ACA Sequences Research Articles

Autologous CD4 T Lymphocytes Modified with a Tat-Dependent, Virus-Specific Endoribonuclease Gene in HIV-Infected Individuals

MazF is an Escherichia coli-derived endoribonuclease that selectively cleaves ACA sequences of mRNA prevalent in HIV. We administered a single infusion of autologous CD4T lymphocytes modified to express a Tat-dependent MazF transgene to 10 HIV-infected individuals (six remaining on antiretroviral therapy [ART]; four undergoing treatment interruption post-infusion) in order to provide a population of HIV-resistant immune cells. In participants who remained on ART, increases in CD4 and CD8 Tcell counts of ~200 cells/mm3 each occurred within 2weeks of infusion and persisted for at least 6months. Modified cells were detectable for several months in the blood and trafficked to gastrointestinal lymph tissue. HIV-1 Tat introduced exvivo to the modified CD4+ Tcells induced MazF expression in both pre- and post-infusion samples, and MazF expression was detected invivo post-viral-rebound during ATI. One participant experienced mild cytokine release syndrome. In sum, this study of a single infusion of MazF-modified CD4T lymphocytes demonstrated safety of these cells, distribution to lymph tissue and maintenance of Tat-inducible MazF endoribonuclease activity, as well as sustained elevation of blood CD4 and CD8 Tcell counts. Future studies to assess effects on viremia and latent proviral reservoir are warranted.

Specific elimination of coxsackievirus B3 infected cells with a protein engineered toxin-antitoxin system.

BackgroundsCoxsackievirus B3 (CVB3) is a member of the family Picornaviridae, and along with polio-viruses, belongs to the Enterovirus genus. The CVB3 genome is composed single-stranded RNA encoding polyproteins, which are cleaved to individual functional proteins by 2A and 3C proteases proteins which have been targeted for drug development. Here, we showed that protease activity required to activate a toxic protein may be used to prevent viral infection.MethodsWe modified the MazE-MazF antitoxin-toxin system of Escherichia coli to fuse a C-terminal fragment of MazE to the N-terminal end of toxin MazF with a linker having a specific protease cleavage site for CVB3. This fusion protein formed a stable dimer and was capable of inactivating the mRNA interferase activity of MazF which cleaves the ACA sequence in mRNA substrates.ResultsThe incubation of 2A proteases with the fusion proteins induced cleavage between the MazE and MazF fragments from the fusion proteins; the subsequent release of MazF significantly inhibited virus replication. Additionally, we note that, CVB3 infected HeLa cells quickly died through a MazF toxin mediated effect before virus protein expression.ConclusionThese findings suggest that the MazEF fusion protein has a strong potential to be developed as an anti-virus therapy following CVB3 infection.

MazF activation causes ACA sequence-independent and selective alterations in RNA levels in Escherichia coli.

Escherichia coli MazF is a toxin protein that cleaves RNA at ACA sequences. Its activation has been thought to cause growth inhibition, primarily through indiscriminate cleavage of RNA. To investigate responses following MazF activation, transcriptomic profiles of mazF-overexpressing and non-overexpressing E. coli K12 cells were compared. Analyses of differentially expressed genes demonstrated that the presence and the number of ACA trimers in RNA was unrelated to cellular RNA levels. Mapping differentially expressed genes onto the chromosome identified two chromosomal segments in which upregulated genes formed clusters, and these segments were absent in the chromosomes of E. coli strains other than K12. These results suggest that MazF regulates selective, rather than indiscriminate, categories of genes, and is involved in the regulation of horizontally acquired genes. We conclude that the primary role of MazF is not only cleaving RNA indiscriminately but also generating a specific cellular state.

Reply to Wade and Laub, 'Concerns about "Stress-Induced MazF-Mediated Proteins in Escherichia coli"'.

We thank Dr. Wade and Dr. Laub for taking the time to offer comments regarding our paper “Stress-Induced MazF-Mediated Proteins in Escherichia coli,” but we disagree with the points raised by them, and we answer all the questions as follows. In response to the first concern raised by Dr. Wade and Dr. Laub, namely, that there is no significant enrichment of ACA sequences upstream of MazF-induced genes, we did not claim that “there is a significant enrichment of ACA sequences upstream of MazF-induced genes.” Rather we claim that MazF-induced genes carry an ACA sequence in the region 100 nucleotides upstream of the AUG initiator of their mRNAs. We did not use the phrase “enrichment of ACA sequences.” Furthermore, as noted our criticized paper (1), our results corroborated previous findings (2) showing that in E. coli, the stress-induced MazF-mediated stress translation machinery (STM) …

