- Email: [email protected]
Electroporation-mediated transformation of Aeromonas hydrophila
Plasmid 54 (2005) 283–287 www.elsevier.com/locate/yplas
Short communication
Electroporation-mediated transformation of Aeromonas hydrophila Hu Fengqing a,b,¤, You Song b a
b
Department of Life Science, Liaoning University, Shenyang 110036, China College of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China Received 27 March 2005, revised 21 April 2005 Available online 20 June 2005 Communicated by Grzegorz Wegrzyn
Abstract A strain of Aeromonas hydrophila producing copolyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate, abbreviated as PHBHHx, was successfully transformed by electroporation. The plasmid used was a broad host range plasmid pBBR1MCS. Electroporation conditions were varied systemically to develop an electroporation protocol. The optimal yield of transformant was approximately 4 £ 102 CFU/g DNA at 12.5 kV/cm and 1000 , resulting in a time constant of approximately 5 ms. The A. hydrophila transformants expressed plasmid-encoded resistance to chloromphenicol. Plasmid DNA in the A. hydrophila transformant was stably maintained. This is the Wrst report of transformation of bacteria A. hydrophila. 2005 Elsevier Inc. All rights reserved. Keywords: Aeromonas hydrophila; Electroporation
1. Introduction The genera Aeromonas is Gram-negative bacteria belonging to Vibrionaceae. Mesophilic, motile Aeromonas hydrophila is ubiguitous and autochthonous aquatic microorganisms occurring in freshwater, sewage, and brachish water (Schubert, 1991), and in chlorinated and unchlorinated drink*
Corresponding author. Fax: +86 24 62202232. E-mail address: [email protected] (H. Fengqing).
0147-619X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2005.04.004
ing water (Havellaar et al., 1992). They also are common contaminants of fresh foods, including Wsh, and other scafoods (Abeyta and Wekell, 1988; Kirov, 1993; Krovacck et al., 1992; Nishikawa and Kishi, 1988). SpeciWes of these genera are known as pathogens for Wsh (Cahill, 1990; Joseph and Carnahan, 1994) and human associated mainly with diarrheal symptoms (Janda, 1991). However, the development of molecular tools for these organisms is far behind the technologies employed for Escherichia coli and similar bacteria. For
284
Short communication / Plasmid 54 (2005) 283–287
instance, transformation of A. hydrophila by electroporation has not been reported up to the present, thus limiting genetic studies of this species. Aeromonas hydrophila WQ was reported to produce copolyesters of 3-hydroxybutyrate (3HB) 3-hydroxyhexanoate (3HHx), abbreviated as PHBHHx, from lauric acid as sole carbon source (Lu et al., 2004). The water insoluble PHBHHx exhibits high molecular weight, thermoplastic and/or elastomeric features as well as other interesting physical and material properties (Deng et al., 2003; Ju et al., 2004). Therefore, they have attracted increasing attention from scientiWc and industrial communities due to their interesting properties including biodegradability, biocompatibility, and piezoelectricity (Deng et al., 2003; Wang et al., 2004; Wu et al., 2000). Polyhydroxyalkanoate (PHA) synthase are the key enzymes of PHA biosynthesis (Rehm and Steinbüchel, 1999). To elucidate the molecular mechanism of PHBHHx accumulation, it is necessary to inactivate PHA synthase gene in A. hydrophila. Considering low eYciency of conjugation, in this study, for the Wrst time, we developed an electroporation protocol for introduction of plasmid DNA into this organism.
2. Materials and methods Aeromonas hydrophila WQ was used as a bacterial host strain and broad host range plasmid pBBR1MCS (Michael et al., 1995) in E. coli JM109 was isolated and used as a plasmid DNA. A. hydrophila WQ was cultivated at 30 °C on Luria–Bertani (LB) medium, while E. coli was grown at 37 °C on LB medium. Chloramphenicol (170 g ml¡1) was added to the medium when needed. In all cases, the cultures were incubated in conical Xask at 200 rpm (NBS, Series 25D, New Brunswick, USA). Extraction of isolation of plasmid, digestion of restriction endonucleases, and agar gel electrophoresis were performed by standard procedures (Sambrook and Russell, 2001) or as recommended by the manufacturers. To obtain optimal electrocompetent cells, A. hydrophila WQ cells were grown on LB medium and successively harvested at the middle of exponential
phase, the end of exponential phase, and the early stationary phase. The cells were quickly placed at 0 °C about 30 min to stop the growth, centrifuged at 2500g and 0 °C for 15 min, and washed twice in equal volume of sterile pyrolized water chilled to 0 °C and twice with 100 ml of sterile 10% glycerol (w/ w) chilled to 0 °C. To ensure the same density of cells harvested in diVerent growth phases, the cell pellet was resuspended in 200–400 l of GYT medium (Sambrook and Russell, 2001) chilled to 0 °C. The cells were dispensed into 40 l aliquots and preserved in refrigerator (¡80 °C).
