Knockout of Targeted Plasmid-Borne β-Lactamase Genes in an Extended-Spectrum-β-Lactamase-Producing Escherichia coli Strain: Impact on Resistance and Proteomic Profile

Resistance to β-lactams is known to be multifactorial, although the underlying mechanisms are not well established. The aim of our study was to develop a system for assessing the phenotypic and proteomic responses of bacteria to antibiotic stress as a result of the loss of selected antimicrobial resistance genes. We applied homologous recombination to knock out plasmid-borne β-lactamase genes (blaOXA-1, blaTEM-1, and blaCTX-M15) in Escherichia coli CCUG 73778, generating knockout clone variants lacking the respective deleted β-lactamases. Quantitative proteomic analyses were performed on the knockout variants and the wild-type strain, using bottom-up liquid chromatography tandem mass spectrometry (LC-MS/MS), after exposure to different concentrations of cefadroxil. Loss of the blaCTX-M-15 gene had the greatest impact on the resulting protein expression dynamics, while losses of blaOXA-1 and blaTEM-1 affected fewer proteins’ expression levels. Proteins involved in antibiotic resistance, cell membrane integrity, stress, and gene expression and unknown function proteins exhibited differential expression. The present study provides a framework for studying protein expression in response to antibiotic exposure and identifying the genomic, proteomic, and phenotypic impacts of resistance gene loss.publishedVersio

TERRA biogenesis, turnover and implications for function

Telomeres are heterochromatic structures at the ends of eukaryotic chromosomes. As other heterochromatin regions, telomeres are transcribed, from the subtelomeric region towards chromosome ends into the long non-coding RNA TERRA. Telomere transcription is a widespread phenomenon as it has been observed in species belonging to several kingdoms of the eukaryotic domain. TERRA is part of telomeric heterochromatin in addition to being present in the nucleoplasm. Here, we review the current knowledge of TERRA structure, biogenesis and turnover. In addition, we discuss presumed roles of this RNA during replication of telomeric DNA, heterochromatin formation and the regulation of telomerase

The ubiquitin pathway regulates multiple steps of Mex67-mediated mRNA export

Dans la levure et chez les eucaryotes supérieurs, les ARN messagers (ARNm) matures sont exportés du noyau vers le cytoplasme par Mex67-Mtr2 (TAP-NXF1 chez les eucaryotes supérieurs), le principal récepteur d'export des ARNm. La faible affinité de ce dernier pour les ARNm implique le besoin d'un adaptateur, Y ra1/REF, seul adaptateur connu de Mex67/TAP jusqu'alors. Ce travail a identifié deux nouveaux adaptateurs pour le recrutement de Mex67 sur les ARNm et a demontré la relevance fonctionnelle de ces interactions pour l'export des ARNm. De plus, nous avons mis en évidence l'importance de la modification post-traductionnelle par l'ubiquitination dans l'export des ARNm. Enfin, nous avons découvert qu'hormis son rôle dans l'export des ARNm, Mex67 avait d'autres fonctions, comme coordonner le recrutement de la machinerie d'export à la transcription et aux étapes précoces de la biogenèse des ARNm, ainsi que d'ancrer les gènes au pore nucléaire

Silencing repetitive DNA

Some RNAs in mammalian cells can help to silence the DNA they are transcribed from

Regulation of mRNP dynamics along the export pathway

The transcription of mRNA is tightly coupled to the concomitant recruitment of mRNA processing and export factors, resulting in the formation of mature and export competent mRNP complexes. This interconnection in gene expression implies extensive spatio-temporal control of mRNP dynamics to prevent mRNA export factors bound to pre-mRNA from functioning at the incorrect time and exporting nascent or incompletely processed pre-mRNAs. Recent discoveries provide molecular understanding of how a broad range of post-translational modifications together with RNA-dependent ATPases coordinate proteins acting at different steps and regulate mRNP assembly and export

Cotranscriptional Recruitment to the mRNA Export Receptor Mex67p Contributes to Nuclear Pore Anchoring of Activated Genes

Transcription activation of some Saccharomyces cerevisiae genes is paralleled by their repositioning to the nuclear periphery, but the mechanism underlying gene anchoring is poorly defined. We show that the nuclear pore complex-associated Mlp1p and the shuttling mRNA export receptor Mex67p contribute to the stable association of the activated GAL10 and HSP104 genes with the nuclear periphery. However, we find no obligatory link between gene positioning and gene expression. Furthermore, gene anchoring correlates with the cotranscriptional recruitment of Mex67p to transcribing genes. Notably, the association of Mex67p with chromatin is not mediated by RNA. Interestingly, a mutant GAL2 gene lacking the coding region is still able to recruit Mex67p upon transcriptional activation and to relocate to the nuclear periphery. Together these data suggest that, at least for GAL2, nascent messenger ribonucleoprotein does not play a major role in gene anchoring and that the early recruitment of Mex67p contributes to gene repositioning by virtue of an RNA-independent process

Antisense RNA stabilization induces transcriptional gene silencing via histone deacetylation in S. cerevisiae

Genome-wide studies in S. cerevisiae reveal that the transcriptome includes numerous antisense RNAs as well as intergenic transcripts regulated by the exosome component Rrp6. We observed that upon the loss of Rrp6 function, two PHO84 antisense transcripts are stabilized, and PHO84 gene transcription is repressed. Interestingly, the same phenotype is observed in wild-type cells during chronological aging. Epistasis and chromatin immunoprecipitation experiments indicate that the loss of Rrp6 function is paralleled by the recruitment of Hda1 histone deacetylase to PHO84 and neighboring genes. However, histone deacetylation is restricted to PHO84, suggesting that Hda1 activity depends on antisense RNA. Accordingly, the knockdown of antisense production prevents PHO84 gene repression, even in the absence of Rrp6. Together, our data indicate that the stabilization of antisense transcripts results in PHO84 gene repression via a mechanism distinct from transcription interference and that the modulation of Rrp6 function contributes to gene regulation by inducing RNA-dependent epigenetic modifications

Perinuclear Mlp proteins downregulate gene expression in response to a defect in mRNA export

The mRNA export adaptor Yra1p/REF contributes to nascent mRNP assembly and recruitment of the export receptor Mex67p. yra1 mutants exhibit mRNA export defects and a decrease in LacZ reporter and certain endogenous transcripts. The loss of Mlp1p/Mlp2p, two TPR-like proteins attached to nuclear pores, rescues LacZ mRNA levels and increases their appearance in the cytoplasm, without restoring bulk poly(A)+ RNA export. Chromatin immunoprecipitation, FISH and pulse-chase experiments indicate that Mlps downregulate LacZ mRNA synthesis in a yra1 mutant strain. Microarray analyses reveal that Mlp2p also reduces a subset of cellular transcripts in the yra1 mutant. Finally, we show that Yra1p genetically interacts with the shuttling mRNA-binding protein Nab2p and that loss of Mlps rescues the growth defect of yra1 and nab2 but not other mRNA export mutants. We propose that Nab2p and Yra1p are required for proper mRNP docking to the Mlp platform. Defects in Yra1p prevent mRNPs from crossing the Mlp gate and this block negatively feeds back on the transcription of a subset of genes, suggesting that Mlps link mRNA transcription and export