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Electroporation Ofalcaligenes Eutrophus With APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1994, p. 3585-3591 Vol. 60, No. 10 0099-2240/94/$04.00+0 Copyright © 1994, American Society for Microbiology Electroporation of Alcaligenes eutrophus with (Mega) Plasmids and Genomic DNA Fragments SAFIEH TAGHAVI, DANIEL VAN DER LELIE,* AND MAX MERGEAY Environmental Division, Flemish Institute for Technological Research, B-2400 Mol, Belgium Received 7 March 1994/Accepted 10 July 1994 Electroporation was used as a tool to explore the genetics of the heavy-metal-resistant strain Alcaligenes eutrophus CH34. A 12.9-kb A. eutrophus-Escherichia coli shuttle vector, pMOL850, was constructed to optimize electroporation conditions. This vector is derived from the E. coli plasmid pSUP202 and contains the replication region of the A. eutrophus megaplasmid pMOL28. Electroporation was used to transform A. eutrophus CH34 derivatives with megaplasmids (sizes up to 240 kb), and transformants were selected for resistance to heavy metals. Electroporation was also performed with endonuclease-digested genomic DNA. Transformation of markers affecting lysine biosynthesis (lysA194) and biosynthesis of the siderophore alcaligin E we,rs observed. Transfer of the nonselected markers pheB332 and aro-333, linked to lysA194, confirmed the intervention of homologous recombination. However, during transformation of ale::TnS-Tc, illegitimate recombination and transposition were also observed as an alternative for the inheritance of the Tn5-Tc markers. Isolates ofAlcaligenes eutrophus have been found in a variety recipients in intra- and intergeneric matings mediated by of biotopes severely polluted with heavy metals or organic pULB113 (12). Plasmids pULB113 and pMOL50, a rear- xenobiotics and therefore seem to be well adapted to the ranged derivative of pMOL28 that displayed chromosome- constraints imposed by such environments (3). The represen- mobilizing activity, were recently used to construct a circular tative strain among the metal-resistant Alcaligenes strains genetic map of the A. eutrophus CH34 chromosome (20). isolated is A. eutrophus CH34, which was isolated from the However, a tool for precise gene mapping is still required, sediment of a zinc factory (15, 16). This facultative chemolitho- since transducing phages are not available in A. eutrophus trophic bacterium carries two megaplasmids, pMOL28 (165 despite intensive searching (5). Therefore, other techniques kb) and pMOL30 (240 kb), bearing multiple resistances to have to be developed to determine the relative positions of heavy metals (for an overview, see reference 4), which can closely linked genetic markers, and as such, electroporation transfer at low frequency. Both plasmids have narrow replica- might be a good alternative. tion ranges and can replicate only in A. eutrophus. Other During the last few years, many papers on the efficient isolates of metallotolerant A. eutrophus were shown to carry transformation of soil bacteria by electroporation as an alter- similar plasmids (3). In soils with high levels of heavy metals, native to other transformation or DNA transfer systems have heterotrophic resistant bacteria closely related to A. eutrophus been published. Examples are the electroporation ofAgrobac- CH34 constitute a substantial fraction of the aerobic micro- terium tumefaciens (18, 29), Agrobacterium rhizogenes (29), flora (3). Therefore, genetic approaches to the study of A. Acetobacter xylinum (8), Pseudomonas aeruginosa (24), and eutrophus CH34 and related strains would be relevant in the Pseudomonas stutzeri (27). Most of these studies used recom- general context of genetic exchanges between microbes in binant plasmids with easily selectable antibiotic markers in- environments with strong selection pressure, an example being stead of endogenous plasmids (8, 18). Only in the case of soils polluted by heavy metals. Many interesting properties of Pseudomonas putida did the authors use a 74.1-kb plasmid A. eutrophus, like multiple resistance to heavy metals, xenobi- conferring resistance to silver (27). otic degradation, and various genes involved in nitrate reduc- In this paper, we describe electroporation of A. eutrophus tion and chemolitotrophy (hydrogenases) (7) are carried by with endogenous megaplasmids with sizes up to 240 kb. To megaplasmids. Therefore, the analysis of newly isolated Alcali- optimize electroporation of A. eutrophus, we first constructed genes strains in general focuses on the endogenous (mega)plas- an Escherichia coli-A. eutrophus shuttle vector, designated mids. This is either done by hybridization or by transferral of pMOL850. This vector is based on the E. coli vector pSUP202 the endogenous megaplasmids to plasmid-free recipients by (23), in which we cloned a replication-competent region of conjugation. Although some megaplasmids may conjugate at pMOL28. high frequencies (4, 20), most either transfer