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

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A New Vector for Insertion of Any DNA Fragment into the Chromosome of Transformable Neisseriae
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  SHORT COMMUNICATIONA New Vector for Insertion of Any DNA Fragment into the Chromosomeof 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, 2000A useful method for inserting any DNA fragment into the chromosome of   Neisseriae  has beendeveloped. The method relies on recombination-proficient vector plasmid pNLE1, a pUC19 deriva-tive 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 specificuptake sequence which is required for natural transformation; (4) a promoter capable of functioningin  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 pathoge-nicity of   Neisseria meningitidis  has been im-paired by the lack of appropriate genetic tools.DNA-mediated transformation of the meningo-coccus is to date the only available tool formolecular genetic manipulation, and it has beenused to target mutations onto chromosomalgenes by allelic replacement with cloned mu-tated 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 systemshave been developed in  Neisseria gonorrhoeae. An autonomously replicating shuttle vector forthe introduction of cloned genes into  N. gonor-rhoeae  by transformation was srcinally ob-tained by fusing the gonococcal 4.2-kb crypticplasmid (Davies and Normark, 1980) with thenaturally occurring 7.2-kb   -lactamase plasmid(Stein  et al.,  1983a,b). However, this vector hasnever been used for genetic studies in menin-gococci. In addition, a system useful for geneticcomplementation of transformable and non-transformable  N. gonorrhoeae  mutants has beendescribed more recently (Kupsch  et al.,  1996).This system relies on introduction of clonedgenes into gonococcal p tetM  25.2 via allelic re-placement and subsequent mobilization by con- jugation to recipients. Also this system hasnever been adapted to meningococci.This paper describes a new integrative vectorcapable of inserting genes or DNA fragmentsinto a definite site within the chromosome of meningococci, gonococci, and several non-pathogenic  Neisseriae,  thereby overcoming theabove-mentioned problems.In order to be suitable for genetic manipula-tion, an ideal vector should (1) be of a relativelysmall size, (2) be able to replicate into theintermediate host with high efficiency, (3) have 1 To whom correspondence should be addressed. Fax:  39 0832 320626. E-mail: alifano@ilenic.unile.it.275 0147-619X/00 $35.00 Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved. Plasmid  44,  275–279 (2000)doi:10.1006/plas.2000.1490, available online at http://www.idealibrary.com on  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 wasobtained by subcloning sequentially (1) a 1480-bp  Sau 3AI fragment, spanning the chromosomal  leuS-dam  regionderived from plasmid pNMdam1 (Bucci  et al.,  1999), and (2) a 859-bp  Kpn I fragment, containing the  ermC   gene (Bucci  et al.,  1999), into the polylinker of pUC19.  E. coli  strain DH5   [F   80d  lacZ   M15  endA1 recA1hsdR17 supE44 thi- 1   gyrA96   ( lacZYA-argF  )  U169 ] was used in cloning procedures. Unique restriction sites 276  SHORT COMMUNICATION  at least one selectable marker, (4) be able totransform the definitive host at a high fre-quency, (5) contain several unique restrictionsites for cloning, (6) be able to drive expressionof cloned genes, and (7) be versatile.All these are properties of plasmid pNLE1(Fig. 1A). It is a pUC19 derivative of 5027 bpharboring (1) a DNA fragment spanning the leuS-dam  region derived from plasmidpNMdam1 (Bucci  et al.,  1999), which is re-quired for its integration into the neisserial chro-mosome by single cross-over, and (2) the struc-tural gene for  Bacillus subtilis  RNA methylase( ermC   ) conferring resistance to macrolide an-tibiotic erythromycin, as a selectable marker inmeningococci (Monod  et al.,  1986). pNLE1also contains an uptake sequence, locateddownstream of the  leuS   gene and required totransform  Neisseria  with high efficiency(Graves  et al.,  1982). Unique restriction sitesare  Hin dIII,  Sph I,  Pst  I,  Xba I,  Bam HI,  Sma I, and  Eco RI. Transcription of cloned DNA fragmentsin the  Sma I site is driven by promoter sequenceslocated 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  Sca I– Pvu II DNA fragmentderived from plasmid