Vibrio cholerae N16961 genome encodes 18 type II Toxin/Antitoxin (TA) systems, all but one located inside gene cassettes of its chromosomal superintegron (SI). This study aims to investigate additional TA systems in this genome. We screened for all two-genes operons of uncharacterized function by analyzing previous RNAseq data. Assays on nine candidates, revealed one additional functional type II TA encoded by the VCA0497-0498 operon, carried inside a SI cassette. We showed that VCA0498 antitoxin alone and in complex with VCA0497 represses its own operon promoter. VCA0497-0498 is the second element of the recently identified dhiT/dhiA superfamily uncharacterized type II TA system. RNAseq analysis revealed that another SI cassette encodes a novel type I TA system: VCA0495 gene and its two associated antisense non-coding RNAs, ncRNA495 and ncRNA496. Silencing of both antisense ncRNAs lead to cell death, demonstrating the type I TA function. Both VCA0497 and VCA0495 toxins do not show any homology to functionally characterized toxins, however our preliminary data suggest that their activity may end up in mRNA degradation, directly or indirectly. Our findings increase the TA systems number carried in this SI to 19, preferentially located in its distal end, confirming their importance in this large cassette array.

Chronic infections and treatment failure are concerning issues in patients with Pseudomonas aeruginosa infections. Persister cell formation in biofilm is considered a key reason for antibiotic resistance and treatment failure. In this study, expression of type II toxin/antitoxin (TA) system genes (relBE, Xre-COG5654, vapBC and Xre-GNAT) in persister cells of biofilm was evaluated in the presence of the antibiotics ciprofloxacin and colistin during exponential and stationary phases.
The impact of antibiotics on biofilm persister cell formation during exponential and stationary phases of P. aeruginosa strains was examined by colony count at different time intervals in the presence of 5-fold minimum inhibitory concentration (MIC) of ciprofloxacin and colistin. Furthermore, expression of relBE, Xre-COG5654, vapBC and Xre-GNAT genes in P. aeruginosa strains undergoing antibiotic treatment for 3.5 h during exponential and stationary phases was examined by RT-qPCR.
Formation of persister cells was observed in biofilms formed by P. aeruginosa strains in the presence of 5 × MIC of ciprofloxacin and colistin compared with the control group after 3.5 h of incubation both during exponential and stationary phases. The number of biofilm persister cells was higher in stationary phase than in exponential phase. According to the findings of RT-qPCR, ciprofloxacin and colistin may induce persister cell formation by enhancing the expression of type II TA systems during stationary and exponential phases.
The result of this study indicate that ciprofloxacin and colistin have the potential to increase persister cells formation in biofilms by influencing the expression of type II TA systems.

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

Chemotherapy is frequently unsuccessful in fully eradicating bacterial biofilm infections. Persisters are a main cause for the failure of antibiotic therapies and are assumed to significantly impact the increased multidrug tolerance and unsuccessful elimination of chronic biofilm infections. Pseudomonas aeruginosa infections are frequently linked to high rates of drug-tolerant persisters, triggering a major challenge to human health. It is crucial to classify persisters to develop novel useful therapeutic strategies to fight infectious diseases. In this study, the mqsR gene was selected as a novel antimicrobial target, and silencing was with antisense peptide nucleic acid (PNA) assay to eradicate the P. aeruginosa persisters. First, they were analysed by experimental procedures. Functionality was assessed by stress conditions. We found that the expression of mqsR (as the toxin) compared with mqsA (as antitoxin) was increased under stress conditions. We demonstrated that when mqsR was targeted and treated with different concentrations of mqsR-PNA after 24 hours; the formation of P. aeruginosa persisters was eradicated. Antisense mqsR-PNA in concentrations of 35 μM or more could eradicate persister cell formation in P. aeruginosa. It was suggested that other toxin-antitoxin loci in P. aeruginosa are examined by antisense PNA to detect their functionality. However, considering the importance of persisters in human infections, ex vivo, in vivo, preclinical and clinical settings should be highlighted.

