Electroporation-Mediated Transformation ofArcanobacterium (Actinomyces) pyogenes

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38, 135–140 (1997) PL971299

ARTICLE NO.

SHORT COMMUNICATION Electroporation-Mediated Transformation of Arcanobacterium (Actinomyces) pyogenes B. Helen Jost,1 Stephen J. Billington, and J. Glenn Songer Department of Veterinary Science and Microbiology, The University of Arizona, Tucson, Arizona 85721 Received January 27, 1997; revised May 28, 1997 Plasmids derived from pNG2 or RSF1010 were introduced into strains of Arcanobacterium (Actinomyces) pyogenes by electroporation. Electroporation conditions were varied systematically to give a maximum electroporation frequency of 3.7 1 105 CFU/mg DNA at 1.5 kV/cm and 246 V, resulting in a time constant of approximately 10 ms. The A. pyogenes transformants expressed plasmid-encoded resistance to chloramphenicol, erythromycin, kanamycin, and streptomycin. The source of incoming DNA affected the growth rate of transformants, but not the electroporation efficiency. This is the first report of genetic transformation of the veterinary pathogen A. pyogenes. q 1997 Academic Press

Arcanobacterium (Actinomyces) pyogenes (Ramos et al., 1997) is a ubiquitous inhabitant of the mucus membranes of the upper respiratory and genital tracts of domesticated animals, notably ruminants and pigs. The organism is associated with a number of suppurative infections in host animals, such as summer mastitis (Lean et al., 1987) and hepatic abscesses (Lechtenberg et al., 1988). Herds infected with A. pyogenes can incur serious economic losses. Little is known about either pathogenesis or the antigens important in immunity to infection with this organism. A. pyogenes elaborates several extracellular ‘‘toxins,’’ including a hemolysin (Funk et al., 1996), several proteases (Schaufuss et al., 1989; Takeuchi et al., 1995), and a DNase (La¨mmler, 1990), and at least two strains produce a neuraminidase (Schaufuss and La¨mmler, 1989). Electroporation, a process which induces the reversible permeability of cell membranes, allows introduction of plasmid DNA into bac1 To whom correspondence should be addressed at Department of Veterinary Science and Microbiology, The University of Arizona, 1117 East Lowell Street, Tucson, AZ 85721. Fax: (520) 621-6366. E-mail: jost@vetsci. microvet.arizona.edu.

teria. This technique has allowed a great leap forward in the study of genetics, especially of Gram-positive organisms, many of which are refractory to the simple chemical methods used to induce competence in Gram-negative species (Chassy and Flickinger, 1987; Allen and Blaschek, 1988; Luchansky et al., 1988; Dunny et al., 1991; Ricci et al., 1994). More specifically, electroporation has also been used with success in members of the family Actinomycetaceae, such as Actinomyces spp. (Yeung and Kozelsky, 1994), Corynebacterium spp. (Serwold-Davis et al., 1987; Liebl et al., 1989; Haynes and Britz, 1990; Songer et al., 1991), Mycobacterium spp. (Hermans et al., 1991), Bifidobacterium spp. (Argnani et al., 1996), and Clavibacter xyli (Metzler et al., 1992), among others. In order to use genetic approaches to more systematically study the virulence attributes of A. pyogenes, we have developed an electroporation protocol for the introduction of plasmid DNA into this organism. This paper investigates the factors which influence the efficiency of this process. BACTERIAL STRAINS AND PLASMIDS The bacterial strains and plasmids used in this study are listed in Table 1. A. pyogenes

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0147-619X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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AND

