Stable electrotransformation of symboint candidate diazotrophic bacterium with plasmids carrying...

INSTITUT PASTEUR/EI.sEVIER Paris 1998

Res. Microbiol. 1998, 149, 361-372

Stable electrotransformation of symbiont candidate diazotrophic bacterium

with plasmids carrying selectable and screenable marker genes

E Kordnyi t*), K. Burg and M. Ber6nyi

Department of Agricultural Research and Biotechnology, Austrian Research Center Seibersdorf,

A-2444 Seibersd¢~rf (Austria)

SUMMARY

Nitrogen-fixing symbioses had been established between the originally asymbiotic soil bacterium Azotobacter vinelandii CCM289 and different lower and higher plant species. Better characterization and further development of such artificial systems require a reliable genetic transformation method for the introduction of marker genes into symbiont candidates. The performance of electroporation was evaluated using pJB3 (4.8 kb), pB!121 (12.8 kb) and pFAJ31.2 (24 kb) plasmid DNAs containing selectable (Ap, Km, Tc) and screenable (gusA, lacZ) marker genes. The adapted methods for the preparation of transformation-competent azotobacters and their electroporation (18 kV/cm electric field strength, 5 ms time constant, 0"C) provided up to 6.8× 10 s transformants per lag plasmid DNA, which is about 103 times the transformation efficiency achieved in control experiments. No electrotransformants were obtained with the 24-kb pFAJ31.2. The size of plasmid DNA did not signifi- cantly affect the efficiency of transformation. Transformants were able to grow at antibiotic concentrations that were 100-200 times greater than the lowest amounts that completely inhibited the growth of wild-type bacteria. A constitutive expression of gusA gene was observed in transformants with the CaMV 35S promoter-gusA fusion containing pBI121, while lacZ expression was not detected under the control of the lac promoter in pJB3 transformants. Electroporated plasmids were reisolated from transformants in their original form, while non-transformed bacteria did not contain indigenous plasmids. PCR amplification and Southern DNA bh~t hybridization showed the integration of plasmid DNA into the host genome as well. Transfor- mants retained their nitrogen-fixing ability and had normal morphological and growth characteristics. Experimental findings proved the stable maintenance of plasmid DNA in azotobacters, making possible the routine transformation and detection of these symbiont candidates.

Kev-words: Electrotransformation, Azotobacter vinelandii, Plasmid, Electroporation; Marker gene, Chromosomal integration, Symbiosis.

Submitted January 4, 1998, accepted March !0, 1998.

(*) Corresponding author; present address: National Institule for Agricultural ',,)uality Contn~l. Budapest, Pf. 18. It-1258 (thmgary).

362 P. KORANYI ET AL.

INTRODUCTION

The major limiting nutrient in modern agro- ecosystems is fixed nitrogen and, even today, its supply is prim~ily restricted to the traditional applications of chemical fertilizers hav- ing their well-known detrimental economic and environmental effects. The increased utilization and extension of natural symbiotic systems were also attempted, but due to the serious technical limitations, large-scale field applications seem unrealistic. Alternatively, the construction of artificial associations between diazotrophic organisms and plants appeared feasible (Berg et al., 1979; Carlson and Cha- left, 1974; Gusev et al., 1986; Vasil et al., 1979). Following up on earlier leads, symbioses of different complexity were established between originally asymbiotic azotobacters and different plant species (Gyurj~in et al., 1995; Kor~inyi et al., 1993). The characterization of these systems suggested that the stable transfer of Nif ÷ diazotrophs is possible into new plant hosts through artificial symbiosis. However, in the absence of specific morphological changes, as in associations with Rhizobium and Frankia, it is rather difficult to study the mechanisms of interactions, bacterial localization and colonization or to reisolate symbionts and mutants, The incorporation of widely used reporter genes (e,g, gusA, l a c Z ) w o u l d greatly facilitate the study of Azotobacter , plant symbioses, as in the case of Azosp i r i l lum-wheat root association (Ars~ne et al., 1994; Vande Broek et al., I993). This and the planned manipula- tions of selected symbiont candidates require an effective and consistent genetic transformation technique.

