Elaboration of an electroporation protocol for Bacillus cereus ATCC 14579.

Nathalie Turgeon1, Christian Laflamme1, Jim Ho2, Caroline Duchaine1,3*.

1 Institut universitaire de cardiologie et de pneumologie, Hôpital Laval, Université Laval,

Quebec City, Québec, Canada

2 Biological Detection Group, Defense Research and Development Canada Suffield, CFB

Suffield, Ralston, Alberta, Canada

3 Département de biochimie et microbiologie, Faculté des sciences et de génie,

Université Laval, Quebec City, Québec, Canada

* Corresponding author. Mailing address: Caroline Duchaine, Ph.D., Centre de recherche,

Hôpital Laval, 2725 Chemin Ste-Foy, Québec, Canada, G1V 4G5. Phone: (418) 656-

8711 ext. 5837. Fax: 418 656-4509. E-mail: Caroline.Duchaine@bcm.ulaval.ca

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Abstract

An electro-transformation procedure was established for Bacillus cereus ATCC 14579.

Using early growth-stage culture and high electric field, the ectroporation efficiency was

up to 2X109 cfu µg-1ml-1 with pC194 plasmid DNA. The procedure was tested with three

other plasmids, of various sizes, replication mechanisms and selection markers, and the

transformation efficiencies ranged between 2X106 to 1X108 cfu µg-1ml-1. The effects of

two wall-weakening agents on electroporation rates were also evaluated. The

transformation rate reached with our procedure is 103 times higher than that previously

obtained with members of Bacillus genus with similar plasmids, and 106 times superior

than that achieved with available protocols for B. cereus. The proposed method is quick,

simple, efficient with small rolling circle plasmids and large theta replicating plasmids

with low copy number per cell, and suitable for many genetic manipulations that are not

possible without high efficiency transformation protocols.

Key words: Bacillus cereus, electroporation, glycine, threonine, wall-weakening.

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Introduction

Bacillus cereus is a gram positive sporulating bacterium commonly found in soil and air.

It is also an opportunistic pathogen that can induce food poisoning (Schoeni and Wong,

2005). Among the B. cereus group, B. cereus is closely related to B. thuringiensis, B.

mycoides and B. anthracis, the causative agent of anthrax (Ash et al., 1991).

Genetic manipulations are useful for various types of studies and applications (Sambrook

and Russell, 2001). DNA can be incorporated into Bacillus cells using several techniques

(cf. review by Bron and Vehmaanperä, 1990). Electroporation is quick and simple

compared to other methods, and results are highly reproducible. However, among

Bacillus species, different electroporation efficiencies have been reported due to the large

variability between methods (Bron and Vehmaanperä, 1990). In recent years, efforts have

been made to increase success rates up to 106 cfu µg-1 for B. subtilis using a combination

of osmoprotective agents and high electric fields (Xue et al., 1999). Similar findings have

been reported for B. pseudofirmus using osmolarity protection followed by treatment with

glycine and high electric field (Ito and Nagane, 2001). These strategies have not been

tested for members of B. cereus group. For B. cereus, the highest electroporation

efficiency is 103 cfu µg-1, being insufficient for many applications such as mutagenesis

(Belliveau and Trevors, 1989).

In the present study we evaluated the influence of modulating growth cycle, electric field

and amino-acid composition (glycine and threonine) to elaborate an electro-

transformation procedure of B. cereus ATCC 14579.

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Material and Methods

Bacterial strains, plasmids and media. Bacterial strains used in this study are

Escherichia coli MC1061 (Wertman et al., 1986), Bacillus cereus ATCC14579, B. cereus

HER-1399 and B. cereus HER-1414. E. coli MC1061 was grown in Luria broth (Difco

Laboratories, Detroit, MI) at 37C, 200 rpm. B. cereus was grown at 37C, 200 rpm in

trypticase soy broth (TSB) (Difco Laboratories) or on trypticase soy agar (TSA) (Difco

Laboratories). When appropriate, 100µg/ml of ampicilin or 20 µg/ml of tetracycline,

chloramphenicol or erythromicine (Sigma-Aldrich, Oakville, Ontario, Canada) were

added.

Plasmids used in this study are listed in table 1. Plasmidic DNA of pLS1 and pAM1 was

purified from E. coli MC1061 using Qiagen plasmid maxi kit (Qiagen, Chatsworth, CA).

