Escherichia coli MazF Leads to the Simultaneous Selective Synthesis of Both “Death Proteins” and “Survival Proteins”

The Escherichia coli mazEF module is one of the most thoroughly studied toxin–antitoxin systems. mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. MazF is an endoribonuclease that leads to the inhibition of protein synthesis by cleaving mRNAs at ACA sequences. Here, using 2D-gels, we show that in E. coli, although MazF induction leads to the inhibition of the synthesis of most proteins, the synthesis of an exclusive group of proteins, mostly smaller than about 20 kDa, is still permitted. We identified some of those small proteins by mass spectrometry. By deleting the genes encoding those proteins from the E. coli chromosome, we showed that they were required for the death of most of the cellular population. Under the same experimental conditions, which induce mazEF-mediated cell death, other such proteins were found to be required for the survival of a small sub-population of cells. Thus, MazF appears to be a regulator that induces downstream pathways leading to death of most of the population and the continued survival of a small sub-population, which will likely become the nucleus of a new population when growth conditions become less stressful

A Differential Effect of E. coli Toxin-Antitoxin Systems on Cell Death in Liquid Media and Biofilm Formation

Toxin-antitoxin (TA) modules are gene pairs specifying for a toxin and its antitoxin and are found on the chromosomes of many bacteria including pathogens. Here we report how each of five such TA systems in E. coli affect bacterial cell death differently in liquid media and during biofilm formation. Of all these systems, only the TA system mazEF mediated cell death both in liquid media and during biofilm formation. At the other extreme, as our results have revealed here, the TA system dinJ-YafQ is unique in that it is involved only in the death process during biofilm formation. Cell death governed by mazEF and dinJ-YafQ seems to participate in biofilm formation through a novel mechanism

Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa3 and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showed that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD+)/NADH ratio via binding of NAD+ to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration.Organismic and Evolutionary Biolog

The relBE2Spn Toxin-Antitoxin System of Streptococcus pneumoniae: Role in Antibiotic Tolerance and Functional Conservation in Clinical Isolates

Type II (proteic) chromosomal toxin-antitoxin systems (TAS) are widespread in Bacteria and Archaea but their precise function is known only for a limited number of them. Out of the many TAS described, the relBE family is one of the most abundant, being present in the three first sequenced strains of Streptococcus pneumoniae (D39, TIGR4 and R6). To address the function of the pneumococcal relBE2Spn TAS in the bacterial physiology, we have compared the response of the R6-relBE2Spn wild type strain with that of an isogenic derivative, ΔrelB2Spn under different stress conditions such as carbon and amino acid starvation and antibiotic exposure. Differences on viability between the wild type and mutant strains were found only when treatment directly impaired protein synthesis. As a criterion for the permanence of this locus in a variety of clinical strains, we checked whether the relBE2Spn locus was conserved in around 100 pneumococcal strains, including clinical isolates and strains with known genomes. All strains, although having various types of polymorphisms at the vicinity of the TA region, contained a functional relBE2Spn locus and the type of its structure correlated with the multilocus sequence type. Functionality of this TAS was maintained even in cases where severe rearrangements around the relBE2Spn region were found. We conclude that even though the relBE2Spn TAS is not essential for pneumococcus, it may provide additional advantages to the bacteria for colonization and/or infection

A Toxin-Antitoxin Module in Bacillus subtilis Can Both Mitigate and Amplify Effects of Lethal Stress

Bacterial type-2 (protein-protein) toxin-antitoxin (TA) modules are two-gene operons that are thought to participate in the response to stress. Previous work with Escherichia coli has led to a debate in which some investigators conclude that the modules protect from stress, while others argue that they amplify lethal stress and lead to programmed cell death. To avoid ambiguity arising from the presence of multiple TA modules in E. coli, the effect of the sole type-2 toxin-antitoxin module of Bacillus subtilis was examined for several types of lethal stress.Genetic knockout of the toxin gene, ndoA (ydcE), conferred protection to lethal stressors that included kanamycin, moxifloxacin, hydrogen peroxide, and UV irradiation. However, at low doses of UV irradiation the ndoA deficiency increased lethality. Indeed, gradually increasing UV dose with the ndoA mutant revealed a crossover response--from the mutant being more sensitive than wild-type cells to being less sensitive. For high temperature and nutrient starvation, the toxin deficiency rendered cells hypersensitive. The ndoA deficiency also reduced sporulation frequency, indicating a role for toxin-antitoxin modules in this developmental process. In the case of lethal antimicrobial treatment, deletion of the toxin eliminated a surge in hydrogen peroxide accumulation observed in wild-type cells.A single toxin-antitoxin module can mediate two opposing effects of stress, one that lowers lethality and another that raises it. Protective effects are thought to arise from toxin-mediated inhibition of translation based on published work. The enhanced, stress-mediated killing probably involves toxin-dependent accumulation of reactive oxygen species, since a deficiency in the NdoA toxin suppressed peroxide accumulation following antimicrobial treatment. The type and perhaps the level of stress appear to be important for determining whether this toxin will have a protective or detrimental effect