Development of mazF-based markerless genome editing system and metabolic pathway engineering in Candida tropicalis for producing long-chain dicarboxylic acids

Candida tropicalis can grow with alkanes or plant oils as the sole carbon source, and its industrial application thus has great potential. However, the choice of a suitable genetic operating system can effectively increase the speed of metabolic engineering. MazF functions as an mRNA interferase that preferentially cleaves single-stranded mRNAs at ACA sequences to inhibit protein synthesis, leading to cell growth arrest. Here, we constructed a suicide plasmid named pPICPJ-mazF that uses the mazF gene of Escherichia coli as a counterselectable marker for the markerless editing of C. tropicalis genes to increase the rate of conversion of oils into long-chain dicarboxylic acids. To reduce the β-oxidation of fatty acids, the carnitine acetyltransferase gene (CART) was deleted using the gene editing system, and the yield of long-chain acids from the strain was increased to 8.27g/L. By two homologous single exchanges, the promoters of both the cytochrome P450 gene and the NADPH-cytochrome P450 reductase gene were subsequently replaced by the constitutively expressed promoter pGAP, and the production of long-chain dicarboxylic acids by the generated strain (C. tropicalis PJPP1702) reached 11.39g/L. The results of fed-batch fermentation showed that the yield of long-chain acids from the strain was further increased to 32.84g/L, which was 11.4 times higher than that from the original strain. The results also showed that the pPICPJ-mazF-based markerless editing system may be more suited for completing the genetic editing of C. tropicalis.

Genome-wide analysis of wheat calcium ATPases and potential role of selected ACAs and ECAs in calcium stress

BackgroundP2- type calcium ATPases (ACAs-auto inhibited calcium ATPases and ECAs-endoplasmic reticulum calcium ATPases) belong to the P- type ATPase family of active membrane transporters and are significantly involved in maintaining accurate levels of Ca2+, Mn2+ and Zn2+ in the cytosol as well as playing a very important role in stress signaling, stomatal opening and closing and pollen tube growth. Here we report the identification and possible role of some of these ATPases from wheat.ResultsIn this study, ACA and ECA sequences of six species (belonging to Poaceae) were retrieved from different databases and a phylogenetic tree was constructed. A high degree of evolutionary relatedness was observed among P2 sequences characterized in this study. Members of the respective groups from different plant species were observed to fall under the same clade. This pattern highlights the common ancestry of P2− type calcium ATPases. Furthermore, qRT-PCR was used to analyse the expression of selected ACAs and ECAs from Triticum aestivum (wheat) under calcium toxicity and calcium deficiency. The data indicated that expression of ECAs is enhanced under calcium stress, suggesting possible roles of these ATPases in calcium homeostasis in wheat. Similarly, the expression of ACAs was significantly different in plants grown under calcium stress as compared to plants grown under control conditions. This gives clues to the role of ACAs in signal transduction during calcium stress in wheat.ConclusionHere we concluded that wheat genome consists of nine P2B and three P2A -type calcium ATPases. Moreover, gene loss events in wheat ancestors lead to the loss of a particular homoeolog of a gene in wheat. To elaborate the role of these wheat ATPases, qRT-PCR was performed. The results indicated that when plants are exposed to calcium stress, both P2A and P2B gene expression get enhanced. This further gives clues about the possible role of these ATPases in wheat in calcium management. These findings can be useful in future for genetic manipulations as well as in wheat genome annotation process.

Deinococcus radiodurans Toxin-Antitoxin MazEF-dr Mediates Cell Death in Response to DNA Damage Stress.

Here we identified a functional MazEF-dr system in the exceptionally stress-resistant bacterium D. radiodurans. We showed that overexpression of the toxin MazF-dr inhibited the growth of Escherichia coli. The toxic effect of MazF-dr was due to its sequence-specific endoribonuclease activity on RNAs containing a consensus 5′ACA3′, and it could be neutralized by MazE-dr. The MazF-dr showed a special cleavage preference for the nucleotide present before the ACA sequence with the order by U>A>G>C. MazEF-dr mediated the death of D. radiodurans cells under sub-lethal dose of stresses. The characteristics of programmed cell death (PCD) including membrane blebbing, loss of membrane integrity and cytoplasm condensation occurred in a fraction of the wild-type population at sub-lethal concentration of the DNA damaging agent mitomycin C (MMC); however, a MazEF-dr mutation relieved the cell death, suggesting that MazEF-dr mediated cell death through its endoribonuclease activity in response to DNA damage stress. The MazEF-dr-mediated cell death of a fraction of the population might serve as a survival strategy for the remaining population of D. radiodurans under DNA damage stress.