Tuberculosis (TB) is a contagious disease caused by Mycobacterium tuberculosis (M. tb). It is regarded as a major health threat all over the world, mainly because of its high mortality and drug-resistant nature. Toxin-antitoxin (TA) systems are modules ubiquitously found in prokaryotic organisms, and the well-studied MazEF systems (MazE means \"what is it?\" in Hebrew) are implicated in the formation of \"persister cells\" in the M. tb pathogen. Here, we report cocrystal structures of M. tb MazF-mt1 and -mt9, two important MazF members responsible for specific mRNA and tRNA cleavages, respectively, in complexes with truncated forms of their cognate antitoxin peptides. These peptides bind to the toxins with comparable affinities to their full-length antitoxins, which would reduce the RNA-cleavage capacities of the toxins in vitro. After structural analysis of the binding modes, we systemically tested the influence of the substitutions of individual residues in the truncated MazE-mt9 peptide on its affinity. This study provides structural insight into the binding modes and the inhibition mechanisms between the MazE/F-mt TA pairs. More importantly, it contributes to the future design of peptide-based antimicrobial agents against TB and potentially relieves the drug-resistance problems by targeting novel M. tb proteins.

The post-translational modification (PTM) serves as an important molecular switch mechanism to modulate diverse biological functions in response to specific cues. Though more commonly found in eukaryotic cells, many PTMs have been identified and characterized in bacteria over the past decade, highlighting the importance of PTMs in regulating bacterial physiology. Several bacterial PTM enzymes have been characterized to function as the toxin component of type II TA systems, which consist of a toxin that inhibits cell growth and an antitoxin that protects the cell from poisoning by the toxin. While TA systems can be classified into seven types based on nature of the antitoxin and its activity, type II TA systems are perhaps the most studied among the different TA types and widely distributed in eubacteria and archaea. The type II toxins possessing PTM activities typically modify various cellular targets mostly associated with protein translation and DNA replication. This review mainly focuses on the enzymatic activities, target specificities, antitoxin neutralizing mechanisms of the different families of PTM toxins. We also proposed that TA systems can be conceptually viewed as molecular switches where the \'on\' and \'off\' state of the system is tightly controlled by antitoxins and discussed the perspective on toxins having other physiologically roles apart from growth inhibition by acting on the nonessential cellular targets.

Small proteins are gaining increased attention due to their important functions in major biological processes throughout the domains of life. However, their small size and low sequence conservation make them difficult to identify. It is therefore not surprising that enterobacterial ryfA has escaped identification as a small protein coding gene for nearly 2 decades. Since its identification in 2001, ryfA has been thought to encode a noncoding RNA and has been implicated in biofilm formation in Escherichia coli and pathogenesis in Shigella dysenteriae Although a recent ribosome profiling study suggested ryfA to be translated, the corresponding protein product was not detected. In this study, we provide evidence that ryfA encodes a small toxic inner membrane protein, TimP, overexpression of which causes cytoplasmic membrane leakage. TimP carries an N-terminal signal sequence, indicating that its membrane localization is Sec-dependent. Expression of TimP is repressed by the small RNA (sRNA) TimR, which base pairs with the timP mRNA to inhibit its translation. In contrast to overexpression, endogenous expression of TimP upon timR deletion permits cell growth, possibly indicating a toxicity-independent function in the bacterial membrane.IMPORTANCE Next-generation sequencing (NGS) has enabled the revelation of a vast number of genomes from organisms spanning all domains of life. To reduce complexity when new genome sequences are annotated, open reading frames (ORFs) shorter than 50 codons in length are generally omitted. However, it has recently become evident that this procedure sorts away ORFs encoding small proteins of high biological significance. For instance, tailored small protein identification approaches have shown that bacteria encode numerous small proteins with important physiological functions. As the number of predicted small ORFs increase, it becomes important to characterize the corresponding proteins. In this study, we discovered a conserved but previously overlooked small enterobacterial protein. We show that this protein, which we dubbed TimP, is a potent toxin that inhibits bacterial growth by targeting the cell membrane. Toxicity is relieved by a small regulatory RNA, which binds the toxin mRNA to inhibit toxin synthesis.

