The small non-coding RNA RsaE influences extracellular matrix composition in Staphylococcus epidermidis biofilm communities

RsaE is a conserved small regulatory RNA (sRNA) which was previously reported to represent a riboregulator of central carbon flow and other metabolic pathways in Staphylococcus aureus and Bacillus subtilis. Here we show that RsaE contributes to extracellular (e)DNA release and biofilm-matrix switching towards polysaccharide intercellular adhesin (PIA) production in a hypervariable Staphylococcus epidermidis isolate. Transcriptome analysis through differential RNA sequencing (dRNA-seq) in combination with confocal laser scanning microscopy (CLSM) and reporter gene fusions demonstrate that S. epidermidis protein- and PIA-biofilm matrix producers differ with respect to RsaE and metabolic gene expression. RsaE is spatiotemporally expressed within S. epidermidis PIA-mediated biofilms, and its overexpression triggers a PIA biofilm phenotype as well as eDNA release in an S. epidermidis protein biofilm matrix-producing strain background. dRNA-seq and Northern blot analyses revealed RsaE to exist as a major full-length 100-nt transcript and a minor processed species lacking approximately 20 nucleotides at the 5'-end. RsaE processing results in expansion of the mRNA target spectrum. Thus, full-length RsaE interacts with S. epidermidis antiholin-encoding lrgA mRNA, facilitating bacterial lysis and eDNA release. Processed RsaE, however, interacts with the 5'-UTR of icaR and sucCD mRNAs, encoding the icaADBC biofilm operon repressor IcaR and succinyl-CoA synthetase of the tricarboxylic acid (TCA) cycle, respectively. RsaE augments PIA-mediated biofilm matrix production, most likely through activation of icaADBC operon expression via repression of icaR as well as by TCA cycle inhibition and re-programming of staphylococcal central carbon metabolism towards PIA precursor synthesis. Additionally, RsaE supports biofilm formation by mediating the release of eDNA as stabilizing biofilm matrix component. As RsaE itself is heterogeneously expressed within biofilms, we consider this sRNA to function as a factor favoring phenotypic heterogeneity and supporting division of labor in S. epidermidis biofilm communities. Author summary Bacterial biofilms are highly organized structures which functionally emulate multicellular organisms, last but not least through heterogeneous gene expression patterns displayed by biofilm subpopulations. Here we analyzed the functions of the non-coding RNA RsaE in Staphylococcus epidermidis biofilm communities. RsaE exerted unexpected influences on S. epidermidis biofilm matrix composition by triggering localized eDNA release and facilitating PIA expression. RsaE accomplishes these effects by targeting mRNAs involved in bacterial lysis control, icaADBC expression and TCA cycle activity, with RsaE undergoing processing to exploit its full target potential. Interestingly, RsaE interaction with lysis-engaged lrgA mRNA is specific for S. epidermidis lrgA, but does not occur with lrgA mRNA from S. aureus, suggesting species-specific differences in staphylococcal lysis control. We speculate that RsaE-mediated bacterial lysis might represent a form of bacterial altruism contributing to biofilm structuring by providing nutrients to neighboring bacterial cells as well as by releasing eDNA as stabilizing biofilm matrix component. Due to its heterogeneous expression, we consider RsaE as a supporting factor that facilitates population diversity. Together, the data give insight into an unanticipated role of sRNAs as players in S. epidermidis biofilm organization.