- Sterile cellulose acetate filters (pore size: 0.2 μm, diam- cence normalized to cell density, the cultures were di- eter: 25 mm, Sartorius Stedim Biotech GmbH, Göttin- luted with SM-Cas to OD600 =1.Next,theOD600 and gen, Germany) were placed on LB agar plates and the the fluorescence intensity (excitation: 470 nm, emission: mating mixtures were pipetted smoothly, with as little 520 nm) were measured using Uvette® cuvettes (Eppen- shearing as possible, to the middle of the filters. These dorf) with a path length of 2 mm in a BioSpectrometer conjugation plates were incubated overnight at 30 °C Fluorescence Photometer (Eppendorf). To calculate the (three plates for each acceptor strain, each containing relative fluorescence, the fluorescence intensity value one mixture of donor/acceptor/helper cultures with the was divided by the measured OD600,takingthepath same OD600). The next day, the filters were carefully re- length of the cuvette into account. Each measurement moved from the LB agar plates and placed into 5 ml was performed in triplicate. Heinze et al. BMC Microbiology (2018) 18:56 Page 9 of 10

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