For the isolation of pT181 from S. aureus and pC194 form B. subtilis, the purification

procedure was preceded by a lysostaphin treatment (25 ng/ml, 15 min, 37C) and a

lysozyme treatment (10mg/ml lysozyme, 30 min, 37C), respectively.

Preparation of electro-competent cells. B. cereus ATCC14579 cells growth was

monitored by measurement of optical density at 600nm using a GeneQuant pro UV/Vis

spectrophotometer (Biochrom Ltd, Cambridge England). When wall-weakening

treatment was performed, 5% glycine or DL-threonine and 250mM sucrose were added

during different stages of growth and incubated 1h, 2h or 3h, 37C, 200 rpm. Cells were

washed five times in electroporation buffer (250mM sucrose, 1mM Hepes, 1mM MgCl2,

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10% glycerol, pH 7,0) and concentrated 150-fold.

Electroporation. Electroporation was performed at 25 µF using a Bio-Rad Gene Pulser

apparatus (Bio-Rad laboratories) equipped with a Bio-Rad pulse controller.

Electroporation was carried out in 2mm cuvettes (Bio-Rad laboratories, Richmond, CA)

where 100µl cells were combined with 1µg DNA (voltage range up to 12.5kV cm -1) or in

1mm electroporation cuvettes with 50µl cells combined with 500ng plasmid, in order to

respect cells/DNA proportions (voltage range between 13 and 25kV cm-1). After

electroporation, cell suspensions were diluted with 1ml of TSB supplemented with

250mM sucrose, 5mM MgCl2, 5mM MgSO4 and incubated for 2h at 37C, 200 rpm to

allow expression of antibiotic resistance markers. Aliquots were spread onto tryptic soy

agar (TSA, Difco Laboratories) supplemented with either 20µg/ml tetracycline,

chloramphenicol or erythromycin (Sigma-Aldrich, Oakville, Ontario, Canada).

Transformants harboring antibiotic resistance were counted following overnight

incubation.

Statistical analyses. The statistical analyses were carried out with Statistical Analytical

Software (SAS). Results were expressed as mean value standard deviation. Data were

analyzed using paired t-test. All reported p-values were declared significant at p < 0.05.

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Results and Discussion

Optimization of growth conditions. We transformed late stage cultures of B. cereus

with pLS1 using the electroporation protocol described by Vehmaanperä (Bron and

Vehmaanperä, 1990). The transformation efficiency was 103 cfu µg-1 ml-1 and is in

agreement with published findings.

Glycine was used to enhance electro-competence of B. cereus as previously described

(Hammes et al., 1973). For many species, the presence of the wall-weakening agent

glycine during bacterial growth improves the transformation rate (Framson et al., 1997;

Helmark et al., 2004; Lee et al., 2002; McDonald et al., 1995). Glycine is incorporated

into interpeptide bridges of peptidoglycan instead of L and D-alanine thereby reduces the

extent of cross-linking of the wall (Hammes et al., 1973). Unfortunately, in the presence

of glycine bacterial growth is highly variable (Buckley et al., 1999; Dunny et al., 1991).

To circumvent this problem, glycine was added to an exponentially growing culture in

order to maximize the effects without compromising cell growth. This method has

previously been used for cryotransformation of B. anthracis (Stepanov et al., 1990) and

electroporation of lactic acid bacteria, streptococci and B. pseudofirmus (Buckley et al.,

1999; Ito and Nagane, 2001; Mason et al., 2005; Turgeon and Moineau, 2001).

Wall-weakening treatments strongly affect the growth of B. cereus. In 10% glycine, total

cellular lysis occurred within one hour even if sucrose was added as isotonic agent. With

5% glycine, the OD remained nearly stable for at least an hour (data not shown). To

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determine the optimal growth stage and incubation time suitable for B. cereus, glycine

and sucrose were added during different stages of growth (Figure 1). Cells were

incubated for 0h, 1h, 2h, or 3h with glycine prior to washing in electroporation buffer and

electroporation with pLS1 DNA, at 12.5 kV cm-1, 200 .

For most of the tested conditions (Figure 1), cells collected in early-stage gave better

electroporation rates than late-stage culture. Treatment with glycine for 1 or 2h provided

similar data; however, with 3h glycine treatment results were highly variable due to cell

lysis in the samples. Under these experimental conditions, optimal results (3X105 cfu µg-1

ml-1) were obtained in early growth stage culture (O.D. 0.2 - 0.5) at 1h incubation with

glycine (Figure 1).