3. Results and discussion Aliquots of cells were thrawed on ice and 3 l plasmid pBBR1MCS (4.7 kb) DNA (approximately 0.1–1 g) was added just prior to electroporation. Electroporation was performed in chilled 0.1 cm cuvettes using a Model JY2000-1 electroporator (SCIENTZ, Ningbo, China). After the electric shock, the bacterial suspension was immediately transferred to an eppendorf tube containing 1000 l of LB medium. Cells of A. hydrophila WQ were incubated about 2–3 h at 30 °C with shaking. After incubation, estimates of survival rate were obtained by spreading aliquots onto nonselective or selective LB agar plates (antibiotics are added when need), respectively. Transformants were presented after 24–48 h. No transformants were obtained in the absence of plasmid DNA. The number of transformants obtained was evaluated. Table 1 clearly shows that optimal electroporation results were obtained with competent cells harvested at early stationary phase. Approximately 2.5 £ 102 transformants were obtained as the maximum yield of transformation when 40 l cell suspension containing 1 £ 108 competent cells from early stationary phase was pulsed at 10 kV/cm with 1 g plasmid DNA. To optimize electroporation conditions, Wrst by studying the eVect of electric Weld strengths for a given pulse time (5 ms). Competent cell of A. hydrophila, prepared at early stationary phase, was transformed with varying the strength of electric Weld (from 0 to 20 kV/cm). As shown in Fig. 1, the maximum value of transformation eYciency was achieved
Short communication / Plasmid 54 (2005) 283–287 Table 1 EVect of growth phase on the transformation of A. hydrophila WQ Growth phase
Cells OD600nm
Transformants (£102/g DNA)
Middle exponential phase End exponential phase Early stationary phase
0.4 0.8 1.0
0.11 1.32 2.57
Aeromonas hydrophila WQ from diVerent growth phases were used for transformation. Three microliters plasmid pBBR1MCS DNA and 40 l competent cells with 10 kV/cm electric Weld strength, 25 F, and 1000 were used for transformation. All experiments were carried out in a Model JY2000-1 electroporator (SCIENTZ, Ningbo, China). The OD was measured at 600 nm with a 722S spectrophotometer (Precision and ScientiWc Instrument, Shanghai, China). All results were repeated three times.
Fig. 1. EVect of electric Weld strengths on survival rate and transformation eYciency of A. hydrophila WQ by electroporation. Forty microliters of electrocompetent cells were mixed with 3 l plasmid DNA. The mixtures were submitted to electroporation in 1 mm cuvettes. Transformants were counted on LB medium supplemented with chloramphenicol (170 g ml¡1). The pulse time was maintained at a constant value (R D 1000 , C D 25 F) and the samples were submitted to electric Welds of 0, 2.5, 5, 7.5, 10, 12.5, 15, and 20 kV/cm. All experiments were carried out in a Model JY2000-1 electroporator (SCIENTZ, Ningbo, China). All results were repeated three times.
