It’s a well known fact that EMBL conferences present the most top-class science from around the world, not only from established researchers but also up-and-coming scientists. In this brand new series we will feature some of the award-winning posters from recent EMBL conferences and symposia.
We begin with 4 of the poster prize winners at the EMBO|EMBL Symposium: New Approaches and Concepts in Microbiology (10-13 July 2019).
De novo selection of peptides that confer antibiotic resistance
Authors: Michael Knopp (1), Jonina Gudmundsdottir (1), Tobias Nilsson (2), Finja König (2), Omar Warsi (1), Fredrika Rajer (1), Pia Ädelroth (2), Dan Andersson (1)
The origin of novel genes and proteins is a fundamental question in evolutionary biology. New genes can originate from different mechanisms including horizontal gene transfer, duplication-divergence and de novo from non-coding DNA sequences. Comparative genomics has generated strong evidence for de novo emergence of genes in various organisms but experimental demonstration of this process has been limited to localized randomization in pre-existing structural scaffolds. This is bypassing the basic requirement of de novo gene emergence, i.e. lack of an ancestral gene. We constructed highly diverse plasmid libraries encoding randomly generated open reading frames and expressed them in Escherichia coli to identify peptides that could confer a beneficial and selectable phenotype in vivo. Selections on antibiotic-containing agar plates resulted in the identification of three inserts that increased aminoglycoside resistance up to 48-fold. Combining genetic and functional analyses, we show that the peptides are highly hydrophobic and that they insert into the membrane, reduce membrane potential, decrease aminoglycoside uptake and thereby confer high-level resistance. This study demonstrates that randomized DNA sequences can encode peptides that confer selective benefits, and illustrates how expression of random sequences could spark the origination of new genes.
(1) Uppsala University, Sweden; (2) Stockholm University, Sweden
Combinatoria genetics approach to prevent and disrupt biofilm-associated infection
Authors: Irina Afonina (1), Kimberly Kline (2), Timothy Lu (3)
Enterococci are opportunistic bacterial pathogens that cause a variety of infections including life-threatening endocarditis, chronic wounds, medical device and urinary tract infections. All of these infections are biofilm-associated, which are intrinsically more tolerant to antimicrobial clearance, and which therefore pose a major challenge in treating these infections. Biofilm formation is multifactorial, requiring multiple factors, which can vary depending on the environment or niche where the bacteria reside. Therefore, to understand the complexity of interactions and factors that contribute to enterococcal biofilms, we are combining CRISPRi technology with rapid DNA assembly to identify gene pairs involved in biofilm formation in different infection niches. We established a dual-vector inducible CRISPRi system for Enterococcus faecalis that targets planktonic and biofilm cells with efficiency resembling that of a gene knock out. We have shown that CRISPRi targeting of constitutively expressed gfp gene on the bacterial chromosome, fully quenches GFP signal within planktonic, early and late biofilm cells. Additionally, we have shown that silencing of the croR gene, required for bacitracin resistance, mimics a croR in-frame deletion phenotype, and both CRISPRi croR and croR show reduced minimal inhibitory concentration to bacitracin compared to uninduced or wild type strains. We are creating combinatorial libraries to identify pairs and quartets of genes of all of the two-component signal transduction systems encoded in E. faecalis, to address the hypothesis that different signals will drive unique biofilm programs in different environmental conditions. This research serves as a platform to rapidly identify combinations of genes involved in enterococcal pathogenesis, including antimicrobial resistance, virulence, and immune invasion.
(1) SMART, Singapore; (2) Nanyang Technological University, Singapore; (3) Massachusetts Institute of Technology, USA
A conserved RNA seed-pairing domain directs small RNA-mediated stress resistance in enterobacteria
Authors: Nikolai Peschek (1), Mona Hoyos (1), Roman Herzog (1), Konrad U. Förstner (2), Kai Papenfort (1)
Small regulatory RNAs (sRNAs) are crucial components of many stress response systems. The envelope stress response (ESR) of Gram-negative bacteria is a paradigm for sRNA-mediated stress management and involves, among other factors, the alternative sigma factor E (σE) and one or more sRNAs. In this study, we identified the MicV sRNA as a new member of the σE regulon in Vibrio cholerae. We show that MicV acts redundantly with another sRNA, VrrA, and that both sRNAs share a conserved seed-pairing domain to regulate multiple target mRNAs. V. cholerae lacking σE displayed increased sensitivity towards antimicrobial substances and overexpression of either of the sRNAs suppressed this phenotype. Laboratory selection experiments using a library of synthetic sRNA regulators revealed that the seed-pairing domain of σE-dependent sRNAs is strongly enriched under membrane-damaging conditions and that repression of OmpA is key for sRNA-mediated stress relief. Together, our work shows that MicV and VrrA act as global regulators in the ESR of V. cholerae and provides evidence that bacterial sRNAs can be functionally annotated by their seed-pairing sequences.
Peschek, N. et al. A conserved RNA seed‐pairing domain directs small RNA‐mediated stress resistance in enterobacteria. The EMBO Journal (2019) DOI:10.15252/embj.2019101650
(1) Ludwig-Maximilians-Universität München, Germany; (2) TH Köln, ZB MED, Germany
The interaction between replication factor DiaA and primary metabolite sedoheptulose-7- phosphate directly regulates DNA replication in Escherichia coli
Authors: Joanna Morcinek-Orlowska (1), Aleksandra Bebel (1), Justyna Galinska (1), Torsten Wladminghaus (2), Anna Zawilak-Pawlik (3), Monika Glinkowska (1)
To proliferate, bacterial cells duplicate their genomes and this process is coordinated with cell growth and division. During the last few decades, various biochemical mechanisms controlling initiation of DNA replication in the model bacterium Escherichia coli have been characterized in detail. However, it remains elusive what constitutes a signal for the growing cell to initiate the next round of chromosomal DNA replication. Here we present evidence that a primary metabolite sedoheptulose 7-phosphate (S7P) binds to a replication factor DiaA and regulates its activity in promoting oligomerization of the DnaA initiator protein. Furthermore, our results show that the cellular level of S7P and the ability of DiaA to interact with the metabolite both influence DNA replication in vivo. S7P is an intermediate in the pentose phosphate pathway, providing building blocks for synthesis of nucleotides and a starting point for production of the outer membrane components. Consequently, we propose a mechanism which links DNA replication with cell growth through primary metabolism.
(1) University of Gdansk, Poland; (2) LOEWE Center for Synthetic Microbiology-SYNMIKRO, Philipps-Universität Marburg, Germany; (3) Polish Academy of Sciences, Poland
Working on your own conference poster? Then check out 10 tips to create a scientific poster people want to stop by .