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. The DNA pellets were washed three times with 1.8 ml of ice-cold 1kb 70% ethanol solution, dried under vacuum, and finally resus- FIG. 1. Restriction maps of pSUP202 and pMOL850. The posi- pended in 100 ,ul of TE buffer (10 mM Tris-HCl, 1 mM EDTA, tions of the functional tetracycline (Tc), ampicillin (Ap), and chloram- pH 8.0). This method yielded highly purified DNA suitable for phenicol (Cm) resistance genes as well as relevant restriction sites are restriction analysis. Plasmid DNA was isolated from E. coli by indicated in the figure. H, Hindlll; B, BamHI; S, Sall; P, PstI; E, the method of Ish-Horowicz and Burke (10), with the modifi- EcoRI; X, XhoI. VOL. 60, 1994 ELECTROPORATION OF A. EUTROPHUS 3587

TABLE 2. Electroporation of A. eutrophus AE587 with pMOL850: effects of growth phase and culture medium

OD660 at time No. of transformantsc: Medium"of harvest" Per p.g of pMOL850 Per pmol of pMOL850 NB 3 0.2 (2.0 ± 0.5) x 106 (1.7 ± 0.4) x 107 NB 3 0.5 (1.1 ± 0.6) x 106 (9.4 ± 1.0) x 106 NB 3 0.8 (0.5 + 0.3) x 106 (4.3 ± 2.5) x 106 NB 3 1.0 (overnight culture) (2.2 + 0.2) x 103 (1.9 ± 0.15) x 104 SOB 0.2 (2.1 + 0.6) x 106 (1.8 ± 0.5) x 107 SOB 0.5 (1.0 + 0.9) x 106 (0.8 ± 0.8) x 107 SOB 0.8 (4.2 ± 0.4) x 106 (3.6 ± 0.3) x 107 SOB 1.3 (overnight culture) (3.5 ± 0.3) x 103 (3.0 ± 0.2) x 104 SOB 0.8 (E. coli DH1OB) (5.1 + 0.9) x i07 (4.3 ± 0.8) x 103 a NB 3, nutrient broth 3. b Electrocompetent cells were prepared and electroporated as described in Materials and Methods. For electroporation, electrocompetent cells were harvested at the indicated OD660 and concentrated to a theoretical OD660 of 120. c The results are the averages of three different transformation experiments plus or minus the standard errors. strain AE587 were grown either in SOB medium (without mants were pooled, and their plasmids were transferred by tri- MgSO4) or in nutrient broth 3. The next morning, 0.5-ml parental mating to plasmid-free A. eutrophus AE104 by use of cultures were diluted in 500 ml of preheated medium and E. coli CM404(pRK2013). This step should select for pSUP202 incubated at 30°C with intense shaking until the desired optical recombinant plasmids, containing pMOL28 replication func- density at 660 nm (OD660) of 0.2, 0.5, or 0.8 was reached. The tions. As a control, pSUP202 was also transferred to AE104. cells were harvested and washed twice with 500 ml of ice-cold With the pSUP202::pMOL28 bank, transconjugants were ob- washing buffer (10% glycerol, 90% H20 [vol/vol]). After the tained at a frequency of 106 transconjugant per recipient. washing steps, the cells were concentrated in washing buffer With pSUP202, transconjugants were also obtained, albeit at a solution to a final OD660 of 120. Aliquots of 40 [LI of electro- very low frequency (10-' transconjugants per recipient), and competent cells and 100 ng of DNA dissolved in 4 RI of H20 analysis of these transconjugants showed the absence of au- per electrotransformation assay were used. By use of the tonomously replicating plasmids, suggesting integration of Bio-Rad Gene Pulser and 2-mm cuvettes, optimal results were pSUP202 in the AE104 chromosome. These trans- obtained with the following settings: voltage, 2.43 kV; capacity, conjugants were not examined further. Transconjugants ob- 25 pF; external resistance, 200 Ql. After electroporation, the tained with the pSUP202::pMOL28 bank were analyzed in cells were mixed with 1 ml of SOC medium and left for 1 h at detail for their plasmids. All recombinant plasmids examined 30°C before being plated. Appropriate dilutions were plated were shown to contain a cloned EcoRI fragment of 4.7 kb, on selective medium to select for transformants and on non- this sometimes in combination with other EcoRI fragments. to counts. selective plates determine the total viable Also, this 4.7-kb EcoRI fragment was found in two orienta- Detection of siderophore production by A. eutrophus. In tions. From these results, we conclude that it contained all order to detect siderophore synthesis by A. eutrophus, the functions required for autonomous replication. The restric- strains were grown in iron-free Tris-minimal salt medium (19). tion map of pMOL850, a pSUP202 derivative containing this After the cultures had reached an OD660 of 0.5, 1 ml of CAS 4.7-kb fragment, is shown in Fig. 1. Southern hybridization was added to 1 ml of culture to solution (chrome azurol S) with pMOL850 confirmed the pMOL28 origin of the cloned monitor the presence of siderophores as described by Schwijn and Neilands (21). fragment (data not shown). Determination of optimal electroporation conditions by use of the shuttle vector pMOL850. Optimal electroporation con- RESULTS ditions were determined for A. eutrophus AE587, a plasmid- Cloning of a replication-competent region from pMOL28. In free, leu-54 mutant of strain CH34. To obtain optimally order to design a cloning vector based on endogenous A. electrocompetent cells, special attention was paid to the eutrophus replication functions, pMOL28 DNA was restricted growth medium and the growth phase in which the cells were with EcoRI and the fragments were ligated to EcoRI-digested, harvested. Electrocompetent cells were prepared and electro- phosphatase-treated pSUP202 DNA. This Tcr cloning vector is porated with pMOL850 obtained from E. coli as described in unable to replicate autonomously in A. eutrophus. The ligation Materials and Methods. The results from these experiments mixture was electroporated into E. coli DH1OB. Tcr transfor- are presented in Table 2. With nutrient broth 3, the best results

