Construction and complementation of the first auxotrophic mutant in the spirochaete Leptospira meyeri

Hélène Bauby, Isabelle Saint Girons and Mathieu Picardeau

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
In bacteria, the first reaction of the tryptophan biosynthetic pathway involves the conversion of chorismate and glutamine to anthranilate by the action of anthranilate synthase, which is composed of the {alpha} (trpE gene product) and {beta} (trpG gene product) subunits. In this study, the tryptophan biosynthetic gene trpE of the spirochaete Leptospira meyeri was interrupted by a kanamycin-resistance cassette by homologous recombination. The trpE double cross-over mutant was not able to grow on solid or in liquid EMJH medium. In contrast, the trpE mutant showed a wild-type phenotype when tryptophan or anthranilate was added to the media, therefore showing that disruption of the L. meyeri trpE gene resulted in tryptophan auxotrophy. The authors have also characterized a second selectable marker that allows the construction of a spectinomycin-resistant L. meyeriE. coli shuttle vector and the functional complementation of the L. meyeri trpE mutant.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The advance of our knowledge with regard to the biology of the Spirochaetales order, which includes the pathogens Borrelia burgdorferi, Treponema pallidum and Leptospira interrogans, has been hampered by the absence of suitable genetic tools (Tilly et al., 2001). Recently, the development of new genetic tools has facilitated the construction of targeted mutants in the saprophytic species Leptospira biflexa. Thus, we have previously disrupted the chromosomal flaB and recA genes of L. biflexa with a kanamycin-resistance marker (Picardeau et al., 2001; Tchamedeu Kameni et al., 2002). However, no targeted mutants have been obtained in other Leptospira species containing saprophytic and pathogenic members.

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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Fig. 1. The tryptophan biosynthetic pathway in E. coli, a model for Leptospira spp.

 

   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains and growth conditions.
L. meyeri serovar semaranga strain Veldrat (Leptospira National Reference Centre, Institut Pasteur, Paris, France) was grown at 30 °C in EMJH (Ellinghausen & McCullough, 1965; Johnson & Harris, 1967) liquid medium or on 1 % agar plates. When necessary, L-tryptophan and anthranilate were used at 1 mM. Kanamycin and spectinomycin were used at 50 µg ml-1.

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 TE1–TE2 and radiolabelled with [{alpha}-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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Table 1. Primers used in this study

 
RNA isolation and RT-PCR.
Total RNA was extracted from exponentially growing cells of L. meyeri (OD420 0·2). After centrifugation (4000 g for 10 min at 4 °C), cells were washed in water, resuspended in 10 ml Trizol reagent (Gibco) and incubated for 5 min at room temperature. Two millilitres of isoamyl chloroform was added. Cells were then shaken and centrifuged. The supernatant was recovered and 5 ml 2-propanol was added. To precipitate RNA, tubes were incubated for 10 min at room temperature. After centrifugation, the RNA was washed with 75 % ethanol. The pellet was dissolved in 1x times; Dnase I buffer (Pharmacia). To remove DNA contamination, the sample was treated with RNase-free DNase I (Pharmacia). RNA was then purified by using the RNeasy mini kit (Qiagen). Reverse transcription of RNA was carried out as described by the manufacturer (SuperScript; Gibco-BRL) by using primer pairs TK1–TK2, TE4–TE2 and TG1–TG2 (Fig. 2, Table 1) at a final concentration of 0·2 µM. After 30 min at 45 °C, reverse transcriptase (RT) was inactivated by incubation at 94 °C for 2 min. PCR conditions were similar to those described above. RNA samples were tested in the presence and absence of RT to test for amplification of contaminant genomic DNA.



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Fig. 2. Genetic analysis of the trpE/trpG locus of L. meyeri. For the inactivation of the L. meyeri trpE gene, the TE1–TE2 amplified product was cloned into a suicide plasmid. The kanamycin-resistance cassette was then inserted into the BstXI restriction site, generating plasmid pTEK, which was used to deliver the mutated allele into L. meyeri. Arrows indicate primers (see Table 1) used for the transcriptional analysis of the L. meyeri trpE and trpG genes (for complete sequence, see Yelton & Peng, 1989). Results of RT-PCR assays with RNA from L. meyeri wild-type and L. meyeri trpE mutant strains are indicated.

