Detection of Very Long Antisense Transcripts by Whole Transcriptome RNA-Seq Analysis of Listeria monocytogenes by Semiconductor Sequencing Technology

Abstract

The Gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a severe food-borne infection characterised by abortion, septicaemia, or meningoencephalitis. L. monocytogenes causes outbreaks of febrile gastroenteritis and accounts for community-acquired bacterial meningitis in humans. Listeriosis has one of the highest mortality rates (up to 30%) of all food-borne infections. This human pathogenic bacterium is an important model organism for biomedical research to investigate cell-mediated immunity. L. monocytogenes is also one of the best characterised bacterial systems for the molecular analysis of intracellular parasitism. Recently several transcriptomic studies have also made the ubiquitous distributed bacterium as a model to understand mechanisms of gene regulation from the environment to the infected host on the level of mRNA and non-coding RNAs (ncRNAs). We have used semiconductor sequencing technology for RNA-seq to investigate the repertoire of listerial ncRNAs under extra- and intracellular growth conditions. Furthermore, we applied a new bioinformatic analysis pipeline for detection, comparative genomics and structural conservation to identify ncRNAs. With this work, in total, 741 ncRNA locations of potential ncRNA candidates are now known for L. monocytogenes, of which 611 ncRNA candidates were identified by RNA-seq. 441 transcribed ncRNAs have never been described before. Among these, we identified novel long non-coding antisense RNAs with a length of up to 5,400 nt e.g. opposite to genes coding for internalins, methylases or a high-affinity potassium uptake system, namely the kdpABC operon, which were confirmed by qRT-PCR analysis. RNA-seq, comparative genomics and structural conservation of L. monocytogenes ncRNAs illustrate that this human pathogen uses a large number and repertoire of ncRNA including novel long antisense RNAs, which could be important for intracellular survival within the infected eukaryotic host.

Initial Transcriptomic Response and Adaption of Listeria monocytogenes to Desiccation on Food Grade Stainless Steel

“…Several other lasRNAs were upregulated in Lm 08-5578, including anti0936 (>22 fold) whose target mRNA lmo0936 was downregulated during desiccation. Identification of anti0936 and its downregulation of lmo0936 was reported by Wehner et al (2014) to be part of the bacterium's response to intracellular growth. In addition the 5.8 kb long anti0605, a σ B induced excludon, which limits transcription of the multidrug efflux pump lmo0605, while serving as coding sequence of lmo0606-08 (Wurtzel et al, 2012), was upregulated (>22 fold) in Lm 08-5578.…”

Comprehensive insights inthe Mycobacterium aviumsubsp.paratuberculosisgenome using new WGS data of sheep strain JIII-386 from Germany

“…Besides the comparative whole-genome sequence (WGS) analysis, in the past decade non-protein-coding fractions of the transcriptome were studied in bacteria ( Sharma et al 2010 ; Lechner et al 2014 ; Wehner, Mannala, et al 2014 ). Regarding Mycobacteria, noncoding RNAs (ncRNAs) were identified in M. tuberculosis and their role in the regulation of the pathogen metabolism was studied ( Arnvig and Young 2009 , 2010 ).…”

A detailed view of the intracellular transcriptome of Listeria monocytogenes in murine macrophages using RNA-seq

“…Non-coding RNA elements are widely implicated in adaptation to different environmental conditions, metabolism, and virulence. The role of regulatory RNA elements ( Johansson et al, 2002 ; Mansjö and Johansson, 2011 ), small RNA ( Christiansen et al, 2006 ; Mandin et al, 2007 ; Nielsen et al, 2008 ; Oliver et al, 2009 ; Toledo-Arana et al, 2009 ; Mraheil et al, 2011 ; Nielsen et al, 2011 ; Sievers et al, 2014 , 2015 ) and antisense RNA ( Toledo-Arana et al, 2009 ; Wurtzel et al, 2012 ; Behrens et al, 2014 ; Wehner et al, 2014 ) was investigated. A new class of regulatory elements, the excludon was defined as long transcript that function as both sense transcript for one gene (or set of genes) as well as antisense transcript for a divergently oriented neighboring gene (or set of genes; Wurtzel et al, 2012 ).…”

“…Despite the introduction of next generation sequencing (NGS) technologies, no study has addressed transcriptional changes in L. monocytogenes in detail. We now revisit the earlier microarray-based work ( Chatterjee et al, 2006 ) complementing it with recent RNA-seq data ( Wehner et al, 2014 ) which exclusively investigated non-coding RNAs of L. monocytogenes under intra- and extracellular growth conditions.…”