Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes

Abstract

Prokaryotic chromosomes code for toxin-antitoxin (TA) loci, often in multiple copies. In E.coli, experimental evidence indicates that TA loci are stress-response elements that help cells survive unfavorable growth conditions. The first gene in a TA operon codes for an antitoxin that combines with and neutralizes a regulatory 'toxin', encoded by the second gene. RelE and MazF toxins are regulators of translation that cleave mRNA and function, in interplay with tmRNA, in quality control of gene expression. Here, we present the results from an exhaustive search for TA loci in 126 completely sequenced prokaryotic genomes (16 archaea and 110 bacteria). We identified 671 TA loci belonging to the seven known TA gene families. Surprisingly, obligate intracellular organisms were devoid of TA loci, whereas free-living slowly growing prokaryotes had particularly many (38 in Mycobacterium tuberculosis and 43 in Nitrosomonas europaea). In many cases, TA loci were clustered and closely linked to mobile genetic elements. In the most extreme of these cases, all 13 TA loci of Vibrio cholerae were bona fide integron elements located in the V.cholerae mega-integron. These observations strongly suggest that TA loci are mobile cassettes that move frequently within and between chromosomes and also lend support to the hypothesis that TA loci function as stress-response elements beneficial to free-living prokaryotes.

Figure 1

Distribution of TA loci in 126 organisms. (A) Number of organisms as a function of their number of TA loci. (B) Number of TA loci in individual genomes as a function of genome size. Filled symbols indicate obligate intracellular organisms.

Figure 2

Phylogenetic tree (Chladogram) of chromosomal RelE, HigB and ParE toxins from enteric bacteria. The RelE, HigB and ParE sequences were retrieved from Table S2 (as GIs) and Table S3 (raw sequences). For comparison, one plasmid-encoded toxin homolog from each group was included in the analysis (RelE of P307, HigB of Rts1 and ParE of RK2). The tree was calculated using Clustal W version 1.83. The lengths of the horizontal lines indicate relative evolutionary distances. A scale bar is also shown.

Figure 3

Chromosomal maps of TA loci in individual chromosomes. Information was derived from Table S2. The maps were created using Vector NTI version 7 (Informax). oriC denotes the origin of replication. All TA loci in the two strains of Mtb were identical except that mazEF-1 and mazEF-7 of Mtb H37Rv were not present in Mtb CDC1551. Note that solitary toxin genes are also shown here as TA loci.

Figure 4

Comparison of the genomic locations of the 23 and 25 vapC loci of S.solfataricus and S.tokodaii. Using BLASTP, each vapC gene of S.solfataricus was paired with its most similar vapC ortholog in S.tokodaii.

Figure 5

All 13 TA loci in the mega-integron of V.cholerae Chromosome II have juxtaposed attC sites. (A) The locations of the 13 TA loci in the V.cholera mega-integron are shown relative to the closest attC site. Arrows indicate direction of transcription of the TA operons. The boundaries of the integron were determined here by the Int4 integrase gene and the right-most attC site (attC-R). attC-L is the leftmost attC site of a total of ∼150 attC sites of the integron. (B) Nucleotide sequences of the attC sites [123 bp elements with inverted repeats at their ends as defined in (56)] located near TA loci in the V.cholerae mega-integron. The coordinates of the attC sites in the V.cholerae genome and their distances to the TA structural genes (start codons of antitoxin genes or stop codons of toxin genes): attC1: 322548..322671, +14 bp; attC2: 327878..328000, +17 bp; attC3: 343101..343224C, +18 bp; attC4: 349509..349631, −3 bp; attC5: 365215..365336, +15 bp; attC6: 369203..369325, +35 bp; attC7: 388220..388342, −80 bp; attC8: 399166..399288, +7 bp; attC9: 415580..415701, −2 bp; attC10: 417953..418074, +18 bp; attC11: 420535..420656, +6 bp; attC12: 426191..426312, +6 bp; attC13: 432894..433016, +18 bp). In all cases but two (higBA-1 and phd/doc-1), the attC sites were located downstream and close to the toxin structural genes.

Figure 6

Comparison of the genetic composition of the relA region of enteric bacteria. Three different genetic contexts of the relA in enterics: (A) E.coli K-12, E.coli O157, E.coli O157 EDL933 and Shigella flexneri 2a; (B) Salmonella typhimurium LT2, S.typhi and S.typhi 2A; (C) E.coli CFT073 and S.flexneri 2a 2457T.