Dataset Information

The vapB-vapC Operon of Acidovorax citrulli Functions as a Bona-fide Toxin-Antitoxin Module.

ABSTRACT: Toxin-antitoxin systems are commonly found on plasmids and chromosomes of bacteria and archaea. These systems appear as biscystronic genes encoding a stable toxin and a labile antitoxin, which protects the cells from the toxin's activity. Under specific, mostly stressful conditions, the unstable antitoxin is degraded, the toxin becomes active and growth is arrested. Using genome analysis we identified a putative toxin-antitoxin encoding system in the genome of the plant pathogen Acidovorax citrulli. The system is homologous to vapB-vapC systems from other bacterial species. PCR and phylogenetic analyses suggested that this locus is unique to group II strains of A. citrulli. Using biochemical and molecular analyses we show that A. citrulli VapBC module is a bona-fide toxin-antitoxin module in which VapC is a toxin with ribonuclease activity that can be counteracted by its cognate VapB antitoxin. We further show that transcription of the A. citrulli vapBC locus is induced by amino acid starvation, chloramphenicol and during plant infection. Due to the possible role of TA systems in both virulence and dormancy of human pathogenic bacteria, studies of these systems are gaining a lot of attention. Conversely, studies characterizing toxin-antitoxin systems in plant pathogenic bacteria are lacking. The study presented here validates the activity of VapB and VapC proteins in A. citrulli and suggests their involvement in stress response and host-pathogen interactions.

SUBMITTER: Shavit R

PROVIDER: S-EPMC4701950 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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The vapB-vapC Operon of Acidovorax citrulli Functions as a Bona-fide Toxin-Antitoxin Module.

Shavit Reut R Lebendiker Mario M Pasternak Zohar Z Burdman Saul S Helman Yael Y

Frontiers in microbiology 20160106

Toxin-antitoxin systems are commonly found on plasmids and chromosomes of bacteria and archaea. These systems appear as biscystronic genes encoding a stable toxin and a labile antitoxin, which protects the cells from the toxin's activity. Under specific, mostly stressful conditions, the unstable antitoxin is degraded, the toxin becomes active and growth is arrested. Using genome analysis we identified a putative toxin-antitoxin encoding system in the genome of the plant pathogen Acidovorax citru ...[more]

PMID: 26779154

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Project description:Toxin-antitoxin (TA) systems are widespread among prokaryotes and can play an important role in the maintenance of genetic elements and pathogen virulence. Their roles in bacterial stress response and antimicrobial persistence currently remain highly controversial. The current study investigated a novel TA system (Aave_1720-Aave_1719) from the phytopathogenic bacterium Acidovorax citrulli, which had characteristics of both type II and type V TA systems. Heterologous expression in Escherichia coli confirmed that Aave_1720 functions as a toxin gene, while Aave_1719 acts as antitoxin gene. The Aave_1720 toxin functions as a ribonuclease, and antitoxin Aave_1719 binds directly to Aave_1720 to inhibit its ribonuclease activity, which is typical of type II TA systems. Interestingly, Aave_1719 itself was found to exhibit ribonuclease activity with a preference for targeting Aave_1720 mRNA, which indicates that it can also neutralize the Aave_1720 toxin in a manner characteristic of type V TA systems. Expression analysis revealed that the Aave_1719 gene is upregulated in response to sodium hypochlorite stress and that deletion of the Aave_1720 gene individually or in combination with Aave_1719 affected biofilm formation, as well as the survival of A. citrulli in response to sodium hypochlorite stress. Taken together, these findings expand our understanding of TA systems revealing a novel mechanism of toxin neutralization that combines aspects of both type II and type V TA systems. IMPORTANCE Bacterial fruit blotch (BFB), which is caused by the seed-borne bacterium Acidovorax citrulli, is a devastating disease affecting cucurbit crops throughout the world. Although seed fermentation and treatment with disinfectants can provide effective management of BFB, they cannot completely guarantee pathogen-free seedstock, which suggests that A. citrulli is a highly stress-resistant pathogen. Toxin-antitoxin (TA) systems are common among a diverse range of bacteria and have been reported to play a role in bacterial stress response. However, there is currently much debate about the relationship between TA systems and stress response in bacteria. The current study characterized a novel TA system (Aave_1720-Aave_1719) from A. citrulli that affects both biofilm formation and survival in response to sodium hypochlorite stress. The mechanism of neutralization differed from typical TA systems as two separate mechanisms were associated with the antitoxin, which exhibited characteristics of both type II and type V TA systems. The Aave_1720-Aave_1719 system described here also constitutes the first known report of a double-ribonuclease TA system in bacteria, which expands our understanding of the range of regulatory mechanisms utilized by bacterial TA systems, providing new insight into the survival of A. citrulli in response to stress.

