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The Importance of the Expendable: Toxin-Antitoxin Genes in Plasmids and Chromosomes.

ABSTRACT: Toxin-antitoxin (TA) genes were first reported in plasmids and were considered expendable genetic cassettes involved in the stable maintenance of the plasmid replicon by interfering with growth and/or viability of bacteria in which the plasmid was lost. TAs were later found in bacterial chromosomes and also in integrated mobile genetic elements; they were proposed to be involved in the bacterial response to stressful situations. At present, 100s of TAs have been identified and classified in up to six families (I to VI), with those belonging to the type II (constituted by two protein components) being the most studied. Based on well-characterized examples of several type II TAs, we discuss in this review that irrespective of their locations in plasmids or chromosomes, TAs functionally overlap as indicated by: (i) in both locations they can mediate the maintenance of genetic elements to which they are physical linked, and (ii) they can induce persistence or virulence in response to stress situations. Examples of functional confluences in homologous TA systems with different locations are also given. We also consider whether the physiological role of TAs is due to their genetic organization as operons or to their inherent properties, like the short lifespan of the antitoxin component.

SUBMITTER: Diaz-Orejas R

PROVIDER: S-EPMC5543033 | biostudies-other | 2017

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:In all Streptococcus mutans strains, 5-13% carry a 5.6-kb plasmid. Despite its frequency, little is known about its mediated functions with most of the information coming from a single study focussing on plasmid pUA140. OBJECTIVE:Here, we describe the sequence and genetic organization of two S. mutans 5.6-kb plasmids, pDC09 and pNC101. RESULTS:Based on PicoGreen dsDNA quantification and Real-Time quantitative PCR (RTQ-PCR), the plasmid copy number was found to range between 10 and 74, depending on the strain tested. In contrast to literature, we identified six instead of five open reading frames (ORFs). While the putative gene products of ORF1 (as a Rep-protein) and ORF2 (as a Mob-protein) could be confirmed as being identical to those from pUA140, the functions of ORF3 (unknown) and ORF 4 (possibly AtpE homologue) could not be further revealed. However, the product of ORF5 showed a fairly high identity (38-50%) and structural similarity (58-74%) to RelE of Streptococcus pneumoniae, Streptococcus equi, and Streptococcus downei. In addition, we identified a functionally corresponding ORF6 encoding a protein with 61-68% identity (81-86% similarity) to the S. equi and S. downei antitoxin of the RelB family. RelE and RelB together form a plasmid-encoded toxin-antitoxin (TA) system, RelBE(plas). Despite its rather limited sequence similarity with chromosomal TA systems in S. mutans (RelBE(chro), MazEF, HicBA), we found similar tertiary structures applying I-Tasser protein prediction analysis. CONCLUSION:Type II-toxins, as the plasmid-encoded RelE, are RNA endonucleases. Depending on their mRNA cleavage activity, they might 1) kill every plasmid-free progeny, thereby stabilizing plasmid transfer at the expense of the host and/or 2) help S. mutans enter a dormant state and survive unfavourable environmental conditions. Whilst a function in plasmid stabilization has been confirmed, a function in persistence under nutritional stress, tested here by inducing amino acid starvation, could not be demonstrated so far.

Project description:BackgroundPseudomonas syringae is a γ-proteobacterium causing economically relevant diseases in practically all cultivated plants. Most isolates of this pathogen contain native plasmids collectively carrying many pathogenicity and virulence genes. However, P. syringae is generally an opportunistic pathogen primarily inhabiting environmental reservoirs, which could exert a low selective pressure for virulence plasmids. Additionally, these plasmids usually contain a large proportion of repeated sequences, which could compromise plasmid integrity. Therefore, the identification of plasmid stability determinants and mechanisms to preserve virulence genes is essential to understand the evolution of this pathogen and its adaptability to agroecosystems.ResultsThe three virulence plasmids of P. syringae pv. savastanoi NCPPB 3335 contain from one to seven functional stability determinants, including three highly active toxin-antitoxin systems (TA) in both pPsv48A and pPsv48C. The TA systems reduced loss frequency of pPsv48A by two orders of magnitude, whereas one of the two replicons of pPsv48C likely confers stable inheritance by itself. Notably, inactivation of the TA systems from pPsv48C exposed the plasmid to high-frequency deletions promoted by mobile genetic elements. Thus, recombination between two copies of MITEPsy2 caused the deletion of an 8.3 kb fragment, with a frequency of 3.8 ± 0.3 × 10- 3. Likewise, one-ended transposition of IS801 generated plasmids containing deletions of variable size, with a frequency of 5.5 ± 2.1 × 10- 4, of which 80% had lost virulence gene idi. These deletion derivatives were stably maintained in the population by replication mediated by repJ, which is adjacent to IS801. IS801 also promoted deletions in plasmid pPsv48A, either by recombination or one-ended transposition. In all cases, functional TA systems contributed significantly to reduce the occurrence of plasmid deletions in vivo.ConclusionsVirulence plasmids from P. syringae harbour a diverse array of stability determinants with a variable contribution to plasmid persistence. Importantly, we showed that multiple plasmid-borne TA systems have a prominent role in preserving plasmid integrity and ensuring the maintenance of virulence genes in free-living conditions. This strategy is likely widespread amongst native plasmids of P. syringae and other bacteria.

Project description:A putative type II toxin-antitoxin (TA) module almost exclusively associated with conjugative IncC plasmids is homologous to the higBA family of TA systems found in chromosomes and plasmids of several species of bacteria. Despite the clinical significance and strong association with high-profile antimicrobial resistance (AMR) genes, the TA system of IncC plasmids remains largely uncharacterized. In this study, we present evidence that IncC plasmids encode a bona fide HigB-like toxin that strongly inhibits bacterial growth and results in cell elongation in Escherichia coli. IncC HigB toxin acts as a ribosome-dependent endoribonuclease that significantly reduces the transcript abundance of a subset of adenine-rich mRNA transcripts. A glycine residue at amino acid position 64 is highly conserved in HigB toxins from different bacterial species, and its replacement with valine (G64V) abolishes the toxicity and the mRNA cleavage activity of the IncC HigB toxin. The IncC plasmid higBA TA system functions as an effective addiction module that maintains plasmid stability in an antibiotic-free environment. This higBA addiction module is the only TA system that we identified in the IncC backbone and appears essential for the stable maintenance of IncC plasmids. We also observed that exposure to subinhibitory concentrations of ciprofloxacin, a DNA-damaging fluoroquinolone antibiotic, results in elevated higBA expression, which raises interesting questions about its regulatory mechanisms. A better understanding of this higBA-type TA module potentially allows for its subversion as part of an AMR eradication strategy. IMPORTANCE Toxin-antitoxin (TA) systems play vital roles in maintaining plasmids in bacteria. Plasmids with incompatibility group C are large plasmids that disseminate via conjugation and carry high-profile antibiotic resistance genes. We present experimental evidence that IncC plasmids carry a TA system that functions as an effective addiction module and maintains plasmid stability in an antibiotic-free environment. The toxin of IncC plasmids acts as an endoribonuclease that targets a subset of mRNA transcripts. Overexpressing the IncC toxin gene strongly inhibits bacterial growth and results in cell elongation in Escherichia coli hosts. We also identify a conserved amino acid residue in the toxin protein that is essential for its toxicity and show that the expression of this TA system is activated by a DNA-damaging antibiotic, ciprofloxacin. This mobile TA system may contribute to managing bacterial stress associated with DNA-damaging antibiotics.

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The Importance of the Expendable: Toxin-Antitoxin Genes in Plasmids and Chromosomes.

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