Chemical and electroporated transformation of Edwardsiella ictaluri using three different plasmids

Authors


  • Editor: Craig Shoemaker

Correspondence: Riccardo Russo, United States Department of Agriculture, Agricultural Research Service, Aquatic Animal Health Research Unit, 990 Wire Road, Auburn, AL 36832, USA. Tel.: +1 334 887 3741; fax: +1 334 887 2983; e-mail: ri.russo@hotmail.com

Abstract

Transfer of DNA by conjugation has been the method generally used for genetic manipulation of Edwardsiella ictaluri because, previously, attempts to transform E. ictaluri by the uptake of naked DNA have apparently failed. We report here the successful transformation of seven strains of E. ictaluri using electroporation and two different chemical procedures [conventional calcium chloride (CaCl2) and ‘one-step’ (polyethylene glycol, dimethyl sulfoxide and MgSO4) protocols]. Seven strains of E. ictaluri were transformed using three different plasmids [pZsGreen, pUC18 and pET-30a(+)]. The highest transformation efficiency was achieved by electroporation (5.5±0.2 × 104 transformants ng−1 plasmid DNA) than with the CaCl2 (8.1±6.1 × 10−1 transformants ng−1 plasmid) and the ‘one-step transformation’ protocol (2.5±2.7 transformants ng−1 plasmid). An efficient transformation by electroporation required only 0.2 ng of plasmid compared with 200 ng required for the CaCl2 and one-step protocols. The plasmids were stably maintained in E. ictaluri grown in the presence of antibiotic for 12 or more passages. The results of this study show that transformation of E. ictaluri by electroporation can be routinely used for the molecular genetic manipulation of this organism, and is a quicker and easier method than transformation performed by conjugation.

Introduction

Edwardsiella ictaluri, the etiologic agent of enteric septicemia in channel catfish, has been recognized as an important bacterial pathogen with a potential to cause appreciable economic loss to the aquaculture industry (Shoemaker et al., 2007; Thune et al., 2007). The bacterium was initially isolated from diseased channel catfish (Hawke, 1979) and characterized by Hawke et al. (1981). Molecular genetic manipulation of E. ictaluri to examine mechanisms of biological and pathogenesis-related phenomena remains an area that is substantially unexplored. Successful transformation of bacteria with foreign genetic information is one such molecular manipulation that is important for the study of host–pathogen interactions (Dhandayuthapani et al., 1995; Mixter et al., 2003; Van der Sar et al., 2003). Transfer of DNA by conjugation mediated by conjugative elements, such as conjugal plasmids or conjugal transposons, requires cell-to-cell contact between donor and recipient cells (Lorenz & Wackernagel, 1994). The latter mode of horizontal DNA transfer between bacteria has been the method generally used for genetic manipulation of E. ictaluri (Lawrence et al., 2001; Maurer et al., 2001), because, previously, attempts to transform E. ictaluri by the uptake of naked DNA have apparently failed (Maurer et al., 2001). In this study, we describe the successful transformation of E. ictaluri by electroporation as well as using calcium chloride (CaCl2) and ‘one-step’ protocols. The results of this study show that transformation of E. ictaluri by electroporation can be routinely used for molecular genetic manipulation of this organism and is a quicker and easier method than transformation performed by conjugation.

Materials and methods

Bacteria and culture conditions

The E. ictaluri strains used in this study and their sources are listed in Table 1. Bacteria were cultured in brain heart infusion (BHI) or Luria–Bertani (LB) (Sambrook & Russell, 2006) broth or agar. Transformed bacteria were plated on synthetic oil-based (SOB) medium (Sambrook & Russell, 2006) containing 25 μg mL−1 kanamycin or 75 μg mL−1 ampicillin and routinely incubated at 26 °C. Bacterial cultures were verified for purity using API 20E test strips (bioMerieux, Hazelwood, MO).

