Abstract
The secretion of biomolecules into the extracellular milieu is a common and well-conserved phenomenon in biology. In bacteria, secreted biomolecules are not only involved in intra-species communication but they also play roles in inter-kingdom exchanges and pathogenicity. To date, released products, such as small molecules, DNA, peptides, and proteins, have been well studied in bacteria. However, the bacterial extracellular RNA complement has so far not been comprehensively characterized. Here, we have analyzed, using a combination of physical characterization and high-throughput sequencing, the extracellular RNA complement of both outer membrane vesicle (OMV)-associated and OMV-free RNA of the enteric Gram-negative model bacterium Escherichia coli K-12 substrain MG1655 and have compared it to its intracellular RNA complement. Our results demonstrate that a large part of the extracellular RNA complement is in the size range between 15 and 40 nucleotides and is derived from specific intracellular RNAs. Furthermore, RNA is associated with OMVs and the relative abundances of RNA biotypes in the intracellular, OMV and OMV-free fractions are distinct. Apart from rRNA fragments, a significant portion of the extracellular RNA complement is composed of specific cleavage products of functionally important structural noncoding RNAs, including tRNAs, 4.5S RNA, 6S RNA, and tmRNA. In addition, the extracellular RNA pool includes RNA biotypes from cryptic prophages, intergenic, and coding regions, of which some are so far uncharacterised, for example, transcripts mapping to the fimA-fimL and ves-spy intergenic regions. Our study provides the first detailed characterization of the extracellular RNA complement of the enteric model bacterium E. coli. Analogous to findings in eukaryotes, our results suggest the selective export of specific RNA biotypes by E. coli, which in turn indicates a potential role for extracellular bacterial RNAs in intercellular communication.
Highlights
While growing either in a natural ecosystem or artificial conditions, bacteria secrete intracellular products into their extracellular milieu (Tseng et al 2009)
We examined whether the outer membrane vesicle (OMV) fraction from E. coli contains RNA, by applying a fluorescent labeling method which was adapted for staining of the OMV fraction (Nicola et al 2009)
RNA is found to be associated with OMV-associated lipids but the observed patterns suggest the presence of nucleoprotein complexes and/ or free extracellular RNA (Fig. 1C)
Summary
While growing either in a natural ecosystem or artificial conditions, bacteria secrete intracellular products into their extracellular milieu (Tseng et al 2009). Secretory products are involved in bacterial social behavior, usually referred to as quorum sensing (Molloy 2010) and in pathogenicity (Lee and Schneewind 2001) and inter-kingdom communication (Hughes and Sperandio 2008; Shen et al 2012; Furusawa et al 2013). Bacteria disseminate secreted products either through their secretory systems via continuous or discontinuous passages across the bacterial membrane or by the release of outer membrane vesicles (OMVs) (Tseng et al 2009; Shen et al 2012). OMVs may be taken up by host cells and may contribute to pathogenesis, as already demonstrated for OMVs derived from Helicobacter pylori, which are taken up by gastric epithelial cells (Parker et al 2010) and enhance the carcinogenic potential of this specific bacterium (Chitcholtan et al 2008)
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