Abstract 4314: Exclusive delivery of mazF in cancer cells by Listeria monocytogenes

Abstract Programmed cell death (PCD), an active process that leads to cell suicide, is a critical mechanism in every organism. MazF, a bacterial ribonuclease protein, can trigger PCD in bacterial cells and also induce Bak-dependent apoptosis in mammalian cells. This bacterial ribonuclease cleaves mRNAs at ACA sequences leading to inhibition of protein synthesis in cells. Hence, we hypothesized that the overexpression of MazF proteins in cancer cells could result in the induction of apoptosis. In the present study, the ACA-less mazF gene was inserted downstream of the Internal Ribosome Entry Site (IRES) and under the control of T7 promoter. The plasmid was electroporated into Listeria monocytogenes harboring pCSA1 that encodes T7 RNA polymerase under the control of the listeria actA promoter. Subsequently, the bacteria were functionalized with Trastuzumab (Herceptin®) to deliver the mazF mRNA into HER2/neu breast cancer cells. The results showed that MazF protein has an ability to induce apoptosis in HER2/neu breast cancer cells. This finding is the first report of the application and delivery of MazF as an anti-cancer agent for the induction of apoptosis in HER2/neu breast cancer cell line. This research suggests cancer therapy that targets only cancer cells but not healthy cells and offers more advantages, e.g., inexpensiveness, target tissue specificity, easy and safe delivery, in comparison to other gene therapy vectors or direct protein administration. Citation Format: Maryam Saffarian, Jeremy Tzeng. Exclusive delivery of mazF in cancer cells by Listeria monocytogenes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4314. doi:10.1158/1538-7445.AM2017-4314

Abstract 3521: The effect of MazF, Escherichia coli ribonuclease, on gastric adenocarcinoma (AGS cells)

Abstract Apoptosis is a gene-directed program that could be inhibited by several factors such as the disruption of the balance between pro-apoptotic and anti-apoptotic proteins, the impaired death receptor signalling or the reduced caspase functions. Several studies have shown that ribonucleases or antibiotics can induce apoptosis in mammalian cells through the shutoff of the protein synthesises. MazF produced by E. coli is a ribonuclease protein that cleaves mRNAs at ACA sequence sites and inhibits their translation. Thus, we hypothesized that the overexpression of MazF proteins in cancer cells could lead to the induction of apoptosis. In the present study, the ACA-less mazF gene was inserted into pSF-T7-EMCV T7 IRES expression plasmid. The mRNAs of mazF gene were synthesized in in vitro conditions, and one μg/ml of the mRNA was transferred into AGS (Gastric adenocarcinoma) cell line. The incidence of apoptosis was detected by conducting caspase 3/7 and Annexin-V assays. The results showed that 99.83% of cells were detached after 18 hours, 0.0086% of cells remained attached without apparent apoptosis, but 0.101% of attached cells were under apoptotic conditions. The results suggest that MazF protein has an ability to induce apoptosis in AGS cells. Since this protein can cleave mRNAs at ACA sites, it could inhibit protein synthesis, reduce the growth rate, and induce apoptosis in cancer cells. This finding is the first report of the application of MazF as an anti-cancer agent for the induction of apoptosis in AGS cell line. Citation Format: Maryam Saffarian, Jeremy Tzeng. The effect of MazF, Escherichia coli ribonuclease, on gastric adenocarcinoma (AGS cells). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3521.

Regression of Solid Tumors by Induction of MazF, a Bacterial mRNA Endoribonuclease

MazF from Escherichia coli is an endoribonuclease that specifically cleaves mRNAs at ACA sequences. Its induction in mammalian cells has been shown to cause programmed cell death. Here we explored if a bacterial MazF-MazE toxin-antitoxin system can be used for gene therapy. For this, we first constructed a tetracycline-inducible MazF expression system in human embryonic kidney cells (T-Rex 293-mazF). Solid tumors were formed by injecting T-Rex 293-mazF cells into nude mice. All 8 mice injected with the cells developed solid tumors, which regressed upon induction of MazF. In 4 mice, tumors completely regressed, while in the remaining 4 mice, tumors reappeared after apparent significant regression, which was found to be due to the lack of presence of functional MazF. Notably, the MazF-mediated regression of the tumors was counteracted by the expression of its cognate antitoxin MazE. These results indicate that a bacterial MazF-MazE toxin-antitoxin system may have potential to be used as a therapeutic tool.