Table 1. Edwardsiella ictaluri isolates used in this study
IsolateSourceOriginNotes
AL-93-58Channel catfishAlabamaAuburn University Fish Diagnostic Laboratory, Auburn University, AL
RE-33Channel catfishAlabamaAvirulent vaccine strain used in commercial vaccine derived from EILO (Klesius & Shoemaker, 1999)
ATCC 33202Channel catfishATCC (type strain)Hawke et al. (1981)
IA-30-NJ#1Tadpole madtom, Noturus gyrinus Klesius et al. (2003)
ALG-03-189Channel catfishAlabamaWilliam Hemstreet, Alabama Fish Farming Center, Greensboro, AL
EILOChannel catfishAlabamaKasornchandra et al. (1987)
S94-1051Channel catfishMississippiAuburn University Fish Diagnostic Laboratory, Auburn University, AL

Plasmids

The plasmids were purchased from commercial sources as follows: (1) pZsGreen (pUC19 derivative, 3330 bp in size, Clontech, Mountain View, CA) was chosen because it encodes a green fluorescent protein derived from the Zoanthus reef coral species and this plasmid has been successfully utilized for in vivo labeling in a variety of studies (Matz et al., 2002; Hutter, 2006; Müller-Taubenberger & Anderson, 2007); (2) pUC18 (2686 bp in size, Fermentas, Glen Burnie, MD), a plasmid M13 derivative with a promoter with several unique restriction enzyme recognition sites and the lacZ gene for β-galactosidase production (Norrander et al., 1983); and (3) plasmid pET-30a(+) (5422 bp in size, Novagen, San Diego, CA), with an inducible (T7 RNA polymerase) expression system placed under the control of a bacteriophage T7 promoter (Studier et al., 1990; Studier, 1991). The pET-30a(+) plasmid has been constructed for efficient protein synthesis in Escherichia coli, utilizing the ColE1 ori sequence derived from pBR322 and the isopropyl-β-d-thiogalactoside-inducible T7/lacO promoter (Studier et al., 1990; Studier, 1991). Plasmids pZsGreen and pUC18 carry an ampicillin resistance marker and are under the control of the lac promoter (Plac), while pET-30a(+) contains a gene specific for kanamycin resistance. These three plasmids were selected for their distinctive characteristics and to assess their potential as efficient vectors for transformation of E. ictaluri.

Preparation of chemically competent E. ictaluri cells using CaCl2

The seven strains of E. ictaluri were cultured in 50 mL of BHI broth at 26 °C until they reached mid log-phase growth (an OD540 nm of 0.8–1.0, corresponding to c. 3 × 109 cells mL−1), typically around 18 h. The cultures were chilled on ice for 10 min and then centrifuged at 3400 g for 15 min at 4 °C. The supernatant was removed and the cells were resuspended in 10 mL of ice-cold 0.1 M CaCl2 and placed on ice for 10 min. The cells were centrifuged again at 3400 g for 15 min at 4 °C. The supernatant was removed and the cells were resuspended in 2 mL of ice-cold 0.1 M CaCl2 and placed on ice. For freezer storage, 70 μL of dimethyl sulfoxide (DMSO) (ATCC, Manassas, VA) was added to the 2 mL of bacterial suspension, and then the vial was mixed and chilled on ice for 15 min. An additional 70 μL of DMSO was added again, and then the cells were aliquoted in 200-μL volumes in cryovials and stored at −80 °C until use.

Transformation of E. ictaluri using CaCl2

A standard CaCl2 transformation protocol (Sambrook & Russell, 2006) was used with essential modifications. Edwardsiella ictaluri from the above frozen stocks (200 μL) (c. 1.4 × 109 cells) were mixed with 200 ng (5 μL) of plasmid DNA. The tubes were flicked to gently suspend the cells and then placed on ice for 10 min followed by exposure to heat shock at 35 °C for 1 min in a water bath. Bacteria were suspended in 1 mL SOB medium, and then the cells were incubated at 26 °C with gentle shaking (100 r.p.m.) for 1 h. Cells were centrifuged at 4000 g for 2 min, the supernatant was discarded, and then the cells were resuspended in 1 mL of phosphate-buffered saline (PBS), pH 7.2. Serial 10-fold dilutions of the bacteria were prepared in PBS and plated in triplicate on SOB agar containing 25 μg mL−1 kanamycin or 75 μg mL−1 ampicillin. The plates were incubated at 26 °C for c. 18 h and the resulting colonies were counted. Controls included nontransformed E. ictaluri and competent E. coli strain DH5α transformed with each of the three plasmids described above using the same protocol.

