A New Vector for Insertion of Any DNA Fragment into the Chromosome of Transformable Neisseriae

Plasmid 44, 275–279 (2000) doi:10.1006/plas.2000.1490, available online at http://www.idealibrary.com on

SHORT COMMUNICATION A New Vector for Insertion of Any DNA Fragment into the Chromosome of Transformable Neisseriae Paola Salvatore,* ,† Giuseppina Cantalupo,* Caterina Pagliarulo,* Maurizio Tredici,‡ Alfredo Lavitola,* Cecilia Bucci,* Carmelo Bruno Bruni,* and Pietro Alifano‡ ,1 *Dipartimento di Biologia e Patologia Cellulare e Molecolare “L. Califano,” Universita` di Napoli “Federico II,” and Centro di Endocrinologia ed Oncologia Sperimentale “G. Salvatore” of the Consiglio Nazionale delle Ricerche, Via S. Pansini 5, 80131 Naples, Italy; †Dipartimento di Scienze Ambientali, Seconda Universita` di Napoli, Via Vivaldi 43, 81100 Caserta, Italy; and ‡Dipartimento di Biologia, Universita` degli Studi di Lecce, Via Monteroni, 73100 Lecce, Italy Received April 20, 2000 A useful method for inserting any DNA fragment into the chromosome of Neisseriae has been developed. The method relies on recombination-proficient vector plasmid pNLE1, a pUC19 derivative containing (1) genes conferring resistance to ampicillin and erythromycin, as selectable markers; (2) a chromosomal region necessary for its integration into the Neisseria chromosome; (3) a specific uptake sequence which is required for natural transformation; (4) a promoter capable of functioning in Neisseria; and (5) several unique restriction sites useful for cloning. pNLE1 integrates into the leuS region of the neisserial chromosome at high frequencies by transformation-mediated recombination. The usefulness of this vector has been demonstrated by cloning the tetracycline-resistance gene (tet) and subsequently inserting the tet gene into the meningococcal chromosome. © 2000 Academic Press Key Words: recombination; ermC; tet; leuS; genetic complementation.

Investigation of the genetic basis of pathogenicity of Neisseria meningitidis has been impaired by the lack of appropriate genetic tools. DNA-mediated transformation of the meningococcus is to date the only available tool for molecular genetic manipulation, and it has been used to target mutations onto chromosomal genes by allelic replacement with cloned mutated genes (Frosch et al., 1990; Stojiljkovic et al., 1995; Tonjum et al., 1995; Pettersson et al., 1998; Swartley et al., 1998; Lewis et al., 1999; Richardson and Stojiljkovic, 1999). In contrast, several shuttle vector systems have been developed in Neisseria gonorrhoeae. An autonomously replicating shuttle vector for the introduction of cloned genes into N. gonorrhoeae by transformation was originally obtained by fusing the gonococcal 4.2-kb cryptic plasmid (Davies and Normark, 1980) with the 1

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naturally occurring 7.2-kb ␤-lactamase plasmid (Stein et al., 1983a,b). However, this vector has never been used for genetic studies in meningococci. In addition, a system useful for genetic complementation of transformable and nontransformable N. gonorrhoeae mutants has been described more recently (Kupsch et al., 1996). This system relies on introduction of cloned genes into gonococcal ptetM25.2 via allelic replacement and subsequent mobilization by conjugation to recipients. Also this system has never been adapted to meningococci. This paper describes a new integrative vector capable of inserting genes or DNA fragments into a definite site within the chromosome of meningococci, gonococci, and several nonpathogenic Neisseriae, thereby overcoming the above-mentioned problems. In order to be suitable for genetic manipulation, an ideal vector should (1) be of a relatively small size, (2) be able to replicate into the intermediate host with high efficiency, (3) have 0147-619X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1. Physical and genetic map of plasmids pNLE1 and derivative pNLET1 and site-specific integration of pNLET1 into the N. meningitidis chromosome. (A) Physical and genetic map of plasmid pNLE1. pNLE1 was obtained by subcloning sequentially (1) a 1480-bp Sau3AI fragment, spanning the chromosomal leuS-dam region derived from plasmid pNMdam1 (Bucci et al., 1999), and (2) a 859-bp KpnI fragment, containing the ermC⬘ gene (Bucci et al., 1999), into the polylinker of pUC19. E. coli strain DH5␣ [F ⫺ ⌽80d lacZ⌬M15 endA1 recA1 hsdR17 supE44 thi-1 ␭ ⫺ gyrA96 ⌬(lacZYA-argF) U169] was used in cloning procedures. Unique restriction sites