PLASMIDS USED

Relevant characteristics Strain A. pyogenes BBR1 A. pyogenes OX8 E. coli DH5a Plasmid pBBR1MCS pEP2 pJGS76

pJRD215 pLTV1 pMAK705 pMMB19 pNG2 pUC4-K pWSK129

Isolated from bovine abscess Porcine isolate F0 F80d/lacZDM15 D(lacZYA-argF)U169 endA1 recA1 hsdR17(rk0 mk/) deoR thi-1 supE44 gyrA96 relA1 4.7-kb, broad host range, CmR 3.1-kb, KmR derivative of pNG2 4.6-kb, EcoRI–SalI fragment (1.5-kb) containing catP from Clostridium perfringens cloned into pEP2, CmR, KmR 10.2-kb, RSF1010 origin, KmR, SmR, Mob/ 20.6-kb, pE194 ts origin, colE1, ApR, CmR, EmR, TcR 5.6-kb, pSC101 ts origin, KmR 8.9-kb, RSF 1010 ts origin, SmR 14.4-kb, broad host range, EmR 3.9-kb, colE1, ApR, KmR 6.7-kb, pSC101 origin, KmR

strains were grown on BHI (Difco) agar plates, supplemented with 5% bovine blood, at 377C and 5% CO2 in a humidified incubator. Liquid cultures of A. pyogenes were grown in BHI broth supplemented with 5% fetal bovine serum (FBS)2 at 377C. Escherichia coli strains were grown on either LB (Difco) agar or in LB broth at 377C. Strains harboring temperaturesensitive plasmids were grown as above at 307C. Antibiotics (Sigma) were added as appropriate; for A. pyogenes strains, 5 mg/ml chloramphenicol (Cm), 15 mg/ml erythromycin (Em), 30 mg/ml kanamycin (Km), 200 mg/ ml streptomycin (Sm); for E. coli strains, 100 mg/ml ampicillin (Ap), 30 mg/ml Cm, 200 mg/ ml Em, 50 mg/ml Km, 50 mg/ml Sm. Plasmid DNA from E. coli strains was prepared using the modified alkaline lysis method of Morelle (1989). Plasmid DNA from A. pyogenes strains was prepared in a similar manner, except that the cells were incubated in 100 mg/ ml lysozyme, 75 mM NaCl, 25 mM EDTA, 20 mM Tris–HCl, pH 8.0, for 30 min at 377C 2 Abbreviations used: FBS, fetal bovine serum; Cm, chloramphenicol; Em, erythromycin; Km, kanamycin; Sm, streptomycin; Ap, ampicillin.

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Marana, AZ Oxford Laboratories, Worthington, MN Bethesda Research Laboratories

Kovach et al., 1994 Radford and Hodgson, 1991 This study

Davison et al., 1987 Camilli et al., 1990 Hamilton et al., 1989 Scholz et al., 1985 Serwold-Davis and Groman, 1986 Pharmacia Wang and Kushner, 1991

prior to cell lysis. DNA to be used for electroporation was ethanol precipitated, washed extensively with 70% ethanol, and resuspended in HPLC grade water. PREPARATION OF ELECTROCOMPETENT CELLS

Cells for electroporation were grown to an OD600 of 0.6 in 200 ml of BHI–5% FBS. The cells were chilled on ice for 10 min, centrifuged at 5000g and 47C for 15 min, and washed twice in an equal volume of 10% glycerol:90% HPLC grade water as above. The cell pellet was resuspended in a final volume of 800 ml (250 times concentration). The cells were dispensed into 20-ml aliquots and stored at 0807C. Frozen cells were approximately threefold less electrocompetent than fresh cells (results not shown). Aliquots of cells were thawed on ice for 20 min and 1–2 ml plasmid DNA (approximately 10 ng–1 mg depending on the plasmid used) was added just prior to electroporation. Electroporation was performed in chilled 0.1-cm cuvettes using a BTX Inc. Electro Cell Manipulator 600, with a constant capacitance of 50 mF. The pulsed

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cells were recovered in 2 ml BHI with 0.3% sucrose and 5% FBS and incubated with shaking at 377C for 2 h or 20 h (for expression of Em resistance). Dilutions were plated onto BHI–blood agar with the appropriate antibiotics and incubated for up to 7 days. Transformants were present by 48–72 h. No transformants were obtained in the absence of plasmid DNA. INITIAL ELECTROPORATION EXPERIMENT