Preparation of electrocompetent E. ictaluri cells

Each E. ictaluri strain was grown at 26 °C in 50 mL of BHI broth until the culture reached mid-log-phase growth as above. Cultures were chilled on ice for 10 min and centrifuged at 3400 g for 15 min at 4 °C. The supernatant was removed and the cells were resuspended in 50 mL of ice-cold distilled water. The bacterial suspensions were centrifuged again at 3400 g for 15 min at 4 °C, the supernatant was discarded and the cells were resuspended in 50 mL of ice-cold 10% glycerol (Sigma-Aldrich, St. Louis, MO). The centrifugation step was repeated once again and the cells were resuspended in 2 mL of ice-cold 10% glycerol. Cells were aliquoted in microcentrifuge tubes (50 μL per tube) and stored frozen at −80 °C until use.

Transformation by electroporation

A standard electroporation protocol (Sambrook & Russell, 2006) was followed with minimal modifications. At the beginning of the study, several plasmid concentrations [pZsGreen and pET-30a(+)] and field strengths were tested in one strain of E. ictaluri (AL-93-58 strain) to optimize the electroporation conditions. To determine the minimal amount of plasmid required for the transformation, plasmid DNA from 0.2 to 200 ng in 5 μL was added to electrocompetent E. ictaluri (c. 2.5 × 109 cells), the tube was gently mixed by flicking and placed on ice. After 1 min, the DNA and cell mixture were transferred to a previously chilled electroporation cuvette (BioRad Laboratories, Hercules, CA) with a 0.1 or 0.2 cm gap. The cuvette was placed in the pulse chamber and electroporation was carried out with a Gene Pulser (BioRad Laboratories) electroporator. The electroporation conditions were 200 Ω, capacitance 25 μF and field strength 12.5 kV cm−1 for 4 ms. After electroporation, 1 mL of SOB medium was immediately added to the bacterial suspension, the content of the cuvette was transferred to a sterile tube and incubated at 26 °C with gentle shaking (100 r.p.m.) for 1 h. The bacterial suspension was centrifuged at 4000 g for 2 min, the supernatant was discarded and the cells were resuspended in 1 mL of sterile PBS, pH 7.2. Serial 10-fold dilutions of the bacteria were prepared in sterile PBS and transformed bacterial counts were carried out in triplicate as described above. Negative controls (E. ictaluri not transformed with plasmid) and positive controls (E. coli DH5α transformed with each plasmid) were plated on the SOB agar without antibiotic (E. ictaluri not transformed with plasmid) or on SOB agar with ampicillin or kanamycin (E. coli DH5α transformed with each plasmid).

After the determination of the minimal amount of plasmid DNA required for the transformation (0.2 ng), E. ictaluri AL-93-58 was exposed to electric fields of 0, 2, 2.5, 5, 7.5, 10, 12.5, 15, 20 and 25 kV cm−1, maintaining a constant pulse time (4 ms), resistance (200 Ω) and capacitance (25 μF). Transformation of the other six strains of E. ictaluri was performed using the minimal amount of plasmid DNA needed and under the optimal electroporation conditions (field strength).

‘One-step’ transformation

A ‘one-step’ transformation protocol described by Chung et al. (1989) was tested, with the single modification of culturing E. ictaluri at 26 °C instead of 37 °C. Competent cells were generated by growing each E. ictaluri strain in 20 mL of BHI broth up to the mid-log phase as described above and chilled on ice for 15 min. Bacteria were centrifuged at 3400 g for 15 min at 4 °C and resuspended in one-tenth of the original volume of ice-cold transformation and storage solution constituted of LB broth with 10% w/v polyethylene glycol, 5% v/v DMSO and 30 mM MgSO4, with a final pH of 6.5. Bacterial suspensions were aliquoted in 100-μL volumes in cryovials and stored frozen at −80 °C until use.