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at least one selectable marker, (4) be able to transform the definitive host at a high frequency, (5) contain several unique restriction sites for cloning, (6) be able to drive expression of cloned genes, and (7) be versatile. All these are properties of plasmid pNLE1 (Fig. 1A). It is a pUC19 derivative of 5027 bp harboring (1) a DNA fragment spanning the leuS-dam region derived from plasmid pNMdam1 (Bucci et al., 1999), which is required for its integration into the neisserial chromosome by single cross-over, and (2) the structural gene for Bacillus subtilis RNA methylase (ermC⬘) conferring resistance to macrolide antibiotic erythromycin, as a selectable marker in meningococci (Monod et al., 1986). pNLE1 also contains an uptake sequence, located downstream of the leuS gene and required to transform Neisseria with high efficiency (Graves et al., 1982). Unique restriction sites are HindIII, SphI, PstI, XbaI, BamHI, SmaI, and EcoRI. Transcription of cloned DNA fragments in the SmaI site is driven by promoter sequences located upstream from the dam gene (Bucci et al., 1999). The complete nucleotide sequence of pNLE1 is available (Accession No. AF276982). To gain evidence of the usefulness of pNLE1, we cloned a 2581-bp ScaI–PvuII DNA fragment derived from plasmid pBR322, spanning the

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structural tetracycline-resistance tet gene, into the SmaI site (Fig. 1B). The resulting plasmid pNLET1 conferred tetracycline resistance (10 ␮g/ml) and erythromycin resistance (125 ␮g/ ml) to the otherwise sensitive Escherichia coli strain DH5␣. pNLET1 was used to transform the N. meningitidis strain BL915 to erythromycin resistance. This procedure led to the isolation of erythromycin-resistant clones (7 ␮g/ml) with a frequency of about 10 ⫺6 (number of transformants/number of viable cells) with 100 ng of plasmid DNA (Table 1). All these transformants were also resistant to tetracycline. The integration of pNLET1 into the chromosomal leuS-drg region (Fig. 1B) was verified by Southern blot analysis using drg-, ermC⬘-, or tet-specific probes (Fig. 1C). Hybridization of EcoRI-digested DNA of the parental strain with the drg probe showed the presence of the expected 6583-bp fragment on the basis of previous mapping and sequencing data (Bucci et al., 1999, and data not shown). In contrast, DNA from the transformed clones revealed an approximately 4200-bp-long fragment. This result was consistent with integration of pNLET1 into the leuS gene leading to production of a 4199-bp fragment. Both the ermC⬘- and the tet-specific probes evidenced a 2942-bp frag-

are indicated. Abbreviations used: Ap, ampicillin-resistance gene of pUC19; ORI, plasmid replication origin of pUC19. (B) Physical and genetic map of the meningococcal leuS-drg region and of plasmid pNLET1. pNLET1 was obtained by subcloning a 2581-bp-long ScaI–PvuII DNA fragment, derived from plasmid pBR322, spanning the structural tetracyclineresistance tet gene, into the SmaI site of pNLE1. Abbreviations used: E, EcoRI; S, SmaI; Sc, ScaI; Pv, PvuII. Abbreviations are the same as in A. In addition, US indicates the neisserial uptake sequence and ppk the structural gene encoding the polyphosphate kinase. Open boxes span the DNA regions used as probes in Southern blot experiments. The drg probe was obtained amplifying a 511-bp-long genomic region from meningococcal strain BL915 by PCR using the oligonucleotides 5⬘-ATAGGCAACAGCGTGCCTGACGG-3⬘ and 5⬘-TCGATCGTATTTTGGTCGCGC-3⬘ as primers. The ermC⬘ probe was obtained by amplifying a 785-bp-long region from plasmid Hermes6a (Kupsch et al., 1996) using the 5⬘ end-labeled oligonucleotides 5⬘-TAATGAACGAGAAAAATATAAAACACAGTC-3⬘ and 5⬘-GGTACACGAAAAACAAGTTAAGGGATGCAG-3⬘. The amplification reactions consisted of 30 cycles including 1 min of denaturation at 94°C, 1 min of annealing at 55°C, and 1–2 min of extension at 72°C. They were carried out in a Perkin-Elmer Cetus DNA Thermal Cycler 480. The 2581-bp-long ScaI–PvuII fragment, containing the tet gene from pBR322, was used as a tet probe. DNA fragments were isolated through polyacrylamide slab gels and recovered by electroelution as described by Sambrook et al. (1989). 5⬘ end labeling of the DNA fragments was performed using the T4 polynucleotide kinase and [␥- 32P]ATP (3000 Ci mmol ⫺1). (C) Southern blot analysis. EcoRI-restricted high-molecular-weight genomic DNAs from the parental N. meningitidis strain BL915 and from the transformed derivatives BL915ET-1, BL915ET-2, and BL915ET-3, prepared as previously described (Bucci et al., 1999), were analyzed by Southern blot according to Sambrook et al. (1989), using drg-, ermC⬘-, and tet-specific probes. Arrows indicate the relative migrations of drg- (6583 and 4199 bp) and tet-ermC⬘-specific (2942 bp) fragments. The sizes of the specific fragments were deduced by running molecular weight ladders in parallel (indicated on the left of the panels).