E. coli-replicated pEP2 plasmid DNA was able to transform A. pyogenes BBR1 or OX8 to Km resistance, using electroporation settings of 18 kV/cm, 246 V and a time constant of approximately 10 ms. However, it was noted that transformants obtained with strain BBR1 were of two types. Colonies with the typical A. pyogenes morphology were present at 0.1–1% of the total transformant number. These transformants grew normally and were visible by 48 h. However, the majority of the transformants were significantly smaller and required approximately 72 h growth to reach similar colony size. This phenomenon was consistently observed with any incoming plasmid DNA prepared from a DH5a host. Plasmid DNA was isolated from both colony types and subjected to restriction enzyme digestion. The plasmid DNA gave identical profiles to pEP2, suggesting that no significant alteration in the DNA had occurred. When small and large colonies were restreaked onto selective media, they grew identically and at normal rates, indicating that the observed phenotype was only transiently expressed. One possible explanation may be that E. coli host DNA modification of the incoming DNA was responsible for the observed differences in growth of the transformants. DEVELOPMENT OF OPTIMAL ELECTROPORATION CONDITIONS

As pEP2 was able to replicate in A. pyogenes BBR1, this plasmid was used in electroporation optimization experiments. However, in order to reduce any potential effects of E. coli host DNA modification, the pEP2 plasmid

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TABLE 2 EFFECT OF PULSE LENGTH ON ELECTROPORATION EFFICIENCY OF A. pyogenes BBR1 WITH pEP2a

Resistance (V)

Time constant (ms)

186 246 360 480 720

8.08 10.62 15.30 20.15 29.50

Average electroporation efficiency (CFU/mg) 1.11 3.11 2.92 1.19 2.67

1 1 1 1 1

105 105 104 104 102

a A. pyogenes BBR1 was transformed with 30 ng pEP2. Field strength and capacitance were held constant at 18 kV/cm and 50 mF, respectively. Each experiment was performed at least twice.

DNA used in optimization experiments was prepared from A. pyogenes BBR1. As the electroporation process is highly dependent on the field strength and duration of pulse or time constant (which is a function of resistance), these two parameters were varied systematically in order to achieve an optimal transformation efficiency. Electroporation efficiency was initially determined using a constant field strength of 18 kV/cm while the time constant was varied (Table 2). A time constant of approximately 10 ms, corresponding to a resistance of 246 V, gave the greatest number of total transformants (Table 2). Electroporation efficiency was then determined as the resistance was held constant at 246 V and the field strength was varied (Table 3). The greatest number of total transformants obtained was 3.7 1 105 CFU/mg of plasmid DNA at 15 kV/ cm and 246 V (Table 3), although there was little difference in electroporation efficiency at field strengths between 15 and 22.5 kV/cm. As in the previous experiment, no transformants were obtained in the absence of plasmid DNA. It is interesting to note that all the transformants obtained in these experiments grew normally and colonies were visible within 48 h. In order to determine the optimal electroporation efficiency, the number of transformants was measured as a function of cell viability following electroporation (Table 4).

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EFFECT OF FIELD STRENGTH ON ELECTROPORATION EFFICIENCY OF A. pyogenes BBR1 WITH pEP2a

Field strength (kV/cm)

Time constant (ms)

15 18 20 22.5 25

10.64 10.53 10.59 10.65 10.80

Average electroporation efficiency (CFU/mg) 3.70 1.13 1.58 1.80 1.34

1 1 1 1 1

The electroporation efficiency was also tested in relation to DNA concentration. The relationship observed between total number of transformants and amount of DNA used was approximately linear within the range tested (3–300 ng), although the electroporation efficiency was slightly decreased at the highest DNA concentration (data not shown).

105 105 105 105 104

HOST DNA RESTRICTION/ MODIFICATION

a

A. pyogenes BBR1 was transformed with 30 ng pEP2. Resistance and capacitance were held constant at 246 V and 50 mF, respectively. Each experiment was performed at least twice.

