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. 2024 Jan 8;18(1):wrae015.
doi: 10.1093/ismejo/wrae015.

VapC10 toxin of the legume symbiont Sinorhizobium meliloti targets tRNASer and controls intracellular lifestyle

Camille Syska  1 , Aurélie Kiers  1 , Corinne Rancurel  1 , Marc Bailly-Bechet  1 , Justine Lipuma  2 , Geneviève Alloing  1 , Isabelle Garcia  1 , Laurence Dupont  1
Affiliations

VapC10 toxin of the legume symbiont Sinorhizobium meliloti targets tRNASer and controls intracellular lifestyle

Camille Syska et al. ISME J. .

Abstract

The soil bacterium Sinorhizobium meliloti can establish a nitrogen-fixing symbiosis with the model legume Medicago truncatula. The rhizobia induce the formation of a specialized root organ called nodule, where they differentiate into bacteroids and reduce atmospheric nitrogen into ammonia. Little is known on the mechanisms involved in nodule senescence onset and in bacteroid survival inside the infected plant cells. Although toxin-antitoxin (TA) systems have been shown to promote intracellular survival within host cells in human pathogenic bacteria, their role in symbiotic bacteria was rarely investigated. S. meliloti encodes several TA systems, mainly of the VapBC family. Here we present the functional characterization, through a multidisciplinary approach, of the VapBC10 TA system of S. meliloti. Following a mapping by overexpression of an RNase in Escherichia coli (MORE) RNA-seq analysis, we demonstrated that the VapC10 toxin is an RNase that cleaves the anticodon loop of two tRNASer. Thereafter, a bioinformatics approach was used to predict VapC10 targets in bacteroids. This analysis suggests that toxin activation triggers a specific proteome reprogramming that could limit nitrogen fixation capability and viability of bacteroids. Accordingly, a vapC10 mutant induces a delayed senescence in nodules, associated to an enhanced bacteroid survival. VapBC10 TA system could contribute to S. meliloti adaptation to symbiotic lifestyle, in response to plant nitrogen status.

Keywords: VapBC toxin–antitoxin; bacteroid viability; nitrogen-fixing symbiosis; root nodule senescence; tRNAse.

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Conflict of interest statement

Authors declare that there are not conflicts of interest.