Initially, a variation of CaCI 2 treatment, devised originally for the transformation of E s c h e r i c h i a col i (Cohen et al., 1972), was adapted to transform the capsule- A. vinelandii OP derivative strain UW with plasmids RP4 (IncP-1 group) and RSFI010 (IncQ group) at a maximal f requency of 5 x 10 -5 transfor- mant/viable cell (David et al., 1981). Later, strains UW, UW 1 and UW 10 were naturally grown to competence by iron limitation which made possible their high frequency (10-3-10 -2 ) transformation with chromosomal DNA (Page, 1982; Page and von Tigerstrom, 1978, 1979, 1982). The modifications of this procedure led to the transformation of A. v ine landi i ATCC 12837 with broad-hos t - range plasmids pRK2501 (lncQ group), RSFI010 and pGSSI5 (lncQ group) at frequencies 102 to 10 3 times those achieved with CaC12 treatment (Glick et al., 1985, 1986). Doran et al. (1987) could transform UWI0 cells with covalently closed circular and open circular forms of RSF1010 derivative plasmid pKT210 and the transformation frequency reached about 10 -4. Bingle (1988) reported on similar frequencies when this strain was transformed with pKT210 containing a cloned azotobacter chromosomal n i f - D N A marker. However, low transformation frequencies, physiological impairments and the loss of plasmids or plasmid-encoded genes were often observed in these experiments. Generally, capsule- strains were used because the natural competence and transformation of encapsulated azotobacters were considered very poor (Page, 1985). On the other hand, the level of competence and the efficiency of transformation were presumed to be strongly related to the strain or culture (Bingle, 1988; Glick et al., 1985),

AG = ~ o t o b a c t e r growth (medium). Ap -- ampicillin.

CaMV = caulif lower mosaic virus. DIG = digoxigenin.

EDTA = ethylenediamine tetraacetic acid. fwd = forward (primer).

Gus = beta-glucuronidase. Km = kanamycin. LAC = beta-galactosidase. LB = Luria-Bertani (medium). OD = optical density.

PCR = polymerase chain reaction. rev = reverse (primer).

SDS = sodium dodecyl sulphate. SSC = saline sodium citrate. Tc = tetracycline.

TE = Tr i s -EDTA (buffer). TF = transformation (medium). UV = ultraviolet.

X-Gal = 5-bromo-4-chloro-3.indolyl-13_D_galactopyranoside" XGluc = 5-bromo-4-chloro-3-indolyi-~-D glucuronide.

ELECTROTRANSFORMA TION OF D1AZOTROPHiC BA CTERIUM 363

which warranted process optimization in each individual case.

In the present study, electrotransformation was utilized for the efficient transfer of plasmids carrying marker genes into the encapsula ted C C M 2 8 9 strain of A. v i n e l a n d i i which had proven to be an excellent symbiont candidate in former experiments, but showed low competence and impairments when it was transformed naturally. Eleetroporation was successfully optimized for several other bacteria (Dunny et al., 1991 ; Lefranqois and Sicard, 1997; McDonald et al.,

1995; Powell et al., 1988; Walker et al., 1996; Wei et al., 1995); however, using the protocol and LB medium, as described by Trevors and Starodub (1990) for the electrotransformation of A. v i n e l a n d i i ATCC 12837 with pRK2501 (resulting in 2.7x 104 transformants/l.tg plasmid DNA), we could not recover CCM289 transformants efficiently and consistently. The selection of opt imal electrical parameters and genetic

markers led to development of an effective, reproducible and generally applicable genetic transformation procedure for this strain, and to the inves- tigation of the physiological effects of plasmid DNA incorporation. Here, we report on the stable transfer of marker genes into azotobacters without detectable debilitating effects, which has great importance in the ease of symbiont candidates introduced into the rather hostile environ- ment provided by their hosts.

MATERIALS AND METHODS

Bacterial strains, plasmids and media

The characteristics of b~,cterial strains and plasmids used are shown in tat,'e I. Nghia (1986) analysed the morphology and growth of several wild- type Azotobacter species including A. vinelandii CCM289 which, on an average, produced 1.39 nmol

Strain or plasmid

Table 1. Bacterial strains and plasmids.

Characteristics Source or reference

A. vinelandii CCM289

E. coli DH5ot S17.1

TGI

Plasmids pBIl21

pBINI9

pFAJ31.2

pLAFR3

pJB3

pKOK5

pUC4

Wild-type capsule + Nif + PHB ÷

F- recA ~80 IacZAM15 pro thi hsdR hsdM ÷ recA Tp r Smr (RP4 2-Tc::Mu-Km: :Tn 7) integrated supE hsdA5 thi A(lac-proAB) F- traD36 proAB + laqlq lacZAMI5

Km r 12.8-kb pBINI9 derivative containing a 1.87-kb gus cassette with CaMV 35S promoter Km r 22-kb RK2 (lncP) derivative carrying a truncated T region and an MI3mpl9 fi'agment Tc r 24-kb pLAFR3 derivative containing a constitutive A. brasilense promoter::gusA fusion Tc r 22-kb RK2 (IncP) derivative with polylinker M13mpl8 and k cos site Apr 4.8-kb RK2 (IncP) derivative carrying a lac cassette Apr Km r 7.4-kb pUC4K derivative carrying a lacZ-Km cassette and a promoterless h,:'Z Ap r Km r 3.9-kb pUC (ColEI) derivative containing an M 13rap7 polylinker

, . .

