Abstract
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.
Highlights
Life in biofilms is a widespread phenomenon in the bacterial world
Biofilm matrix variants display varying carbon flux gene expression patterns To get an insight into the molecular basis of varying biofilm matrix production in S. epidermidis, we analyzed the transcription profiles of the PS2 variant in comparison to the PS10 variant (PIA biofilm matrix) during growth in batch cultures by dRNAseq
Interactive visualization of the complete data set is shown in S1 Fig. In the polysaccharide intercellular adhesin (PIA) producing PS10 variant, transcription of genes of galactose and pyruvate metabolism, glycolysis/ gluconeogenesis and the tricarboxylic acid (TCA) cycle were downregulated
Summary
Life in biofilms is a widespread phenomenon in the bacterial world. Many planktonically living bacteria are capable of aggregating on surfaces and to each other by encasing in a self-produced extracellular matrix, making biofilm formation a common and in some species even the preferred bacterial lifestyle. The mechanisms underlying the generation and maintenance of diversity in biofilms and, more general, in bacterial communities, seem to be manifold. They may involve regulatory pathways (including quorum-sensing circuits), reversible and non-reversible genetic events, and stochastic variations during transcription and translation, resulting in fluctuations of protein levels [3]. Biofilm formation of S. epidermidis typically occurs on the inert surfaces of medical devices and involves the production of a self-produced extracellular matrix [5]. The bacterium was demonstrated to switch spontaneously between protein- and PIA-mediated biofilm matrix production (and vice versa), suggesting a high degree of phenotypic heterogeneity within S. epidermidis biofilm communities [7, 8, 10, 14,15,16].
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