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The spirochetal chpK-chromosomal toxin–antitoxin locus induces growth inhibition of yeast and mycobacteria

Mathieu Picardeau, Corinne Le Dantec, Guy-Franck Richard, Isabelle Saint Girons
DOI: http://dx.doi.org/10.1016/S0378-1097(03)00848-6 277-281 First published online: 1 December 2003

Abstract

Toxin–antitoxin systems encoded by bacterial plasmids and chromosomes typically consist of a toxin that inhibits growth of the host cell and a specific antitoxin. In this report, the chpK gene from the chromosomal toxin–antitoxin locus of the spirochete Leptospira interrogans was studied in both prokaryotic and eukaryotic systems. Cloning of the the spirochetal chpK gene into a mycobacterial expressing vector led to dramatic reductions of transformation efficiency in both Mycobacterium smegmatis and Mycobacterium bovis BCG. However, few mycobacterial transformants were obtained. This result could be due to plasmid structural modifications leading to disruption of chpK expression, suggesting that L. interrogans ChpK is highly toxic for mycobacteria. Presence of the L. interrogans chpK gene was also found to inhibit cell growth of the yeast Saccharomyces cerevisiae. These results show that ChpK possesses a broad activity against both prokaryotes and eukaryotes, suggesting that the cellular target of the toxin is conserved in these organisms.

Keywords
  • Leptospira
  • Toxin–antitoxin locus
  • chpK
  • Plasmid instability
  • Growth inhibition

1 Introduction

Toxin–antitoxin (TA) loci are organized into operons in which typically the first gene encodes the antitoxin and the second gene encodes the toxin. The product of the toxin gene is long lived and toxic, while the product of the antitoxin gene is short lived and neutralizes the toxin by protein–protein interaction. Since their discovery in bacterial plasmids as maintenance systems, TA loci have been identified in many prokaryotic (including archaeal) chromosomes [1]. The relBE loci constitute the largest chromosomal TA gene family in prokaryotes and most of the studies have been done on the relBE gene pair in Escherichia coli. Thus, it has been shown that the RelE toxin inhibits protein synthesis by cleavage of ribosome bound mRNA in response to nutritional stress [2,3]. This induces inhibition of cell growth but without cell killing [4]. In addition, it has been shown that the E. coli RelE toxin inhibits growth of the yeast Saccharomyces cerevisiae and human cells [5,6]. Similar results were obtained with Kid/PemK toxin system of E. coli plasmid R1 [7]. These findings may have several applications such as the selective killing of cancer cells [7]. Recently, we have identified a chromosome-encoded TA locus in the pathogenic spirochete Leptospira interrogans [8]. This locus belongs to the chp TA gene family, which is distinct from the relBE loci [1]. We have shown that the expression of the L. interrogans toxin (ChpK) is toxic to E. coli cells and coexpression of the L. interrogans antitoxin (ChpI) neutralizes the toxin in E. coli [8]. Since little information is available on TA loci from species other than E. coli, we were interested to determine if the spirochetal ChpK toxin is active in phylogenetically non-related organisms such as mycobacteria and yeast.

2 Materials and methods

2.1 Bacterial strains and growth conditions

L. interrogans serogroup icterohaemorrhagiae strain Lai (Leptospira National Reference Center, Institut Pasteur, Paris, France) was grown at 30°C in EMJH [9,10] liquid medium or on 1% agar plates. The yeast strain used was S. cerevisiae FYBL1-23D (MATαura3Δ851 his3Δ200 trp1Δ63). Yeast cells were grown in liquid or solid SC-ura. When necessary, doxycycline was added at 1 µg ml−1. E. coli was grown in Luria–Bertani medium. Mycobacteria were selected on solid 7H11 medium supplemented with 20 µg ml−1 kanamycin.

2.2 DNA manipulations

Genomic DNA of mycobacteria was extracted as previously described [11]. For Southern blot analysis, digested DNA was subjected to electrophoresis overnight, transferred onto a nylon membrane, and hybridized with (α-32P) dATP labelled probe under stringent conditions as previously described [11]. All polymerase chain reaction (PCR) amplifications were 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), and a final cycle of extension of 10 min at 72°C.