BACKGROUND: Persistence is a natural phenomenon whereby a subset of a population of isogenic bacteria either grow slow or become dormant conferring them with the ability to withstand various stresses including antibiotics. In a clinical setting bacterial persistence often leads to the recalcitrance of various infections increasing the treatment time and cost. Additionally, some studies also indicate that persistence can also pave way for the emergence of resistant strains. In a laboratory setting this persistent phenotype is enriched in nutritionally deprived environments. Consequently, in a batch culture the late stationary phase is enriched with persistent bacteria. The mechanism of persister cell formation and its regulation is not well understood. Toxin-antitoxin (TA) systems have been implicated to be responsible for bacterial persistence and rifampicin is used to treat highly persistent bacterial strains. The current study tries to explore a possible interaction between rifampicin and the MazEF TA system that furthers the former\'s success rate in treating persistent bacteria.
RESULTS: In the current study we found that the population of bacteria in the death phase of a batch culture consists of metabolically inactive live cells resembling persisters, which showed higher membrane depolarization as compared to the log phase bacteria. We also observed an increase in the expression of the MazEF TA modules in this phase. Since rifampicin is used to kill the persisters, we assessed the interaction of rifampicin with MazEF complex. We showed that rifampicin moderately interacts with MazEF complex with 1:1 stoichiometry.
CONCLUSIONS: Our study suggests that the interaction of rifampicin with MazEF complex might play an important role in inhibition of persisters.

The bacterial toxin-antitoxin (TA) system regulates cell growth under various environmental stresses. Mycobacterium tuberculosis, the causative pathogen of tuberculosis (TB), has three HigBA type II TA systems with reverse gene organization, consisting of the toxin protein HigB and labile antitoxin protein HigA. Most type II TA modules are transcriptionally autoregulated by the antitoxin itself. In this report, we first present the crystal structure of the M. tuberculosis HigA3 antitoxin (MtHigA3) and MtHigA3 bound to its operator DNA complex. We also investigated the interaction between MtHigA3 and DNA using NMR spectroscopy. The MtHigA3 antitoxin structure is a homodimer that contains a structurally well conserved DNA-binding domain at the N-terminus and a dimerization domain at the C-terminus. Upon comparing the HigA homologue structures, a distinct difference was found in the C-terminal region that possesses the β-lid, and diverse orientations of two helix-turn-helix (HTH) motifs from HigA homologue dimers were observed. The structure of MtHigA3 bound to DNA reveals that the promoter DNA is bound to two HTH motifs of the MtHigA3 dimer presenting 46.5° bending, and the distance between the two HTH motifs of each MtHigA3 monomer was increased in MtHigA3 bound to DNA. The β-lid, which is found only in the tertiary structure of MtHigA3 among the HigA homologues, causes the formation of a tight dimerization network and leads to a unique arrangement for dimer formation that is related to the curvature of the bound DNA. This work could contribute to the understanding of the HigBA system of M. tuberculosis at the atomic level and may contribute to the development of new antibiotics for TB treatment.

Toxin-antitoxin (TA) systems, which regulate many important cellular processes, are abundantly present in prokaryotic organisms. MazEF is a common type of TA system implicated in the formation of \"persisters cells\" of the pathogen Mycobacterium tuberculosis, which contains 10 such systems. However, the exact function and inhibition mode of each MazF protein are not quite understood. Here, we report four high-resolution crystal structures of MazF-mt1 in various forms, including one in complex with MazE-mt1. The toxin displayed two unique interlocked loops that allow the formation of a tight dimer. These loops would open upon interacting with the MazE-mt1 antitoxin mediated by the last two helices of MazE-mt1. With our structure-based design, a mutant that could bind to the antitoxin with an enhanced affinity was produced. Combined crystallographic and biochemical studies further revealed that the binding affinity of MazE-mt1 to MazF-mt1 was mainly attributed to its α3 helical region, while the terminal helix η1 contributes very little or even negatively to the association of the pair, in stark contrast to the MazEF-mt9 system. This study provides structural insight into the binding mode and the inhibition mechanism of the MazE/F-mt1 TA pair, which may reflect the functional differences between different TA systems.