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N-acetylcysteine selectively antagonizes the activity of imipenem in Pseudomonas aeruginosa by an OprD-mediated mechanism

Antimicrob Agents Chemother. 2015; pii: AAC.00017-15.

Rodríguez-Beltrán J, Cabot G, Valencia EY, Costas C, Bou G, Oliver A, Blázquez J.

Antimicrob Agents Chemother. 2015; pii: AAC.00017-15The modulating effect of N-acetylcysteine (NAC) on the activity of different antibiotics has been studied in Pseudomonas aeruginosa. Our results demonstrate that, in contrast to previous reports, only the activity of imipenem is clearly affected by NAC.

Minimal inhibitory concentration and checkerboard determinations indicate that the NAC-based modulation of imipenem activity is mainly dependent on OprD. SDS-PAGE of OMPs after NAC treatments demonstrates that NAC does not modify the expression of OprD, suggesting that NAC competitively inhibits the uptake of imipenem through OprD. Similar effects on imipenem activity were obtained with P. aeruginosa clinical isolates. Our results indicate that imipenem-susceptible P. aeruginosa strains become resistant upon simultaneous treatment with NAC and imipenem. Moreover, the generality of the observed effects of NAC on antibiotic activity was assessed with two additional bacterial species, Escherichia coli and Acinetobacter baumannii.

Caution should be taken during treatments as the activity of imipenem may be modified by physiologically attainable concentrations of NAC, particularly during intravenous and nebulized regimes.

Global variability in gene expression and alternative splicing is modulated by mitochondrial content

Genome Res. 2015; pii: gr.178426.114.

Guantes R, Rastrojo A, Pires das Neves R, Lima A, Aguado B, Iborra FJ.

Genome Res. 2015; pii: gr.178426.114Noise in gene expression is a main determinant of phenotypic variability. Increasing experimental evidence suggests that genome-wide cellular constraints largely contribute to the heterogeneity observed in gene products. It is still unclear, however, which global factors mostly affect gene expression noise, and to which extent. Since eukaryotic gene expression is an energy demanding process, differences in the energy budget of each cell could determine gene expression differences.

Here, we quantify the contribution of mitochondrial variability (a natural source of ATP variation) to global variability in gene expression. We find that changes in mitochondrial content can account for ~50% of the variability observed in protein levels. This is the combined result of the effect of mitochondria dosage on transcription and translation apparatus content and activities. Moreover, we find that mitochondrial levels have a large impact on alternative splicing, thus modulating both the abundance and type of mRNAs. A simple mathematical model where mitochondrial content simultaneously affects transcription rate and splicing site choice can explain the alternative splicing data. The results of this study show that mitochondrial content (and/or probably function) influence mRNA abundance, translation and alternative splicing which ultimately impacts on cellular phenotype.

Endogenous Arabidopsis messenger RNAs transported to distant tissues

Nature Plants 2015; doi: 10.1038/nplants.2015.25.

Thieme CJ, Rojas-Triana M, Stecyk E, Schudoma C, Zhang W, Yang L, Miñambres M, Walther D, Schulze WX, Paz-Ares J, Scheible WR, Kragler F.

Nature Plants 2015; doi: 10.1038/nplants.2015.25The concept that proteins and small RNAs can move to and function in distant body parts is well established. However, non-cell-autonomy of small RNA molecules raises the question: To what extent are protein-coding messenger RNAs (mRNAs) exchanged between tissues in plants?

Here we report the comprehensive identification of 2,006 genes producing mobile RNAs in Arabidopsis thaliana. The analysis of variant ecotype transcripts that were present in heterografted plants allowed the identification of mRNAs moving between various organs under normal or nutrient-limiting conditions. Most of these mobile transcripts seem to follow the phloem-dependent allocation pathway transporting sugars from photosynthetic tissues to roots via the vasculature. Notably, a high number of transcripts also move in the opposite, root-to-shoot direction and are transported to specific tissues including flowers. Proteomic data on grafted plants indicate the presence of proteins from mobile RNAs, allowing the possibility that they may be translated at their destination site. The mobility of a high number of mRNAs suggests that a postulated tissue-specific gene expression profile might not be predictive for the actual plant body part in which a transcript exerts its function.

Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase

Nucleic Acids Res. 2015; pii: gkv204.

Morin JA, Cao FJ, Lázaro JM, Arias-Gonzalez JR, Valpuesta JM, Carrascosa JL, Salas M, Ibarra B.

Nucleic Acids Res. 2015; pii: gkv204During DNA replication replicative polymerases move in discrete mechanical steps along the DNA template. To address how the chemical cycle is coupled to mechanical motion of the enzyme, here we use optical tweezers to study the translocation mechanism of individual bacteriophage Phi29 DNA polymerases during processive DNA replication. We determine the main kinetic parameters of the nucleotide incorporation cycle and their dependence on external load and nucleotide (dNTP) concentration.

The data is inconsistent with power stroke models for translocation, instead supports a loose-coupling mechanism between chemical catalysis and mechanical translocation during DNA replication. According to this mechanism the DNA polymerase works by alternating between a dNTP/PPi-free state, which diffuses thermally between pre- and post-translocated states, and a dNTP/PPi-bound state where dNTP binding stabilizes the post-translocated state. We show how this thermal ratchet mechanism is used by the polymerase to generate work against large opposing loads (∼50 pN).

Distinct type I and type II toxin-antitoxin modules control Salmonella lifestyle inside eukaryotic cells

Sci Rep. 2015; 5: 9374.

Lobato-Márquez D, Moreno-Córdoba I, Figueroa V, Díaz-Orejas R, García-Del Portillo F.

Sci Rep. 2015; 5: 9374Toxin-antitoxin (TA) modules contribute to the generation of non-growing cells in response to stress. These modules abound in bacterial pathogens although the bases for this profusion remain largely unknown. Using the intracellular bacterial pathogen Salmonella enterica serovar Typhimurium as a model, here we show that a selected group of TA modules impact bacterial fitness inside eukaryotic cells.

We characterized in this pathogen twenty-seven TA modules, including type I and type II TA modules encoding antisense RNA and proteinaceous antitoxins, respectively. Proteomic and gene expression analyses revealed that the pathogen produces numerous toxins of TA modules inside eukaryotic cells. Among these, the toxins HokST, LdrAST, and TisBST, encoded by type I TA modules and T4ST and VapC2ST, encoded by type II TA modules, promote bacterial survival inside fibroblasts. In contrast, only VapC2ST shows that positive effect in bacterial fitness when the pathogen infects epithelial cells.

These results illustrate how S. Typhimurium uses distinct type I and type II TA modules to regulate its intracellular lifestyle in varied host cell types. This function specialization might explain why the number of TA modules increased in intracellular bacterial pathogens.