Unité de Bactériologie Moléculaire et Médicale, Institut Pasteur, 28 rue du docteur Roux, 75724 Paris Cedex 15, France
Correspondence
Mathieu Picardeau
mpicard{at}pasteur.fr
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ABSTRACT |
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INTRODUCTION |
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Charon et al. (1974) demonstrated that Leptospira spp. have the potential to synthesize the essential amino acids and that most of them were synthesized by biosynthetic pathways similar to those used in Escherichia coli. In previous genetic studies, several leptospiral genes have been isolated by functional complementation of E. coli mutants. This methodology has allowed the identification of some amino acid biosynthetic genes such as asd, aroD, dapD, metX, metY, trpE, proA and leuB (Baril et al., 1992
; Belfaiza et al., 1998
; Richaud et al., 1990
; Yelton & Cohen, 1986
). In order to test our genetic exchange system in the saprophytic species L. meyeri and to study the tryptophan biosynthetic pathway (Fig. 1
), we constructed a L. meyeri trpE mutant. This study also describes the use of a second selectable marker, the spectinomycin-resistance cassette, that allows complementation studies in Leptospira spp.
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METHODS |
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DNA manipulations.
Plasmids from E. coli were recovered using a Qiaprep Spin miniprep kit (Qiagen). Genomic DNA of L. meyeri was extracted as previously described (Picardeau et al., 2001). For Southern blot analysis, DNA of representative clones of L. meyeri was digested with EcoRI, subjected to electrophoresis overnight, and transferred onto a nylon membrane. The L. meyeri trpE gene was amplified with primers TE1TE2 and radiolabelled with [
-33P]dATP (Megaprime, Amersham). The membrane was then hybridized with the labelled probe under stringent conditions as previously described (Picardeau et al., 2001
). Amplification with appropriate primers (Table 1
) was achieved using one cycle of denaturation (94 °C, 5 min), followed by 35 cycles of amplification consisting of denaturation (94 °C, 30 s) annealing (55 °C, 30 s), and primer extension (72 °C, 1 min 30 s), and a final cycle of extension of 10 min at 72 °C.
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Complementation of the L. meyeri trpE mutant.
To complement the L. meyeri tryptophan (trpE : : Km) auxotroph, we tested the spectinomycin-resistance cassette of Staphylococcus aureus (Chary et al., 1997; Murphy et al., 1985
) as a second selectable marker. As previously described (Bono et al., 2000
), the promoter of the B. burgdorferi flgB gene was amplified with primers FLG5 and FLG3 (Table 1
) (introducing NdeI and PstI restriction sites at the 3' end of the promoter) and cloned into the SmaI site of pGEM7Zf(+) (Promega), resulting in plasmid pGFB. The promoterless spc gene amplified from pVK61 (Chary et al., 1997
) using primers SN and SP (Table 1
) was cloned into pCR2.1-TOPO (Invitrogen), resulting in plasmid pCRS. The spc gene was released from pCRS with NdeI and NsiI restriction enzymes and inserted into the NdeIPstI sites of pGFB, to make pGFBS. The PflgBspc was amplified from pGFBS with primers FLG5 and SP (Table 1
), then inserted into the SmaI site of pGKLep4, resulting in plasmid pGKLS (Fig. 3
). Spectinomycin-resistant colonies were obtained after electroporation of pGKLS into L. meyeri. We then cloned the trpE gene (amplified product of TE1TE2 starting at position -102 upstream of the start codon and ending at position +225 downstream of the stop codon, Fig. 2
) into the FspI sites of pGKLS, resulting in plasmid pGLStrpE (Fig. 3
). Plasmid pGSLtrpE was used to transform the L. meyeri trpE mutant, by electroporation.
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RESULTS AND DISCUSSION |
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Use of a second selectable marker and complementation of the L. meyeri trpE mutant
To date, Leptospira spp. have only one reliable marker, the E. faecalis kanamycin-resistance cassette (Saint Girons et al., 2000). To confirm that the auxotrophy of L. meyeri was due to the inactivation of the trpE gene, a second selectable marker was tested for complementation experiments. We used the spectinomycin-resistance gene (spc) from S. aureus (Chary et al., 1997
). This cassette was chosen because its sequence shows a G+C content (35 mol%) similar to that of Leptospira spp. (3439 mol%). In addition, similarly to most of the markers used in spirochaetes (Hardham & Rosey, 2000
), the spc gene is an antibiotic-resistance cassette from a Gram-positive bacterium. Finally, this cassette appears to be expressed in B. burgdorferi (Sartakova et al., 2001a
). To enhance its transcription in spirochaetes, the promoterless spc gene was linked to the promoter region of the B. burgdorferi flgB gene as previously described (Bono et al., 2000
) and cloned into the L. meyeriE. coli shuttle vector pGKLep4 (Fig. 3
). Electroporation of pGKLS into L. meyeri resulted in spectinomycin-resistant (Spcr) colonies. The MIC of transformants was >128 µg ml-1 (versus <2 µg ml-1 for the wild-type strain). This suggests that the borrelial promoter of flgB (Bono et al., 2000
) is recognized in L. meyeri. We then cloned the L. meyeri trpE gene into pGKLS (Fig. 3
) and the constructs were transferred into L. meyeri trpE mutant by electrotransformation. While transformants containing the vector alone were not able to grow on EMJH medium without tryptophan, the Spcr transformants containing the trpE gene recovered the ability to grow on EMJH medium (data not shown). Plasmid DNAs extracted from several Spcr L. meyeri transformants were analysed after amplification in E. coli and showed restriction profiles similar to those from the original plasmid constructs (data not shown). These results show that the Kanr auxotroph was complemented by the wild-type genes present on the shuttle vector containing the Spcr marker. Only two other spirochaetal genes have been inactivated and complemented so far: the T. denticola flgE (Chi et al., 2002
) and the B. burgdorferi flaB (Sartakova et al., 2001b
) genes.
Concluding remarks
We report here the characterization of the first auxotroph in Leptospira spp. (as well as the first mutant in L. meyeri) and the identification of a second selectable marker, the spectinomycin-resistance cassette, for genetic manipulations of Leptospira spp. We also demonstrate that tryptophan prototrophy is essential for survival of Leptospira spp. in an environment lacking amino acids, such as EMJH medium. During the completion of this work, the genomic sequence of L. interrogans serogroup icterohaemorrhagiae pathogenic strain Lai (Chinese Human Genome Center, Shanghai, China; see http://www.chgc.sh.cn/gn/ and http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/micr.html) was released (S. Ren, G. Fu, X. Jiang & 18 other authors, unpublished). Sequence analysis of the genome of the pathogen L. interrogans shows a tryptophan pathway similar to that of E. coli, as shown in Fig. 1. Interestingly, analyses of the genomic sequences of the evolutionarily related spirochaetes T. pallidum and B. burgdorferi have shown that genes encoding enzymes of the tryptophan pathway are absent (Fraser et al., 1997
, 1998
). In fact, these spirochaetes have a minimal genome that lacks genes encoding the enzymes for most biosynthetic pathways, and are naturally auxotrophs for all amino acids. The prospect of soon possessing genetic tools for pathogenic strains of Leptospira could allow the construction of a L. interrogans trpE mutant as a putative candidate for vaccines. Indeed, auxotrophs could be vaccine candidates as previously demonstrated in many pathogens such as Corynebacterium pseudotuberculosis, Mycobacterium tuberculosis and Legionella pneumophila (Mintz et al., 1988
; Simmons et al., 1997
; Smith et al., 2001
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 21 October 2002;
revised 6 December 2002;
accepted 13 December 2002.
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