TABLE 3. Electroporation of A. eutrophus AE587 and CH34 with megaplasmids

Size (kb) Origin of the Selected No. of transformants: Plasmid plasmid DNA marker Per ,ug of DNA Per pmol of DNA pMOL850 12.9 E. coli Tcr (5.0 ± 0.6) x 106 (4.3 ± 0.5) x 107 pMOL28 165 AE126 Nir (5.0 ± 1.2) x 102 (5.4 ± 1.3) x 104 pMOL50 210 AE3 Nir (2.5 ± 1.0) X 102 (3.4 ± 1.4) x 104 pMOL30 238 AE128 Znr (2.0 ± 0.8) X 102 (3.2 ± 1.3) x 104 " The results presented in this table are the averages of three different transformation experiments plus or minus the standard. After electroporation, the presence of the intact megaplasmids was confirmed by testing for the presence of the other heavy-metal resistance markers and by restriction analysis (see also Fig. 2). 3588 TAGHAVI ET AL. APPL. ENVIRON. MICROBIOL.

A. eutrophus, strain AE587 was transformed with pMOL28, pMOL50, or pMOL30, having sizes of 165, 210, and 238 kb, respectively. The nickel resistance markers of pMOL28 and pMOL50 and the zinc resistance marker of pMOL30 were used for selection of the transformants. The results are pre- sented in Table 3. It was possible to obtain transformants resistant to the selective heavy metal with all three megaplasmids tested, but the transformation efficiencies were considerably lower than those observed with pMOL850 (a difference of 104/[Lg of DNA and of 103/pmol of DNA). This may be due to the large difference in plasmid size. The presence of the intact megaplas- mid was verified by testing of the other heavy-metal resistance markers present on the megaplasmids, as well as by EcoRI restriction analysis of 10 independently obtained transformants for each of the megaplasmids. The results of the restriction analysis for several transformants containing pMOL28 or pMOL50 are shown in Fig. 2. All transformants tested con- tained the intact megaplasmid. This is the first report of electroporation of bacteria with megaplasmids that have sizes FIG. 2. EcoRI restriction analysis of pMOL28 and pMOL50 after of 165 kb or more. transformation into A. eutrophus AE587. Lanes: MI, 1-kb ladder; M2, Electroporation ofA. eutrophus AE587 and CH34 with linear HindIII-digested X; 1, pMOL28 isolated from AE126; 2 to 4, pMOL28 fragments of chromosomal DNA: gene replacement and trans- isolated from AE587 transformants; 5, pMOL50 isolated from AE3; 6 position. We tried to transform A. eutrophus with chromo- to 8, pMOL50 isolated from AE587 transformants. somal DNA fragments, since this technique could be very useful for strain construction or for precise chromosomal mapping. Total DNA of A. eutrophus AE1093 and AE1152, were obtained with cells harvested in the beginning of the two alcaligin E siderophore mutants of CH34 that were exponential growth phase (OD66t0 = 0.2). The number of obtained after transposon mutagenesis with Tn5-Tc, was iso- transformants decreased with increasing OD of the culture. On lated and used in the electroporation experiments. Before the contrary, with SOB medium the best results were obtained electroporation, the chromosomal DNAs were digested with when the cells were