Although there was a slightly higher number of transformants per viable cell at 18 kV/cm (2.89 1 1003), this was similar to the ratio observed at 15 kV/cm (2.28 1 1003). However, as there was less cell death when 15 kV/ cm was used, there were more total transformants obtained with this field strength (Table 4). In addition, use of the lower field strength may be more practical as it can potentially result in less arcing due to salt or other impurities in the plasmid DNA. Hence, electroporation conditions of 15 kV/cm and 246 V, resulting in a time constant of approximately 10 ms, were considered optimal.

There was less than a threefold decrease in electroporation efficiency if plasmid DNA replicated in DH5a was used (data not shown), suggesting that there is no significant host restriction of incoming DNA. However, the observation of transformants with differential growth rates suggested that E. coli-specified DNA modification may have an effect on transformation. One possible explanation is that some component of the A. pyogenes DNA replication process cannot efficiently replicate incoming DNA modified by methylases encoded by DH5a. Therefore, expression of Km resistance may be reduced due to gene copy number effects and hence the transformants grow more slowly. In addition, DNA modification can affect host transcription (van der Woude and Low, 1994), also resulting in reduced expression of Km resistance or plasmid encoded replication proteins. The smaller

TABLE 4 EFFECT

Field strength (kV/cm) 15 18 25 No pulse

OF

FIELD STRENGTH

ON

Viable count (CFU/ml) 1.62 3.90 2.25 2.12

1 1 1 1

108 107 107 108

NUMBER

OF

TRANSFORMANTS/VIABLE CELLa

% Cell deathb

Average electroporation efficiency (CFU/mg)

Average number of transformants/ viable cell

23.6 81.6 89.4 0

3.70 1 105 1.13 1 105 1.34 1 104 0

2.28 1 1003 2.89 1 1003 5.96 1 1004 0

a

A. pyogenes BBR1 was transformed with 30 ng pEP2. Resistance and capacitance were held constant at 246 V and 50 mF, respectively. Following the pulse, the cells were recovered in 2 ml BHI with 0.3% sucrose and 5% FBS and incubated for 2 h at 377C, and dilutions were plated in order to determine number of transformants and viable counts. Each experiment was performed at least twice. b Cell death was measured as the number of cells surviving the electroporation pulse compared with nonpulsed cells.

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number of transformants with normal growth rates may represent a proportion of the bacterial population which have mutations in genes involved in DNA replication or transcription. Plasmid replication in such mutants may not be affected by the E. coli-specified DNA modification. It appeared that this finding may be strain specific as strain OX8 did not exhibit differential growth of colonies when transformed with E. coli-replicated DNA. OTHER PLASMIDS WHICH REPLICATE IN A. pyogenes

In addition to pEP2, the ability of other plasmids to replicate in A. pyogenes was tested. pJRD215 was able to replicate in A. pyogenes BBR1 and OX8, and both the Km and the Sm resistance genes were expressed. pMMB19, a temperature-sensitive derivative of RSF1010, was also able to replicate in A. pyogenes BBR1 and plasmid replication occurred in a temperature-dependent manner (data not shown). In addition, pNG2, the parental plasmid of pEP2, replicated in A. pyogenes BBR1 and OX8, with expression of the Em resistance gene. However, following electroporation, a 2-h incubation was insufficient for expression of Em resistance and an incubation time of 6–20 h was required for expression of this resistance. The catP gene from C. perfringens was cloned into pEP2 and the recombinant plasmid, pJGS76, was used to transform A. pyogenes BBR1. Chloramphenicol resistant transformants were obtained, indicating that the catP gene was expressed in A. pyogenes. In all cases, plasmids were isolated from the transformants and their identity was confirmed by restriction digestion. Under optimal electroporation conditions, transformants were not obtained with pBBR1MCS, pLTV1, pMAK705, pUC4-K, or pWSK129. In these experiments, 500 ng – 1 mg of plasmid DNA was used to transform A. pyogenes BBR1 competent cells and each experiment was performed at least twice. This paper describes genetic transformation of the veterinary pathogen A. pyogenes. In summary, transformation of A. pyogenes by electroporation with plasmid DNA was

