We report the discovery of a simple environmental sensing mechanism for biofilm formation in the bacterium Bacillus subtilis that operates without the involvement of a dedicated RNA or protein. Certain serine codons, the four TCN codons, in the gene for the biofilm repressor SinR caused a lowering of SinR levels under biofilm-inducing conditions. Synonymous substitutions of these TCN codons with AGC or AGT impaired biofilm formation and gene expression. Conversely, switching AGC or AGT to TCN codons upregulated biofilm formation. Genome-wide ribosome profiling showed that ribosome density was higher at UCN codons than at AGC or AGU during biofilm formation. Serine starvation recapitulated the effect of biofilm-inducing conditions on ribosome occupancy and SinR production. As serine is one of the first amino acids to be exhausted at the end of exponential phase growth, reduced translation speed at serine codons may be exploited by other microbes in adapting to stationary phase.DOI: http://dx.doi.org/10.7554/eLife.01501.001

Staphylococcus aureus is a leading cause of both nosocomial and community-acquired infection. Biofilm formation at the site of infection reduces antimicrobial susceptibility and can lead to chronic infection. During biofilm formation, a subset of cells liberate cytoplasmic proteins and DNA, which are repurposed to form the extracellular matrix that binds the remaining cells together in large clusters. Using a strain that forms robust biofilms in vitro during growth under glucose supplementation, we carried out a genome-wide screen for genes involved in the release of extracellular DNA (eDNA). A high-density transposon insertion library was grown under biofilm-inducing conditions, and the relative frequency of insertions was compared between genomic DNA (gDNA) collected from cells in the biofilm and eDNA from the matrix. Transposon insertions into genes encoding functions necessary for eDNA release were identified by reduced representation in the eDNA. On direct testing, mutants of some of these genes exhibited markedly reduced levels of eDNA and a concomitant reduction in cell clustering. Among the genes with robust mutant phenotypes were gdpP, which encodes a phosphodiesterase that degrades the second messenger cyclic-di-AMP, and xdrA, the gene for a transcription factor that, as revealed by RNA-sequencing analysis, influences the expression of multiple genes, including many involved in cell wall homeostasis. Finally, we report that growth in biofilm-inducing medium lowers cyclicdi-AMP levels and does so in a manner that depends on the gdpP phosphodiesterase gene.Staphylococcus aureus | biofilm | eDNA | cyclic-di-AMP

The signal recognition particle (SRP) recognizes polypeptide chains bearing a signal sequence as they emerge from the ribosome, and then binds its membrane-associated receptor (SR), thereby delivering the ribosome-nascent chain complex to the endoplasmic reticulum in eukaryotic cells and the plasma membrane in prokaryotic cells. SRP RNA catalytically accelerates the interaction of SRP and SR, which stimulates their guanosine triphosphatase (GTPase) activities, leading to dissociation of the complex. We found that although the catalytic activity of SRP RNA appeared to be constitutive, SRP RNA accelerated complex formation only when SRP was bound to a signal sequence. This crucial control step was obscured because a detergent commonly included in the reaction buffer acted as a signal peptide mimic. Thus, SRP RNA is a molecular switch that renders the SRP-SR GTPase engine responsive to signal peptide recruitment, coupling GTP hydrolysis to productive protein targeting.Secretory and transmembrane proteins are delivered to the membrane cotranslationally by the signal recognition particle (SRP) and its membrane-associated receptor (SR) (1). SRP recognizes signal sequences as they emerge from the ribosome (2) and then associates with SR at the membrane where the ribosome is transferred to the translocon. The guanosine triphosphatase (GTPase) domains of SRP and SR mediate this interaction cycle (3). Interaction of SRP with SR leads to the reciprocal stimulation of their GTPase activities, and GTP hydrolysis dissociates the complex (4,5). In Escherichia coli, SR is a single protein, FtsY, and SRP consists of 4.5S RNA and a single protein, Ffh (6). 4.5S RNA catalyzes the interaction of Ffh and FtsY, accelerating both on and off rates by a factor of more than 100 (7). If the energy of GTP hydrolysis is to be harnessed for protein targeting, recruitment of targeting substrates by SRP should be coupled to the SRP-SR interaction cycle. Both signal sequences and 4.5S RNA bind to the M domain of Ffh, which suggests that the catalytic activity of 4.5S RNA could be responsive to signal sequence binding (8). However, under typical assay conditions, 4.5S RNA is constitutively active, negating this role for the RNA (4,7,9,10). A small amount of the nonionic detergent octaethyleneglycol dodecylether (C 12 E 8 ) has been used in assays for SRP function, including kinetic characterization of the Ffh-FtsY interaction (4,

The interaction of the signal-recognition particle (SRP) with its receptor (SR) mediates co-translational protein targeting to the membrane. SRP and SR interact via their homologous core GTPase domains and N-terminal four-helix bundles (N domains). SRP–SR complex formation is slow unless catalyzed by SRP’s essential RNA component. We show that truncation of the first helix of the N domain (helix N1) of both proteins dramatically accelerates their interaction. SRP and SR with helix N1 truncations interact at nearly the RNA-catalyzed rate in the absence of RNA. NMR spectroscopy and analysis of GTPase activity show that helix N1 truncation in SR mimics the conformational switch caused by complex formation. These results demonstrate that the N-terminal helices of SRP and SR are autoinhibitory for complex formation in the absence of SRP RNA, suggesting a mechanism for RNA-mediated coordination of the SRP–SR interaction.