pBR322, spanning thestructural tetracycline-resistance  tet   gene, intothe  Sma I site (Fig. 1B). The resulting plasmidpNLET1 conferred tetracycline resistance (10  g/ml) and erythromycin resistance (125   g/ ml) to the otherwise sensitive  Escherichia coli strain DH5  . pNLET1 was used to transformthe  N. meningitidis  strain BL915 to erythromy-cin resistance. This procedure led to the isola-tion of erythromycin-resistant clones (7   g/ml)with a frequency of about 10  6 (number of transformants/number of viable cells) with 100ng of plasmid DNA (Table 1). All these trans-formants were also resistant to tetracycline. Theintegration of pNLET1 into the chromosomal leuS-drg  region (Fig. 1B) was verified bySouthern blot analysis using  drg-, ermC   -,  or tet  -specific probes (Fig. 1C). Hybridization of   Eco RI-digested DNA of the parental strain withthe  drg  probe showed the presence of the ex-pected 6583-bp fragment on the basis of previ-ous mapping and sequencing data (Bucci  et al., 1999, and data not shown). In contrast, DNAfrom the transformed clones revealed an ap-proximately 4200-bp-long fragment. This resultwas consistent with integration of pNLET1 intothe  leuS   gene leading to production of a4199-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 srcin of pUC19. (B)Physical and genetic map of the meningococcal  leuS-drg  region and of plasmid pNLET1. pNLET1 was obtained bysubcloning a 2581-bp-long  Sca I– Pvu II DNA fragment, derived from plasmid pBR322, spanning the structural tetracycline-resistance  tet   gene, into the  Sma I site of pNLE1. Abbreviations used: E,  Eco RI; S,  Sma I; Sc,  Sca I; Pv,  Pvu II. Abbreviationsare the same as in A. In addition, US indicates the neisserial uptake sequence and  ppk   the structural gene encoding thepolyphosphate kinase. Open boxes span the DNA regions used as probes in Southern blot experiments. The  drg  probe wasobtained amplifying a 511-bp-long genomic region from meningococcal strain BL915 by PCR using the oligonucleotides5  -ATAGGCAACAGCGTGCCTGACGG-3   and 5  -TCGATCGTATTTTGGTCGCGC-3   as primers. The  ermC    probewas obtained by amplifying a 785-bp-long region from plasmid Hermes6a (Kupsch  et al.,  1996) using the 5   end-labeledoligonucleotides 5  -TAATGAACGAGAAAAATATAAAACACAGTC-3   and 5  -GGTACACGAAAAACAAGTTA-AGGGATGCAG-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 Cycler480. The 2581-bp-long  Sca I– Pvu II fragment, containing the  tet   gene from pBR322, was used as a  tet   probe. DNA fragmentswere 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 [   - 32 P]ATP (3000 Ci mmol  1 ).(C) Southern blot analysis.  Eco RI-restricted high-molecular-weight genomic DNAs from the parental  N. meningitidis  strainBL915 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 onthe left of the panels). 277 SHORT COMMUNICATION  ment, thus confirming insertion of the intactplasmid DNA into the chromosome.Because the  tet   gene in pNLET1 was clonedtogether with its natural promoter, to test theactivity of the  dam  promoter in pNLE1, weengineered derivative plasmids in which the  tet  gene was cloned, without its promoter. Theresulting plasmids, pNLET2 and pNLET3, wereobtained by inserting a 2035-bp  Hin dIII– Pvu II(after blunting its ends) DNA fragment into the Sma I site, in opposite directions with respect tothe  leuS-dam  region. pNLET2, harboring the  tet  gene starting immediately downstream of the dam  promoter, but not pNLET3, conferred tet-racycline 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   genewas driven by the  dam  promoter in pNLET2.To test the versatility and the efficiency of thesystem, we transformed several meningococcalstrains with pNLET1. The results of the trans-formation experiments demonstrated that clonesresistant to both erythromycin and tetracyclinecould be isolated with frequencies ranging fromabout 10  6 to 10  8 with 100 ng of plasmidDNA, depending on the competence of the in-dividual strains (Table 1). Moreover, as the  leuS  region is also present and well conservedthroughout the evolution both in gonococci andin several nonpathogenic  Neisseriae  (unpub-lished results), the system is suitable for geneticanalysis in these related species. ACKNOWLEDGMENTS We thank Dr. V. Roberti for technical support and J. C.Chapalain (Hopital d’Instruction des Arme´es, BREST NA-VAL, France) and Dr. J. M. Alonzo (Institut Pasteur, Paris,France) for providing us with meningococcal strains. Thiswork was partially supported by a grant from the MURST-PRIN program (D.M. No. 503 DAE-UFFIII, 18/10/1999)and from the MURST-CNR Biotechnology program L. 95/ 95. REFERENCES Bucci, C., Lavitola, A., Salvatore, P., Del Giudice, L.,Massardo, D. R., Bruni, C. B., and Alifano, P. (1999).Hypermutation in pathogenic bacteria: Frequent phasevariation in meningococci is a phenotypic trait of a spe-cialized mutator biotype.  