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The hok (host killing) and sok (suppressor of killing) genes (hok/sok) efficiently maintain the low-copynumber plasmid R1. To investigate whether the hok/sok locus evolved as a phage-exclusion mechanism, Escherichia coli cells that contain hok/sok on a pBR322-based plasmid were challenged with T1, T4, T5, T7, and phage. Upon infection with T4, the optical density of cells containing hok/sok on a high-copy-number plasmid continued to increase whereas the optical density for those lacking hok/sok rapidly declined. The presence of hok/sok reduced the efficiency of plating of T4 by 42% and decreased the plaque size by ϳ85%. Single-step growth experiments demonstrated that hok/sok decreased the T4 burst size by 40%, increased the time to form mature phage (eclipse time) from 22 to 30 min, and increased the time to cell lysis (latent period) from 30 to 60 min. These results further suggest that single cells exhibit altruistic behavior.Recent advances in the study of killer loci and phage exclusion suggest that the two areas may be closely related (22,29,39 (11,15,24,26,35) by killing plasmid-free daughter cells. Each killer system contains a gene that encodes a protein toxin and a gene that inhibits the toxin from being expressed (15). Antisense mRNA killer systems use fold-back inhibition and a small unstable antisense mRNA to prevent translation of the toxin in plasmid-bearing cells (44). A halt in transcription of antisense mRNA due to plasmid loss leads to a rapid decline in its concentration, allowing expression of the killer peptide (14). Proteic killer systems overexpress a labile antitoxin that masks the toxin. A halt in synthesis of antitoxin due to plasmid loss leads to a decline in antitoxin concentration, allowing the toxin to poison the cell.Plasmid loss is not the only event that can prevent transcription of antisense mRNA and induce suicide systems. Temperature shock, amino acid deprivation (1), antibiotics (e.g., rifampin [16]), and phage infection all cause a sudden change in protein synthesis (23). This suggests that cell killing might be induced during phage infection. Furthermore, restriction-modification systems are generally believed to have evolved to inhibit phage infection; however, some plasmid-borne restriction-modification systems have been shown recently to mediate plasmid stability by postsegregational killing (22). This also suggests that plasmid-stabilizing killer loci may also function to exclude phage and might explain why the chromosome contains several killer loci (such as relB Phage-exclusion systems (other than superinfectivity) have been found in phage, plasmids, and cryptic phage elements (29,42). The best-studied systems are exclusion of T4 rII mutants by rex of lysogens (2, 42), exclusion of T4 by lit of the cryptic DNA element e14 (3, 42), exclusion of polynucleotide kinase and RNA-ligase mutants of T4 by prr of cryptic DNA element prr (41, 46), and exclusion of T7 by pif of plasmid F (10, 29). The rex system consists of two proteins, RexA and RexB, which form a membrane comp...

BackgroundEnergy from remote methane reserves is transformative; however, unintended release of this potent greenhouse gas makes it imperative to convert methane efficiently into more readily transported biofuels. No pure microbial culture that grows on methane anaerobically has been isolated, despite that methane capture through anaerobic processes is more efficient than aerobic ones.ResultsHere we engineered the archaeal methanogen Methanosarcina acetivorans to grow anaerobically on methane as a pure culture and to convert methane into the biofuel precursor acetate. To capture methane, we cloned the enzyme methyl-coenzyme M reductase (Mcr) from an unculturable organism, anaerobic methanotrophic archaeal population 1 (ANME-1) from a Black Sea mat, into M. acetivorans to effectively run methanogenesis in reverse. Starting with low-density inocula, M. acetivorans cells producing ANME-1 Mcr consumed up to 9 ± 1 % of methane (corresponding to 109 ± 12 µmol of methane) after 6 weeks of anaerobic growth on methane and utilized 10 mM FeCl3 as an electron acceptor. Accordingly, increases in cell density and total protein were observed as cells grew on methane in a biofilm on solid FeCl3. When incubated on methane for 5 days, high-densities of ANME-1 Mcr-producing M. acetivorans cells consumed 15 ± 2 % methane (corresponding to 143 ± 16 µmol of methane), and produced 10.3 ± 0.8 mM acetate (corresponding to 52 ± 4 µmol of acetate). We further confirmed the growth on methane and acetate production using 13C isotopic labeling of methane and bicarbonate coupled with nuclear magnetic resonance and gas chromatography/mass spectroscopy, as well as RNA sequencing.ConclusionsWe anticipate that our metabolically-engineered strain will provide insights into how methane is cycled in the environment by Archaea as well as will possibly be utilized to convert remote sources of methane into more easily transported biofuels via acetate.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0397-z) contains supplementary material, which is available to authorized users.