Purkinje University, Brno, Czech Republic

S. Valla Simon et aL, 1983

T.J. Gibson

Jefferson et aL, 1987

Bevan, 1984

Vande Brock et al., 1993

Staskowicz et ai., 1987

S. Valla

Kokotek and l.otz, 1989

Veiera and Messing, 1982

. , .


ethylene/10 s cell/h and had a generation time of 2.31 h. Among others, the nitrogenase activity and growth rate of this strain provided optimal conditions for its symbiotic coexistence inside the cells and intercellular spaces of plants. Its stocks and selected ~ s f o ~ a n t s were maintained on nitrogen- free medium (Newton et aL, 1953)at 30°C. Azoto- bacters were ~ansformed in 10% glycerol solution or liquid transformation (TF) medium and then selected on solid azotobacter growth (AG) medium (Glick et al., 1985; Page and yon Tigerstrom, 1979). E. coil strains were routinely cultured in LB medium at 37°C (Miller, 1972). Antibiotics added into the above media to grow transformed bacteria were kanamycin 50 ~tg/ml, tetracycline 20 [tg/ml and ampiciilin 100 ~tg/ml, depending on the particular plasmid. To detect Azotobacter strains expressing gusA or lacZ genes, the selection media were supplemented with 50 ~tg/ml 5-bromo-4-chloro-3-indolyl-~-D, glucuronide (X-Glue) or 5-bromo-4-chloro- 3-indolyi-l~-D-galactopyranoside (X-Gal), respectively. Gus activity of transformants was quantitated spectrophotometrically using the substrate p-nitro- phenyl-[i-D-glucuronide as described by Jefferson (1987).

Isolation of plasmid and genomic DNA

Plasmid DNA was prepared from E. coli and A. vinelandii by the alkali lysis method and purified further with phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation (Maniatis et al., 1982). The rapid screening of plasmids present in transformed azotobacters was performed with agarose gel electmphoresis of minipreps obtained by the "PlasmidPURE" DNA "Miniprep Kit" (Sigma Chemical Co., St. Louis, MO), To isolate genomic DNA from Azotobacter, the protocol of Marmur (1961) was modified. After low speed centrifuga- tion, the bacterial pellet was resuspended in Tris- EDTA (TE)pH 8.0 buffer containing 0.5 % SDS and 50 ~tg/ml proteinase K and incubated for 3 h at 50°C. After phenollchloroform/isoamyl alcohol extraction, a 0. l volume of 3 M sodium acetate was added to the aqueous phase. DNA was then precipi- tated with isopropanol, washed with 70% ethanol, dried briefly on air and dissolved in TE buffer. DNA quantification was made using a "Hoefer TKO 100 Mini-Fluorometer" (Hoefer Scientific Instruments, San Francisco, CA) as recommended by the manu- facturer.

Transformation of azotobacters

E!ectroporation was performed with an "E. coli Pulser" (Bio-Rad Laboratories) permitt ing the adjustment of voltage in the range of 0.20-2.50 kV.

The apparatus operated at a fixed time constant of 5 ms and is meant for use with samples of high resistance (2000-5000 ~). To prepare such suspen- sions of recipient cells, bacteria were grown with vigorous shaking to an OD o 0 of approximately 0 4-

2 . o" 0.5 at 30°C. Cells were harvested by centnfugauon at 5,000 rpm for 10 min at 0°C and resuspended in ice-cold 10 % sterile glycerol of the same quantity as the original culture volume. This step was ~epeated three times using a half and a quarter the original volume, and finally 2-4 ml of the glycerol solution. The final cell concentration reached was about 7.0× 107 eell/ml and 40-[Ltl aliquots were frozen in liquid nitrogen and stored at -70°C prior to use; a 40-~tl cell suspension was mixed with 1 ~tl plasmid DNA (300 ng/l.tl) in a sterile "Gene Pulser/E. coli Pulser" cuvette (Bio-Rad Laboratories, Hercules, CA) with an interelectrode distance of 0.1 cm, and one pulse was applied delivering 18.0 kV/cm field strength for approximately 5 ms; 1 ml AG medium was added immediately to the cuvette and resuspended cel ls were incubated wi th shaking at 200 rpm for 1 h at 30°C. Bacteria were then plated onto selective AG media containing inhibitory concentrations of antibiotics.

In control experiments, t ransformat ion was induced by iron and molybdenum deprivation using the modified protocol of Renaud et al. (1989). Azot- obacters present in 50 lal TF medium (OD620=0.5) were mixed with 10 lal plasmid DNA (300 ng/lal) and incubated for 1 h at 30°C. After resuspension in TF medium, they were further incubated for 2 h and then plated onto selective media.

Confirmation of transformants

Plasmid preparations obtained from transformed and non-transformed bacteria were analysed first by electrophoresis in 1.0 % agarose gel at 80 V constant voltage and ethidium bromide staining, and then by PCR using primers specific to plasmid reporter genes, and following Southern transfer and hybridization with digoxigenin (DIG)-labelled probes.

