METHODS AND PROTOCOLS

An efficient transformation method for Bacillus subtilis DB104

Ljubica Vojcic & Dragana Despotovic &

Ronny Martinez & Karl-Heinz Maurer &

Ulrich Schwaneberg

Received: 18 December 2011 /Revised: 15 February 2012 /Accepted: 17 February 2012# Springer-Verlag 2012

Abstract Bacillus subtilis strains are used for extracellularexpression of enzymes (i.e., proteases, lipases, and cellu-lases) which are often engineered by directed evolution forindustrial applications. B. subtilis DB104 represents an at-tractive directed evolution host since it has a low proteolyticactivity and efficient secretion. B. subtilis DB104 is ham-pered like many other Bacillus strains by insufficient trans-formation efficiencies (≤103 transformants/μg DNA). Afterinvestigating five physical and chemical transformation pro-tocols, a novel natural competent transformation protocolwas established for B. subtilis DB104 by optimizing growthconditions and histidine concentration during competencedevelopment, implementing an additional incubation step inthe competence development phase and a recovery stepduring the transformation procedure. In addition, the influ-ence of the amount and size of the transformed plasmidDNA on transformation efficiency was investigated. Thenatural competence protocol is “easy” in handling andallows for the first time to generate large libraries (1.5×105

transformants/μg plasmid DNA) in B. subtilisDB104 withoutrequiring microgram amounts of DNA.

Keywords B. subtilisDB104 . Directed evolution . Naturalcompetence . Transformation protocol

Introduction

Directed evolution is a powerful algorithm to improve en-zyme properties in iterative cycles of diversity generationand screening. A typical directed evolution experiment com-prises three major steps: (1) diversity generation, (2) screen-ing to identify improved mutants out of a large pool ofvariants, and (3) isolating the gene encoding for the im-proved protein variant (Tee and Schwaneberg 2007). Thefirst step in a directed evolution campaign includes thegeneration of a DNA mutant library and its subsequenttransformation into the host organism. Advances in screen-ing technologies like flow cytometry-based screening sys-tems (Aharoni et al. 2005; Prodanovic et al. 2011; Tu et al.2011) allow a throughput of up to 108 variants so thattransformation efficiencies are increasingly becoming thelimiting step in directed evolution experiments.

B. subtilis is used as host for production of secretoryproteins especially proteases and lipases which have a signif-icant market value (Westers et al. 2004). Low transformationefficiencies are a main challenge when usingBacillus strains indirected evolution campaigns. In order to ensure an efficientand secreted expression of the target enzyme in a Bacillus host,it is essential to perform the directed evolution directly in theBacillus production strain. B. subtilisDB104 is one of themostused strains for the production of industrially important extra-cellular enzymes, especially subtilisin proteases. B. subtilisDB104 is a derivative of B. subtilis 168 Marburg strain(Kawamura and Doi 1984), generated by lesions in the genes

Electronic supplementary material The online version of this article(doi:10.1007/s00253-012-3987-2) contains supplementary material,which is available to authorized users.

L. Vojcic :D. Despotovic : R. Martinez :U. Schwaneberg (*)Lehrstuhl für Biotechnologie, RWTH Aachen University,Worringerweg 1,52074 Aachen, Germanye-mail: u.schwaneberg@biotec.rwth-aachen.de

K.-H. MaurerInternational Research Laundry & Home Care, Biotechnology,Henkel AG & Co. KGaA,40191 Düsseldorf, Germany

K.-H. MaurerAB Enzymes GmbH,Feldbergstraße 78,64293 Darmstadt, Germany

Appl Microbiol BiotechnolDOI 10.1007/s00253-012-3987-2

coding for the alkaline protease (aprA3) and the neutral prote-ase (nprE18). As a consequence, B. subtilis DB104 shows lessthan 4% of the extracellular protease activity of B. subtilis 168Marburg strain, which is a key advantage when proteases areexpressed heterologously. B. subtilis DB104 requires histidineas an essential amino acid which is usually supplemented in thegrowth medium (Kawamura and Doi 1984).