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Transformation Efficiency of Meningococcal Strains with Plasmid pNLET1

Strains Serotyping a BL2 BF9 BL915 BL911 BL892 BF52

B:NT B:NT B:NT:P1.5 B:NT:P1.9 B:4:P1.4 B:NT

Transformation efficiency b with pNLET1 Source c 3.3 ⫻ 10 ⫺6 4.2 ⫻ 10 ⫺8 5.4 ⫻ 10 ⫺7 9.4 ⫻ 10 ⫺7 1.9 ⫻ 10 ⫺6 5.5 ⫻ 10 ⫺8

i i ii ii ii iii

Note. Meningococcal strains were cultured on chocolate agar (Becton–Dickinson) or on GC agar or broth supplemented with 1% (v/v) Polyvitox (Bio-Merieux) at 37°C in 5% CO 2. Transformations were performed according to Frosch et al. (1990) by using 100 ng of plasmid DNA. Transformants were selected on GC agar base supplemented with erythromycin (7 ␮g/ml) or tetracycline (0.1 ␮g/ml) when requested. a NT, not typed. b Values indicate numbers of transformants/numbers of viable cells and are means of three independent experiments. c i, II policlinico, Universita` di Napoli, Italy; ii, Institut Pasteur, Paris, France; iii, Hoˆpital d’Instruction des Arme´e, BREST NAVAL, France.

ment, thus confirming insertion of the intact plasmid DNA into the chromosome. Because the tet gene in pNLET1 was cloned together with its natural promoter, to test the activity of the dam promoter in pNLE1, we engineered derivative plasmids in which the tet gene was cloned, without its promoter. The resulting plasmids, pNLET2 and pNLET3, were obtained by inserting a 2035-bp HindIII–PvuII (after blunting its ends) DNA fragment into the SmaI site, in opposite directions with respect to the leuS-dam region. pNLET2, harboring the tet gene starting immediately downstream of the dam promoter, but not pNLET3, conferred tetracycline resistance to both E. coli strain DH5␣ and N. meningitidis strain BL915. As the dam promoter is located immediately downstream of the transcription terminator of the leuS gene (Bucci et al., 1999), transcription of the tet gene was driven by the dam promoter in pNLET2. To test the versatility and the efficiency of the system, we transformed several meningococcal strains with pNLET1. The results of the trans-

formation experiments demonstrated that clones resistant to both erythromycin and tetracycline could be isolated with frequencies ranging from about 10 ⫺6 to 10 ⫺8 with 100 ng of plasmid DNA, depending on the competence of the individual strains (Table 1). Moreover, as the leuS region is also present and well conserved throughout the evolution both in gonococci and in several nonpathogenic Neisseriae (unpublished results), the system is suitable for genetic analysis in these related species. ACKNOWLEDGMENTS We thank Dr. V. Roberti for technical support and J. C. Chapalain (Hopital d’Instruction des Arme´es, BREST NAVAL, France) and Dr. J. M. Alonzo (Institut Pasteur, Paris, France) for providing us with meningococcal strains. This work was partially supported by a grant from the MURSTPRIN program (D.M. No. 503 DAE-UFFIII, 18/10/1999) and from the MURST-CNR Biotechnology program L. 95/ 95.

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