PCR amplifications were performed in a "Hybaid Thermal Reactor" (Hybaid Ltd., Teddington, UK) using standard reaction components. Forward (fwd) and reverse (rev) primers were designed and synthe- sized or selected according to the gus and lac sequences present in pBIl21 and pJB3, respectively. For lacZ, 20 mer primer LacZ1 fwd (5'-CTG-CGC- AAC-TGT-TGG-GAA-GG-3') and 22 mer primer S- 22 rev (5'-TCA-CAC-AGG-AAA-CAG-CTA-TGA- C-3') were used, whereas gusA was amplified by 18 mer primers pot I fwd (5'-TGA-CGT-AAG-GGA- CGA-CGC-3') and pot2 rev (5'-AAT-CAT-CGC- AAG-ACC-GGC-3'). These sequences were specific to the corresponding structural genes with the excep-

E L E C T R O T R A N S F O R M A T I O N OF D I A Z O T R O P H I C BACTERIUM 365

tion of sequencing primer S-22 which recognized the last 17 bases of lac operator and the first 5 of lacZ Amplifications were achieved by "DynaZyme II" thermostable DNA polymerase (Finnzymes Oy, Espoo, Finland) in 30 cycles of denaturation and extension at 95°C and 72°C, respectively. Annealing temperatures depended on the Tm values of primers and 57°C proved suitable for gus- and 48°C for lac- amplifying sequences. PCR products were checked on 1.0% agarose gel containing ethidium bromide.

Plasmid reporter gene-specific DIG-labelled probes were used in Southern blot hybridization with Azotobacter genomic DNA Bell restriction fragments. PCR-generated probe sequences purified on "QIA- quick-spin" PCR columns (Qiagen, Hilden, Germany) were labelled with DIG-dUTP as described in the "DIG DNA Labeling and Detection Kit" protocol of Boehringer (Mannheim, Germany). Genomic DNA restriction fragments were separated and blotted according to standard procedures (Maniatis et al., 1982). "Duralon-UV" (Strategene, La Jolla, CA) non- charged nylon membranes were used and the transferred fragments were cross-linked with a "Strata- linker UV Crosslinker" (Stratagene, La Joila, CA). Prehybridization (1 h) and hybridization (6 h) were performed at 68°C using the same buffer (5xSSC, 1.0% blocking reagent, 0.1% N-lauroylsarcosine and 0.02 % SDS). After hybridization with about 60 ng labelled DNA, filters were washed with 2 ×SSC, 0.1% SDS (2x5 min) and0.1×SSC, 1.0% SDS (2× 15 min) and used for direct detection vf hybridized DNA. Hybrids were visualized by enzyme- linked immunoassay using an antibody conjugate and subsequent enzyme-catalysed colour reaction.

General characterization of transformants

Colony morphology was examined on AG plates and growth rates were determined by OD measure- ments at 620 nm in liquid AG and N-free AG cultures. Media were supplemented with antibiotics as described above. Microscopy and nitrogen fixation assay were performed by previously reported methods (Kor~inyi et al., 1993).

RESULTS

Electroporation of A. v inelandi i with plasmid DNA

To obtain high electrotransfommtion frequencies and to determine optimal plasmid/cell ratios, a relatively low cell density was used in combination with high DNA concentration. Electropora-

tion conditions were adapted to a viable cell number of 7 . 0 x 10 7 cell/ml which was routinely reached by the employed harvesting protocol. Transformation frequencies, in contrast to those observed in natural transformations, were not influenced by the OD of culture within the log phase of growth; however, to ensure a more direct comparison with natural transformation, cells were grown to an OD620 of about 0.5 corresponding to the early-log phase. Transformation frequency increased with increasing DNA concentration over several orders of magnitude, but above the level of 600-900 ng DNA per 10 .7 cell (680 ng for pJB3, 960 ng for pBll21 and about 800 ng for pKOK5 derivatives), transformation efficiency decreased markedly. Based on these results, the ideal plasmid/cell ratio seemed to be about 104 (1.36× 104 for pJB3, 1.05×104 for pKOK5 and 7 2 2 × 103 for pBI121 derivatives). Transformations with pFAJ31.2 and pKOK5 derivatives were performed only with the covalently-closed circular forms, because digestions with mapped restriction endonucleases led to the fragmentation of these plasm~ds. It was not possible to obtain consistent and reproducible results with linearized pBl 121, while circular and Clal- digested pJB3 DNAs transformed the bacteria with similar frequencies.