at 12.5 kV/cm and approximately 4£ 102 transformants/g plasmid DNA was obtained with only 20% of survival rate for competent cells. The transformation eYciency was elevated when increasing the strength of electric Welds, whereas the strength of
285
electric Weld higher than 12.5 kV/cm resulted in the marked decrease of transformation eYciency. Survival rate of competent cells was always declined along with the increase of electric Weld strength. No transformants were obtained at the strength of electric Welds less than or equal to 5 kV/cm. Using the same strain and plasmid, pulse time was varied from 0 to 15 ms for a given electric Weld strength (12.5 kV/cm). Fig. 2 shows the eVect of pulse time on survival rate and transformation eYciency of A. hydrophila WQ for a given electric Weld strengpth (12.5 kV/cm). A narrow optimum was found for a pulse time of 5 ms with approximately 400 transformants/g plasmid DNA. Fewer transformants were obtained at lower or higher values of pulse time. Using optimized conditions, electroporation of suicide plasmid pFH5 (6.5 kb) harboring the disrupted PHBHHx synthase gene (phaC::Cm) transformed A. hydrophila WQ, through an in vivo homologous recombination process, PHBHHx synthase gene (phaC) of A. hydrophila
Fig. 2. EVect of pulse time on survival rate and transformation eYciency of A. hydrophila WQ by electroporation. Forty microliters of electrocompetent cells were mixed with 3 l plasmid DNA. The mixtures were submitted to electroporation in 1 mm cuvettes. Transformants were counted on LB medium supplemented with chloramphenicol (170 g ml¡1). The strength of electric Weld was maintained at a constant value (12.5 kV/cm) and the samples were submitted to pulse time of 0, 2.5, 5, 10, and 15 ms. All experiments were carried out in a Model JY2000-1 electroporator (SCIENTZ, Ningbo, China). All results were repeated three times.
286
Short communication / Plasmid 54 (2005) 283–287
genome was replaced by the disrupted PHBHHx synthase gene, and chloramphenicol resistance gene was integrated into the genome of A. hydrophila, resulting in PHBHHx synthase negative mutant. The mutant resisting chloramphenicol could be easily screened and obtained from selective LB agar plate (170 g ml¡1 chloramphenicol was added). This result strongly proved that A. hydrophila WQ, via a electroporation procedure, is amenable to genetic manipulation in a way similar to E. coli. Usually, it was diYcult to transfer DNA into A. hydrophila by conjugation especially when the size of plasmid DNA was larger than 6 kb, and the eYciency of conjugation was only 1.0 £ 10¡8. The results presented in this paper are the Wrst presentation that A. hydrophila WQ can be directly transformed by the well-known broad host range plasmid pBBR1MCS. We attempted to transfer this plasmid into A. hydrophila WQ using the conventional transformation methods. Although transformation eYciency for A. hydrophila WQ by electroporation is not as eYcient as that of E. coli, electroporation of A. hydrophila WQ is easier and more eYcient than triparental conjugal transfer method. The plasmid pBBR1MCS in the transformant of A. hydrophila WQ stably maintained after 100 generations of cultivation. In addition, all the transformants were determined to maintain the plasmid pBBR1MCS as monomeric forms and this structural stability was further conWrmed by the restriction endonuclease cleavage analysis. To our knowledge, this is the Wrst report of genetic manipulation of A. hydrophila and certainly the Wrst 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 and also facilitate the application of recombinantDNA technology to A. hydrophila.
Acknowledgments This research was supported by the National Nature Science Foundation for Distinguished Young Scholars (Grant No. 30225001). We are grateful to Professor Chen G.Q. of Tsinghua Uni-
versity (Beijing, China) for kindly providing us with A. hydrophila strain WQ.