harvested at the end of the exponential EcoRI. This enzyme does not cut within Tn5-Tc, and restric- growth phase (OD660 = 0.8). For both media, the numbers of tion should therefore result in Tn5-Tc containing fragments transformants dropped dramatically when overnight cultures flanked by chromosomal DNA. Both AE587 and CH34 were were used to prepare electrocompetent cells. As a control, electroporated with the EcoRI-digested chromosomal DNAs, electrocompetent cells of E. coli DH1OB were also trans- and TCr clones were selected. Transformants were obtained for formed with 100 ng of pMOL850 DNA. This resulted in 5.1 x both strains with either type of chromosomal DNA, albeit at 107 transformants per p.g of DNA, indicating that electrotrans- low frequencies (Table 4). Subsequently, the transformants formation is about 10 times more efficient for E. coli DH10B were analyzed for their siderophore phenotype in order to than for A. eutrophus AE587, given that the total number of determine the type of event at the basis of the integration of surviving cells after electroporation was the same for both the TCr marker: gene replacement would result in a TCr Sid- species. For further electrotransformation experiments, A. phenotype, while transposition or illegitimate recombination eutrophus was grown in SOB medium (without Mg) and would most likely give rise to a TCr Sid' phenotype. The results harvested at an OD660) of 0.8. of this analysis are also presented in Table 4. Electroporation of A. eutrophus AE587 with the megaplas- Electroporation of AE587 with DNA fragments of strain mids pMOL28, pMOL50, and pMOL30. To examine whether AE1093 resulted exclusively in transformants with a TCr Sid+ electroporation could be used to introduce megaplasmids in phenotype, suggesting transposition or illegitimate recombina-

TABLE 4. Electroporation of A. eutrophus AE587 and CH34 with EcoRI-linearized chromosomal DNA No. of Origin of EcoRI-digested No. of No. of mecha- Straln transformed chromosomal DNA (relevant transformants/p.g transformants Relevant genotype of nisms of genotype for transformation) of DNA" tested transformants insertion" T GR AE587 (leui-54) AE1093 (aleAl093::Tn5-Tc) 15 + 6 75 leu-54 Tn5-Tc ale' 75 0 AE587 (leu-54) AE1152 (aleBl152::Tn5-Tc) 10 + 4 50 lei-54 aleBl152::Tn5-Tc 44 leu-54 Tn5-Tc ale' 6 CH34 (pMOL28, pMOL30) AEI 152 (aleBI152::Tn5-Tc) 25 + 9 100 aleB1152::Tn5-Tc 89 Tn5-Tc ale + 11 "Average of four experiments plus or minus the standard error. h The plasmids in the CH34 transformants were unchanged. Event being at the basis of the recovery of the selected TCr marker (TCr, present on Tn5-Tc). T, transposition or in some specific cases illegitimate recombination, identified by a Sid' phenotype; GR, gene replacement, identified by a Sid- phenotype. Transformants were selected for resistance to tetracycline. Subsequently, they were examined for KMr (encoded by Tn5-Tc), heavy-metal resistance (only for CH34 transformants), and the ability to synthesize the alcaligin E siderophore. VOL. 60, 1994 ELECTROPORATION OF A. EUTROPHUS 3589