For transformation, the bacteria were thawed and 100 μL (c. 2.5 × 109 cells) of E. ictaluri was mixed with plasmid DNA (200 ng in 5 μL) and held at room temperature for 10 min. The bacterial suspension was added to 1 mL of SOB medium and incubated at 26 °C with gentle shaking (100 r.p.m.) for 1 h. The bacteria were centrifuged at 4000 g for 2 min, the supernatant was discarded and cells were resuspended in 1 mL of sterile PBS, pH 7.2. Serial 10-fold dilutions of the bacteria were prepared and transformed cell numbers were assessed as described above. Negative controls were nontransformed E. ictaluri and positive controls were E. coli strain DH5α transformed with each of the plasmids. These bacteria were plated on SOB selective media containing the appropriate antibiotic (negative controls were grown in media without antibiotic) and incubated at 26 °C for c. 18 h before counting the colonies.

Transformation efficiency

The transformation efficiency of all three procedures was calculated as the number of transformed bacterial colonies per nanogram of plasmid DNA (Chung et al., 1989; Fengqing & Song, 2005).

Plasmid isolation and characterization

Isolation of pZsGreen, pUC18 and pET-30a(+) plasmids from transformed E. ictaluri strain AL-93-58 (bacteria grown for five and 10 passages) and E. coli DH5α was performed using the PureLinkt HiPure Plasmid DNA Purification Kit for Miniprep (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Native plasmids of E. ictaluri, pEI1 (4807 bp) and pEI2 (5643 bp) (Fernandez et al., 2001) were isolated from nontransformed bacteria and used for comparison. The native plasmids of E. ictaluri and the plasmids pZsGreen, pUC18 and pET-30a(+) used for transformation were isolated from each respective bacterium (E. ictaluri AL-93-58 and/or E. coli DH5α) and were digested with the restriction enzyme EcoRI (New England Biolabs, Ipswitch, MA) or with a combination of the restriction enzymes, SalI and SmaI, and the digested products were run on a 1% agarose gel to verify that the bacteria were transformed with the appropriate plasmid.

Plasmid stability

The E. ictaluri AL-93-58 and RE-33 transformed with any of the three plasmids were grown under selection in the presence of ampicillin or kanamycin in BHI agar for 12 or more successive passages (E. ictaluri transformed with pZsGreen were grown for >40 passages) to determine the plasmid stability. The E. ictaluri AL-93-58 and RE-33 transformed with pZsGreen were also grown in BHI agar without addition of ampicillin to determine the plasmid stability under nonselective conditions.

Results and discussion

Transformation of cells has become an important molecular tool to genetically manipulate bacteria to study gene function and complex cellular processes. The two most widely used techniques for introduction of foreign DNA into bacteria are treatment of cells with chemicals such as CaCl2 or chlorides of other elements, or by exposure of cells to an electric field by a process known as electroporation (Lorenz & Wackernagel, 1994). In this study, we used both chemicals and electroporation to transform six strains of E. ictaluri isolated from channel catfish and a strain isolated from a Tadpole madtom (Noturus gyrinus) (Table 1). Our present study showed that E. ictaluri could be successfully transformed by electroporation. The transformation efficiency was higher with the electroporation procedure (5.7±1.3 × 104, 5.6±1.7 × 104 and 5.3±0.5 × 104 transformants ng−1 of pZsGreen, pUC18 and pET-30a(+), respectively) than with the CaCl2 (4.7±0.7 × 10−1, 4.6±2.1 × 10−1 and 1.5±0.3 transformants ng−1 of pZsGreen, pUC18 and pET-30a(+), respectively) and the ‘one-step transformation’ protocols (1.5±0.3, 4.8±2.0 × 10−1 and 5.6±2.3 transformants ng−1 of pZsGreen, pUC18 and pET-30a(+), respectively) (Table 2). An efficient transformation by electroporation required only 0.2 ng of plasmid DNA compared with 200 ng required for the CaCl2 and one-step protocols. The efficiency of transformation using the electroporation protocol was a linear function of the DNA concentration (Table 3), as yet observed in other studies (Dower et al., 1988). The optimal conditions for electroporation were a field strength from 15 to 20 kV cm−1 (obtaining, respectively, 6.4±0.4 × 105 and 7.3±1.0 × 105 transformants) for 4 ms, 200 Ω and a capacitance of 25 μF using a 0.1-cm (gap) electroporation cuvette (Fig. 1). Transformants obtained by the three methods contained intact plasmid molecules as revealed by digestion with restriction enzymes of isolated plasmids.