References Abeyta Jr., C., Wekell, M.M., 1988. Potential sources of Aeromonas hydrophila. J. Food Safety 9, 11–22. Cahill, M.M., 1990. Bacterial Xora of Wshes: a reviews. Microb. Ecol. 19, 21–41. Deng, Y., Lin, X.S., Zheng, Z., Deng, J.G., Chen, J.C., Ma, H., Chen, G.Q., 2003. Poly (hydroxybutyrate-co-hydroxyhexanoate) promoted production of extracellular matrix of articular cartilage chondrocytes in vitro. Biomaterials 24, 4273–4281. Havellaar, A.H., Schets, F.M., van Silhout, A., W.H., Wieten, G., and Van den Kooij., 1992. Typing of Aeromonas strains from patients with diarrhea and from drinking water. J. Appl. Bacteriol. 72, 435–444 Janda, J.M., 1991. Recent advances in the study of the taxonomy, pathogenicity, and infectious syndromes associated with the genes Aeromonas. Clin. Microbiol. Rev. 4, 397– 410. Joseph, S.W., Carnahan, A., 1994. The isolation, identiWcation, and systematics of the motilc Aeromonas species. Ann. Rev. Fish Dis. 4, 315–343. Ju, X., Guo, B.H., Zhang, Z.M., Zhou, J.J., Jiang, Y., Yan, S., Li, L., Wu, Q., Chen, G.Q., Schultz, J.M., 2004. Direct AFM observation of crystal twisting and organization in banded spherulites of chiral poly (3-hydroxybutyrate-co-3hydroxyhexanoate). Macromolecules 37, 4118–4123. Kirov, S.M., 1993. The public health signiWcance of Aeromonas spp. in foods. Int. J. Food Microbiol. 20, 179–198. Krovacck, K., Fanis, A., Baloda, S.B., Petenz, M., Lundberg, T., Mansson, I., 1992. Prevalence and characterization of Aeromonas spp. isolated from foods in Uppsala. Sweden. Food Microbiol. 9, 29–36. Lu, X.Y., Wu, Q., Zhang, W.J., Zhang, G., Chen, G.Q., 2004. Molecular cloning of polyhydroxyalkanoate synthesis operon from Aeromonas hydrophila and its expression in Escherichia coli. Biotechnol. Progr. 20, 1332–1336. Michael, E.K., Philip, H.E., Hill, D.S., Gregory, T.R., Michael, A.F., 1995. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying diVerent antibioticsresistance cassettes. Gene 166, 175–176. Nishikawa, Y., Kishi, T., 1988. Isolation and characterization of motilc Aeromonas from human, food and environmental speciWces. Epidemiol. Infect. 101, 213–223. Rehm, B.H.A., Steinbüchel, A., 1999. Biochemical and genetic analysis of PHA synthases and other proteins required for PHA synthesis. Int. J. Biol. Macromol. 25, 3–19. Sambrook, J., Russell, D.W., 2001. Molecular Cloning: a Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Schubert, R.H.W., 1991. Aeromonas and their signiWcance as potential pathogens in water. J. Appl. Bacteriol. 70, 131S–135S.
Short communication / Plasmid 54 (2005) 283–287 Wang, Y.W., Wu, Q., Chen, G.Q., 2004. Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) scaVolds with good biocompatibility for osteoblast proliferation and diVerentiation. Biomaterials 25 (4), 669–675.
287
Wu, Q., Sun, S.Q., Yu, P., Chen, G.Q., 2000. Enviromental dependence of microbial synthesis of polyhydroxyalkanoates. Acta Polym. Sin. 6, 751–756.
Short communication
Electroporation-mediated transformation of Aeromonas hydrophila Hu Fengqing a,b,¤, You Song b a
b
Department of Life Science, Liaoning University, Shenyang 110036, China College of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China Received 27 March 2005, revised 21 April 2005 Available online 20 June 2005 Communicated by Grzegorz Wegrzyn
Abstract A strain of Aeromonas hydrophila producing copolyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate, abbreviated as PHBHHx, was successfully transformed by electroporation. The plasmid used was a broad host range plasmid pBBR1MCS. Electroporation conditions were varied systemically to develop an electroporation protocol. The optimal yield of transformant was approximately 4 £ 102 CFU/g DNA at 12.5 kV/cm and 1000 , resulting in a time constant of approximately 5 ms. The A. hydrophila transformants expressed plasmid-encoded resistance to chloromphenicol. Plasmid DNA in the A. hydrophila transformant was stably maintained. This is the Wrst report of transformation of bacteria A. hydrophila. 2005 Elsevier Inc. All rights reserved. Keywords: Aeromonas hydrophila; Electroporation
1. Introduction The genera Aeromonas is Gram-negative bacteria belonging to Vibrionaceae. Mesophilic, motile Aeromonas hydrophila is ubiguitous and autochthonous aquatic microorganisms occurring in freshwater, sewage, and brachish water (Schubert, 1991), and in chlorinated and unchlorinated drink*
Corresponding author. Fax: +86 24 62202232. E-mail address: [email protected] (H. Fengqing).