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achieved at a frequency of 3.7 1 105 CFU/ mg. Although this frequency may not allow for direct cloning in A. pyogenes, this is not a significant obstacle as both the shuttle vectors pEP2 and pJRD215 contain polylinkers which allow easy cloning of fragments in E. coli. A number of E. coli promoters are expressed in A. pyogenes, such as the Tn5 Km resistance gene promoter and the pBR322 tetracycline resistance gene promoter, found upstream of the Km and Sm resistance genes of pJRD215, respectively (Davison et al., 1987), and the Tn903 Km resistance gene promoter found in pEP2 (Radford and Hodgson, 1991). To our knowledge, this is the first report of genetic manipulation of A. pyogenes and certainly the first report of transformation in this species. The results of this study provide basic genetic tools with which to begin development of more advanced recombinant DNA methodologies, such as allelic exchange and transposon mutagenesis. These techniques will allow investigation of the virulence factors important in the pathogenesis of A. pyogenes. ACKNOWLEDGMENTS We are grateful to M. M. Bagdasarian, Michigan State University, M. E. Kovach, Louisiana State University, S. R. Kushner, University of Georgia, D. A. Portnoy, University of Pennsylvania, J. I. Rood, Monash University, and M. K. Yeung, The University of Texas Health Science Center, for providing plasmids.

REFERENCES Allen, S. P., and Blaschek, H. P. (1988). Electroporationinduced transformation of intact cells of Clostridium perfringens. Appl. Environ. Microbiol. 54, 2322–2324. Argnani, A., Leer, R. J., van Luijk, N., and Pouwles, P. H. (1996). A convenient and reproducible method to genetically transform bacteria of the genus Bifidobacterium. Microbiology 142, 109–114. Camilli, A., Portnoy, D. A., and Youngman, P. (1990). Insertional mutagenesis of Listeria monocytogenes with a novel Tn917 derivative that allows direct cloning of DNA flanking transposon insertions. J. Bacteriol. 172, 3738–3744. Chassy, B. M., and Flickinger, J. L. (1987). Transformation of Lactobacillus casei by electroporation. FEMS Microbiol. Lett. 44, 17–177. Davison, J., Heusterspreute, M., Chevalier, N., Vinh,

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H.-T., and Brunel, F. (1987). Vectors with restriction site banks. V. pJRD215, a wide-host-range cosmid vector with multiple cloning sites. Gene 51, 275–280. Dunny, G. M., Lee, L. N., and LeBlanc, D. J. (1991). Improved electroporation and cloning vector system for Gram-positive bacteria. Appl. Environ. Microbiol. 57, 1194–1201. Funk, P. G., Staats, J. J., Howe, M., Nagaraja, T. G., and Chengappa, M. M. (1996). Identification and partial characterization of an Actinomyces pyogenes hemolysin. Vet. Microbiol. 50, 129–142. Hamilton, C. M., Aldea, M., Washburn, B. K., Babitzke, B. K., and Kushner, S. R. (1989). New method for generating deletions and gene replacements in Escherichia coli. J. Bacteriol. 171, 4617–4622. Haynes, J. A., and Britz, M. L. (1990). The effect of growth conditions of Corynebacterium glutamicum on the transformation frequency obtained by electroporation. J. Gen. Microbiol. 136, 255–263. Hermans, J., Martin, C., Huijberts, G. N. M., Goosen, T., and de Bont, J. A. M. (1991). Transformation of Mycobacterium aurum and Mycobacterium smegmatis with the broad host-range Gram-negative cosmid vector pJRD215. Mol. Microbiol. 5, 1561–1566. Kovach, M. E., Phillips, R. W., Elzer, P. H., Roop, R. M., II, and Peterson, K. M. (1994). pBBR1MCS: A broadhost-range cloning vector. BioTechniques 16, 800–802. La¨mmler, C. (1990). Untersuchungen zu mo¨glichen ¨ berPathogenita¨tsfaktoren von Actinomyces pyogenes U sichtsreferat. Berl. Mu¨nch. Tiera¨rztl. Wschr. 103, 121– 125. Lean, I. J., Edmondson, A. J., Smith, G., and Villanueva, M. (1987). Corynebacterium pyogenes mastitis outbreak in inbred heifers in a California dairy. Cornell Vet. 77, 367–373. Lechtenberg, K. F., Nagaraja, T. G., Leipold, H. W., and Chengappa, M. M. (1988). Bacteriologic and histologic studies of hepatic abscesses in cattle. Am. J. Vet. Res. 49, 58–62. Liebl, W., Bayerl, A., Schein, B., Stillner, U., and Schleifer, K. H. (1989). High efficiency electroporation of intact Corynebacterium glutamicum cells. FEMS Microbiol. Lett. 65, 299–304. Luchansky, J. B., Muriana, P. M., and Klaenhammer, T. R. (1988). Application of electroporation for transfer of plasmid DNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Bacillus, Staphylococcus, Enterococcus and Propionibacterium. Molec. Microbiol. 2, 637–646. Metzler, M. C., Zhang, Y.-P., and Chen, T.-A. (1992). Transformation of the Gram-positive bacterium Clavibacter xyli subsp. cynodontis by electroporation with plasmids from the IncP incompatibility group. J. Bacteriol. 174, 4500–4503.

