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Characterization of HicAB toxin-antitoxin module of Sinorhizobium meliloti.

ABSTRACT: BACKGROUND:Toxin-antitoxin (TA) systems are little genetic units generally composed of two genes encoding antitoxin and toxin. These systems are known to be involved in many functions that can lead to growth arrest and cell death. Among the different types of TA systems, the type II gathers together systems where the antitoxin directly binds and inhibits the toxin. Among these type II TA systems, the HicAB module is widely distributed in free-living Bacteria and Archaea and the toxin HicA functions via RNA binding and cleavage. The genome of the symbiotic Sinorhizobium meliloti encodes numerous TA systems and only a few of them are functional. Among the predicted TA systems, there is one homologous to HicAB modules. RESULTS:In this study, we characterize the HicAB toxin-antitoxin module of S. meliloti. The production of the HicA of S. meliloti in Escherichia coli cells abolishes growth and decreases cell viability. We show that expression of the HicB of S. meliloti counteracts HicA toxicity. The results of double hybrid assays and co-purification experiments allow demonstrating the interaction of HicB with the toxin HicA. Purified HicA, but not HicAB complex, is able to degrade ribosomal RNA in vitro. The analysis of separated domains of HicB protein permits us to define the antitoxin activity and the operator-binding domain. CONCLUSIONS:This study points out the first characterization of the HicAB system of the symbiotic S. meliloti whereas HicA is a toxin with ribonuclease activity and HicB has two domains: the COOH-terminal one that binds the operator and the NH2-terminal one that inhibits the toxin.

SUBMITTER: Thomet M

PROVIDER: S-EPMC6327479 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Characterization of HicAB toxin-antitoxin module of Sinorhizobium meliloti.

Thomet Manon M Trautwetter Annie A Ermel Gwennola G Blanco Carlos C

BMC microbiology 20190110 1

<h4>Background</h4>Toxin-antitoxin (TA) systems are little genetic units generally composed of two genes encoding antitoxin and toxin. These systems are known to be involved in many functions that can lead to growth arrest and cell death. Among the different types of TA systems, the type II gathers together systems where the antitoxin directly binds and inhibits the toxin. Among these type II TA systems, the HicAB module is widely distributed in free-living Bacteria and Archaea and the toxin Hic ...[more]

PMID: 30630415

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Project description:l-Carnitine is a trimethylammonium compound mostly known for its contribution to fatty acid transport into mitochondria. In bacteria, it is synthesized from γ-butyrobetaine (GBB) and can be used as a carbon source. l-Carnitine can be formed directly by GBB hydroxylation or synthesized via a biosynthetic route analogous to fatty acid degradation. However, this multistep pathway has not been experimentally characterized. In this work, we identified by gene context analysis a cluster of l-carnitine anabolic genes next to those involved in its catabolism and proceeded to the complete in vitro characterization of l-carnitine biosynthesis and degradation in Sinorhizobium meliloti The five enzymes catalyzing the seven steps that convert GBB to glycine betaine are described. Metabolomic analysis confirmed the multistage synthesis of l-carnitine in GBB-grown cells but also revealed that GBB is synthesized by S. meliloti To our knowledge, this is the first report of aerobic GBB synthesis in bacteria. The conservation of l-carnitine metabolism genes in different bacterial taxonomic classes underscores the role of l-carnitine as a ubiquitous nutrient.IMPORTANCE The experimental characterization of novel metabolic pathways is essential for realizing the value of genome sequences and improving our knowledge of the enzymatic capabilities of the bacterial world. However, 30% to 40% of genes of a typical genome remain unannotated or associated with a putative function. We used enzyme kinetics, liquid chromatography-mass spectroscopy (LC-MS)-based metabolomics, and mutant phenotyping for the characterization of the metabolism of l-carnitine in Sinorhizobium meliloti to provide an accurate annotation of the corresponding genes. The occurrence of conserved gene clusters for carnitine metabolism in soil, plant-associated, and marine bacteria underlines the environmental abundance of carnitine and suggests this molecule might make a significant contribution to ecosystem nitrogen and carbon cycling.

Project description:UnlabelledEscherichia coli regulates its metabolism to adapt to changes in the environment, in particular to stressful downshifts in nutrient quality. Such shifts elicit the so-called stringent response, coordinated by the alarmone guanosine tetra- and pentaphosphate [(p)ppGpp]. On sudden amino acid (aa) starvation, RelA [(p)ppGpp synthetase I] activity is stimulated by binding of uncharged tRNAs to a vacant ribosomal site; the (p)ppGpp level increases dramatically and peaks within the time scale of a few minutes. The decrease of the (p)ppGpp level after the peak is mediated by the decreased production of mRNA by (p)ppGpp-associated transcriptional regulation, which reduces the vacant ribosomal A site and thus constitutes negative feedback to the RelA-dependent (p)ppGpp synthesis. Here we showed that on sudden isoleucine starvation, this peak was higher in an E. coli strain that lacks the 10 known mRNase-encoding toxin-antitoxin (TA) modules present in the wild-type (wt) strain. This observation suggested that toxins are part of the negative-feedback mechanism to control the (p)ppGpp level during the early stringent response. We built a ribosome trafficking model to evaluate the fold increase in RelA activity just after the onset of aa starvation. Combining this with a feedback model between the (p)ppGpp level and the mRNA level, we obtained reasonable fits to the experimental data for both strains. The analysis revealed that toxins are activated rapidly, within a minute after the onset of starvation, reducing the mRNA half-life by ∼30%.ImportanceThe early stringent response elicited by amino acid starvation is controlled by a sharp increase of the cellular (p)ppGpp level. Toxin-antitoxin module-encoded mRNases are activated by (p)ppGpp through enhanced degradation of antitoxins. The present work shows that this activation happens over a very short time scale and that the activated mRNases negatively affect the (p)ppGpp level. The proposed mathematical model of (p)ppGpp regulation through the mRNA level highlights the importance of several feedback loops in early (p)ppGpp regulation.

Dataset Information

Characterization of HicAB toxin-antitoxin module of Sinorhizobium meliloti.

Publications

Characterization of HicAB toxin-antitoxin module of Sinorhizobium meliloti.

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