Sustained Inhibition of HIV-1 Replication by Conditional Expression of theE. coli-Derived Endoribonuclease MazF in CD4+T cells

Gene therapy using a Tat-dependent expression system of MazF, an ACA nucleotide sequence-specific endoribonuclease derived from Escherichia coli, in a retroviral vector appears to be an alternative approach to the treatment of human immunodeficiency virus type 1 (HIV-1) infection. MazF can cleave HIV-1 RNA, since it has more than 240 ACA sequences. Significant inhibition of viral replication, irrespective of HIV-1 strains, was observed in CD4(+) T cells that had been transduced with the MazF-expressing retroviral vector (MazF-T cells). The growth and viability of MazF-T cells were not affected by HIV-1 infection. Interestingly, the infectivity of HIV-1 produced from MazF-T cells was found to be lower than that from control CD4(+) T cells. A long-term culture experiment with HIV-1-infected cells revealed that viral replication was always lower in MazF-T cells than in CD4(+) T cells transduced with or without a control vector for more than 200 days. MazF was expressed and mainly localized in the cytoplasm of the infected cells. Unlike in CD4(+) T cells, the expression level of Tat gradually decreased rather than increased in MazF-T cells after HIV-1 infection. As a consequence, the expression level of MazF appeared to be well regulated and sustained during HIV-1 infection in MazF-T cells. Furthermore, the levels of cellular mRNA were not affected by HIV-1 infection. Thus, the Tat-dependent MazF expression system has great potential for inhibition of HIV-1 replication in vivo without apparent toxicity and may be able to avoid the emergence of resistant strains.

Generation of selenoprotein with glutathione peroxidase activity by chemical modification of the single-chain variable fragment expressed in a single-protein production system and its antioxidant ability

Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. Single-chain variable fragment (scFv) can be converted into seleniumcontaining single-chain variable fragment (Se-scFv) by chemical modification of the hydroxyl groups in scFv, thus Se-scFv possesses GPX activity and becomes a prodrug. To improve the expression of scFv and simplify its purification steps, Single-protein production (SPP) system was used to express scFv and chemical modification was used to synthesize Se-scFv. Therefore, we must construct a new scFv-WCD1-lessACA gene, which can express its mRNA not containing any ACA sequences and express its amino acid sequence of target protein (scFv) being same to scFv-WCD1. In this way, the scFv-WCD1-lessACA can be only expressed in SPP system and no other background proteins in the cells could be expressed. The expression results showed that high level of scFv-WCD1-lessACA synthesis was at least sustained for 96 h in the virtual absence of background protein synthesis. Then, selenocysteine (Sec) was incorporated into the scFv-WCD1-lessACA by chemical modification and resulted in Se-scFv-WCD1-lessACA. The enzymatic characteristics of Se-scFv-WCD1-lessACA were determined. GPX activity was 2,563 U/μmol, its binding constant for GSH was 0.687 ×105/mol. Moreover, Se-scFv-WCD1-lessACA was confirmed to have a strong antioxidant ability to protect mitochondria against oxidative damage induced by Vc/Fe2+ (mitochondrial damage model), suggesting that Se-scFv-WCD1-lessACA has potential application for protection of mitochondrial damage induced by reactive oxygen species (ROS).

Clostridium difficile MazF Toxin Exhibits Selective, Not Global, mRNA Cleavage

Clostridium difficile is an important, emerging nosocomial pathogen. The transition from harmless colonization to disease is typically preceded by antimicrobial therapy, which alters the balance of the intestinal flora, enabling C. difficile to proliferate in the colon. One of the most perplexing aspects of the C. difficile infectious cycle is its ability to survive antimicrobial therapy and transition from inert colonization to active infection. Toxin-antitoxin (TA) systems have been implicated in facilitating persistence after antibiotic treatment. We identified only one TA system in C. difficile strain 630 (epidemic type X), designated MazE-cd and MazF-cd, a counterpart of the well-characterized Escherichia coli MazEF TA system. This E. coli MazF toxin cleaves mRNA at ACA sequences, leading to global mRNA degradation, growth arrest, and death. Likewise, MazF-cd expression in E. coli or Clostridium perfringens resulted in growth arrest. Primer extension analysis revealed that MazF-cd cleaved RNA at the five-base consensus sequence UACAU, suggesting that the mRNAs susceptible to cleavage comprise a subset of total mRNAs. In agreement, we observed differential cleavage of several mRNAs by MazF-cd in vivo, revealing a direct correlation between the number of cleavage recognition sites within a given transcript and its susceptibility to degradation by MazF-cd. Interestingly, upon detailed statistical analyses of the C. difficile transcriptome, the major C. difficile virulence factor toxin B (TcdB) and CwpV, a cell wall protein involved in aggregation, were predicted to be significantly resistant to MazF-cd cleavage.