Transformation efficiency and cell survival were also related to the electrical variables. The high resistance of samples and the brief pulse length made possibte successful electrotransfor- mations with exponentially decaying fields of very high amplitude. The number of transformants showed a nearly linear increase with the field strength up to 18 kV/cm, but above this value the survival of cells decreased dramatically when 0. l-cm cuvettes were used. Consequently, and for practical reasons of simple routine application, a peak field strength setting of 18.0 kV/cm and intact circular plasmids in about 1.0 ~tg DNA/I() v cell concentration were used in the subsequcnt experiments. Table II shows the efficiency and frequency values obtained during the transtbrmation of A. vinelandii with plasmids carrying selectable and screenable marker genes. With plasmid pFAJ31.2, electrotransformants were not detected when it v, as used in the 1.0 ng-l.0 lag/107 cell ~angc, while in the control experiments, colony


Table II. The efficiency and frequency of electroporation and control transformation of A. vinelandii with different plasmids carrying selectable and screenable marker genes.

Markers Plasmid Selectable Screenable

Control transformation Efficiency Frequency

Electrotransformation Efficiency Frequency

pFAJ31.2 Tc r Gus 1.42x 10 2 2.58x 10 -4 - - m pBll21 Km r Gus - - - - 2.57× 10 4 2.70x 10 -2 pKOK5 Apr Km r Lac 1.22x l02 2.22x l 0 -4 2.41× 105 2.53× l0 -i pJB3 Apr Lac 4.87x 10 2 8.94x 10 -4 6.80× 10 "s 7.14X 10 -I

The efficiency of transformation is defined as the number of transformants per microgram plasmid DNA. The frequency of transformation is defined as transformation efficiency per total number of viable cells.

formation was observed on plates containing 10.0 gg per ml tetracycline. These cells, similarly to ~ 3 natural transformants, formed slow-grow- ing colonies on selective plates and did not show any detectable reporter gene activity. Circular or linearized pBll21 DNA preparations were inef- fective in control experiments even when transformation mixtures were transferred into AG medium with subinhibitory kanamycin concentration. Successful transformations with pFAJ31.2, pBll21 and pJB3 were otherwise verified by the growth on media amended with 10.0 lag per ml tetracycline, 50.0 lag per ml kanamycin and 100.0 ktg per ml ampicillin, respectively. Each of these antibiotics (in 1.0 ktg per ml concentration) completely i~ibi ted the growth of wild-type A. vinelandii. The use of subinhibitory antibiotic concentrations accelerated the appearance and growth of elec~transformants, but did not influence their frequency. They were detected within 48 h after plating into selective media and the postpulse incu- bation time had no effect on the number of viable transformants when cultures were shaken vigour- ously during this period. Transformation efficiency increased with the decrease in plasmid size, but always exceeded about 10 3 times the efficiency of controls (table II).

of reporter genes. Non-transformed bacteria, iron-limited controls and electrotransformants derived from pJB3 did not show colour reactions characteristic of the expression of gusA or lacZ. Electrotransformants originating from pBl 121 were surrounded by blue zones on X-Gluc plates, indicating the presence of Gus activity. Its mean value, determined in three independent experiments in t r ipl icate , was 1.20 nmol nitro- phenol/min/107 cells. One possible explanation for this gusA expression was the unexpected effectiveness of the CaMV 35S promoter in Azot- obacter. E. ccli and Azosipirilum brasilense pro- moters, on the other hand, did not facilitate the expression of downstream structure genes.

LacZ activity had already been demonstrated in Azotobacter, and promoter probe vector pKOK5 was used in some experiments to prove that the developed electroporation procedure can also ensure the expression of this gene. To this end, Azotobacter genomic DNA was partially digested with SaulllA and the resulting fragments ligated into the BamHl sites of the plasmid pre- ceding its lac sequences. Alter electroporation, blue and white colonies were selected on X-Gal plates containing Ap and Kin.

Detection of reporter gene activity

Translbrmants selected according to the acquisition of stable antibiotic resistance were further screened for the presence and expression

Confirmation of plasmid and host authenticity

Electrophoretic analysis of plasmid isolates proved the presence of transforming vectors in the selected transformants and showed that the original bacterial strain does not harbour any kind

ELECTROTRANSFORMATION OF DIAZOTROPHIC BACTERIUM 367

of plasmids. In the case of pBIl21 and pJB3 transformants, plasmid bands were present which migrated with the same mobility as the covalently closed circular forms of the original vectors. This was also true for bacteria transformed with linearized pJB3.

The utility of PCR-based techniques was evaluated for the rapid screening of plasmid identity and reporter gene integration into the bacterial genome. Primers potl and pot2 were designed to amplify gusA present in pBIl21, cloned between the CaMV 35S promoter and nopa l in synthase t e rmina to r regions of pBINI9, whereas LacZI and $22 were suitable for the amplification of iacZ and the terminal region of the lac operator . Primer pairs potl +pot2 and LacZl +$22 detected a 1.87- kbp gusA fragment in electrotransformants originating from pBI121, and a 232-bp lacZ fragment in those derived from pJB3 (fig. 1).