Transformation of Bacillus strains can be accomplished bynatural competence or artificial methods. Natural competencefor DNA uptake is in Gram-positive and Gram-negative bac-teria a physiologically and genetically determined trait inresponse to environmental stress (Hamoen et al. 2003). Thedevelopment of competence in B. subtilis is, in part, dictatedby nutritional conditions and growth stage. In addition, thedevelopment of natural competence is strain specific due todivergent structure of the quorum sensing components whichcontrol development of natural competence (Tran et al. 2000).Transformation based on natural competence allows DNAuptake from various sources, for instance phage DNA, plas-mid DNA, and chromosomal DNA. DNA uptake occurs by acommon pathway through the following stages: binding, frag-mentation, uptake, as well as additionally integration andreplication/or mismatch repair in the case of chromosomalDNA (Dubnau 1991). Reported transformation efficiency forB. subtilis 168 strain reached 106 transformants/μg chromo-somal DNA (Anagnostopoulos and Spizizen 1961). Artificialtransformation methods employ, in contrast to the abovemen-tioned natural competence methods, physical and chemicaltreatments such as electroporation or addition of polyethyleneglycol or mannitol (Brigidi et al. 1990; Chang and Cohen1979) (Table 1). The technically least demanding method,reported as “simple and rapid method” (Table 1, no. 1), allowstransformation on solid media by overlaying plated Bacilluscells with chromosomal or plasmid DNA (2–3 μg). This agarplate-based transformation method is described to yield ap-proximately 100–200 transformants/μg DNA (Hauser andKaramata 1994). An advancement of the agar plate-basedtransformation method was achieved by adding DNA in pro-toplast lysates (Table 1, no. 2), yielding a transformationefficiency of 2.3×103/μg chromosomal DNA (Akamatsuand Taguchi 2001); this transformation efficiency decreased

100 times when plasmid DNAwas transformed. For transfor-mation of plasmid DNA, an electroporation method (Table 1,no. 3) with a transformation efficiency of 104/μg DNA wasdeveloped (Brigidi et al. 1990). Electroporation combinedwith a previous treatment of the cells with glycine (Table 1,no. 4) increased efficiency to 1.7×106 transformants/μg plas-mid DNA. Preparation and transformation of B. subtilis pro-toplasts (Table 1, no. 5) in presence of polyethylene glycolyielded up to 1.4×107 transformants/μg plasmid DNA(Chang and Cohen 1979). All methods summarized inTable 1 were investigated, yielding, in case of B. subtilisDB104, up to 5×103 transformants/μg plasmid DNA(Table 1, no. 5). The transformation efficiency represents abottleneck in the directed evolution of proteases when B.subtilis DB104 is used as an expression host. In thisreport, a highly efficient transformation method based ondevelopment of natural competence in B. subtilis DB104was optimized, resulting in an efficiency of 1.5×105 trans-formants/μg plasmid DNA. The developed protocol isbased on transformation protocol for B. subtilis 168 strainemploying chromosomal DNA (Anagnostopoulos andSpizizen 1961).

Materials and methods

Chemicals

All chemicals were purchased from AppliChem (Darmstadt,Germany) or Carl Roth GmbH (Karlsruhe, Germany) exceptcasamino acids (ForMediumTM, Norfolk, UK).

Materials

The cell culture was cultivated in a Certomat® RM shaker(Sartorius Stedim Biotech GmbH, Goettingen, Germany).The amount of DNA in the experiments was quantifiedby using a NanoDrop photometer (ND-1000; NanoDropTechnologies, Wilmington, DE, USA). Plasmid isolationkit was purchased from Qiagen (Hilden, Germany).Optical density of the cell culture at 600 nm (OD600) was

Table 1 Summary of reported artificial methods for transformation of Bacillus species

No. Transformation method Bacillus strain Transformants/μg DNA

Remarks

1 Solid media B. subtilis 168/W23 100–200 2–3 μg of DNA needed

2 Solid media in protoplast lysates B. subtilis AYG2 2.3×103 Optimized for chromosomal DNA

3 Electroporation of intact Bacillus cells B. subtilis PB1424 1×104 Low survival rate due to applied voltage

4 Electroporation with glycine treatment B. pseudofirmus OF4 1.69×106 Survival rate due to applied voltage (2–16%)

5 Protoplasts Derivatives of B. subtilisMarburg 168 strain

4×107 2–3 days recovery time

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measured using a BioPhotometer plus photometer(Eppendorf AG, Hamburg, Germany).