Intramolecular Regulation of the Sequence-Specific mRNA Interferase Activity of MazF Fused to a MazE Fragment with a Linker Cleavable by Specific Proteases

The genomes of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) consist of single-stranded RNA encoding polyproteins, which are processed to individual functional proteins by virus-encoded specific proteases. These proteases have been used as targets for drug development. Here, instead of targeting these proteases to inhibit viral infection, we utilized the protease activity to activate a toxic protein to prevent viral infection. We engineered the MazE-MazF antitoxin-toxin system of Escherichia coli to fuse a C-terminal 41-residue fragment of antitoxin MazE to the N-terminal end of toxin MazF with a linker having a specific protease cleavage site for either HIV PR (HIV-1 protease), NS3 protease (HCV protease), or factor Xa. These fusion proteins formed a stable dimer (instead of the MazF(2)-MazE(2)-MazF(2) heterohexamer in nature) to inactivate the ACA (sequence)-specific mRNA interferase activity of MazF. When the fusion proteins were incubated with the corresponding proteases, the MazE fragment was cleaved from the fusion proteins, releasing active MazF, which then acted as an ACA-specific mRNA interferase cleaving single-stranded MS2 phage RNA. The intramolecular regulation of MazF toxicity by proteases as demonstrated may provide a novel approach for preventive and therapeutic treatments of infection by HIV-1, HCV, and other single-stranded RNA viruses.

Discovery of Pyrobaculum small RNA families with atypical pseudouridine guide RNA features.

In the Eukarya and Archaea, small RNA-guided pseudouridine modification is believed to be an essential step in ribosomal RNA maturation. While readily modeled and identified by computational methods in eukaryotic species, these guide RNAs have not been found in most archaeal genomes. Using high-throughput transcriptome sequencing and comparative genomics, we have identified ten novel small RNA families that appear to function as H/ACA pseudouridylation guide sRNAs, yet surprisingly lack several expected canonical features. The new RNA genes are transcribed and highly conserved across at least six species in the archaeal hyperthermophilic genus Pyrobaculum. The sRNAs exhibit a single hairpin structure interrupted by a conserved kink-turn motif, yet only two of ten families contain the complete canonical structure found in all other H/ACA sRNAs. Half of the sRNAs lack the conserved 3'-terminal ACA sequence, and many contain only a single 3' guide region rather than the canonical 5' and 3' bipartite guides. The predicted sRNA structures contain guide sequences that exhibit strong complementarity to ribosomal RNA or transfer RNA. Most of the predicted targets of pseudouridine modification are structurally equivalent to those known in other species. One sRNA appears capable of guiding pseudouridine modification at positions U54 and U55 in most or all Pyrobaculum tRNAs. We experimentally tested seven predicted pseudouridine modifications in ribosomal RNA, and all but one was confirmed. The structural insights provided by this new set of Pyrobaculum sRNAs will augment existing models and may facilitate the identification and characterization of new guide sRNAs in other archaeal species.

Selective Translation of Leaderless mRNAs by Specialized Ribosomes Generated by MazF in Escherichia coli

Escherichia coli (E. coli) mazEF is a stress-induced toxin-antitoxin (TA) module. The toxin MazF is an endoribonuclease that cleaves single-stranded mRNAs at ACA sequences. Here, we show that MazF cleaves at ACA sites at or closely upstream of the AUG start codon of some specific mRNAs and thereby generates leaderless mRNAs. Moreover, we provide evidence that MazF also targets 16S rRNA within 30S ribosomal subunits at the decoding center, thereby removing 43 nucleotides from the 3' terminus. As this region comprises the anti-Shine-Dalgarno (aSD) sequence that is required for translation initiation on canonical mRNAs, a subpopulation of ribosomes is formed that selectively translates the described leaderless mRNAs both in vivo and in vitro. Thus, we have discovered a modified translation machinery that is generated in response to MazF induction and that probably serves for stress adaptation in Escherichia coli.