 
Construction of a L. meyeri trpE mutant.
The trpE (1714 bp) locus of L. meyeri was amplified by PCR with primer pairs TE1–TE2 (Fig. 2, Table 1). The amplified product was then inserted into the SmaI site of pUC19, resulting in plasmid pTE. The Enterococcus faecalis kanamycin-resistance cassette was amplified as previously described (Picardeau et al., 2001) and inserted into the blunt-ended site BstXI of trpE to generate plasmid pTEK (trpE : : Km). The plasmid was then subjected to UV irradiation and used to deliver the inactivated allele into L. meyeri as previously described (Picardeau et al., 2001). Optimal conditions (maximum number of transformants) were obtained for 1 µg of circular plasmid DNA treated with low UVB or UVC irradiation (1 mJ cm-2) using cells from an early exponential-phase culture (OD420 0·2, corresponding to 4x108 bacteria ml-1). Kanamycin-resistant colonies were picked and tested for the insertion of the kanamycin-resistance cassette into the target gene by PCR with primers TK1 and TK2 (Table 1) and Southern blot analysis as previously described (Picardeau et al., 2001).

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 NdeI–PstI 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 TE1–TE2 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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Fig. 3. Construction of shuttle vectors pGKLS and pGLStrpE. Relevant restriction sites are indicated. The spectinomycin-resistant cassette (Spc) was amplified and cloned into SmaI-cut pGKLep4, to generate pGKLS. We then cloned the trpE gene (trpE) into the FspI sites of pGKLS (then releasing the kanamycin-resistance cassette, Km), resulting in plasmid pGLStrpE.

 

   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Generation of a tryptophan-auxotrophic mutant in L. meyeri
In E. coli, the synthesis of the aromatic amino acids phenylalanine, tyrosine and tryptophan shares a common pathway leading to the synthesis of the branch point compound chorismate (Pittard, 1996). The first reaction of the tryptophan pathway involves the conversion of chorismate and glutamine to anthranilate by the action of anthranilate synthase (encoded by the trpE and trpG genes) (Fig. 1). The sequential actions of the products of the trpD, trpF and trpC genes lead to the penultimate compound in the pathway, indoleglycerol phosphate. The last step is catalysed by tryptophan synthase (encoded by the trpA and trpB genes) (Pittard, 1996) (Fig. 1). The trpE and trpG genes of L. meyeri were previously isolated by complementation of an E. coli trpE mutant (Yelton & Cohen, 1986). The suicide plasmid carrying the L. meyeri inactivated trpE gene, pTEK, was treated by UV irradiation prior to electroporation. UV irradiation of the plasmids yielded up to 100-fold more transformants compared to non-treated DNA. However, no double cross-over event was detected for the trpE gene (0/40 tested colonies, all had undergone a single cross-over event), suggesting that auxotrophs were not able to grow on EMJH medium, which is a complex medium and includes undefined components. Therefore, further transformation experiments were done in the presence of tryptophan. This resulted in the identification of double cross-over events for the trpE gene of L. meyeri (10/50 tested colonies). Confirmation of allelic exchange was obtained by PCR and Southern hybridization (Fig. 4a); an increase in size of either the PCR product or the hybridizing fragment by 1·1 kb is due to the insertion of the kanamycin-resistant cassette into the chromosomal trpE locus. The trpE mutants resulting from a double cross-over event were unable to grow on solid or in liquid medium (Fig. 4b) lacking supplemental amino acids. Addition of tryptophan restored growth of the L. meyeri trpE mutant at the wild-type rate (Fig. 4b). This result shows that L. meyeri is able to acquire amino acids from its environment by taking up free amino acids. A wild-type phenotype was also obtained by the addition of anthranilate (data not shown), further confirming that the L. meyeri TrpE protein is involved in the first step of the tryptophan biosynthetic pathway, leading to anthranilate (Fig. 1).



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Fig. 4. (a) Evidence for homologous recombination with the L. meyeri trpE gene. PCR (with primers TK1 and TK2, left panel) and Southern blot analysis (hybridized with the trpE probe, right panel) of representative clones. Lanes: 1, L. meyeri wild-type strain; 2, single cross-over recombinant strain; 3, double cross-over recombinant strain. Sizes of the fragments of the molecular mass marker are indicated on the left. (b) Growth curve of L. meyeri: {bullet}, parental strain; {square}, trpE mutant; {blacktriangleup}, trpE mutant supplemented with tryptophan.

 
The trpE and trpG genes are separated by only 68 bp and could form an operon (Yelton & Peng, 1989). However, no amplification between trpE and trpG was obtained by RT-PCR with RNA from either trpE mutant or wild-type strains (Fig. 2). Interestingly, inactivation of trpE does not affect the transcription of trpG in the L. meyeri trpE mutant (Fig. 2). Taken together these results suggest that trpE and trpG do not form an operon and that trpG possesses its own promoter.

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. (34–39 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).


   ACKNOWLEDGEMENTS
 
We thank Guoping Zhao, Zhu Chen, Shuangxi Ren and the Chinese Human Genome Center (Shanghai, China) for sharing data on the genome sequence of L. interrogans prior to publication. We thank F. C. Cabello for the gift of the plasmid pVK61. This work received support from the Institut Pasteur and Programme de Recherches Avancées franco-chinois (PRA B00-05).


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 21 October 2002; revised 6 December 2002; accepted 13 December 2002.



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