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Effect of herbicide combinations on Bt-maize rhizobacterial diversity

J Microbiol Biotechnol. 2014; doi: 10.4014/jmb.1405.05054.

Valverde JR, Marin S, Mellado RP.

J Microbiol Biotechnol. 2014; doi: 10.4014/jmb.1405.05054Reports of herbicide resistance events are proliferating worldwide leading to new cultivation strategies using combinations of pre-emergence and post-emergence herbicides. We analysed the impact during a one-year cultivation cycle of several herbicide combinations on the rhizobacterial community of glyphosate-tolerant Bt-maize and compared them to those of the untreated or glyphosate-treated soils. Samples were analysed using pyrosequencing of the V6 hypervariable region of the 16S rRNA gene.

The sequences obtained were subjected to taxonomic, taxonomy-independent and phylogeny-based diversity studies followed by a statistical analysis using principal components analysis and hierarchical clustering with jackknife statistical validation. The resilience of the microbial communities was analysed by comparing their relative composition at the end of the cultivation cycle. The bacterial communites from soil subjected to a combined treatment with mesotrione plus s-metolachlor followed by glyphosate were not statistically different from those treated with glyphosate or the untreated ones. The use of acetochlor plus terbuthylazine followed by glyphosate and the use of aclonifen plus isoxaflutole followed by mesotrione clearly affected the resilience of their corresponding bacterial communities. The treatment with pethoxamid followed by glyphosate resulted in an intermediate effect. The use of glyphosate alone seems to be the less aggressive one for bacterial communities. Should a combined treatment be needed, the combination of mesotrione and s-metolachlor shows the next best final resilience.

Our results show the relevance of comparative rhizobacterial community studies when novel combined herbicide treatments are deemed necessary to control weed growth.

Characterization of a novel Zn2+-dependent intrinsic imipenemase from Pseudomonas aeruginosa

J Antimicrob Chemother. 2014; pii: dku267.

Fajardo A, Hernando-Amado S, Oliver A, Ball G, Filloux A, Martinez JL.

J Antimicrob Chemother. 2014; pii: dku267Objectives: Previous work showed that PA5542 inactivation increases Pseudomonas aeruginosa 59.20 susceptibility to carbapenems. The objective of the current study was to purify PA5542, to determine its role in carbapenem resistance and to analyse the kinetic constants of this putative new β-lactamase.

Methods: PA5542 was cloned and expressed in Escherichia coli. The enzyme was purified by affinity as a GST fusion protein and, after that, cleaved to remove the GST tag. β-Lactamase activity was measured spectrophotometrically using imipenem as substrate. Susceptibility to antibiotics was determined by Etest. Zn2+ was added when needed. The expression levels of PA5542, ampC, poxB, mexA and oprD were determined by real-time RT–PCR.

Results: Lack of PA5542 increases P. aeruginosa 59.20 susceptibility to carbapenems and its overexpression reduces E. coli susceptibility to these β-lactams. PA5542 is highly conserved in all sequenced P. aeruginosa strains. The clinical isolate 59.20 is resistant to imipenem (MIC >32 mg/L) and to meropenem (MIC 24 mg/L) and presents high-level expression of PA5542 in comparison with the wild-type strain PAO1. Spectrophotometric analyses showed that PA5542 is a Zn2+-dependent imipenemase. Analysis of the PA5542 sequence indicates that it does not belong to the classical categories of β-lactamases.

Conclusions PA5542 encodes a new Zn2+-dependent imipenemase. The presence of PA5542 in all sequenced P. aeruginosa genomes, maintaining the synteny and without adjacent gene-mobility elements, indicates that it belongs to the P. aeruginosa core genome. High PA5542 expression in 59.20 suggests it may contribute to the resistance to carbapenems of this P. aeruginosa clinical isolate.

WIP is necessary for matrix invasion by breast cancer cells

Eur J Cell Biol. 2014;. pii: S0171-9335(14)00104-6.

García E, Machesky LM, Jones GE, Antón IM.