In Vivo Safety and Persistence of Endoribonuclease Gene-Transduced CD4+ T Cells in Cynomolgus Macaques for HIV-1 Gene Therapy Model

BackgroundMazF is an endoribonuclease encoded by Escherichia coli that specifically cleaves the ACA sequence of mRNA. In our previous report, conditional expression of MazF in the HIV-1 LTR rendered CD4+ T lymphocytes resistant to HIV-1 replication. In this study, we examined the in vivo safety and persistence of MazF-transduced cynomolgus macaque CD4+ T cells infused into autologous monkeys.Methodology/Principal FindingsThe in vivo persistence of the gene-modified CD4+ T cells in the peripheral blood was monitored for more than half a year using quantitative real-time PCR and flow cytometry, followed by experimental autopsy in order to examine the safety and distribution pattern of the infused cells in several organs. Although the levels of the MazF-transduced CD4+ T cells gradually decreased in the peripheral blood, they were clearly detected throughout the experimental period. Moreover, the infused cells were detected in the distal lymphoid tissues, such as several lymph nodes and the spleen. Histopathological analyses of tissues revealed that there were no lesions related to the infused gene modified cells. Antibodies against MazF were not detected. These data suggest the safety and the low immunogenicity of MazF-transduced CD4+ T cells. Finally, gene modified cells harvested from the monkey more than half a year post-infusion suppressed the replication of SHIV 89.6P.Conclusions/SignificanceThe long-term persistence, safety and continuous HIV replication resistance of the mazF gene-modified CD4+ T cells in the non-human primate model suggests that autologous transplantation of mazF gene-modified cells is an attractive strategy for HIV gene therapy.

Bacillus subtilis MazF-bs (EndoA) is a UACAU-specific mRNA interferase

MazF is an mRNA interferase which cleaves mRNAs at a specific sequence. Here, we show that in contrast to MazF-ec from Escherichia coli, which specifically cleaves ACA sequences, MazF-bs from Bacillus subtilis is an mRNA interferase that specifically cleaves a five-base sequence, UACAU. MazF homologues widely prevailing in Gram-positive bacteria were found to be highly homologous to MazF-bs, suggesting that they may also have similar cleavage specificity. This cleavage site is over-represented in the B. subtilis genes associated with biosynthesis of secondary metabolites, suggesting that MazF-bs may be involved in the regulation of the production of secondary metabolites.

The Escherichia coli Extracellular Death Factor EDF Induces the Endoribonucleolytic Activities of the Toxins MazF and ChpBK

Escherichia coli (E. coli) mazEF is a toxin-antitoxin (TA) stress-induced module that mediates cell death requiring the quorum-sensing pentapeptide NNWNN designated EDF (extracellular death factor). E. coli toxin MazF is a sequence-specific endoribonuclease cleaving single-stranded mRNAs at ACA sequences. E. coli ChpBK, a toxin homologous to MazF, is a sequence-specific endoribonuclease cleaving single-stranded mRNAs at ACA, ACG, and ACU sequences. Here we report that, in vitro, the signaling molecule EDF significantly amplifies the endoribonucleolytic activities of both MazF and ChpBK. EDF also overcomes the inhibitory activity of the antitoxins MazE over the toxin MazF and ChpBI over ChpBK. EDF sequence is important for both functions. Moreover, direct sequence-specific binding of EDF to MazF has been confirmed. Peptide-protein modeling revealed parallel contacts between EDF-MazF and MazE-MazF. These findings are intriguing, since most known quorum-sensing molecules monitor gene expression on the transcriptional level, while EDF monitors posttranscriptionally.

Production of membrane proteins for NMR studies using the condensed single protein (cSPP) production system.

In the Single Protein Production (SPP) method, all E. coli cellular mRNAs are eliminated by the induction of MazF, an ACA-specific mRNA interferase. When an mRNA for a membrane protein, engineered to have no ACA sequences without altering its amino acid sequence, is induced in the MazF-induced cells, E. coli is converted into a bioreactor producing only the targeted membrane protein. Here we demonstrate that three prokaryotic inner membrane proteins, two prokaryotic outer membrane proteins, and one human virus membrane protein can be produced at very high levels, and assembled in appropriate membrane fractions. The condensed SPP (cSPP) system was used to selectively produce isotope-enriched membrane proteins for NMR studies in up to 150-fold condensed culture without affecting protein yields, providing more than 99% cost saving for isotopes. As a novel application of the cSPP system for studies of membrane proteins prior to purification we also demonstrate, for the first time, fast detergent screening by microcoil NMR and well-resolved NMR spectra of several targeted integral membrane proteins obtained without purification.