Figures

Figure 1
Figure 1
vapBC10 operon encodes a TA system; (A) multiple sequence alignment of VapC10 toxin of S. meliloti with previously characterized VapC homologs in other bacteria; the VapC10 protein was compared to various PIN domain VapC toxins whose structure has been previously solved by crystallography; the sequence alignment was realized using PROMALS3D webserver [40]; the positions of the alpha helices (α) and beta sheets (β) deduced by PROMALS3D are indicated above the alignment; the four conserved acidic amino acid residues required for the catalytic activity of the PIN domain of VapC toxins are indicated by stars at the bottom of the alignment; (B) comparison of the 3D structure of VapC toxins; the structure of S. meliloti VapC10 was predicted using TrRosetta [41] and that of VapC homologs was extracted from Protein Data Bank (PDB accession: VapC30 of Mycobacterium tuberculosis “4XGQ” [42]; VapC1 of H. influenzae “6NKL” [43]; VapC of S. flexneri “3TND” [44]; VapCLT2 of S. Typhimurium “6IFC” [45]); all the 3D models were visualized using Pymol software; (C, D) toxicity assay of the VapC10 protein; E. coli DH5α was transformed with pBAD24 (Control) or pBAD24-vapC10 (vapC10) plasmids (Table S1), and induced with 1% of arabinose (+ ara) or not (− ara); (C) colony-forming unit (CFU) per ml at 1 h postinduction; cells grown until OD600nm= 0.4 were induced by arabinose (T0) for 1 h, serially diluted as indicated, and spotted on LB glucose agar plates; (D) kinetics of viability (CFU/ml); (E) antitoxicity assay of VapB10 protein; E. coli BL21 (DE3) pLysS was transformed with pRSF1b-vapB10 (IPTG inducible), pBAD24-vapC10 (arabinose inducible) or co-transformed with both plasmids; cells were grown until OD600nm = 0.15, induced by arabinose and IPTG at the indicated time (arrow), and their growth capacity was followed by OD600nm measurement for 3 h; results shown are a representative example of three independent biological replicates; the means of the technical duplicates from each condition are shown on the viability (1D) and growth (1E) curves.
Figure 2
Figure 2
VapC10 cleaves tRNASer(GGA) and tRNASer(UGA) in the CU(U/G)G^AA anticodon loop in E. coli; (A, D) mapping of sequencing reads identified using the MORE RNA-seq method on E. coli genome; examples correspond to one replicate obtained on the serX (A) and the serT (D) tRNAs; genes are represented by arrows and reads by lines; color dots in the reads represent punctual nucleotide differences; the 5′ RNA moieties generated by VapC10 cleavage correspond to the 1 093 072-nucleotide position of serX gene (A) and the 1 027 132 nucleotide position of serT gene (D) on the E. coli BW25113∆6 genome (vertical lines); (B, E) histograms representing the fold change of sequencing reads between the induced and noninduced VapC10 conditions, in the tRNASer(GGA) (serX and serW) (B) and in the tRNASer(UGA) (serT) (E), at each nucleotide position; the cleavage site is shown in bold letters in the tRNA sequences, and the anticodon is underlined; the specific position of VapC10 cleavage in the anticodon loop is shown in the secondary structure of the corresponding E. coli tRNAs; the numbers shown below the histograms indicate the nucleotide positions of serX and serT tRNAs; (C, F) predicted structures of tRNASer and best homologs of E. coli tRNASer in S. meliloti; the best homologs of the VapC10-targeted tRNAs, specified by serX and serT in E. coli, are SMc02915 and SMc01243, respectively; the corresponding specific position of VapC10 cleavage in the anticodon loop is shown by an arrow in the secondary structure of the corresponding tRNAs in E. coli and S. meliloti; the consensus cleavage site is shown in bold letters in the tRNA sequences.
Figure 3
Figure 3
Transcripts of the fixation zone, rich in specific serine codons; (A) recognition of the six serine codons by the four tRNASer and their relative codon usage in S. meliloti; (B) transcripts rich in serine codons translated by tRNASer(UGA) and tRNASer(GGA), predicted to be impacted by VapC10 cleavage; a Venn diagram was used to identify genes encoding mRNAs containing at least 4 rare UCA codon (named serT/SMc01243 list), and mRNAs containing at least 12 frequent UCC codon or containing at least 4 rare UCU codon (named serX/SMc02915 list), in the specifically and highly expressed genes (n ≥ 1500 reads) of the fixation Zone III (named ZIII+ list); (C) transcripts rich in serine codons translated by tRNASer(GCU) and tRNASer(CGA), predicted to be not impacted by VapC10 cleavage; a Venn diagram was used to identify genes encoding mRNAs containing at least 12 frequent AGC codon or containing at least 4 rare AGU codon (named serV/SMc02409 list), and mRNAs containing at least 12 frequent UCG codon (named serU/SMc03779 list), in the specifically and highly expressed genes (n ≥ 1500 reads) of the fixation Zone III (named ZIII+ list); lists of genes are shown in File S1; FixC, oxidoreductase involved in nitrogenase functioning; FixI1, cation transporter ATPase subunit; NifA, transcriptional activator of nitrogen fixation genes; NifB, nitrogenase molybdenum-cofactor synthesis protein; NifD, nitrogenase molybdenum-iron protein alpha chain; NifK, nitrogenase molybdenum-iron protein beta chain; NifE, nitrogenase molybdenum-cofactor synthesis protein; NoeA and NoeB, host specific nodulation proteins.