0147-619X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2005.04.004
ing water (Havellaar et al., 1992). They also are common contaminants of fresh foods, including Wsh, and other scafoods (Abeyta and Wekell, 1988; Kirov, 1993; Krovacck et al., 1992; Nishikawa and Kishi, 1988). SpeciWes of these genera are known as pathogens for Wsh (Cahill, 1990; Joseph and Carnahan, 1994) and human associated mainly with diarrheal symptoms (Janda, 1991). However, the development of molecular tools for these organisms is far behind the technologies employed for Escherichia coli and similar bacteria. For
284
Short communication / Plasmid 54 (2005) 283–287
instance, transformation of A. hydrophila by electroporation has not been reported up to the present, thus limiting genetic studies of this species. Aeromonas hydrophila WQ was reported to produce copolyesters of 3-hydroxybutyrate (3HB) 3-hydroxyhexanoate (3HHx), abbreviated as PHBHHx, from lauric acid as sole carbon source (Lu et al., 2004). The water insoluble PHBHHx exhibits high molecular weight, thermoplastic and/or elastomeric features as well as other interesting physical and material properties (Deng et al., 2003; Ju et al., 2004). Therefore, they have attracted increasing attention from scientiWc and industrial communities due to their interesting properties including biodegradability, biocompatibility, and piezoelectricity (Deng et al., 2003; Wang et al., 2004; Wu et al., 2000). Polyhydroxyalkanoate (PHA) synthase are the key enzymes of PHA biosynthesis (Rehm and Steinbüchel, 1999). To elucidate the molecular mechanism of PHBHHx accumulation, it is necessary to inactivate PHA synthase gene in A. hydrophila. Considering low eYciency of conjugation, in this study, for the Wrst time, we developed an electroporation protocol for introduction of plasmid DNA into this organism.
2. Materials and methods Aeromonas hydrophila WQ was used as a bacterial host strain and broad host range plasmid pBBR1MCS (Michael et al., 1995) in E. coli JM109 was isolated and used as a plasmid DNA. A. hydrophila WQ was cultivated at 30 °C on Luria–Bertani (LB) medium, while E. coli was grown at 37 °C on LB medium. Chloramphenicol (170 g ml¡1) was added to the medium when needed. In all cases, the cultures were incubated in conical Xask at 200 rpm (NBS, Series 25D, New Brunswick, USA). Extraction of isolation of plasmid, digestion of restriction endonucleases, and agar gel electrophoresis were performed by standard procedures (Sambrook and Russell, 2001) or as recommended by the manufacturers. To obtain optimal electrocompetent cells, A. hydrophila WQ cells were grown on LB medium and successively harvested at the middle of exponential
phase, the end of exponential phase, and the early stationary phase. The cells were quickly placed at 0 °C about 30 min to stop the growth, centrifuged at 2500g and 0 °C for 15 min, and washed twice in equal volume of sterile pyrolized water chilled to 0 °C and twice with 100 ml of sterile 10% glycerol (w/ w) chilled to 0 °C. To ensure the same density of cells harvested in diVerent growth phases, the cell pellet was resuspended in 200–400 l of GYT medium (Sambrook and Russell, 2001) chilled to 0 °C. The cells were dispensed into 40 l aliquots and preserved in refrigerator (¡80 °C).
3. Results and discussion Aliquots of cells were thrawed on ice and 3 l plasmid pBBR1MCS (4.7 kb) DNA (approximately 0.1–1 g) was added just prior to electroporation. Electroporation was performed in chilled 0.1 cm cuvettes using a Model JY2000-1 electroporator (SCIENTZ, Ningbo, China). After the electric shock, the bacterial suspension was immediately transferred to an eppendorf tube containing 1000 l of LB medium. Cells of A. hydrophila WQ were incubated about 2–3 h at 30 °C with shaking. After incubation, estimates of survival rate were obtained by spreading aliquots onto nonselective or selective LB agar plates (antibiotics are added when need), respectively. Transformants were presented after 24–48 h. No transformants were obtained in the absence of plasmid DNA. The number of transformants obtained was evaluated. Table 1 clearly shows that optimal electroporation results were obtained with competent cells harvested at early stationary phase. Approximately 2.5 £ 102 transformants were obtained as the maximum yield of transformation when 40 l cell suspension containing 1 £ 108 competent cells from early stationary phase was pulsed at 10 kV/cm with 1 g plasmid DNA. To optimize electroporation conditions, Wrst by studying the eVect of electric Weld strengths for a given pulse time (5 ms). Competent cell of A. hydrophila, prepared at early stationary phase, was transformed with varying the strength of electric Weld (from 0 to 20 kV/cm). As shown in Fig. 1, the maximum value of transformation eYciency was achieved
Short communication / Plasmid 54 (2005) 283–287 Table 1 EVect of growth phase on the transformation of A. hydrophila WQ Growth phase
Cells OD600nm
Transformants (£102/g DNA)
Middle exponential phase End exponential phase Early stationary phase
0.4 0.8 1.0
0.11 1.32 2.57
Aeromonas hydrophila WQ from diVerent growth phases were used for transformation. Three microliters plasmid pBBR1MCS DNA and 40 l competent cells with 10 kV/cm electric Weld strength, 25 F, and 1000 were used for transformation. All experiments were carried out in a Model JY2000-1 electroporator (SCIENTZ, Ningbo, China). The OD was measured at 600 nm with a 722S spectrophotometer (Precision and ScientiWc Instrument, Shanghai, China). All results were repeated three times.