Table 2.   Efficiency of transformation (number of transformed colonies ng−1 plasmid DNA) of Edwardsiella ictaluri
Transformation methodPlasmidStrain of Edwardsiella ictaluri
AL-93-58RE-33ATCC 33202NJ #1ALG-03-158Average ± SD
  1. For each transformation method, nanograms of plasmid used is reported within parentheses. Only five strains of Edwardsiella ictaluri were used to determine the efficiency of transformation, but all seven bacterial strains reported in Table 1 were successfully transformed with the three plasmids.

Electroporation (0.2 ng plasmid) (c. 2.5 × 109 cells)pZsGreen5.3 ± 0.4 × 1043.6 ± 0.4 × 1046.1 ± 0.4 × 1046.3 ± 0.5 × 1047.0 ± 0.5 × 1045.7 ± 1.3 × 104
pUC187.0 ± 0.4 × 1043.4 ± 0.3 × 1046.7 ± 0.4 × 1044.1 ± 0.5 × 1046.8 ± 0.6 × 1045.6 ± 1.7 × 104
pET-30a(+)5.5 ± 0.4 × 1044.7 ± 0.3 × 1045.5 ± 0.4 × 1046.0 ± 0.4 × 1044.9 ± 0.5 × 1045.3 ± 0.5 × 104
CaCl2 (200 ng plasmid) (c. 1.4 × 109 cells)pZsGreen3.5 ± 1.3 × 10−14.5 ± 1.3 × 10−15.0 ± 1.5 × 10−15.4 ± 0.8 × 10−14.9 ± 1.0 × 10−14.7 ± 0.7 × 10−1
pUC187.8 ± 1.8 × 10−13.0 ± 1.0 × 10−14.3 ± 1.5 × 10−12.4 ± 1.0 × 10−15.2 ± 1.3 × 10−14.6 ± 2.1 × 10−1
pET-30a(+)2.0 ± 0.21.4 ± 0.31.3 ± 0.21.4 ± 0.31.5 ± 0.21.5 ± 0.3
One-step protocol (200 ng plasmid) (c. 2.5 × 109 cells)pZsGreen1.5 ± 0.21.8 ± 0.11.0 ± 0.11.9 ± 0.51.4 ± 0.31.5 ± 0.3
pUC187.0 ± 0.5 × 10−13.8 ± 0.4 × 10−15.0 ± 0.6 × 10−12.8 ± 0.7 × 10−17.5 ± 0.7 × 10−14.8 ± 2.0 × 10−1
pET-30a(+)9.0 ± 0.33.5 ± 0.24.2 ± 0.16.9 ± 0.24.4 ± 0.35.6 ± 2.3
Table 3.   Efficiency of transformation (number of transformed colonies ng−1 plasmid DNA) by electroporation of Edwardsiella ictaluri strain AL-93-58 using different amounts of pZsGreen and pET-30a(+) plasmids
PlasmidAmount of plasmid used for the transformation by electroporation (ng)
0.2220200
pZsGreen6.1 ± 0.8 × 1043.8 ± 0.4 × 1047.2 ± 1.1 × 1045.8 ± 1.2 × 104
pET-30a(+)5.2 ± 0.6 × 1047.0 ± 0.7 × 1044.3 ± 0.7 × 1046.3 ± 1.5 × 104
Figure 1.

