Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 15, Num. 4, 1998

Santiago Dueñas, @ Rolando Pajón, Maité Delgado, Erick C Nogueiras, Alejandro Martín

Centro de Ingeniería Genética y Biotecnología. AP 6162, CP 10600, Ciudad de La Habana, Cuba. Fax: (53-7) 218070; E-mail: ricardo.silva@cigb.edu.cu

Code Number: BA98040
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ABSTRACT

In this paper we describe an electroporation protocol for Neisseria meningitidis. Plasmid vectors up to 12.5 kb, containing a selection marker, flanked by genomic regions, were constructed and used to select electroporation conditions. When nonpiliated B385 cells, suspended in electroporation buffer (glycerol 15%; MgCl2 1 mM; Sucrose 272 mM; HEPES 1 mM; pH 7.2), were mixed with 200 ng of plasmid DNA and a single pulse of 11.5 kV/cm, 400W and 25 mF was applied (in a BioRad Gene Pulser II), transformation efficiencies up to 6.5 x 104 transformants/mg and survival rates of up to 80% were reached. The generation with this method, of an lpdA neisserial knock-out mutant, is also shown. To date this is the first electroporation report in meningococci.

Keywords: electroporation, N. meningitidis, transformation efficiency

En este artículo describimos un protocolo de electroporación para Neisseria meningitidis. Varios plasmidios de hasta 12,5 kb, conteniendo regiones genómicas interrumpidas por un marcador de selección, fueron construidos y usados para establecer las condiciones de electroporación. Cuando se mezclaron células de B385 no piliadas, suspendidas en el tampón de electroporación (15% glicerol; 1 mM MgCl2; 272 mM sacarosa; 1 mM HEPES; pH 7,2), con 200 ng de ADN plasmídico, se obtuvieron eficiencias de transformación que alcanzaron 6,5 x 104 transformantes/mg con porcientos de supervivencia alrededor de 80. También se muestra la generación, a través de este método, de un mutante de N. menigitidis para el gen lpdA. Este constituye el primer reporte describiendo la técnica de electroporación aplicada a meningococo.

Palabras claves: electroporación, N. meningitidis, eficiencia de transformación

Transformation is currently the most feasible option for exploring the genetics of Neisseria meningitidis, given that transducing phages, or efficient conjugative systems, are not available for the Neisseriaceae [1]. Like other bacterial species, this organism is naturally competent for genetic transformation. However, standard molecular techniques for transformation, used in Escherichia coli, are hardly applicable for N.meningitidis. The in vitro transformation of meningococci is dependent on a number of bacterial functions, most notably: the host piliation status, and the presence of species-specific sequence motifs in the transforming DNA. Both factors affect the first stages of DNA transfer, during which nucleic acids traverse the bacterial envelope [2, 3]. Unfortunately, even as few as two rounds of laboratory culture passages can render neisserial strains refractory to transformation, due to the quick appearance and take over of non-piliated, non competent variants [4]. This situation is sometimes compounded by the absence of uptake motifs in the genetic constructions under study.

Previously reported transformation in Neisseria gonorrhoeae, using electroporation, support this technique as well suited for overcoming the above limitations [5]. Many species have been transformed by using this methodology [6, 7]. Campylobacter jejuni [8] and E. coli [9] have served as models for field strength, time constant (pulse duration), temperature, host cells, electroporation media, and DNA concentration optimization. However, despite the relatively simple concepts that apply to electroporation, the relatively complicated, or unknown parameters, inherent to each biological system under study demand a close monitoring of electroporation conditions. In this article we describe the development of a novel and simple electroporation protocol to transform nonpiliated variants of the N. meningitidis strain B385.

Bacterial strains used in this study included XL-1 Blue, an E. coli K12 strain, and B385p-, a nonpiliated variant from N.meningitidis strain B385 (B4:P1.15). Meningococcal cells were grown in a Brain Heart Infusion (BHI) broth, or on BHI agar plates, at 37°C in a candle jar. XL-1 Blue was grown in a Luria-Bertani (LB) broth. When needed, Ampicillin, or Kanamycin, was added at a final concentration of 50 mg/mL.

The methods used for in vitro manipulation of DNA (plasmid purification, restriction analysis, Southern blots) were performed according to Sambrook, et al. [10]. Chromosomal DNA extraction from meningococci was performed following the procedure described by Cornelissen et al, 1992 [11].

Expanded colonies from the transformation plates, were inoculated into 5 mL of BHI cultures overnight at 37°C. The following day, the cultures were harvested and 20 mg of total cellular proteins were used for SDS-PAGE [12] and Western blotting [13] with MAb114, a monoclonal antibody elicited against the C-terminal region of the LpdA protein. The immune complexes were visualized by using 3-amino-9 ethyl-carbazole.

pM-150: pUC18-derived recombinant plasmids obtained from subcloning a 9 kb KpnI-SalI library fragment from N. meningitidis, interrupted by a 1.2 kb Kanamycin resistance marker from pUC4K [14].

pM-110: pUC18-derived recombinant plasmid containing one copy of the 1.2 kb Kanamycin resistance marker, from pUC4K, flanked by 207 and 253 bp from 5' and 3' regions of the lpdA gene.

pM-84: Recombinant plasmid containing one intact copy of the lpdA gene, in pUC18.