2.3 Cloning of L. interrogans chpK into expression vectors

The complete coding sequences (from the putative start codon to the downstream region of the stop codon) of L. interrogans chpK was amplified by Taq polymerase (Amersham) with primer pairs BLK1 (5′-AAAGGATCCATGATTCGT-3′)–CKP (5′-ACTGCAGGAGTGAGGATTG-3′) and pKM1 (5′-CGGGATCCATGATTCGTGGTGAAATTTG-3′)–pKM2 (5′-CGCTGCAGAATCGTAATTAGGCTAAATA-3′), and inserted into pCR2.1-TOPO by using the TOPO TA cloning kit (Invitrogen). BamHI and PstI restriction sites, which were added at the 5′ ends of the sense and antisense primers, respectively, are underlined. After gel purification of BamHI–PstI digestion, the PCR products BLK1-CKP and pKM1-pKM2 were inserted into the BamHI–PstI sites of the S. cerevisiae expression vector pCMha190 (J. Boyer and B. Dujon, in preparation), which is derived from pCM190 [12] and the mycobacterial expression vector pMIP12 [11], to generate plasmid pCMKL and pMIPKL, respectively. Similarly, the Mycobacterium celatum pemK was amplified with primers P5 and P6 [11], then inserted into pCMha190, to generate plasmid pCMKM. Plasmids from E. coli were recovered using a Qiaprep Spin miniprep kit (Qiagen). Yeast and mycobacterial cells were transformed as described previously [11,13]. DNA sequencing was performed on plasmid constructs in order to check the constructions.

3 Results and discussion

3.1 Chromosome-encoded TA loci are widely spread in prokaryotes

The spirochetes represent an ancient and diverse group of prokaryotes possessing a number of unique characteristics. However, these bacteria are poorly studied genetically. A BLAST search reveals that TA loci are widely spread among prokaryotic chromosomes [1], including the spirochete L. interrogans. A phylogenetic analysis for toxin proteins of chromosome-encoded TA loci is shown in Fig. 1. Multiple phylogenetic methods were used to determine the consistency of the topology tree. The spirochetal ChpK clustered with those of other bacteria, as well as pemK plasmid-encoded toxins, a result consistent with the fact that chp loci are structurally and functionally similar to pem loci [1]. The L. interrogans ChpK protein shares more than 44% identity with one of the ChpK homologs (rv1991c or C-Mtub1 in Fig. 1) of Mycobacterium tuberculosis. The RelE homologs constitute a gene family distinct from the ChpK homologs and they are also found in species of the Archae domain (Fig. 1) [1]. To test whether ChpK is toxic in both prokaryotic and eukaryotic cells, the L. interrogans chpK gene has been introduced under the control of a strong mycobacterial promoter (pblaF*) and an inducible promoter (tetO) in mycobacteria and yeast, respectively.

Figure 1

Phylogenetic tree of the ChpK, PemK, and RelE homologs from prokaryotes. The ClustalX program was used to generate alignment with sequences available from selected members of prokaryotes. The tree was calculated by using the distance method. The different species from which the ChpK (C), PemK (P), and RelE (R) homologs were derived are indicated by the following letters and numbers: Bacillus subtilis (Bsub), Clostridium acetobutylicum (Cace), E. coli (Ecol1 and Ecol2), E. coli plasmids R100 (Ecolp1) and P307 (Ecolp2), Haemophilus influenzae (Hinf), L. interrogans (Lint), Listeria monocytogenes (Lmon), M. celatum plasmid pCLP (Mcel), M. tuberculosis (Mtub1 and Mtub2), Pseudomonas syringae (Psyr), Salmonella typhimurium (Styp), Vibrio cholerae (Vcho), and the archae Pyrococcus horikoshii (Phor) and Archaeoglobus fulgidus (Aful).