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Morelle, G. (1989). A plasmid extraction procedure on a miniprep scale. Focus 11, 7–8. Radford, A. J., and Hodgson, A. L. (1991). Construction and characterization of a Mycobacterium-Escherichia coli shuttle vector. Plasmid 25, 149–153. Ramos, C. P., Foster, G., and Collins, M. D. (1997). Phylogenetic analysis of the genus Actinomyces based on 16S rRNA gene sequences: description of Arcanobacterium phocae sp. nov., Arcanobacterium bernardiae comb. nov., and Arcanobacterium pyogenes comb. nov. Int. J. Syst. Bacteriol. 47, 46–53. Ricci, M. L., Manganelli, R., Berneri, C., Orefici, G., and Pozzi, G. (1994). Electrotransformation of Streptococcus agalactiae with plasmid DNA. FEMS Microbiol. Lett. 119, 47–52. Schaufuss, P., and La¨mmler, C. (1989). Characterization of the extracellular neuraminidase produced by Actinomyces pyogenes. Zbl. Bakt. 271, 28–35. Schaufuss, P., Sting, R., and La¨mmler, C. (1989). Isolation and characterization of an extracellular protease of Actinomyces pyogenes. Zbl. Bakt. 271, 452–459. Scholz, P., Haring, V., Scherzinger, E., Lurz, R., Bagdasarian, M. M., Schuster, H., and Bagdasarian, M. (1985). Replication determinants of the broad host range plasmid RSF1010. Basic Life Sci. 30, 243–259. Serwold-Davis, T. M., and Groman, N. B. (1986). Mapping and cloning of Corynebacterium diphtheriae plasmid pNG2 and characterization of its relatedness to plasmids from skin coryneforms. Antimicrob. Agents Chemother. 30, 69–72. Serwold-Davis, T., Groman, N., and Rabin, M. (1987). Transformation of Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum, and Escherichia coli with the C. diphtheriae plasmid pNG2. Proc. Natl. Acad. Sci. USA 84, 4964–4968. Songer, J. G., Hilwig, R. W., Leeming, M. N., Iandolo, J. J., and Libby, S. J. (1991). Transformation of Corynebacterium pseudotuberculosis by electroporation. Am. J. Vet. Res. 52, 1258–1261. Takeuchi, S., Kaidoh, T., and Azuma, R. (1995). Assay of proteases from Actinomyces pyogenes isolated from pigs and cows by zymography. J. Vet. Med. Sci. 57, 977–979. van der Woude, M. W., and Low, D. A. (1994). Leucineresponsive regulatory protein and deoxyadenosine methylase control the phase variation and expression of the sfa and daa pili operons in Escherichia coli. Mol. Microbiol. 11, 605–618. Wang, R. F., and Kushner, S. R. (1991). Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene 100, 195–199. Yeung, M. K., and Kozelsky, C. S. (1994). Transformation of Actinomyces spp. by a Gram-negative broadhost-range plasmid. J. Bacteriol. 176, 4173–4176. Communicated by G. Dunny

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