Optimization of electroporation conditions. Electroporation parameters were further

optimized as follows. Experiments were performed with pLS1 DNA using the optimal

growth parameters defined in the previous section (O.D. 0.2 - 0.4 with 1h incubation in

glycine-sucrose). Electroporation parameters were set as shown in Figure 2. The best

transformation efficiency was obtained using high electric field at 200, 20kV cm-1.

Transformation efficiency declined markedly at higher voltages. Using this procedure, a

transformation efficiency of 9X105 cfu µg-1 ml-1 was obtained for B. cereus.

Comparison with other studies. To validate the procedure and to compare results with

other studies, transformation with plasmid pC194 was tested. This plasmid has the same

replication mechanism and a similar size than pUB110 (McKenzie et al., 1986), the

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plasmid used in B. subtilis and B. pseudofirmus where transformation efficiencies of 106

cfu µg-1 have been achieved. The transformation rate obtained using our procedure (O.D.

0.2-0.5, 1h incubation with 5% glycine, 20 kV cm-1, 200Ω) was 5X108 cfu µg-1 ml-1 with

pC194 plasmid DNA (compared to 9X105 cfu µg-1 ml-1 with pLS1 DNA). This result is

100 times greater than that obtained in earlier studies with other Bacillus sp. using

osmolarity protection, glycine treatment, high electric field and pUB110 DNA (Ito and

Nagane, 2001; Xue et al., 1999). Bacterial growth during the 2h of recovery after

electroporation cannot be responsible of this enhancement because a 2h or 3h of recovery

was used in previous studies (Ito and Nagane, 2001; McDonald et al., 1995; Xue et al.,

1999).

Comparison of wall-weakening treatments. Previous studies document that DL-

threonine is an effective wall-weakening agent for B. subtilis (McDonald et al., 1995).

We compared the effects of DL-threonine and glycine as described above. Furthermore,

to determine the influence of wall-weakening treatment on electroporation rates,

replicates of the procedure were done and statistical analyses were performed. Cells were

grown until OD 0.2-0.4 was reached and collected without treatment or incubated 1h with

5% glycine or DL-threonine and 250mM sucrose. OD was monitored during wall-

weakening treatment to ensure that cellular lysis did not occur. Experiments were done

using three different cell batches. Electroporation treatments were performed three to six

times for each cell lot as described previously, with pC194 plasmid DNA and high

electric field (25 µF, 20 kV cm-1, 200Ω). The greatest transformation efficiencies were

obtained in the absence of wall-weakening agent (control) and were up to 2X109 cfu µg-1

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ml-1 compared with 5X108 cfu µg-1 ml-1 for glycine treatment and 2X108 cfu µg-1 ml-1 for

DL-threonine treatment (Figure 3). To our knowledge, this level of electro-competence is

the highest ever achieved for the Bacillus genus.

Paired T-test performed on data from all experimental conditions documented a

significant and reproducible difference (p 0.05). Under these conditions, DL-threonine

reduced the transformation rates of B. cereus by 50% compared to glycine-treatment, and

glycine reduced the transformability by 71% compared to untreated cells. Wall-

weakening treatment did not improve transformation rates with pLS1 DNA either (data

not shown). Thus, it is apparent that under weak electric field, glycine positively affects

transformation rate (Figure 1). With high electric field, the presence of wall-weakening

agents decreases electro-transformability of B. cereus (Figure 3). As such, glycine and

DL-threonine treatment does not confer a positive effect on transformation rate or reduce

the overall burden of work. Consequently, we would recommend use of the

electroporation protocol without addition of wall weakening agents.

Transformation efficiencies with various strains plasmids. Many factors influence

transformation efficiency, namely size and replication mechanism of the plasmid used.

We compared the electroporation efficiencies obtained with four different plasmids,

harboring various size, selection marker, copy number per cell and replication mechanism

(Table 2). The highest results were obtained with the smallest plasmid harboring a

chloramphenicol selection marker (pC194). High electroporation efficiency was also

obtained with the larger plasmid carrying an erythromycin resistance gene

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(pMTL500Eres). The lowest transformation rates were obtained with plasmids containing

tetracycline resistance marker. In this case, for the same plasmid size, the plasmid with a

high copy number (pT181) gave better results than the plasmid with a low copy number

(pLS1).