1 2 3 4 5 6 7 8 9 10 11 12 M Transformants of AE587 with AE1093 DNA showed a ie0"-. A hybridization pattern (after EcoRI and Sall digestion) that 0,£D differed from the pattern found with AE1093 DNA (Fig. 3, compare lanes 9 to 11 with lanes 12). In one of the three transformants tested, only one Sall fragment hybridized with the internal TnS-Tc BglII fragment (Fig. 3B, lane 10), suggest- ing that illegitimate recombination and rearrangements were at the basis of the integration of the Tcr marker. For the other transformants tested, three Sall fragments hybridized with the Tn5-Tc probe but, except for the Tn5-Tc Sall internal frag- ment, these fragments had sizes which differ from those detected in the AE1093 DNA, indicating that transposition of Tn5-Tc must have occurred after transformation with AE1093 DNA. Similar results were obtained with the Tcr Sid' trans- formants isolated after transformation with AE1152 DNA (results not shown). Electroporation with linear fragments of chromosomal DNA: gene linkage. In order to test the use of electroporation in gene mapping as an alternative for transduction in A. eutrophus, we studied the linkage between the closely linked pheB332 and 1 2 3 4 5 6 7 8 9 10 11 12 M aro-333 markers, with the lysA194 marker (20). Chromosomal DNA of CH34 was digested with XbaI or DraI and electropo- rated into A. eutrophus AE332 (pheB332 lysA194) and AE333 (aro-333 lysA194). XbaI and Dral were chosen to linearize the chromosomal DNA, since pulsed-field gel electrophoresis combined with hybridization studies had shown that lysA194, pheB332, and aro-333 were all located on a 235-kb XbaI or a 245-kb Dral fragment (data not shown). The results of the transformations, together with the percentages of linkage between the markers, are presented in Table 5 and show that both thepheB332 and the aro-333 markers are closely linked to the lysA194 marker. However, from these results it is impossi- ble to determine the relative positions of these markers on the chromosome. An interesting observation was made with regard to the replacement of the aro-333 marker. In addition to transfor- mants that had become prototrophic for Aro, a second class of transformants, which were Aro bradytrophic, was found. In FIG. 3. Hybridization analysis of A. eutrophus CH34 and AE587 contrast to the Aro prototrophics, none of the Aro bradytro- Tcr transformants obtained after transformation with strain AE1093 or phics was Lys prototrophic. This indicates that a second aro strain AEI 152 EcoRI-linearized chromosomal DNA. For analysis, the locus, perhaps aro-240, which can partly complement the chromosomal DNAs of the transformants were digested with EcoRI Aro-333 auxotrophy after transformation, is present on the A. (A) or Sall (B). As a probe, the 32P-labeled internal BglII fragment of eutrophus chromosome. Aro bradytrophy was not observed Tn5-Tc was used. Lanes: M, marker (1-kb DNA ladder); 1 to 4, total DNA of CH34 transformants (Sid- Tcr) obtained after transformation with transformants first selected for Lys prototrophy. with AE1152 DNA; 5, total DNA of AE1152; 6, total DNA of CH34; 7, chromosomal DNA of AE587 transformant obtained after transfor- DISCUSSION mation with AE1152 DNA; 8, chromosomal DNA of AE587; 9 to 11, chromosomal DNA AE587 transformants (Sid- Tcr) obtained after In this paper, we describe electrotransformation as a genetic transformation with AE1093 DNA; 12, total DNA of AE1093. tool for the study ofA. eutrophus and its plasmids, which often carry genes that encode functions with important environmen- tal applications. These functions concern the ability to degrade tion. In contrast, electroporation with AE1152 DNA resulted certain haloaromatic compounds as well as the resistance to in a Tcr Sid- phenotype in 90% of the transformants tested, heavy metals. The ability of A. eutrophus strains to degrade suggesting that homologous recombination was the common haloaromatics was shown to originate from plasmids of the mechanism of inheriting the Tcr marker. To confirm the IncP type (22). chromosomal integrity of the different classes of transfor- The genetics of the heavy-metal resistance megaplasmids of mants, their total DNA was isolated, digested with EcoRI or A. eutrophus are poorly studied: about 20% of their genetic Sall, and subsequently hybridized with the internal BglII content is occupied by heavy-metal resistance mechanisms, but fragment of Tn5-Tc. The hybridizations are shown in Fig. 3A no information is available about the other 80%. However, (EcoRI digestion) and Fig. 3B (Sall digestion). important functions involved in plasmid replication, plasmid Tcr Sid- transformants in both CH34 and AE587, which maintenance, and plasmid transfer will be present on this were obtained with AE1152 DNA, showed the same hybrid- DNA, justifying a detailed study of these megaplasmids. ization patterns after EcoRI and Sall digestion as AE1152. As A. eutrophus CH34 and its derivatives have been proven to expected, only one EcoRI fragment and three Sall fragments be good host strains for the heterologous expression of genes (the upper 4.2-kb band which hybridizes a doublet) hybridized that encode functions with environmental applications, like the with the internal Tn5-Tc BglII fragment. degradation of organic xenobiotics. This is in contrast to E. 3590 TAGHAVI ET AL. APPL. ENVIRON. MICROBIOL.