Fig. 1. EVect of electric Weld strengths on survival rate and transformation eYciency of A. hydrophila WQ by electroporation. Forty microliters of electrocompetent cells were mixed with 3 l plasmid DNA. The mixtures were submitted to electroporation in 1 mm cuvettes. Transformants were counted on LB medium supplemented with chloramphenicol (170 g ml¡1). The pulse time was maintained at a constant value (R D 1000 , C D 25 F) and the samples were submitted to electric Welds of 0, 2.5, 5, 7.5, 10, 12.5, 15, and 20 kV/cm. All experiments were carried out in a Model JY2000-1 electroporator (SCIENTZ, Ningbo, China). All results were repeated three times.
at 12.5 kV/cm and approximately 4£ 102 transformants/g plasmid DNA was obtained with only 20% of survival rate for competent cells. The transformation eYciency was elevated when increasing the strength of electric Welds, whereas the strength of
285
electric Weld higher than 12.5 kV/cm resulted in the marked decrease of transformation eYciency. Survival rate of competent cells was always declined along with the increase of electric Weld strength. No transformants were obtained at the strength of electric Welds less than or equal to 5 kV/cm. Using the same strain and plasmid, pulse time was varied from 0 to 15 ms for a given electric Weld strength (12.5 kV/cm). Fig. 2 shows the eVect of pulse time on survival rate and transformation eYciency of A. hydrophila WQ for a given electric Weld strengpth (12.5 kV/cm). A narrow optimum was found for a pulse time of 5 ms with approximately 400 transformants/g plasmid DNA. Fewer transformants were obtained at lower or higher values of pulse time. Using optimized conditions, electroporation of suicide plasmid pFH5 (6.5 kb) harboring the disrupted PHBHHx synthase gene (phaC::Cm) transformed A. hydrophila WQ, through an in vivo homologous recombination process, PHBHHx synthase gene (phaC) of A. hydrophila
Fig. 2. EVect of pulse time on survival rate and transformation eYciency of A. hydrophila WQ by electroporation. Forty microliters of electrocompetent cells were mixed with 3 l plasmid DNA. The mixtures were submitted to electroporation in 1 mm cuvettes. Transformants were counted on LB medium supplemented with chloramphenicol (170 g ml¡1). The strength of electric Weld was maintained at a constant value (12.5 kV/cm) and the samples were submitted to pulse time of 0, 2.5, 5, 10, and 15 ms. All experiments were carried out in a Model JY2000-1 electroporator (SCIENTZ, Ningbo, China). All results were repeated three times.
286
Short communication / Plasmid 54 (2005) 283–287
genome was replaced by the disrupted PHBHHx synthase gene, and chloramphenicol resistance gene was integrated into the genome of A. hydrophila, resulting in PHBHHx synthase negative mutant. The mutant resisting chloramphenicol could be easily screened and obtained from selective LB agar plate (170 g ml¡1 chloramphenicol was added). This result strongly proved that A. hydrophila WQ, via a electroporation procedure, is amenable to genetic manipulation in a way similar to E. coli. Usually, it was diYcult to transfer DNA into A. hydrophila by conjugation especially when the size of plasmid DNA was larger than 6 kb, and the eYciency of conjugation was only 1.0 £ 10¡8. The results presented in this paper are the Wrst presentation that A. hydrophila WQ can be directly transformed by the well-known broad host range plasmid pBBR1MCS. We attempted to transfer this plasmid into A. hydrophila WQ using the conventional transformation methods. Although transformation eYciency for A. hydrophila WQ by electroporation is not as eYcient as that of E. coli, electroporation of A. hydrophila WQ is easier and more eYcient than triparental conjugal transfer method. The plasmid pBBR1MCS in the transformant of A. hydrophila WQ stably maintained after 100 generations of cultivation. In addition, all the transformants were determined to maintain the plasmid pBBR1MCS as monomeric forms and this structural stability was further conWrmed by the restriction endonuclease cleavage analysis. To our knowledge, this is the Wrst report of genetic manipulation of A. hydrophila and certainly the Wrst 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 and also facilitate the application of recombinantDNA technology to A. hydrophila.