Eur J Cell Biol. 2014;. pii: S0171-9335(14)00104-6Actin filament assembly and reorganisation during cell migration and invasion into extracellular matrices is a well-documented phenomenon. Among actin-binding proteins regulating its polymerisation, the members of the WASP (Wiskott Aldrich Syndrome Protein) family are generally thought to play the most significant role in supporting cell invasiveness.

In situ, cytosolic N-WASP (neural WASP) is associated with a partner protein termed WIP (WASP Interacting Protein) that is bound to the N-terminal domain of N-WASP. Despite much effort, rather little is known about the role of WIP in regulating N-WASP and consequent actin-filament assembly. Even less is known about the function of WIP within the specialised cell adhesion and attachment structures known as podosomes and invadopodia. In particular, whilst the interaction of WIP with known participants in the development and maturation of invadopodia such as N-WASP, the Arp2/3 complex and cortactin has been described, little is known concerning the direct contribution of WIP to invadopodia and its potential role as a regulator of cancer cell invasion.

In this report, we use 2D and 3D culture systems to describe the role played by WIP in modulating the morphology and invasiveness of metastatic breast cancer cells in vitro, as well as its effect on the process of mesenchymal–epithelial transition (MET) seen in these cells. We demonstrate that WIP is necessary for invadopodium formation and matrix degradation by basal breast cancer cells, but not sufficient to induce invasiveness in luminal cells.

Yeast mitochondrial RNAP conformational changes are regulated by interactions with the mitochondrial transcription factor

Nucleic Acids Res. 2014; pii: gku795.

Drakulic S, Wang L, Cuéllar J, Guo Q, Velázquez G, Martín-Benito J, Sousa R, Valpuesta JM.

Nucleic Acids Res. 2014; pii: gku795Mitochondrial RNA polymerases (MtRNAPs) are members of the single-subunit RNAP family, the most well-characterized member being the RNAP from T7 bacteriophage. MtRNAPs are, however, functionally distinct in that they depend on one or more transcription factors to recognize and open the promoter and initiate transcription, while the phage RNAPs are capable of performing these tasks alone. Since the transcriptional mechanisms that are conserved in phage and mitochondrial RNAPs have been so effectively characterized in the phage enzymes, outstanding structure-mechanism questions concern those aspects that are distinct in the MtRNAPs, particularly the role of the mitochondrial transcription factor(s).

To address these questions we have used both negative staining and cryo-EM to generate three-dimensional reconstructions of yeast MtRNAP initiation complexes with and without the mitochondrial transcription factor (MTF1), and of the elongation complex. Together with biochemical experiments, these data indicate that MTF1 uses multiple mechanisms to drive promoter opening, and that its interactions with the MtRNAP regulate the conformational changes undergone by the latter enzyme as it traverses the template strand.

Failure of cell cleavage induces senescence in tetraploid primary cells

Mol Biol Cell. 2014; pii: mbc.E14-03-0844.

Panopoulos A, Pacios-Bras C, Choi J, Yenjerla M, Sussman MA, Fotedar R, Margolis RL.

Mol Biol Cell. 2014; pii: mbc.E14-03-0844Tetraploidy can arise from various mitotic or cleavage defects in mammalian cells, and inheritance of multiple centrosomes induces aneuploidy when tetraploid cells continue to cycle. Arrest of the tetraploid cell cycle is therefore potentially a critical cellular control.

We report here that primary rat embryo fibroblasts (REF52) and human foreskin fibroblasts (HFF) become senescent in tetraploid G1 following drug or siRNA induced failure of cell cleavage. In contrast, T-antigen transformed REF52 and p53+/+ HCT116 tumor cells rapidly become aneuploid by continuing to cycle following cleavage failure. Tetraploid primary cells quickly become quiescent, as determined by loss of the Ki-67 proliferation marker, and of the FUCCI late cell cycle marker geminin. Arrest is not due to DNA damage, as the γ-H2AX DNA damage marker remains at control levels after tetraploidy induction. Arrested tetraploid cells finally become senescent, as determined by SA-β-galactosidase activity. Tetraploid arrest is dependent on p16INK4a expression, as siRNA suppression of p16INK4a bypasses tetraploid arrest, permitting primary cells to become aneuploid.

We conclude that tetraploid primary cells can become senescent without DNA damage, and that induction of senescence is critical to tetraploidy arrest.