TABLE 5. Electroporation of A. eutrophus AE332 and A. eutrophus AE333 with linear chromosomal CH34 DNA fragments for linkage studies between the lysA194, pheB332, and aro-333 genetic determinants

Restriction enzyme used Selected No. of transformants/ Unselected Strain for DNA digestion phenotypea ,ug of DNA" marker % Cotransfer' A. eutrophus AE332 (pheB332 1ysA194) XbaI Phe+ (1.0 ± 0.2) x 103 lysA194 100 XbaI Lys+ (1.2 ± 0.3) x 103 phe332 98 XbaI Phe+ Lys+ (1.1 + 0.2) x 103 Dral Phe+ (4.5 + 0.5) X 102 lysA 194 100 Dral Lys+ (5.1 ± 0.6) X 102 pheA332 98 DraI Phe+ Lys+ (4.8 ± 0.7) x 102 A. eutrophus AE333 (aro-333 lysA4194) XbaI Aro+ (4.8 + 0.7) x 102 lysA 194 100 XbaI Lys+ (4.6 + 0.5) x 102 aro-333 100 XbaI Aro+ Lys+ (5.2 ± 0.4) x 102 DraI Aro+ (4.2 + 0.5) x 102 lysA 194 100 DraI Lys+ (5.2 + 0.6) X 102 aro-333 100 DraI Aro+ Lys+ (3.8 + 0.7) x 102 With strain AE333, in addition to Aro prototrophic transformants, Aro bradytrophs were observed at similar frequencies with both XbaI- and Dral-digested DNAs. "The results are averages of three experiments plus or minus the standard errors. 'Colonies (n = 100) were tested for complementation of both markers to determine the percent cotransfer. coli, which often fails to express such functions. Therefore, we these experiments and that the Tn5-Tc-containing fragments were prompted to develop new genetic tools forA. eutrophus in only form a minor fraction (±+0.4%) of the DNA. In E. coli, the order to better understand its genetics and to make it more transformation with linear DNA is barely detectable unless accessible to genetic manipulation. recB recC sbcB strains (2), which lack the RecBCD enzyme The electrotransformation of A. eutrophus depended on exonuclease V (26), the major nuclease for degrading linear several parameters: the quality of the DNA used for transfor- DNA in E. coli (25), are used. Since A. eutrophus CH34 can be mation, the growth medium, and the growth phase of the cells. transformed with linear chromosomal DNA fragments, it is The physical transformation parameters were very similar to conceivable that this strain lacks an equivalent of the RecBCD those used for E. coli as determined with the pMOL850 shuttle pathway. vector. The transformation of megaplasmids was considerably Until now, no phages have been found for A. eutrophus less efficient than when pMOL850 was employed (a difference CH34 (5). This makes transformation of A. eutrophus with of 10,000-fold per p.g of DNA or 1,000-fold per pmol of DNA). linearized chromosomal DNA fragments an attractive alterna- This is to be expected because of the large difference in tive to transduction, although a high frequency of illegitimate plasmid size. Small effects of plasmid size on the transforma- recombination may bias linkage data. The occurrence of both tion efficiency were also observed in comparisons of the results Aro prototrophics and bradytrophics after transformation of obtained with different megaplasmids. AE333 with CH34 genomic DNA serves as an example of a In all cases tested, examination of the heavy-metal resistance cryptic or silent marker that is expressed after illegitimate markers and restriction analysis revealed that the transfor- recombination. The results presented in this paper show that mants contained an unaltered megaplasmid. This is in contrast chromosomal DNA fragments can be introduced into A. to earlier results obtained with A. eutrophus AS2 plasmids eutrophus by electroporation. Electroporation with smaller similar in size to the CH34 megaplasmids (3). The reason for linear chromosomal DNA fragments should allow precise this difference probably resides in the DNA isolation proce- mapping of closely linked markers. Finally, the development of dure: in the previous study, the DNA band containing all the genetic tools forA. eutrophus will be helpful in the construction nicked plasmid forms of the different megaplasmids was iso- of new strains and in the characterization of its plasmids. lated from an agarose gel and used for transformation. This would explain why not only a pMOL28-like plasmid was found ACKNOWLEDGMENTS after electroporation with this DNA, but also deleted forms of We thank M. Faelen and S. Wuertz for fruitful discussion and for this and other megaplasmids (3). critical review of the manuscript. We also thank R. Leysen and H. Tas We succeeded in transforming A. eutrophus CH34 and its for allowing S. Taghavi to prepare her Ph.D. thesis. derivatives with linear chromosomal DNA fragments. 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