These sequences were amplified from both genomic and plasmid DNA preparations and were found to be identical to those present in the original transforming vehicles. GusA and lacZ could not be amplified from non-transformed A. vinelandii DNA.

To check the reliability of PCR data and to study the integration of plasmid DNA into the wild-type A. vinelandii genome, Southern analysis was performed with the use of probes containing gus and lac sequences. The results of control hybridizations with reporter genes and plasmids carrying them indicated that the probes were specific. Souther.~ analyses, in accordance with PCR results, showed the integration of plasmid sequences into the host chromosome (fig. 2.). In the case of pBll21 transformants, the gus probe hybridized to two Bcll fragments with molecular sizes of 1.9 and 2.2 kb. The lac probe hybridized to 2.1- and 3.8-kb

l a c gus A B A B

Fig. 1. PCR products derived from wild-type and transformed A. vinelandii separated in 1.0% agarose gel and

stained with ethidium bromide.

Oligonucleotide primer pairs LacZl fwd+S22 rev and potl fwd+pot2 rev were used to amplify 232-bp lac and 1.87-kbp gus fragments from plasmid and genomic DNA isolates originating from wild-type azotobacters and those electrotransformed with pJB3 and pBI121. Lanes: 1 =original plasmid pJB3; 2 and 6=genomic DNA of wild-type azotobacter used in transformation experiments; 3=plasmid pJB3 reisolated from transformants; 4=genomic DNA isolated from bacteria transformed with pJB3; 5=original plasmid pBll21 ; 7=plasmid pBll21 reisolated from transformants; 8=genomic DNA isolated from bacteria transformed with pBll21. Size position markers of 2,176, 1,766, 1,320, 1,033, 653, 517, 453, 394, 298, 234/220 and 154 bp are shown in the middle.

-2.2kb

-1.gkb

Fig. 2. Southern blot analysis of wild-type (A) and electrotransformed (B) azotobacters to confirm the chromosomal incorporation of iacZ and gusA reporter genes introduced into bacteria by pJB3 and pBll21 plasmids,

respectively.

Bcll-digested genomic DNA was probed on a Dura- ion-UV no, n-charged nylon membrane with DIG-labelled lac- and gus-specific probe sequences generated by PCR.


Bcll fragments of pJB3 transformants. Hybrid- ization patterns of bacteria transformed with the same plasmids were similar when the same restriction endonuclease-probe combination was used. ~ e results of some additional South- ern analyses using BamHI and Hindl l l were in line with these observations (data not pre- sented). It was also shown that the genomic DNA of non-transformed A. v i n e l a n d i i CCM289 did not contain any regions with significant homology to the reporter genes.

Side effects of transformations

In natural transformants, similar debilitating effects were observed as described previously (Glick e t al . , 1985, 1986; Renaud et al . , 1989). They grew very slowly only on solid media supplemented with N, and showed a decreased cell size, altered cell and colony morphology and the lack of reporter gene activity. Microscopically, the transformed cells were spherical rather than ovoid and generally half the size of wild-type bacteria. The colonies of pFAJ31.2 and pJB3 transformants were mostly clear, large and mucoid. Eiectrotrans- formants could not be differentiated from wild-type bacteria according to their general cell and colony morphology and growth characteristics (fig. 3). In liquid AG medium, A. v i n e l a n d i i CCM289 and its transformants showed a relatively long lag period (about 24 h)followed by a typical log phase (generation time, about 2.3 h). In N-free liquid media, contrary to the rapid growth on solid media, this strain proliferated very poorly and cell density was a nearly linear function of time (generation time, about 7.5 h). Electrotransfor- mants grew similarly in N-free AG medium with indistinguishable lag and log phases. On AG and N-free AG media, they fixed atmospheric nitrogen similarly, showed Lac or Gus activity and, according to the appearance of a bright yellow-green colour, produced azotobactin. Loss of plasmids or marker gene expression was not observed during the pro- longed cultivation of electrotransformants with antibiotic selection.

1.2

1

0,8

mE 0,6

0,4

0,2

0

0

o-

i i i i i ~ t q 8 16 18 20 22 24 26 28 30

Time, h

. . . . b i

- _ - - C

B

0,5 ; 0,45 ~

0,4 -

l 0,35 . I

0,3 . .~ • " " ~ ~ a ! 0,25 .... ~ ~"-

0,2 . . _ ~ . . . . . b

0,15 .,..,..¢~ ...- - - - - - - c

0,1 0,05

0 i t J i

0 8 16 18 20 22 24 26 28 30

Time, h

Fig. 3. The growth kinetics of A. vinelandii CCM289 on AG medium (A) and N-free AG medium (B).

a = non- t ransformed cells ; b = pBl 121 - t ransformed cells; c =pJB3-transformed cells.