3.2 Instability of the L. interrogans chpK gene in mycobacteria

Expression vector pMIP12 [11] is a pAL5000-derived plasmid that replicates in the fast-growing species M. smegmatis and in the slow-growing species such as M. bovis BCG and the pathogen M. tuberculosis. Cloning of the L. interrogans toxin-encoding gene into pMIP12, leading to plasmid pMIPKL, leads to dramatic reductions of transformation efficiency in both M. smegmatis and M. bovis BCG (Table 1). After pMIPKL transformation, all the plasmids recovered from M. bovis BCG and M. smegmatis are rearranged plasmids (Fig. 2). Previous studies have also described structural instability of plasmids in mycobacteria [1416]. Analysis of plasmid restriction patterns shows some apparently identical DNA rearrangements (Fig. 2), suggesting that the mechanism responsible for the structural instability is not random. In contrast, plasmids recovered from pMIP12 transformants did not show DNA rearrangements. Similarly, transformation of both M. smegmatis and M. bovis BCG with pMIP12 carrying a mutated allele of L. interrogans chpK (introduction of a point mutation into the chpK coding sequence), did not result in plasmid instability (data not shown). This result suggests that the expression of chpK gene was responsible for plasmid instability. Structural modifications may abolish expression of a functional ChpK protein which is highly toxic for mycobacteria.

View this table:
Table 1

Transformation of Mycobacterium spp. to kanamycin resistance by plasmid carrying the L. interrogans chpK gene (pMIPKL) and plasmid control pMIP12

Plasmid (cloned gene)Recipient strainTransformants (μg−1 DNA)DNA rearrangement
pMIP12M. smegmatis mc21552.1×104no
PMIPKL (chpK)M. smegmatis mc21553yes
pMIP12M. bovis BCG strain Pasteur9×102no
pMIPKL (chpK)M. bovis BCG strain Pasteur1yes
  • Data represent the mean of three experiments. Negative controls with no DNA resulted in the absence of KmR colonies.

  • The number given (one KmR colony) is the total of three experiments.

Figure 2

Southern blot analysis of PvuII-digested DNA of M. smegmatis transformed with pMIPKL (carrying the L. interrogans chpK gene). Genomic DNA in all lanes is digested with PvuII and probed with pMIPKL. Lanes 1–6: individual colonies of M. smegmatis transformants; lane 7: original plasmid pMIPKL.

3.3 Growth inhibition of yeast cells harboring the L. interrogans chpK gene

A tetracycline-regulatable promoter system [12] was used for the expression of L. interrogans chpK in S. cerevisiae. Expression from the tetO promoter is regulated by tetracycline or derivatives such as doxycycline. In the presence of antibiotic in the medium (1 µg ml−1 doxycycline), expression from the tetO promoter is negligible [12], and no change in growth rate was detected between yeast cells harboring the empty plasmid and cells containing the plasmid carrying the L. interrogans chpK gene (data not shown). In contrast, in cultures growing in the absence of antibiotic, an obvious inhibitory effect on growth was observed for yeast cells harboring the L. interrogans chpK gene, in comparison to non-chpK-containing cells (Fig. 3). The growth rate of S. cerevisiae harboring the expression vector with a mutated allele of chpK was not affected. In a previous study, we have identified a pem (for ‘plasmid emergency maintenance’) locus, consisting of pemK (for ‘killer protein’) and pemI (for ‘inhibitory protein’) on a mycobacterial plasmid [11]. The M. celatum PemK shares 30% identity with the L. interrogans ChpK (Fig. 1). To compare toxicity of other toxins in this system, the M. celatum pemK was expressed in S. cerevisiae. Although less important, an inhibitory effect was also observed when the M. celatum pemK gene was expressed in S. cerevisiae (Fig. 3). Similar results were obtained in solid media (data not shown).

Figure 3

Growth of S. cerevisiae cells harboring the L. interrogans chpK gene. Growth of yeast cells harboring pCMha190 (open circles), pCMha190 carrying the L. interrogans chpK gene (filled triangles), and pCMha190 carrying the M. celatum pemK gene (filled squares). The mean of two independent experiments was plotted with the corresponding standard deviations.

4 Conclusions

In conclusion, we have shown that the L. interrogans ChpK inhibits cell growth in E. coli [8], mycobacteria, and yeast. This may have applications in controlling cell proliferation [7] or growth of pathogenic bacteria such as the tubercle bacilli. The physiological role of chp loci remains unclear, as well as the cellular target of ChpK proteins. ChpK might function by inhibiting protein synthesis and/or translation as previously demonstrated for some bacterial toxins of TA loci [1]. Our results suggest that the cellular target of ChpK is conserved in both prokaryotes and eukaryotes.

Acknowledgements

We thank V. Vincent and B. Dujon for their support. We thank J. Boyer for the generous gift of plasmid pCMha190.

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