Evaluation of anti-biofilm activity of acidic amino acids and synergy with ciprofloxacin on Staphylococcus aureus biofilms

Acidic amino acids, aspartic acid (Asp) and glutamic acid (Glu) can enhance the solubility of many poorly soluble drugs including ciprofloxacin (Cip). One of the mechanisms of resistance within a biofilm is retardation of drug diffusion due to poor penetration across the matrix. To overcome this challenge, this work set to investigate novel counter ion approach with acidic amino acids, which we hypothesised will disrupt the biofilm matrix as well as simultaneously improve drug effectiveness. The anti-biofilm activity of D-Asp and D-Glu was studied on Staphylococcus aureus biofilms. Synergistic effect of combining D-amino acids with Cip was also investigated as a strategy to overcome anti-microbial resistance in these biofilms. Interestingly at equimolar combinations, D-Asp and D-Glu were able to significantly disperse (at 20 mM and 40 mM) established biofilms and inhibit (at 10 mM, 20 mM and 40 mM) new biofilm formation in the absence of an antibiotic. Moreover, our study confirmed L-amino acids also exhibit anti-biofilm activity. The synergistic effect of acidic amino acids with Cip was observed at lower concentration ranges (<40 mM amino acids and <90.54 µM, respectively), which resulted in 96.89% (inhibition) and 97.60% (dispersal) reduction in CFU with exposure to 40 mM amino acids. Confocal imaging indicated that the amino acids disrupt the honeycomb-like extracellular DNA (eDNA) meshwork whilst also preventing its formation

The ζ Toxin Induces a Set of Protective Responses and Dormancy

The ζε module consists of a labile antitoxin protein, ε, which in dimer form (ε2) interferes with the action of the long-living monomeric ζ phosphotransferase toxin through protein complex formation. Toxin ζ, which inhibits cell wall biosynthesis and may be bactericide in nature, at or near physiological concentrations induces reversible cessation of Bacillus subtilis proliferation (protective dormancy) by targeting essential metabolic functions followed by propidium iodide (PI) staining in a fraction (20–30%) of the population and selects a subpopulation of cells that exhibit non-inheritable tolerance (1–5×10−5). Early after induction ζ toxin alters the expression of ∼78 genes, with the up-regulation of relA among them. RelA contributes to enforce toxin-induced dormancy. At later times, free active ζ decreases synthesis of macromolecules and releases intracellular K+. We propose that ζ toxin induces reversible protective dormancy and permeation to PI, and expression of ε2 antitoxin reverses these effects. At later times, toxin expression is followed by death of a small fraction (∼10%) of PI stained cells that exited earlier or did not enter into the dormant state. Recovery from stress leads to de novo synthesis of ε2 antitoxin, which blocks ATP binding by ζ toxin, thereby inhibiting its phosphotransferase activity

E. coli Histidine Triad Nucleotide Binding Protein 1 (ecHinT) Is a Catalytic Regulator of D-Alanine Dehydrogenase (DadA) Activity In Vivo

Histidine triad nucleotide binding proteins (Hints) are highly conserved members of the histidine triad (HIT) protein superfamily. Hints comprise the most ancient branch of this superfamily and can be found in Archaea, Bacteria, and Eukaryota. Prokaryotic genomes, including a wide diversity of both Gram-negative and Gram-positive bacteria, typically have one Hint gene encoded by hinT (ycfF in E. coli). Despite their ubiquity, the foundational reason for the wide-spread conservation of Hints across all kingdoms of life remains a mystery. In this study, we used a combination of phenotypic screening and complementation analyses with wild-type and hinT knock-out Escherichia coli strains to show that catalytically active ecHinT is required in E. coli for growth on D-alanine as a sole carbon source. We demonstrate that the expression of catalytically active ecHinT is essential for the activity of the enzyme D-alanine dehydrogenase (DadA) (equivalent to D-amino acid oxidase in eukaryotes), a necessary component of the D-alanine catabolic pathway. Site-directed mutagenesis studies revealed that catalytically active C-terminal mutants of ecHinT are unable to activate DadA activity. In addition, we have designed and synthesized the first cell-permeable inhibitor of ecHinT and demonstrated that the wild-type E. coli treated with the inhibitor exhibited the same phenotype observed for the hinT knock-out strain. These results reveal that the catalytic activity and structure of ecHinT is essential for DadA function and therefore alanine metabolism in E. coli. Moreover, they provide the first biochemical evidence linking the catalytic activity of this ubiquitous protein to the biological function of Hints in Escherichia coli