Bacterial strain and plasmids

The bacterial strain used in this study was B. subtilis DB104(Kawamura and Doi 1984). Plasmids used for transforma-tion were as follows: (1) pC194 (2,910 bp; cat. no. 4393;DSMZ, Braunschweig, Germany); (2) pHY300PLK, aBacillus–Escherichia coli shuttle vector (4,870 bp; TakaraBio Inc., Shiga, Japan); (3) pHY300Car, derivative ofpHY300PLK vector containing a subtilisin Carlsberg(6,221 bp); and (4) pHCMC04 Bacillus–E. coli shuttlevector (8,089 bp) (Nguyen et al. 2005). The final concen-trations of antibiotics in agar plates were 15 μg/ml tetracy-cline for cells containing pHY300PLK/pHY300Carplasmids, and 10 μg/ml or 5 μg/ml chloramphenicol forcells containing the pC194 or the pHCMC04 plasmid.

Protocol

Media composition

Starvation medium 1 (SM1) contains 0.2% ammoniumsulfate, 1.4% dipotassium hydrogen phosphate, 0.6% potas-sium dihydrogen phosphate, 0.07% sodium citrate, 0.5%glucose, 0.02% magnesium sulfate heptahydrate, 0.2% yeastextract, and 0.025% casamino acids. All the componentswere mixed together and autoclaved. Starvation medium 2(SM2) is less rich in nutrients and includes 0.2% ammoniumsulfate, 1.4% dipotassium hydrogen phosphate, 0.6% potas-sium dihydrogen phosphate, 0.07% sodium citrate, 0.5%glucose, 0.08% magnesium sulfate heptahydrate, 0.1% yeastextract, 0.01% casamino acids, and 0.05% calcium chloride.All components of SM2 medium were mixed and auto-claved together. Histidine solution was sterilized by filtra-tion using 0.2-μm filters (PuradiscTM 25 mm, cat. no. 6780-2502; GE Healthcare, Munich, Germany).

Preparation of competent cells

B. subtilis DB104 cells were spread on a LB agar platewithout antibiotics (37 °C; for 9 h). Antibiotic-free SM1media was inoculated by transferring a single colony andsubsequent cultivation (37 °C; 250 rpm for 14–16 h).Overnight culture was diluted in SM1 medium by adjustingoptical density at 600 nm (OD600) to 0.5 (approximately2.9×107 cells/ml) in a volume of 10 ml and incubated (37 °C; 200 rpm for 3 h). The cell culture volume was doubled byaddition of SM2 medium and subsequently supplementedwith varied histidine concentrations (final concentration 0,10, 50, 200, 500, and 1,000 μg/ml; total volume 20 ml) andincubated (37 °C; 300 rpm for 2 h). After the treatment, B.

subtilis DB104 cells were competent for approximately 1 h.Histidine concentration of 200 μg/ml (final concentration)was used in final protocol.

Transformation procedure

Five hundred microliters of the competent cells was mixedwith varied amounts of plasmid DNA (pHY300Car—2, 5,10, 20, 40, 100, 250, 500, 1,000 ng) and incubated (37 °C;200 rpm for 30 min). In order to recover the cells, 300 μl offresh LB medium was supplemented and competent B. sub-tilis DB104 were additionally incubated (37 °C; 200 rpm for30 min). Two hundred microliters of B. subtilis DB104 cellsuspension was subsequently spread on LB agar plates withselective antibiotic (tetracycline, final concentration15 μg/ml).

Results

An efficient protocol for B. subtilis DB104 strain for trans-forming plasmid DNA was developed which represents anextension of earlier work by Anagnostopoulos and Spizizen(1961). Table 2 summarizes the optimization steps duringcompetence development and transformation procedures.The protocol optimization comprises steps such as omittingcentrifugation, adapting incubation times, and optimizationof the cell growth, transformation times, and histidine con-centration. Subsequently, the influence of plasmid DNAamount and size on transformation efficiency wereinvestigated.