at low frequency We also used electroporation for the transformation of A. or are not transferable at all. Also, conjugation might be eutrophus CH34 and its derivatives with endonuclease-digested hindered by the absence of markers to counterselect the donor chromosomal DNA. This technique, which is a good tool for strain. Earlier, we recognized that A. eutrophus CH34 was the introduction of short DNA fragments, can be used for amenable to genetic manipulation by conjugation and that strain construction and might be an alternative for transduc- auxotrophic mutants of this strain, obtained by temperature- tion in A. eutrophus. induced mutagenesis and mortality (17, 20, 28), could serve as MATERIALS AND METHODS * Corresponding author. Phone: 32-14-333111, ext. 5116. Fax: 32- Bacterial strains, plasmids, and media. The bacterial strains 14-320372. and plasmids used are described in Table 1. A. eutrophus and 3585 3586 TAGHAVI ET AL. APPL. ENVIRON. MICROBIOL. TABLE 1. Bacterial strains and plasmids Strain or plasmid Relevant marker(s) Plasmid(s) or property of plasmid Origin or reference' A. eutrophus CH34 (Wild type) pMOL28, pMOL30 15, 16 AE3 aut-3 pMOL50 4 AE332 lysA194 pheB332 20 AE333 lysA4194 aro-333 20 AE587 leu-54 This study AE1093 aleA1093::Tn5-Tc pMOL28, pMOL30 M. A. Khan AE1152 aleB1152::TnS-Tc pMOL28, pMOL30 M. A. Khan AE126 pMOL28 16 AE128 pMOL30 16 E. coli CM404 Leu- Pro- Thi- Lac- Kmr pRK2013 6 Plasmids pSUP202 Apr Tcr Cmr Mob' Tra- ColEl origin unable to replicate in A. eutrophus 23 pRK2013 Kmr Tra+ Unable to replicate in A. eutrophus 6 pMOL850 Apr Tcr Mob' Tra- A. eutrophus-E. coli shuttle vector This study pMOL28 Nir Cor CrO4r Hgr 16 pMOL30 Cd' Znr Cor Hgr Cur 16 pMOL50 Nir Cor CrO4r Hgr Enlarged derivative of pMOL28 derepressed in 4 self-transfer E. coli were cultured and plated at 30 and 37°C, respectively, in cation that after addition of the lysis solution (0.1 N NaOH, 869 broth supplemented with 2 mM CaCl2 and on 869 agar 1% SDS, pH 12.8) the mixture was gently mixed for 15 min at (14). Resistance to heavy-metal salts was tested on a minimal room temperature before being cooled down on ice for 5 min. medium as described before (16, 19). If necessary, amino acids Total DNA was isolated from A. eutrophus as described for were added at a concentration of 40 ,ug/ml. For plasmid DNA Bacillus subtilis according to the method of Bron and Venema extraction from A. eutrophus, cells were grown in nutrient (1). broth 3 (Difco Laboratories) without selective pressure. Elec- Restriction enzymes, phosphatase, and T4 DNA ligase were trocompetent cells were prepared with cultures grown either in purchased from Gibco BRL and used as recommended by the SOB medium (9) without Mg2+ or in nutrient broth 3. After supplier. Molecular cloning techniques were performed as electroporation, bacteria were resuspended and incubated for described by Maniatis et al. (13). 1 h in SOC medium (9). Tetracycline-resistant clones of E. coli Conjugations. Solid-surface matings between A. eutrophus and A. eutrophus were selected on 869 agar supplemented with and E. coli strains were performed as described by Lejeune et 20 jLg of the antibiotic per ml. al. (12). E. coli CM404(pRK2013) (6) was used as the helper DNA isolation and manipulation. A modified method based strain in triparental matings involving the mobilizable plasmid on the alkaline lysis procedure from Kado and Liu (11) was pMOL850. employed for the isolation of megaplasmid DNA from A. Electroporation of E. coli and A. eutrophus. Electrocompe- eutrophus. Cells grown overnight in 15 ml of nutrient broth 3 tent cells of E. coli were prepared and transformed with the were harvested and washed with 5 ml of E buffer (40 mM Bio-Rad Gene Pulser (Bio-Rad Laboratories, Richmond, Ca- Tris-acetate, 2 mM EDTA, pH 7.9). After washing, the cells lif.) according to the manufacturer's instructions. To obtain were resuspended in 1 ml of E buffer and 2 ml of freshly electrocompetent cells of A. eutrophus, overnight cultures of prepared lysis solution (40 mM Tris, 1% sodium dodecyl sulfate [SDS]; final pH, 12.6 with 5 N NaOH) was added. The mixture was incubated for 20 min at room temperature under S gentle agitation and subsequently incubated at 68°C for 75 H B/ P E H ItI min. After incubation, 0.5 ml of 4 M NaCl and 8 ml of -* - > - =4 pSUP202 phenol-chloroform (1:1, H20 saturated, pH 4.8) were added and the suspension was gently mixed for 30 min at room Tc Ap Cm temperature. After centrifugation in a Sorvall SS34 rotor (30 min, 12,000 rpm at 4°C) and storage for 2 h at 4°C, the aqueous S x phase was transferred into another tube and extracted with 2 H B/ P E H/ S B E H ml of diethyl ether. The upper phase was discarded, and the ILII I H_/I l l l residual ether was evaporated for 20 min at 37°C. Aliquots (1.8 > - I ml) of the DNA solution were transferred and centrifuged for TC Ap P p pMOL850 3 h in a Ti 50 rotor (40,000 rpm, 4°C) to pellet the DNA.
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