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Quorum sensing (QS) is a communication mechanism between bacteria that allows specific processes to be controlled, such as biofilm formation, virulence factor expression, production of secondary metabolites and stress adaptation mechanisms such as bacterial competition systems including secretion systems (SS). These SS have an important role in bacterial communication. SS are ubiquitous; they are present in both Gram-negative and Gram-positive bacteria and in Mycobacterium sp. To date, 8 types of SS have been described (T1SS, T2SS, T3SS, T4SS, T5SS, T6SS, T7SS, and T9SS). They have global functions such as the transport of proteases, lipases, adhesins, heme-binding proteins, and amidases, and specific functions such as the synthesis of proteins in host cells, adaptation to the environment, the secretion of effectors to establish an infectious niche, transfer, absorption and release of DNA, translocation of effector proteins or DNA and autotransporter secretion. All of these functions can contribute to virulence and pathogenesis. In this review, we describe the known types of SS and discuss the ones that have been shown to be regulated by QS. Due to the large amount of information about this topic in some pathogens, we focus mainly on Pseudomonas aeruginosa and Vibrio spp.

Most bacterial cells are stressed, and as a result, some become tolerant to antibiotics by entering a dormant state known as persistence. The key intracellular metabolite that has been linked to this persister state is guanosine tetraphosphate (ppGpp), the alarmone that was first linked to nutrient stress. In Escherichia coli, ppGpp redirects protein production during nutrient stress by interacting with RNA polymerase directly and by inhibiting several proteins. Consistently, increased levels of ppGpp lead to increased persistence; but, the mechanism by which elevated ppGpp translates into persistence has not been determined. Hence, we explored persistence in the absence of ppGpp so that the underlying mechanism of persister cell formation could be explored. We found that persister cells still form, although at lower levels, in the absence of ppGpp. Additionally, the toxin/antitoxin systems that we investigated (MqsR, MazF, GhoT, and YafQ) remain able to increase persistence dramatically in the absence of ppGpp. By overproducing each E. coli protein from the 4287 plasmid vectors of the ASKA library and selecting for increased persistence in the absence of ppGpp (via a relA spoT mutant), we identified five new proteins, YihS, PntA, YqjE, FocA, and Zur, that increase persistence simply by reducing cell growth.

Toxin/antitoxin (TA) systems are prevalent in most bacterial and archaeal genomes, and one of the emerging physiological roles of TA systems is to help regulate pathogenicity. Although TA systems have been studied in several model organisms, few studies have investigated the role of TA systems in pseudomonads. Here, we demonstrate that the previously uncharacterized proteins HigB (unannotated) and HigA (PA4674) of Pseudomonas aeruginosa PA14 form a type II TA system in which antitoxin HigA masks the RNase activity of toxin HigB through direct binding. Furthermore, toxin HigB reduces production of the virulence factors pyochelin, pyocyanin, swarming, and biofilm formation; hence, this system affects the pathogencity of this strain in a manner that has not been demonstrated previously for TA systems.

SUMMARYPathogens that infect the gastrointestinal and respiratory tracts are subjected to intense pressure due to the environmental conditions of the surroundings. This pressure has led to the development of mechanisms of bacterial tolerance or persistence which enable microorganisms to survive in these locations. In this review, we analyze the general stress response (RpoS mediated), reactive oxygen species (ROS) tolerance, energy metabolism, drug efflux pumps, SOS response, quorum sensing (QS) bacterial communication, (p)ppGpp signaling, and toxin-antitoxin (TA) systems of pathogens, such as , spp., spp., spp., , spp., spp., spp., and , all of which inhabit the gastrointestinal tract. The following respiratory tract pathogens are also considered:, ,, , and Knowledge of the molecular mechanisms regulating the bacterial tolerance and persistence phenotypes is essential in the fight against multiresistant pathogens, as it will enable the identification of new targets for developing innovative anti-infective treatments.