Optimization of the growth time of B. subtilis DB104

B. subtilis develops its natural competence at the end ofexponential growth phase with the expression of the comKgene (Hamoen et al. 2003). In order to determine the timerequired for the cells to enter into a stationary phase, thegrowth of B. subtilis DB104 in SM1 and SM2 medium wasmonitored. Overnight culture (37 °C; 200 rpm for 14–16 h)of B. subtilis DB104 in SM1 medium was diluted with SM1medium until OD600 reached a value of 0.5. B. subtilisDB104 cells were grown (37 °C; 200 rpm) and OD600 wasmonitored every 30 min. Fig. 1 shows the growth curve ofB. subtilis DB104 in SM1 medium (closed square) reachinga stationary phase after 3 h. At the end of exponential phase(3 h of growth time), the total cell culture volume wasdiluted 1:1 with SM2 medium and supplemented with var-ied histidine concentrations (final concentration 0, 10, 50,200, 500, and 1,000 μg/ml). Upon dilution with SM2 me-dium (SM1/SM2, open squares), the end of exponentialphase was reached after 2 h of growth and different histidineconcentrations show no detectable effect on growth of B.

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subtilis DB104. After optimization, B. subtilis DB104 cellsreach a state of natural competence after a cultivation timeof 3 h in SM1 and of 2 h in SM1/SM2 medium.

Influence of histidine concentration on transformationefficiency

To investigate the dependence of different histidine concen-trations on transformation efficiency of B. subtilis DB104,the concentration range of histidine as supplement to SM1/SM2 medium was varied (final concentration 0, 10, 50, 200,500, and 1,000 μg/ml). Fig. 2 shows that concentrations ofhistidine up to 500 μg/ml influenced the transformationefficiency of B. subtilis DB104 with the range varying from8.0×104 to 1.5×105. At higher concentrations of histidine

(1,000 μg/ml), the competence of B. subtilis DB104 de-creased to 4.5×104. In the final protocol, 200 μg/ml histi-dine was used.

Dependence of different shuttle vectors on thetransformation efficiency of B. subtilis DB104 cells

Three different shuttle expression vectors for Bacillus(pC194, pHY300PLK, and pHCMC04) were used for trans-formation of natural competent B. subtilis DB104 in order toinvestigate the dependence of vector size and antibioticresistance on the transformation efficiency. The used plas-mids ranged from 2.9 to 8.1 kb [pC194 (2.9 kb),pHY300PLK (4.9 kb), pHY300Car (6.2 kb), pHCMC04(8.1 kb)]. The highest number of transformants was obtained

Table 2 Comparison of the developed plasmid transformation protocol for B. subtilis DB104 to the transformation protocol of Spizizen for B.subtilis 168 strain (Anagnostopoulos and Spizizen 1961)

Spizizen’s protocol Optimized protocol for B. subtilis DB104

Organism: B. subtilis 168 Organism: B. subtilis DB104

Overnight culture on an LB agar plate Overnight culture in SM1 liquid medium

108 cells/ml inoculums cultivated in SM1 mediumcontaining essential tryptophan (50 μg/ml)

Dilution in SM1 medium (no essential histidine amino acidsin medium) to reach 107 cells/ml (OD600 ~0.5)

Time of incubation: 4 h Time of incubation: 3 h

Centrifugation of the cells No centrifugation step

1:10 dilution in SM2 medium containing tryptophan (5 μg/ml) 1:1 dilution in SM2 medium containing histidine (200 μg/ml)

No additional incubation step in SM2 medium Incubation for additional 2 h (37 °C; 300 rpm without DNA;final volume 20 ml)

Chromosomal DNA added to the cell culture followed by incubationfor 90 min (37 °C; rpm not defined, final volume 1 ml)

Plasmid DNA added to the cell culture followed by incubationfor 30 min (37 °C; 200 rpm; final volume 0.8 ml)

No recovery step in LB medium Recovery of the cells with 300 μl fresh LB medium for 30 min(37 °C; 200 rpm)

Plated on agar plates with selective antibiotic Plated on agar plates with selective antibiotic