Those results showed that chloramphenicol and erythromycin are more suitable selection

markers for the electro-transformation of B. cereus. With the appropriate selection

marker, the described electroporation protocol gave high transformation efficiencies,

even for large theta replicating plasmids with low copy number per cell (Table 2).

Interesting results were also obtained with two other strains of B. cereus (6X106 ufc µg-1

ml-1 with HER-1399 and 3X106 ufc µg-1 ml-1 with HER-1414 with pC194 DNA). Those

results are 103 times superior than previously reported for B. cereus strains. Therefore,

this protocol can increase the electro-transformation efficiencies for several strains of B.

cereus, but growth and electroporation parameters should be adapted to each specific

strain, as previously reported for other bacterial species (Buckley et al., 1999).

This paper describes an electro-transformation method for Bacillus cereus ATCC14579

with results of 2X109 cfu µg-1 ml-1; this level of transformation is the highest ever

reported for Bacillus sp. The method is quick, simple, highly reproducible, appropriate

for many plasmids and suitable for many genetic manipulations that are not possible

without high efficiency transformation protocols. In addition, this procedure may be

useful for other Bacillus strains that are refractory to electroporation.

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Acknowledgements

We are very grateful to Gloria del Solar, Marie-Françoise Noirot-Gros, Sleem Khan and

Laurent Jannière for generous provision of the pLS1, pC194, pT181 and pMTL500Eres

plasmids. We also acknowledge Felix d’Hérelle Reference Center for Bacterial Viruses

for providing B. cereus strains HER-1399 and HER-1414. We thank the members of the

Groupe de recherche en santé respiratoire for helpful discussions. NT is the recipient of a

postdoctoral fellowship from the Groupe de recherche en santé respiratoire. CD

acknowledges a CIHR/IRSST scholarship. This work was funded by Defense Research

and Development Canada Suffield.

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References

Ash, C., Farrow, J.A., Dorsch, M., Stackebrandt, E., Collins, M.D., 1991. Comparative analysis of Bacillus anthracis, Bacillus cereus, and related species on the basis of reverse transcriptase sequencing of 16S rRNA. Int. J. Syst. Bacteriol. 41, 343-346.

Belliveau, B.H., Trevors, J.T., 1989. Transformation of Bacillus cereus vegetative cells by electroporation. Appl. Environ. Microbiol. 55, 1649-1652.

Bron, S., Vehmaanperä, J., 1990. Electrotransformation of B. amyloliquefaciens/B. subtilis. In Molecular biological methods for Bacillus, pp. 156-157. Edited by Harwood, C., Cutting, S. Chichester: John Wiley and sons.

Buckley, N.D., Vadeboncoeur, C., LeBlanc, D.J., Lee, L.N., Frenette, M., 1999. An effective strategy, applicable to Streptococcus salivarius and related bacteria, to enhance or confer electroporation competence. Appl. Environ. Microbiol. 65, 3800-3804.

Dunny, G.M., Lee, L.N., LeBlanc, D.J., 1991. Improved electroporation and cloning vector system for gram-positive bacteria. Appl. Environ. Microbiol. 57, 1194-1201.

Framson, P.E., Nittayajarn, A., Merry, J., Youngman, P., Rubens, C.E., 1997. New genetic techniques for group B streptococci: high-efficiency transformation, maintenance of temperature-sensitive pWV01 plasmids, and mutagenesis with Tn917. Appl. Environ. Microbiol. 63, 3539-3547.

Hammes, W., Schleifer, K.H., Kandler, O., 1973. Mode of action of glycine on the biosynthesis of peptidoglycan. J. Bacteriol. 116, 1029-1053.

Helmark, S., Hansen, M.E., Jelle, B., Sorensen, K.I., Jensen, P.R., 2004. Transformation of Leuconostoc carnosum 4010 and evidence for natural competence of the organism. Appl. Environ. Microbiol. 70, 3695-3699.

Horinouchi, S., Weisblum, B., 1982. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J. Bacteriol. 150, 815-825.

Ito, M., Nagane, M., 2001. Improvement of the electro-transformation efficiency of facultatively alkaliphilic Bacillus pseudofirmus OF4 by high osmolarity and glycine treatment. Biosci. Biotechnol. Biochem. 65, 2773-2775.

