The inverse autotransporters of Yersinia ruckeri, YrInv and YrIlm, contribute to biofilm formation and virulence

Yersinia ruckeri causes enteric redmouth disease (ERM) that mainly affects salmonid fishes and leads to significant economic losses in the aquaculture industry. An increasing number of outbreaks and the lack of effective vaccines against some serotypes necessitates novel measures to control ERM. Importantly, Y. ruckeri survives in the environment for long periods, presumably by forming biofilms. How the pathogen forms biofilms and which molecular factors are involved in this process, remains unclear. Yersinia ruckeri produces two surface‐exposed adhesins, belonging to the inverse autotransporters (IATs), called Y. ruckeri invasin (YrInv) and Y. ruckeri invasin‐like molecule (YrIlm). Here, we investigated whether YrInv and YrIlm play a role in biofilm formation and virulence. Functional assays revealed that YrInv and YrIlm promote biofilm formation on different abiotic substrates. Confocal microscopy revealed that they are involved in microcolony interaction and formation, respectively. The effect of both IATs on biofilm formation correlated with the presence of different biopolymers in the biofilm matrix, including extracellular DNA, RNA and proteins. Moreover, YrInv and YrIlm contributed to virulence in the Galleria mellonella infection model. Taken together, we propose that both IATs are possible targets for the development of novel diagnostic and preventative strategies to control ERM

Deciphering the modulation of gene expression by type I and II interferons combining 4sU-tagging, translational arrest and in silico promoter analysis

Interferons (IFN) play a pivotal role in innate immunity, orchestrating a cell-intrinsic anti-pathogenic state and stimulating adaptive immune responses. The complex interplay between the primary response to IFNs and its modulation by positive and negative feedback loops is incompletely understood. Here, we implement the combination of high-resolution gene-expression profiling of nascent RNA with translational inhibition of secondary feedback by cycloheximide. Unexpectedly, this approach revealed a prominent role of negative feedback mechanisms during the immediate (≤60 min) IFNα response. In contrast, a more complex picture involving both negative and positive feedback loops was observed on IFNγ treatment. IFNγ-induced repression of genes associated with regulation of gene expression, cellular development, apoptosis and cell growth resulted from cycloheximide-resistant primary IFNγ signalling. In silico promoter analysis revealed significant overrepresentation of SP1/SP3-binding sites and/or GC-rich stretches. Although signal transducer and activator of transcription 1 (STAT1)-binding sites were not overrepresented, repression was lost in absence of STAT1. Interestingly, basal expression of the majority of these IFNγ-repressed genes was dependent on STAT1 in IFN-naïve fibroblasts. Finally, IFNγ-mediated repression was also found to be evident in primary murine macrophages. IFN-repressed genes include negative regulators of innate and stress response, and their decrease may thus aid the establishment of a signalling perceptive milieu.Fil: Trilling, Mirko. Universitat Duisburg - Essen; AlemaniaFil: Bellora, Nicolás. Parque de Investigación Biomédica de Barcelona; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Patagonia Norte. Instituto de Investigación en Biodiversidad y Medioambiente; ArgentinaFil: Rutkowski, Andrzej J.. University of Cambridge; Reino UnidoFil: de Graaf, Miranda. University of Cambridge; Reino UnidoFil: Dickinson, Paul. University Of Edinburgh; Reino UnidoFil: Robertson, Kevin. University Of Edinburgh; Reino UnidoFil: Da Costa, Olivia Prazeres. Universitat Technical Zu Munich; AlemaniaFil: Ghazal, Peter. University Of Edinburgh; Reino UnidoFil: Friedel, Caroline C.. Ludwig-Maximilians-University Munich; AlemaniaFil: Albà, M. Mar. Institució Catalana de Recerca I Estudis Avancats; España. Parque de Investigación Biomédica de Barcelona; EspañaFil: Dölken, Lars. University of Cambridge; Reino Unid

Autoregulation of the Drosophila Noncoding roX1 RNA Gene

Most genes along the male single X chromosome in Drosophila are hypertranscribed about two-fold relative to each of the two female X chromosomes. This is accomplished by the MSL (male-specific lethal) complex that acetylates histone H4 at lysine 16. The MSL complex contains two large noncoding RNAs, roX1 (RNA on X) and roX2, that help target chromatin modifying enzymes to the X. The roX RNAs are functionally redundant but differ in size, sequence, and transcriptional control. We wanted to find out how roX1 production is regulated. Ectopic DC can be induced in wild-type (roX1+ roX2+) females if we provide a heterologous source of MSL2. However, in the absence of roX2, we found that roX1 expression failed to come on reliably. Using an in situ hybridization probe that is specific only to endogenous roX1, we found that expression was restored if we introduced either roX2 or a truncated but functional version of roX1. This shows that pre-existing roX RNA is required to positively autoregulate roX1 expression. We also observed massive cis spreading of the MSL complex from the site of roX1 transcription at its endogenous location on the X chromosome. We propose that retention of newly assembled MSL complex around the roX gene is needed to drive sustained transcription and that spreading into flanking chromatin contributes to the X chromosome targeting specificity. Finally, we found that the gene encoding the key male-limited protein subunit, msl2, is transcribed predominantly during DNA replication. This suggests that new MSL complex is made as the chromatin template doubles. We offer a model describing how the production of roX1 and msl2, two key components of the MSL complex, are coordinated to meet the dosage compensation demands of the male cell

Differential effect of H1 variant overexpression on cell cycle progression and gene expression.

To identify functional differences among non-allelic variants of the mammalian H1 linker histones a system for the overexpression of individual H1 variants in vivo was developed. Mouse 3T3 cells were transformed with an expression vector containing the coding regions for the H1c or H10 variant under the control of an inducible promoter. Stable, single colony transformants, in which the normal stoichiometry of H1 variants was perturbed, displayed normal viability, unaltered morphology and no long-term growth arrest. However, upon release from synchronization at different points in the cell cycle transformants significantly overproducing H10 exhibited transient inhibition of both G1 and S phase progression. Overexpression of H1c to comparable levels had no effect on cell cycle progression. Analysis of transcript levels for several cell cycle-regulated and housekeeping genes indicated that overexpression of H10 resulted in significantly reduced expression of all genes tested. Surprisingly, overexpression of H1c to comparable levels resulted in either a negligible effect or, in some cases, a dramatic increase in transcript levels. These results support the suggestion that functional differences exist among H1 variants