Fig. 1 Growth curve of B.subtilis DB104 in SM1 medium(filled squares) and SM1/SM2medium (open squares).Optical density (OD600)monitored at 600 nm infunction of time. The cells weregrown (37 °C, 200 rpm) and 1-ml aliquots were taken forOD600 measurements every30 min. In order to ensure thatOD600 reading is within thelinear range (0.1–0.5) of accu-racy of photometer, the cellsuspension was diluted prior tothe measurement

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using pHY300Car (1.5×105 transformants/μg DNA)(Table 3). This result indicates that the size of the usedplasmid does not correlate with the transformation effi-ciency. In case of the antibiotics (chloramphenicol andtetracycline), 5- to 10-fold higher transformation efficien-cy could be obtained using plasmids bearing the tetracy-cline resistance cassette.

Dependence of amount of plasmid DNAon the transformation efficiency of B. subtilis DB104 cells

Transformation efficiencies of B. subtilis DB104 cells weredetermined as a function of DNA amount (pHY300Car—2,5, 10, 20, 40, 100, 250, 500, and 1,000 ng). Results pre-sented in Fig. 3 indicate the correlation between transfor-mation efficiency and DNA amount (range of 2–1,000 ng).Results also showed a linear relationship between the DNAamount used for transformation and the total number oftransformants indicating single-hit kinetics (see ElectronicSupplementary Material Fig. 1). An optimum was foundbetween 5 and 10 ng of plasmid DNA. At plasmid amountshigher than 40 ng, the transformation efficiency declinescontinuously from 7×104 to 4×103 transformants/μg plas-mid DNA.

Discussion

Advances in screening technologies with throughputs up to108 variants (Prodanovic et al. 2011; Tu et al. 2011) enablenovel directed evolution strategies for instance using highmutational loads. In order to match throughput screening,efficient transformation protocols are necessary to generatea sufficient number of variants, especially in non-E. colihosts. The use of Bacillus strains as protein expression hostsis especially important for industrially used hydrolases (pro-teases and/or lipases) which have to be secreted into mediafor high level production. B. subtilis DB104 represents anattractive directed evolution host due to its low proteolyticactivity and efficient secretion.

Transformation protocols for Bacillus strains shown inTable 1 were investigated for B. subtilis DB104, yielding atransformation efficiency of up to 5×103 variants/μg plas-mid DNA using protoplast method (Table 1, no. 5). Theprotoplast method required after transformation a 3-dayrecovery phase and resulted in a growth of high number ofB. subtilis DB104 cells without plasmid DNA despite ofantibiotic use. The obtained transformation efficiency andrequirement of high DNA concentrations (microgram range;Table 1, nos. 1, 2, and 5) made the physical and chemicaltransformation methods not suitable for directed evolutionin B. subtilis DB104. Based on Spizizen’s protocol fortransformation of chromosomal DNA by natural compe-tence into the Bacillus 168 strain, a novel transformationprotocol for B. subtilis DB104 was developed to transformplasmid DNA. After various optimizations [growth phase,histidine/DNA concentration, shuttle vectors (size, resis-tance/four constructs)], the highest transformation efficiencyof B. subtilis DB104 was obtained by addition of plasmid

Fig. 2 Transformation efficiency (transformants per microgram ofplasmid DNA0cfu/μg) at varied concentrations of histidine supple-mented (0, 10, 50, 200, 500, and 1,000 μg/ml) to SM1/SM2 medium.Error bars represent standard deviation from the mean value betweenthree triplicate experiments

Table 3 Influence of plasmid resistance (CmR/TcR) and size on thetransformation efficiency of B. subtilis DB104

Plasmid Size (kb) Phenotypea Transformants/μg plasmid DNA

pC194 2.9 CmR 1.3×104

pHY300PLK 4.9 TcR 1.2×105

pHY300Car 6.2 TcR 1.5×105

pHCMC04 8.1 CmR 2.2×104

aCmR chloramphenicol resistance, TcR tetracycline resistance

Fig. 3 Transformation efficiency (transformants per microgram ofDNA0cfu/μg) in function of amount of DNA (ng). B. subtilisDB104 cells were transformed with different amounts of plasmidDNA (pHY300Car—2, 5, 10, 20, 40, 100, 250, 500, and 1,000 ng).Error bars represent standard deviation from the mean value betweentriplicate experiments

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DNA at the end of the exponential growth phase withefficiencies up to 1.5×105 transformants/μg DNA. Theobtained efficiency is two orders of magnitude higher com-pared to that obtained after transformation of B. subtilisDB104 with any of the methods summarized in Table 1.The obtained result is in concordance with a study ofDubnau (1991) reporting that natural competence is devel-oped at the end of exponential phase.