Mol. Cell  3,  435–445.Davies, J. K., and Normark, S. (1980). A relationship be-tween plasmid structure, structural lability, and sensitiv-ity to site specific endonucleases in  Neisseria gonor-rhoeae. Mol. Gen. Genet.  177,  251–260.Frosch, M., Schultz, E., Glenn-Calvo, E., and Meyer, T. F.(1990). Generation of capsule-deficient  Neisseria menin-gitidis  strains by homologous recombination.  Mol. Mi-crobiol.  4,  1215–1218.Graves, J. F., Biswas, G. D., and Sparling, P. F. (1982).Sequence-specific DNA uptake in transformation of   Neis-seria gonorrhoeae. J. Bacteriol.  152,  1071–1077.Kupsch, E.-M., Aubel, D., Gibbs, C. P., Kahrs, A. F., Rudel,T., and Meyer, T. F. (1996). Construction of Hermesshuttle vectors: A versatile system useful for geneticcomplementation of transformable and non-transform-able  Neisseria  mutants.  Mol. Gen. Genet.  250,  558–569.Lewis, L. A., Gipson, M., Hartman, K., Ownbey, T.,Vaughn, J., and Dyer, D. W. (1999). Phase variation of HpuAB and HmbR, two distinct haemoglobin receptorsof   Neisseria meningitidis  DNM2.  Mol. Microbiol.  32, 977–989.Monod, M., Denoya, C., and Dubnau, C. (1986). Sequenceand properties of pIM13, a macrolide-lincosamide-strep-togramin B resistance plasmid from  Bacillus subtilis. J. Bacteriol.  167,  138–147.Pettersson, A., Prinz, T., Umar, A., van der Biezen, J., andTABLE 1Transformation Efficiency of Meningococcal Strainswith Plasmid pNLET1Strains Serotyping a Transformationefficiency b with pNLET1 Source c BL2 B:NT 3.3  10  6 iBF9 B:NT 4.2  10  8 iBL915 B:NT:P1.5 5.4  10  7 iiBL911 B:NT:P1.9 9.4  10  7 iiBL892 B:4:P1.4 1.9  10  6 iiBF52 B:NT 5.5  10  8 iii  Note.  Meningococcal strains were cultured on chocolateagar (Becton–Dickinson) or on GC agar or broth supple-mented with 1% (v/v) Polyvitox (Bio-Merieux) at 37°C in5% CO 2 . Transformations were performed according toFrosch  et al.  (1990) by using 100 ng of plasmid DNA.Transformants were selected on GC agar base supplementedwith 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 experi-ments. c i, II policlinico, Universita` di Napoli, Italy; ii, InstitutPasteur, Paris, France; iii, Hoˆpital d’Instruction des Arme´e,BREST NAVAL, France. 278  SHORT COMMUNICATION  Tommassen, J. (1998). Molecular characterization of Lbp, the second lactoferrin-binding protein of   Neisseriameningitidis. Mol. Microbiol.  27,  599–610.Richardson, A. R., and Stojiljkovic, I. (1999). HmbR, ahemoglobin-binding outer membrane protein of   Neisseriameningitidis,  undergoes phase variation.  J. Bacteriol. 181,  2067–2074.Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). “Mo-lecular Cloning. A Laboratory Manual,” 2nd ed. ColdSpring Harbor Laboratory Press, Cold Spring Harbor,NY.Stein, D. C., Silver, L. E., Clark, V. L., and Young, F. E.(1983a). Construction and characterization of a new shut-tle vector, pLES2, capable of functioning in  Escherichiacoli  and  Neisseria gonorrhoeae. Gene  25,  241–247.Stein, D. C., Young, F. E., Tenover, F. C., and Clark, V. L.(1983b). Characterization of a chimeric  -lactamase plas-mid of   Neisseria gonorrhoeae  which can function in  Escherichia coli. Mol. Gen. Genet.  189,  79–84.Stojiljkovic, I., Hwa, V., de Saint Martin, L., O’Gaora, P.,Nassif, X., Heffron, F., and So, M. (1995). The  Neisseriameningitidis  haemoglobin receptor: Its role in iron utili-zation and virulence.  Mol. Microbiol.  15,  531–541.Swartley, J. S., Lin, J. J., Miller, Y. K., Martin, L. E.,Edupuganti, S., and Stephens, D. S. (1998). Character-ization of the gene cassette required for biosynthesis of the (alpha1  3   6)-linked  N  -acetyl- D -mannosamine-1-phosphate capsule of serogroup A  Neisseria meningitidis. J. Bacteriol.  180,  1533–1539.Tonjum, T., Freitag, N. E., Namork, E., and Koomey, M.(1995). Identification and characterization of   pilG,  ahighly conserved pilus-assembly gene in pathogenic  Neisseria. Mol. Microbiol.  16,  451–464. Communicated by M. Espinosa 279 SHORT COMMUNICATION
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