Biofilms are associated with a wide variety of bacterial infections and pose a serious problem in clinical medicine due to their inherent resilience to antibiotic treatment. Within biofilms, persister cells comprise a small bacterial subpopulation that exhibits multidrug tolerance to antibiotics without undergoing genetic change. The low frequency of persister cell formation makes it difficult to isolate and study persisters, and bacterial persistence is often attributed to a quiescent metabolic state induced by toxins that are regulated through toxin-antitoxin systems. Here we mimic toxins via chemical pretreatments to induce high levels of persistence (10 to 100%) from an initial population of 0.01%. Pretreatment of Escherichia coli with (i) rifampin, which halts transcription, (ii) tetracycline, which halts translation, and (iii) carbonyl cyanide m -chlorophenylhydrazone, which halts ATP synthesis, all increased persistence dramatically. Using these compounds, we demonstrate that bacterial persistence results from halted protein synthesis and from environmental cues.

Expanding the bacterial toxin-antitoxin system classes to a fifth class, GhoST was found to be involved in maintenance of persister cells, dormant cells that are tolerant of antibiotics. GhoS is the antitoxin, an endoribonuclease that cleaves the toxin mRNA ghoT, whose gene product is a membrane-lytic protein. Among bacterial toxin-antitoxin systems, to date no antitoxin has been identified that functions by cleaving toxin mRNA. Here we show that YjdO (renamed GhoT) is a membrane lytic peptide that causes ghost cell formation (lysed cells with damaged membranes) and increases persistence (persister cells are tolerant to antibiotics without undergoing genetic change). GhoT is part of a new toxin-antitoxin system with YjdK (renamed GhoS) because in vitro RNA degradation studies, quantitative real-time reverse-transcription PCR and whole-transcriptome studies revealed that GhoS masks GhoT toxicity by cleaving specifically yjdO (ghoT) mRNA. Alanine substitutions showed that Arg28 is important for GhoS activity, and RNA sequencing indicated that the GhoS cleavage site is rich in U and A. The NMR structure of GhoS indicates it is related to the CRISPR-associated-2 RNase, and GhoS is a monomer. Hence, GhoT-GhoS is to our knowledge the first type V toxin-antitoxin system where a protein antitoxin inhibits the toxin by cleaving specifically its mRNA.

Antigenic or mitogenic stimulation of T cells induces the secretion of an array of protein hormones that regulate immune responses. Molecular cloning has contributed strongly to our present understanding of the nature of this regulation. A complementary DNA (cDNA) library prepared from a cloned concanavalin A-activated mouse T-helper cell line was screened for abundant and induction-specific cDNA's. One such randomly chosen cDNA was found to encode mouse preproenkephalin messenger RNA (mRNA). Preproenkephalin mRNA represented about 0.4 percent of the mRNA in the activated cell line but was absent in resting cells of this line. Other induced T-helper cell lines have 0.1 to 0.5 percent of their mRNA as preproenkephalin mRNA. Induced T-helper cell culture supernatants have [Met]enkephalin-immunoreactive material. The production by activated T cells of a peptide neurotransmitter identifies a signal that can potentially permit T cells to modulate the nervous system.

The toxin-antitoxin pair MqsR and MqsA are linked to biofilm formation, quorum sensing and motility, but their specific role in these and other cellular processes is unclear. The demonstration that MqsA directly represses transcription of rpoS, encoding the master regulator of the stress response, provides a unifying explanation. Although it is well recognized that bacteria respond to environmental stress through global networks, the mechanism by which stress is relayed to the interior of the cell is poorly understood. Here we show that enigmatic toxin-antitoxin systems are vital in mediating the environmental stress response. Specifically, the antitoxin MqsA represses rpoS, which encodes the master regulator of stress. Repression of rpoS by MqsA reduces the concentration of the internal messenger 3,5-cyclic diguanylic acid, leading to increased motility and decreased biofilm formation. Furthermore, the repression of rpoS by MqsA decreases oxidative stress resistance via catalase activity. Upon oxidative stress, MqsA is rapidly degraded by Lon protease, resulting in induction of rpoS. Hence, we show that external stress alters gene regulation controlled by toxin-antitoxin systems, such that the degradation of antitoxins during stress leads to a switch from the planktonic state (high motility) to the biofilm state (low motility).

Summary: Since persister cells cause chronic infections and since Escherichia coli toxin MqsR increases persisters, we used protein engineering to increase the toxicity of MqsR to gain insights into persister cell formation. Through two amino acid replacements that increased the stability of MqsR, toxicity and persistence were increased. A whole‐transcriptome study revealed that the MqsR variant increased persistence by repressing genes for acid resistance, multidrug resistance and osmotic resistance. Corroborating these microarray results, deletion of rpoS , as well as the genes that the master stress response regulator RpoS controls, increased persister formation dramatically to the extent that nearly the whole population became persistent. Furthermore, wild‐type cells stressed by prior treatment to acid or hydrogen peroxide increased persistence 12 000‐fold. Whole‐transcriptome analyses of persister cells generated by two different methods (wild‐type cells pretreated with hydrogen peroxide and the rpoS deletion) corroborated the importance of suppressing RpoS in persister cell formation. Therefore, the more toxic MqsR increases persistence by decreasing the ability of the cell to respond to antibiotic stress through its RpoS‐based regulation of acid resistance, multidrug resistance and osmotic resistance systems.

The nitrilases are enzymes that convert nitriles to the corresponding acid and ammonia. They are members of a superfamily, which includes amidases and occur in both prokaryotes and eukaryotes. The superfamily is characterized by having a homodimeric building block with a αββα–αββα sandwich fold and an active site containing four positionally conserved residues: cys, glu, glu and lys. Their high chemical specificity and frequent enantioselectivity makes them attractive biocatalysts for the production of fine chemicals and pharmaceutical intermediates. Nitrilases are also used in the treatment of toxic industrial effluent and cyanide remediation. The superfamily enzymes have been visualized as dimers, tetramers, hexamers, octamers, tetradecamers, octadecamers and variable length helices, but all nitrilase oligomers have the same basic dimer interface. Moreover, in the case of the octamers, tetradecamers, octadecamers and the helices, common principles of subunit association apply. While the range of industrially interesting reactions catalysed by this enzyme class continues to increase, research efforts are still hampered by the lack of a high resolution microbial nitrilase structure which can provide insights into their specificity, enantioselectivity and the mechanism of catalysis. This review provides an overview of the current progress in elucidation of structure and function in this enzyme class and emphasizes insights that may lead to further biotechnological applications.