The robustness of the transformation method was deter-mined by investigating whether supplementing essentialhistidine affects the growth rate and transformation efficien-cy of B. subtilis DB104. In addition, the influence of theemployed plasmid system and the amount of transformedplasmid DNA on transformation efficiency was determined.Varied histidine concentrations showed no effect on growthcurve of B. subtilis DB104 but influenced transformationefficiency (Fig. 2). A histidine concentration of 200 μg/mlwas finally selected for B. subtilis DB104 transformation.Table 3 shows that the transformation efficiency of B. sub-tilis DB104 does not depend on the plasmid size. For in-stance, the empty vector pHY300PLK (4.9 kb) andharboring a Carlsberg protease gene (pHY300Car; 6.2 kb)have a similar transformation efficiency (Table 3; 1.2 vs.1.5×105 variants/μg plasmid DNA). Interestingly, the trans-formation efficiency for the tetracycline resistant shuttlevectors (pHY300PLK, pHY300Car) is 5- to 10-fold highercompared to the pC194 and pHCMC04 vectors which usechloramphenicol for selection. Independence of transforma-tion efficiency from the vector size can be explained bydifferences in DNA uptake between natural competent andphysically treated (voltage) Bacillus cells (Brigidi et al.1990). In case of natural competence uptake, the double-stranded DNA is first digested to single-stranded DNA byNucA nuclease (Hamoen et al. 2003), taken up throughpilin-like structures at the Bacillus surface, and transportedas single-stranded DNA across the membrane, complemen-tary strand synthesis, and nick repair (Dubnau 1999; Kidaneet al. 2009). This “reconstruction” could introduce deletionsor repeats into the transformed DNA, which is undesired inmost molecular biology work. Approaches to avoid DNAdamage include the uptake of plasmid DNA by competentcells harboring a partially homologous resident “helper”plasmid to rescue the incoming DNA (Haima et al. 1990).The use of “helper” plasmid increases the probability ofcorrect reconstruction of the input plasmid and potentiallyincreases the transformation efficiency.

To asses if the uptake mechanism used by Bacillus com-petent cells in this work was prone to damage the trans-formed plasmid DNA, a physical analysis of the plasmidisolated from 28 transformants was performed to investigatepossible deletions and/or mutations and to test the suitabilityof the transformation method for directed evolution. Therestriction pattern (two restriction enzymes, six fragments)

of the transformed recovered plasmid and sequencing anal-ysis of the promoter-gene insert showed no changes com-pared to the input transformed plasmid (see ElectronicSupplementary Material Fig. 2). The obtained results indi-cate that the transformed plasmid remains intact aftertransformation.

The natural competence transformation protocols allowin contrast to many other artificial transformation protocols(Table 1, nos. 1, 2, and 5) to transform efficiently lowamounts of plasmid DNA. An optimum was found between5 and 10 ng of plasmid DNA (Fig. 3) and can likely beattributed to the number of specific receptors (~50) (Dubnau1999) on the Bacillus surface which can be regarded aslimiting factor for the natural uptake of high amounts ofplasmid DNA.

In summary, a transformation protocol was developed forB. subtilis DB104 as an expression host in directed proteaseevolution experiments. The natural competence protocolallows for the first time to generate large libraries (~105

transformants/μg plasmid DNA) in B. subtilis DB104 andis furthermore robust, fast, and simple in handling. We hopethat the current protocol can be extended to other Bacillusstrains and will be used to encourage researchers to performdirected evolution experiments using Bacillus species asexpression hosts.

Acknowledgments This work was supported by the German Gov-ernment through the Bundesministerium für Bildung and Forschung(Bioindustrie-2021, FKZ0315250) and Henkel AG & Co. KGaA.

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