DISCUSSION

Artificial symbioses between azotobacters and plants were originally characterized using techni- cally sophisticated and sometimes less reliable techniques of electron microscopy and gas chromatography (Gyurjdn et al., 1995; Korfinyi et al., 1993). Suitable reporter systems, used with success in several other bacterial species, should assist the detection and localization of symbiotic azotobacters. However, traditional transformation methods of CaCI 2 treatment or iron limitation may lead to low transformation frequencies (David et al., 1981), the morphological and metabolic debilitation of bacteria (Glick et al., 1985, 1986; Renaud et al., 1989) or the loss of plasmids and marker genes (Doran et al., 1987; Renaud et al., 1989; Robinson et al., 1986) even in the case of capsule- strains. The competence of encapsulated azotobacters is low and consequently the natural transformation of A. vinelandii CCM289 was also poor. The only documented azotobacter

ELECTROTRANSFORMA TION OF D1AZOTROPHIC BA CTERIUM 369

electrotransformation procedure (Trevors and Starodub, 1990), proved unsatisfactory, possibly due to the differences in strains, media and electrical parameters. To overcome these difficulties, a new electroporation protocol was elaborated for the transformation of the symbiont candidate strain with plasmids carrying selectable and screenable genes. The gradual concent ra t ion of cel ls in glycerol and the employed media and electrical conditions led to the introduction of differe,at plasmids with the exception of pFAJ31.2, which may be the consequence of the rather large size of this vehicle. The frequency of transformation was lower than with E. coil ( i 09-10 l0 transformants per ~tg DNA), but generally reached or even exceeded the values obtained in other Azoto- bacter transformation experiments. In addition to the above m e n t i o n e d factors , this is explained by the high resistance of samples, the exponentially decaying electrical field of high initial peak voltage and the short pulse length. Other reports had also emphasized the impor tance of these electr ical parameters (Dower et al., 1988; Miller et al., 1988). The plasmid to cell ratio also played a decisive role, and with the selection of optimal values (about 10 4 plasmid per cell), which seemed to corre- late with each other on a molar basis, maximal transformation frequencies were reached. For the 12.8-kb pBI 121, this ratio was 7 x 10 3, while for the 11.1-kb pRK2501, a similar value (2× 103 ) was calculated based on the results of Trevors and Starodub (1990). In the well-documented natural azotobacter transformations, as deduced from the experimental data (Glick et al., 1985; Doran et al., 1987; Bingle, 1988), at least one order of magni tude higher ratios (104-105) had been required to obtain maximal transformation frequencies.

Following the electroporation of plasmids pBll21, pJB3 and pKOK5 derivatives into azotobacters, stable acquisition and maintenance of resistance determinants were observed and transformants could tolerate 100-200 times the lowest inhibitory concentrations of the relevant antibiotics. The CaMV 35S promoter of pBl121 and Azotobacter genomic sequences with promoter functions in pKOK5 made possible the expres-

sion of gusA and lacZ in the electrotransformants, while reporter gene activity was not observed under the control of E. coli and Azospirillum pro- moters. Natural transformants exhibited obvi- ously impaired morphology and metabolic functions and did not show any reporter gene activity. These observations may be explained by the pro- longed iron and molybdenum deprivation arising during transformation alone or in combination with the metabolic load often accompanying foreign plasmid replication and heterologous gene expression (Glick, 1995) or by an incomplete genetic transformation which may include the uptake of foreign DNA without its perfect integration and expression. Electrotransformants expressed resistance and reporter genes without the development of debilitating effects. The similar growth characteristics of wild-type and transformed bacteria and their azotobactin production both in the presence and absence of fixed nitrogen contradicted some of the earlier observations (Glick et al., 1985) and indicated the metabolic intactness of electrotransformants. These findings are quite remarkable because azotobacters forced into symbiotic partnerships should retain their competitiveness under conditions which may be harsher than in the natural habitats.

In former experiments, the introduction of plasmids into A. vinelandii had led to plasmid establishment without chromosomal integration (Glick et aL, 1985, 1986), the integration of plasmid-encoded genes into the host chromosome accompanied by the loss of plasmid as an independent replicon (Doran et al., 1987; Renaud et al., 1989; Robinson et aL, 1986), or the parallel occurrence of plasmid establishment and integration (Bingle, 1988). Sequence homology-facili- tated recombination had also been regarded as a prerequisite for plasmid establishment, but later it became evident that the initial establishment of plasmids in some A. vinelandii strains was possible in the absence of homologous plasmid and chromosome sequences - - and consequently without recombination (Bingle, 1988; Doran et

al., 1987).