Acknowledgments This research was supported by the National Nature Science Foundation for Distinguished Young Scholars (Grant No. 30225001). We are grateful to Professor Chen G.Q. of Tsinghua Uni-
versity (Beijing, China) for kindly providing us with A. hydrophila strain WQ.
References Abeyta Jr., C., Wekell, M.M., 1988. Potential sources of Aeromonas hydrophila. J. Food Safety 9, 11–22. Cahill, M.M., 1990. Bacterial Xora of Wshes: a reviews. Microb. Ecol. 19, 21–41. Deng, Y., Lin, X.S., Zheng, Z., Deng, J.G., Chen, J.C., Ma, H., Chen, G.Q., 2003. Poly (hydroxybutyrate-co-hydroxyhexanoate) promoted production of extracellular matrix of articular cartilage chondrocytes in vitro. Biomaterials 24, 4273–4281. Havellaar, A.H., Schets, F.M., van Silhout, A., W.H., Wieten, G., and Van den Kooij., 1992. Typing of Aeromonas strains from patients with diarrhea and from drinking water. J. Appl. Bacteriol. 72, 435–444 Janda, J.M., 1991. Recent advances in the study of the taxonomy, pathogenicity, and infectious syndromes associated with the genes Aeromonas. Clin. Microbiol. Rev. 4, 397– 410. Joseph, S.W., Carnahan, A., 1994. The isolation, identiWcation, and systematics of the motilc Aeromonas species. Ann. Rev. Fish Dis. 4, 315–343. Ju, X., Guo, B.H., Zhang, Z.M., Zhou, J.J., Jiang, Y., Yan, S., Li, L., Wu, Q., Chen, G.Q., Schultz, J.M., 2004. Direct AFM observation of crystal twisting and organization in banded spherulites of chiral poly (3-hydroxybutyrate-co-3hydroxyhexanoate). Macromolecules 37, 4118–4123. Kirov, S.M., 1993. The public health signiWcance of Aeromonas spp. in foods. Int. J. Food Microbiol. 20, 179–198. Krovacck, K., Fanis, A., Baloda, S.B., Petenz, M., Lundberg, T., Mansson, I., 1992. Prevalence and characterization of Aeromonas spp. isolated from foods in Uppsala. Sweden. Food Microbiol. 9, 29–36. Lu, X.Y., Wu, Q., Zhang, W.J., Zhang, G., Chen, G.Q., 2004. Molecular cloning of polyhydroxyalkanoate synthesis operon from Aeromonas hydrophila and its expression in Escherichia coli. Biotechnol. Progr. 20, 1332–1336. Michael, E.K., Philip, H.E., Hill, D.S., Gregory, T.R., Michael, A.F., 1995. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying diVerent antibioticsresistance cassettes. Gene 166, 175–176. Nishikawa, Y., Kishi, T., 1988. Isolation and characterization of motilc Aeromonas from human, food and environmental speciWces. Epidemiol. Infect. 101, 213–223. Rehm, B.H.A., Steinbüchel, A., 1999. Biochemical and genetic analysis of PHA synthases and other proteins required for PHA synthesis. Int. J. Biol. Macromol. 25, 3–19. Sambrook, J., Russell, D.W., 2001. Molecular Cloning: a Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Schubert, R.H.W., 1991. Aeromonas and their signiWcance as potential pathogens in water. J. Appl. Bacteriol. 70, 131S–135S.
Short communication / Plasmid 54 (2005) 283–287 Wang, Y.W., Wu, Q., Chen, G.Q., 2004. Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) scaVolds with good biocompatibility for osteoblast proliferation and diVerentiation. Biomaterials 25 (4), 669–675.
287
Wu, Q., Sun, S.Q., Yu, P., Chen, G.Q., 2000. Enviromental dependence of microbial synthesis of polyhydroxyalkanoates. Acta Polym. Sin. 6, 751–756.