300 mL of the BHI broth were inoculated from a fresh plate of B385p- to a final OD640nm = 0.1 (in Photoelectric colorimeter, Model AE330, EOW, LTD) and grown at 37°C to exponential phase (OD640nm = 0.8-0.9). Cells were harvested by centrifugation (6000 rpm, 4°C, 5 min), suspended in 150 mL of ice-cold electroporation buffer (EB: glycerol 15%; MgCl2 1mM; Sucrose 272 mM; HEPES 1 mM; pH7.2), and washed twice with 40 and 20mL in the same EB. The cells were suspended in a final volume of 400 mL EB. High density aliquots (over 109 colony forming units (cfu)/mL) of cell suspension (40mL), were frozen at -70°C or mixed with 1 mL of undigested plasmid DNA in a TE 0.1X solution, and transferred immediately to a 0.2cm electrode gap pre-chilled Gene Pulser cuvette (0.1cm electrode gap cuvettes were used to reach field strengths above 12.5 kV/cm), just prior to the electric pulse with the BioRad Gene Pulser II. The parameters were set in the following ranges: Voltage, 8.0-14 kV/cm; Capacitance, 25-50 mF; Resistance, 100-600 W. After pulsing, 1 mL of BHI was immediately added to the cells, followed by 3 h of incubation at 37°C before plating and selection for Kanamycin resistance.

The cells were plated both onto a selective medium for the detection of plasmid recipients and onto a non selective medium for the determination of survival rate. Transformants were visible 16-20 h after plating, and individual colonies were further isolated by serially streaking twice onto a selective medium before additional analysis.

The effect of different field strengths on the electroporation efficiency of B385p- was determined using 1 mg of the plasmid pM-150, a single pulse with a capacitance of 25 mF and a resistance of 400W resulting in pulse lengths of 6-9 ms.

The best transformation efficiency was obtained using a single pulse of 10-12.5 kV/cm. When the above conditions were used, strain B385p- consistently gave 103 transformants/mg with a 60% survival rate. Higher field strengths (13-15 kV/cm) resulted in survival rates lower than 30% and thus a decrease in overall transformation efficiency. Lower values (8-9.5 kV/cm), while yielding higher survival rates (90%), were unable to achieve the same electroporation frequency, even when longer pulse lengths were used (data not shown).

Figure 1 shows the influence of pulse length in transformation efficiency, using the above conditions and a constant field strength of 12.5 kV/cm. The best pulse time peaks were around 8.31 ms, with a drop in transformation efficiency above 8.78 ms.

To ascertain the dependence of the electroporation efficacy on the total amount of DNA, several assays were set up using a single pulse of 11.5 kV/cm with a capacitance of 25 mF and a resistance of 400 W. The results for varying amounts of DNA are shown in Figure 2. As can be seen, there is a steep increase in efficiency which levels off at 50 ng and starts to decrease above 200 ng. Pulse lengths of 6.3 to 7.7 ms were reached within the useful 50-200 ng range. This further improvement allowed us to obtain more than 104 transformants/mg of plasmid DNA.

We applied these parameters to obtain neisserial lpdA knock-out mutants. A plasmid containing a Kanamycin resistance marker flanked by 207 and 253 bp from 5' and 3' regions of the lpdA gene (pM-110, see Materials and Methods) was used to electroporate B385p-. Since pM-110 does not replicate in meningococci, Kanamycin resistant transformants can be rescued only in the event of a single (insertion and gene duplication) or double (allelic replacement) recombination between the sequences flanking the selection marker and their genomic counterparts. All eight transformants tested by Western blot with MAb114 (Figure 3) failed to express the protein, suggesting that allelic replacement had occurred in all cases. This conclusion was confirmed by Southern blot analysis of 4 transformants (Figure 4) with an internal 300 bp ClaI fragment from the lpdA obtained from pM-84 as a probe.

We searched for an efficient electrotransformation protocol of N. meningitidis as a feasible way for the generation of knock-out insertional mutants of chromosomal genes. As a model we used a 9 kb KpnI-SalI library fragment from N. meningitidis B385 interrupted by a Kanamycin resistance marker. With pM-150, the best results were obtained by using cells grown on a liquid BHI medium, harvested during the early exponential phase, washed and suspended in the referred electroporation buffer, in aliquots of cell density above 109 cfu/mL, which were mixed with 50-200 ng of DNA in a low conductivity solution. A single electric pulse of 11.5kV field strength reaching nearly 8 ms in length with a capacitance and resistance of 25 mF and 400W, respectively, was applied.

Field strength and pulse length were selected according to a compromise between transformation efficiency and survival rate. Extreme values of these electrical parameters drastically reduced cell viability or failed to induce high DNA acquisition, respectively.

No transformants were obtained when older cultures were used (data not shown). A similar decrease in transformation efficiency has been reported for Streptococcus pyogenes [15]. Negative results were also obtained when cells were taken directly from plates for electroporation, probably because lower viable cell densities were reached (data not shown).

The number of transformants dropped dramatically when amounts of DNA higher than 200 ng were added. A possible explanation to this phenomenon could be the occurrence of cotransformation events affecting cell physiology. However, we can not rule out the presence of contaminating elements in our DNA preparations as a probable cause, since the influence of alternative DNA purification methods was not evaluated.

Using the conditions established in this work (50-200 ng of DNA, 11.5 kV/cm, 400 W, 25 mF in a BioRad Gene Pulser II), transformation efficiencies of up to 6.5 x 104 transformants/mg and survival rates of up to 80% were reached. It should be noted that the efficiency could be even higher if replicative vectors are used [16]. Another important point to investigate is the influence of size, topology, and source of DNA (linear DNA, single stranded DNA, PCR-derived DNA, etc.), in this process.

The generation of lpdA mutants by electroporation was extremely easy, avoiding a large process of selection for piliated competent variants, which is the alternative method. Our results prove the feasibility of electroporation as a methodology for the genetic manipulation of laboratory-adapted strains of N.meningitidis, where standard molecular techniques for transformation are hardly applicable and neither transducing phages nor efficient conjugative systems are available. The development of genetic tools for this species can be very helpful in the construction of new strains for vaccine development.

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