The establishment of foreign plasmids was demonstrated in the selected electrotransformants, and Southern analysis revealed the parallel


integration of reporter genes into the host chromosome - - possibly by site-specific recombination, since transformants harbouring the same plasmid had similar hybridization patterns. Com- plementary regions were clearly outside the struc-

genes, since probes specific to reporter genes failed to reveal any significant homology with the wild-type genome, and the CCM289 strain had no natural resistance to the antibiotics used. The parallel occurrence of plasmid establishment and chromosomal integration of plasmid-encoded sequences did not resulted in host impairment, which may be explained by the integration of plasmid sequences at functionally less important loci, This was also emphasized by Renaud et al. (1989) as a key factor in successful transformation, although the hypothesis needs further experimental proof. Southern analysis also supported the usefulness of PCR amplifications which, in addition to confirming the presence of reporter genes on plasmids isolated from electrotransformants, could detect the chromosomal integration of foreign genes without producing false-positives.

The method described made possible the introduction of useful foreign genes into A. vinelandii and may promote the practical utilization of gus fusions in the related symbiosis research, as in other plant systems (Jefferson et al., 1987; Vande Brock et al,, 1993). The lack of metabolic stress after ~ s f o r m a t i o n and the chromosomal integration of desired genes under the control of appropriate regulatory elements may be among the factors which explain the success of the devised procedure. Glick and his coworkers reported on the development of metabolic impair- merits after natural transformation of wild-type encapsulated A. v inelandi i ATCC 12837 with broad-host-range plasmids and explained this phenomenon by foreign plasmid replication and gene expression. Using the same method for the transformation of wi ld- type encapsulated CCM289, transformants with similar impaired morphology and metabolic functions appeared. These findings suggest that encapsulated azotobacters are more sensitive to the treatments employed during natural transformation. How- ever, the observed effects may not be closely related to plasmid replication or plasmid gene

expression, since the same plasmids were used in the electroporation experiments which provided transformants with normal functions. Capsule- OP derivatives did not show significant impairment of nitrogen-fixing ability after natural transformation (Bingle, 1988), while the loss of plasmids was f requent ly observed in similar experiments (Doran et al., 1987; Renaud et al., 1989; Robinson et al., 1986) making plasmid replication and plasmid gene expression ab ovo impossible. These observations also suggest that plasmid establishment, compared to the appropriate chromosomal integrat ion of plasmid sequences, may have only a secondary role in successful azotobacter transformation - - at least in the case of the reporter genes used here.

Acknowledgements

We thank Svein Valla (UNIGEN Center for Molecular Biology, Norway) for providing the plasmid pJB3, Florence Ars~ne (Institute Pasteur, France) for providing the plasmid pKOK5 and Veede Keijers (CUL, F.A. Janssens Laboratory of Genetics, Belgium) for providing the plasmid pFAJ31.2.

This work was supported in part by the OTKA NO 25651 grant.

Electrotransformation stable d'un candidat symbionte diazotrophique

avec des plasmides porteurs de gimes marqueurs permettant un criblage sElectif

Nous avons 6valu6 les capacit6s de l'61ectropora- tion en utilisant les ADNs des plasmides pJB3 (4,8 kb), pBll21 (12,8 kb) et pFAJ31,2 (24 kb) contenant des g~nes marqueurs qui permettent la s61ection (Ap, Km et Tc) et le criblage (gusA et lacZ). Les m6thodes de pr6paration de Azotobacter vinelandii CCM289 comp6tent et d'61ectroporation (18 kV/cm, 5 ms, 0°C) ont fourni 6,8x 105 transformants par ~tg d'ADN plasmidique, ce qui represente une efficacit6 de transformation 1.000 fois sup6- rieure ,~ celle des t6moins. Aucun 61ectrotransfor- mant n'a 6t6 obtenu avec le plasmide pFAJ31,2. La taille de I'ADN plasmidique n'influence pas signifi- cativement l'efficacit6 de transformation. Les transformants sont capables de croitre en pr6sence de concentrations d'antibiotiques 100 h 200 plus fortes que celles qui inhibent la croissance des bact6ries sauvages. Une expression constitutive du g6ne gusA est observ6e chez des transformants pBll21 avec

E L E C T R O T R A N S F O R M A T I O N OF D I A Z O T R O P H I C B A C T E R I U M 371

des fusions promoteur CaMV 35S-gusA alors que l ' e x p r e s s i o n de l a c Z n ' e s t pas dEcelEe sous le contr61e du promoteur lac chez les transformants pJB3. Les plasmides d'Electroporation sont rEisoiEs chez les t ransformants dans leur forme originale alors que les bactEdes non transformEes ne contien- nent pas de plasmides indig/mes. La PCR et l 'hybri- dation de type Southern dEmontrent l'intEgration de I 'ADN plasmidique dans le gEnome de l 'h6te. Les transformants gardent leur aptitude h fixer i 'azote et ils ont des caract~res normaux de morphologie et de croissance. La stabilitE de i ' A D N plasmidique de Azotobacter peut assurer ia transformation ainsi que la detection de tels candidats symbiontes.

Mots-clC, s : Transformation, Azotobacter vinelandii, Plasmide, Electroporat ion; G~nes marqueurs, IntEgration chromosomique, Symbiose.

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