Figure 4
Figure 4
Symbiotic phenotype of vapC10-induced nodules; M. truncatula plants were inoculated with the WT or the vapC10 mutant strains of S. meliloti, and the physiological symbiotic parameters were analyzed at 3- and 6- wpi; (A) phenotype of WT and vapC10 mutant-infected plants; 15 plants inoculated with WT (left) or vapC10 (right) strains are shown; scale bar= 5 cm; (B) weight of aerial plant part (g) (N=3, n=15); (C) nitrogenase activity per plant determined by ARA (N=3; n=5); (D) number of nodules per plant (N=3; n=5); each measure was realized on a three-plant pool and then reported by plant; (E) nitrogenase activity per nodule determined by ARA (N=3; n=5); the nomenclature (N=x) refers to the number of biological replicates and (n=x) refers to the number of measures obtained per replicate; the bars of standard deviation followed by a same letter did not differ significantly; a summary of the P value is shown in Table S3.
Figure 5
Figure 5
vapC10-induced nodules have a delayed senescence at 6 wpi; WT and vapC10-induced nodules were harvested at 6 wpi, on the 5 cm below the crown; (A) stereomicroscope observation of nodules; the indicated percentage corresponds to the relative size of the senescence zone compared to the nodule full length; (B) percentage of fixing (top) and senescent (bottom) nodules; (C) senescence zone length in WT and vapC10-induced nodules; (D) nodule size of WT and vapC10-induced nodules; (E) relative expression of senescence markers in vapC10-induced nodules by RT-qPCR: cysteine protease 6 (CP6), VPE; the gene expression is relative to the expression in the WT-induced nodules; error bars indicate the standard error of four biological replicates F; bacteroid viability in WT and vapC10-induced nodules; WT and vapC10 mutant-induced nodules, semi-thin longitudinal sections (120 μm), were stained with live/dead fluorescent dyes (Live/Dead BacLight probe, Invitrogen) and observed by confocal microscopy (ZEISS LSM; objective 10×); healthy bacteroids are green, and damaged bacteroids are red; I : meristematic Zone, II : infection Zone, III: fixation Zone, IV: senescence zone; scale bars: 500 μm; observations and measures shown in (A–D) were realized on binocular magnifier (Leica MZFLIII, 0.8X) and treated on Axiovision LE software; these experiments were repeated on three biological replicates and one representative result is shown; the RT-qPCR statistical analysis was performed by a t-test; the representation of P values is as follow: ns, nonsignificant, *<.05, **<.01, ***<.005.
Figure 6
Figure 6
Model of VapC10 toxin role during symbiosis; schematic model summarizing the consequences of vapC10 mutation (right panel) and deduced role of an active VapC10 toxin in a WT context (left panel) on bacteroid viability, nodule functioning, and host plant phenotype, during symbiotic interaction of S. meliloti with M. truncatula; the vapC10 mutant induces a lower number of root nodules, but more performant in their nitrogen fixation capacity (improved bacteroid viability and delayed nodule senescence), compared to the WT strain; thus, the global nitrogen supply to the plant is satisfied, explaining a comparable plant yield obtained with the two interacting strains; in a WT context, the VapC10 toxin could act as a post-transcriptional regulator by cleaving specific tRNASer, altering the translation of symbiotic proteins involved in nitrogenase synthesis and functioning; this proteome reprogramming could limit nitrogen fixation and bacterial viability and therefore initiate bacteria and nodule senescence; the VapC10 activity could be associated to the nitrogen plant status; rectangles symbolize host plant-infected cells inside the nodules; the vacuoles (v) and the Golgi apparatus (g) are shown, the nucleus is represented by a circle containing 64 copies of the genome due to its endoreduplication (64C); bacteroids (b) are represented by rod-shaped and Y-shaped cells, the differentiated forms of bacteria (24C indicates the bacterial genome copies after endoreduplication); the pink and green colors indicate cells or nodule zones that are nitrogen fixing or senescent, respectively; ZIII: nitrogen fixation zone of the nodule; ZIV: senescence zone of the nodule; FixC, oxidoreductase regulator of nitrogenase; FixI1, ATPase; NifA, transcriptional activator of nitrogenase; NifB, FeMo cofactor biosynthesis protein; NifE, nitrogenase molybdenum-cofactor synthesis protein; NoeA and NoeB, host-specific nodulation proteins.
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