Project description:Acidovorax citrulli causes bacterial fruit blotch (BFB) of cucurbits, a disease that threatens the cucurbit industry worldwide. Despite the economic importance of BFB, little is known about pathogenicity and fitness strategies of the bacterium. We have observed the phenomenon of phenotypic variation in A. citrulli. Here we report the characterization of phenotypic variants (PVs) of two strains, M6 and 7a1, isolated from melon and watermelon, respectively. Phenotypic variation was observed following growth in rich medium, as well as upon isolation of bacteria from inoculated plants or exposure to several stresses, including heat, salt and acidic conditions. When grown on nutrient agar, all PV colonies possessed a translucent appearance, in contrast to parental strain colonies that were opaque. After 72 h, PV colonies were bigger than parental colonies, and had a fuzzy appearance relative to parental strain colonies that are relatively smooth. A. citrulli colonies are generally surrounded by haloes detectable by the naked eye. These haloes are formed by type IV pilus (T4P)-mediated twitching motility that occurs at the edge of the colony. No twitching haloes could be detected around colonies of both M6 and 7a1 PVs, and microscopy observations confirmed that indeed the PVs did not perform twitching motility. In agreement with these results, transmission electron microscopy revealed that M6 and 7a1 PVs do not produce T4P under tested conditions. PVs also differed from their parental strain in swimming motility and biofilm formation, and interestingly, all assessed variants were less virulent than their corresponding parental strains in seed transmission assays. Slight alterations could be detected in some DNA fingerprinting profiles of 7a1 variants relative to the parental strain, while no differences at all could be seen among M6 variants and parental strain, suggesting that, at least in the latter, phenotypic variation is mediated by slight genetic and/or epigenetic alterations.

Project description:UnlabelledMammalian prions are PrP proteins with altered structures causing transmissible fatal neurodegenerative diseases. They are self-perpetuating through formation of beta-sheet-rich assemblies that seed conformational change of cellular PrP. Pathological PrP usually forms an insoluble protease-resistant core exhibiting beta-sheet structures but no more alpha-helical content, loosing the three alpha-helices contained in the correctly folded PrP. The lack of a high-resolution prion structure makes it difficult to understand the dynamics of conversion and to identify elements of the protein involved in this process. To determine whether completeness of residues within the protease-resistant domain is required for prions, we performed serial deletions in the helix H2 C terminus of ovine PrP, since this region has previously shown some tolerance to sequence changes without preventing prion replication. Deletions of either four or five residues essentially preserved the overall PrP structure and mutant PrP expressed in RK13 cells were efficiently converted into bona fide prions upon challenge by three different prion strains. Remarkably, deletions in PrP facilitated the replication of two strains that otherwise do not replicate in this cellular context. Prions with internal deletion were self-propagating and de novo infectious for naive homologous and wild-type PrP-expressing cells. Moreover, they caused transmissible spongiform encephalopathies in mice, with similar biochemical signatures and neuropathologies other than the original strains. Prion convertibility and transfer of strain-specific information are thus preserved despite shortening of an alpha-helix in PrP and removal of residues within prions. These findings provide new insights into sequence/structure/infectivity relationship for prions.ImportancePrions are misfolded PrP proteins that convert the normal protein into a replicate of their own abnormal form. They are responsible for invariably fatal neurodegenerative disorders. Other aggregation-prone proteins appear to have a prion-like mode of expansion in brains, such as in Alzheimer's or Parkinson's diseases. To date, the resolution of prion structure remains elusive. Thus, to genetically define the landscape of regions critical for prion conversion, we tested the effect of short deletions. We found that, surprisingly, removal of a portion of PrP, the C terminus of alpha-helix H2, did not hamper prion formation but generated infectious agents with an internal deletion that showed characteristics essentially similar to those of original infecting strains. Thus, we demonstrate that completeness of the residues inside prions is not necessary for maintaining infectivity and the main strain-specific information, while reporting one of the few if not the only bona fide prions with an internal deletion.

Dataset Information

The vapB-vapC Operon of Acidovorax citrulli Functions as a Bona-fide Toxin-Antitoxin Module.

Publications

The vapB-vapC Operon of Acidovorax citrulli Functions as a Bona-fide Toxin-Antitoxin Module.

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