 Effect of the electric field strengths on the survival rate of Edwardsiella ictaluri strain AL-93-58 not transformed with foreign DNA plasmid (dotted line, bacteria grown on solid agar without antibiotic) and on the transformation efficiency using 0.2 ng of the pZsGreen plasmid (solid line, bacteria grown on solid agar with the addition of ampicillin). Similar results were observed for the transformation efficiency using the pET-30a(+) plasmid (data not shown). Survival data are expressed as the percent of counted CFU for each electric field strength compared with the number of CFU of the sample not exposed to an electric field (0 kV cm−1). Efficiency data are expressed as the percent of counted transformed CFU for each electric field strength compared with the highest number of transformed CFU observed. The electroporations were effectuated, maintaining a constant pulse time (4 ms), resistance (200 Ω) and capacitance (25 μF).

Previous attempts to transform E. ictaluri have met with little or no success, and endogenous host restriction endonucleases have been postulated as a likely obstacle to transformation (Maurer et al., 2001; Thune et al., 2007). For this reason, Maurer et al. (2001) optimized a conjugation procedure for transfer of foreign DNA into E. ictaluri using a kanamycin-resistant plasmid (pLOF-Km) construct with a mobilizable transposon (Tn10) as a suicide vector. Lawrence et al. (2001) successfully used this conjugation procedure to transform wild-type E. ictaluri strain 93–146 by mixing donor E. coli SM10 λpir pLOFKm with the recipient in the presence of 10 mM MgSO4 to construct a lipopolysaccharide O side-chain (O-antigen) mutant strain of E. ictaluri. The conjugation procedure has also been used for transformation of E. ictaluri strain 93–146 with the pAKlux plasmid containing a bioluminescent reporter (from Photorhabdus luminescence) for noninvasive monitoring of disease progression in channel catfish (Karsi et al., 2006).

The transformation efficiency of E. ictaluri observed in our study was lower than that reported in literature for E. coli (Dower et al., 1988; Inoue et al., 1990; Sambrook & Russell, 2006), but these strains of E. coli have been selected for providing a high transformation efficiency. In a study with Aeromonas hydrophila, Fengqing & Song (2005) reported that the transformation efficiency obtained by electroporation was similar to our study (4 × 102 CFU μg−1 DNA). The transformed E. ictaluri strains, grown under selection in the presence of ampicillin or kanamycin, stably maintained the plasmid DNA over several generations (12 or more successive passages; data not shown). The E. ictaluri AL-93-58 and RE-33 transformed with pZsGreen maintain the plasmid even when grown in BHI agar without ampicillin. We have also transformed E. ictaluri strain AL-93-58 with the pDsRed vector (Clontech) carrying the DsRed2 (Discosoma sp. coral fluorescent protein) reporter gene to monitor the interactions of channel catfish macrophages with E. ictaluri in in vitro and in vivo studies following vaccination (Russo et al., 2009). This and other studies conducted in our laboratory with E. ictaluri transformed with either the pZsGreen or the pDsRed2 plasmid and injected in channel catfish demonstrated that E. ictaluri is able to maintain the pZsGreen or the pDsRed2 plasmids even when not grown under selective pressure. In these studies, we detected fluorescent bacteria inside the fish for a few weeks, and the fluorescent signal increased with an increase in the concentration of the bacterial count in the body of the fish. Our studies clearly show that E. ictaluri was successfully transformed and that electroporation is an efficient procedure for transformation of this species. Furthermore, transformation of E. ictaluri by electroporation is a quicker and easier method than transformation performed by conjugation (Chen & Dubnau, 2004; Chen et al., 2005). Thus, E. ictaluri transformed with foreign DNA, including specific mutants generated by transformation, could be employed as useful tools to study the virulence potential of this bacterium, its invasive pathways and other important host–pathogen interactions that remain to be elucidated.

Acknowledgements

This work was supported by the US Department of Agriculture, Agricultural Research Service, Current Research Information Systems project no. 6420-32000-020-00D. The authors gratefully acknowledge the technical assistance of Mrs Elizabeth Peterman. This research was conducted in compliance with all relevant federal guidelines and institutional policies.

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