Khan, S.A., Novick, R.P., 1983. Complete nucleotide sequence of pT181, a tetracycline-resistance plasmid from Staphylococcus aureus. Plasmid 10, 251-259.

Lacks, S., Lopez, P., Greenberg, B., Espinosa, M., 1986. Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1. J. Mol. Biol. 192, 753-765.

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Mason, C.K., Collins, M.A., Thompson, K., 2005. Modified electroporation protocol for Lactobacilli isolated from the chicken crop facilitates transformation and the use of a genetic tool. J. Microbiol. Methods 60, 353-363.

McDonald, I.R., Riley, P.W., Sharp, R.J., McCarthy, A.J., 1995. Factors affecting the electroporation of Bacillus subtilis. J. Appl. Bacteriol. 79, 213-218.

McKenzie, T., Hoshino, T., Tanaka, T., Sueoka, N., 1986. The nucleotide sequence of pUB110: some salient features in relation to replication and its regulation. Plasmid 15, 93-103.

Sambrook, J., Russell, D.W., 2001. Molecular cloning: a laboratory manual, 3 edn. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press.

Schoeni, J.L., Wong, A.C., 2005. Bacillus cereus food poisoning and its toxins. J. Food Prot. 68, 636-648.

Stepanov, A.S., Puzanova, O.B., Dityatkin, S., Loginova, O.G., Ilyashenko, B.N., 1990. Glycine-induced cryotransformation of plasmids into Bacillus anthracis. J. Gen. Microbiol. 136, 1217-1221.

Swinfield, T.J., Janniere, L., Ehrlich, S.D., Minton, N.P., 1991. Characterization of a region of the Enterococcus faecalis plasmid pAM beta 1 which enhances the segregational stability of pAM beta 1-derived cloning vectors in Bacillus subtilis. Plasmid 26, 209-221.

Turgeon, N., Moineau, S., 2001. Isolation and characterization of a Streptococcus thermophilus plasmid closely related to the pMV158 family. Plasmid 45, 171-183.

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Xue, G.-P., Johnson, J.S., Dalrymple, B.P., 1999. High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis. J. Microbiol. Methods 34, 183-191.

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Table 1Plasmids used in this studyPlasmid Relevant characteristics ReferencespC194 Staphylococcus aureus, Cmr, 2.9 kb (Horinouchi and Weisblum, 1982)pLS1 Streptococcus agalactiae, pMV158 mob derivative, Tcr, 4.4 kb (Lacks et al., 1986)pMTL500Eres Enterococcus faecalis, pAM1 derivative, Ampr, Emr, 7.1 kb (Swinfield et al., 1991)pT181 Staphylococcus aureus, Tcr, 4.4 kb (Khan and Novick, 1983)Note. Ampr, ampicilin resistance; Cmr, chloramphenicol resistance; Emr, erythromycin resistance; Tcr, tetracycline resistance

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Table 2Transformation efficiencies obtained with various plasmidsPlasmids Plasmid

copy numberReplication mechanism

Size(kb)

Antibioticselection

Transformationefficiencies

pC194 Medium RC 2.9 Cm 8X108

pLS1 Low RC 4.4 Tc 2X106

pT181 High RC 4.4 Tc 8X106

pMTL500Eres Low 7.1 Em 1X108

Note. , theta replication mechanism; Cm, chloramphenicol; Em, erythromycin; RC, rolling circle replication mechanism; Tc, tetracycline

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Figure 1. Relationship between numbers of transformants obtained per microgram of

pLS1 DNA and per milliliter of competent cells and cell density. Cells were grown until

the optical density indicated was obtained and then further incubated with glycine for 0h

(), 1h (), 2h (), and 3h (). Electroporations were performed with low electric field

(200 , 12.5 kV cm-1).

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Figure 2. Optimization of electroporation parameters. Relationship between numbers of

transformants obtained per microgram of pLS1 DNA and per milliliter of competent cells

and voltage. Electroporations were performed in early growth stage cultures (O.D. 0.2-

0.4) incubated for 1h glycine, at the voltages indicated and at resistance levels of 100

(), 200 () or 400 ().

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Figure 3. Comparison of different wall-weakening treatments with untreated cells.

Transformation rates obtained with pC194 DNA under high electric field (200 , 20 kV

cm-1) with 3 different batches (, , ) of untreated cells (control), glycine treated, and

DL-threonine treated cells. Standard deviation and p-value are displayed on the graphic.

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