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TAC from Mycobacterium tuberculosis: a paradigm for stress-responsive toxin–antitoxin systems controlled by SecB-like chaperones

Bacterial type II toxin–antitoxins (TAs) are two-component systems that modulate growth in response to specific stress conditions, thus promoting adaptation and persistence. The major human pathogen Mycobacterium tuberculosis potentially encodes 75 TAs and it has been proposed that persistence induced by active toxins might be relevant for its pathogenesis. In this work, we focus on the newly discovered toxin–antitoxin–chaperone (TAC) system of M. tuberculosis, an atypical stress-responsive TA system tightly controlled by a molecular chaperone that shows similarity to the canonical SecB chaperone involved in Sec-dependent protein export in Gram-negative bacteria. We performed a large-scale genome screening to reconstruct the evolutionary history of TAC systems and found that TAC is not restricted to mycobacteria and seems to have disseminated in diverse taxonomic groups by horizontal gene transfer. Our results suggest that TAC chaperones are evolutionary related to the solitary chaperone SecB and have diverged to become specialized toward their cognate antitoxins. Electronic supplementary material The online version of this article (doi:10.1007/s12192-012-0396-5) contains supplementary material, which is available to authorized users.


Taxonomic tree and distribution of solitary SecB versus TAC/AC
The taxonomic tree of bacterial species was generated from the available complete genomes (October 2011) on the basis of the NCBI taxonomic classification (http://www.ncbi.nlm.nih.gov/taxonomy). The tree was then edited with iTol (http://itol.embl.de/index.shtml) and annotated with the solitary SecB chaperones and TAC/AC systems identified. For the sake of clarity, only one strain per species was retained, giving 901 genomes in the tree. Therefore, in some species other strains may not possess the system.

Horizontal Gene Transfer detection
We used Alien Hunter (AH) software (Vernikos and Parkhill 2006) to identify regions that have unusual sequence composition in terms of k-mers for various values of k (called interpolated variable order). An alien region is one whose AH score is above a genomedependent and automatically calculated threshold based on the sequence composition of the whole genome (termed background composition). AH was run on all the 52 complete genomes possessing a TAC or AC system. Predictions were visualized using Artemis (Rutherford et al. 2000). In some cases, AH predictions could be reinforced when the region encoded other HGT signatures such as tRNA, integrase, transposase, phage genes, plasmid genes or TA genes.

MCL analyses
A graph was built in which nodes correspond to proteins and weighted edges represent the BLASTP log(e-value) obtained between a pair of proteins. Graph partitioning -detection of communities of highly connected nodes -was performed using the Markov clustering program. Sequences used correspond to the proteins whose locus tags are given in table S1.
For SecB chaperones we used the seed of the Pfam02556, corresponding to a subset of 114 representative sequences. Two sequences were removed: one from an archaea, since our analysis focuses on bacteria, and one that our analysis identified as part of a TAC system. The homology relationship was inferred when an e-value less than or equal to 10 -5 was observed between two chaperone or toxin sequences and 10 -3 between two antitoxin sequences.
Partitioning was performed by using an inflat factor of 1.2.

Determination of antitoxin and toxin families
On the base of MCL results, we used RPS-BLAST (Altschul et al. 1990) to detect conserved domains describing each community. The five MCL antitoxin communities, namely HigA, HicB, MqsA, MqsA-like and HTH-like are respectively described by the conserved domains pfam01381, pfam05534, tigr03830, a partial tigr03830 (no CXXCG N-terminal motifs) and a partial and weakly conserved pfam01381. MCL partition leads to 7 toxin communities from which 3 correspond to the pfam05973 that characterizes the HigB toxin of M. tuberculosis, thus these sequences were annotated as HigB toxins. Repartition of HigB toxins in the three communities globally follows taxonomic groups. The MqsR family was named according to the annotation of one of the members of the corresponding community. All toxins associated to HicB antitoxins presented the pfam07927 conserved domain. This domain characterizes the sequences homologues to the YcfA protein from E. coli. Since the BLASTP best hits for this protein are annotated as HicA toxins, we named this family HicA. Two toxin communities did not present any conserved domain, but were considered as potential new families. In some cases, putative toxin sequences were excluded from the MCL analysis, but were either described by conserved domains corresponding to the HigB or HicA families, either presented low sequence similarity with proteins annotated as toxins.

Strains and media
In vivo assays were performed in the strain Escherichia coli W3110 ∆secB::Cm R (Ullers et al. 2007). Bacteria were grown at 37°C in LB medium supplemented when necessary with ampicillin (100 µg/mL) or kanamycin (100 µg/mL).

In vivo assays
The in vivo assays were performed as described (Bordes et al. 2011) with minor modifications.
For complementation of Rv1957 function in controlling HigBA, the E. coli W3110 ∆secB strain co-transformed with plasmids pMPMK6-HigBA and pSE380∆NcoI, pSE380-Rv1957 or pSE380-SmegB, were grown to mid-log phase in LB containing 0.2% glucose, kanamycin and ampicillin. Cultures were serially diluted and spotted on LB-ampicillin-kanamycin agar plates with or without arabinose and IPTG inducers as indicated. Plates were incubated at 37°C overnight. For complementation of the SecB chaperone activity at low temperature, fresh transformants of E. coli W3110 ∆secB containing pSE380∆NcoI, pSE380-SecB, pSE380-Rv1957 or pSE380-SmegB were grown to mid-log phase at 37°C in LB containing 0.2% glucose and ampicillin. Cultures were serially diluted and spotted on LB-ampicillin plates with or without IPTG as indicated and incubated at 37°C overnight or at 16°C for 5 days.