Regulation of major bacterial survival strategies by transcripts sequestration in a membraneless organelle.

TmaR, the only known pole-localizer protein in Escherichia coli, was shown to cluster at the cell poles and control localization and activity of the major sugar regulator in a tyrosine phosphorylation-dependent manner. Here, we show that TmaR assembles by phase separation (PS) via heterotypic interactions with RNA in vivo and in vitro. An unbiased automated mutant screen combined with directed mutagenesis and genetic manipulations uncovered the importance of a predicted nucleic-acid-binding domain, a disordered region, and charged patches, one containing the phosphorylated tyrosine, for TmaR condensation. We demonstrate that, by protecting flagella-related transcripts, TmaR controls flagella production and, thus, cell motility and biofilm formation. These results connect PS in bacteria to survival and provide an explanation for the linkage between metabolism and motility. Intriguingly, a point mutation or increase in its cellular concentration induces irreversible liquid-to-solid transition of TmaR, similar to human disease-causing proteins, which affects cell morphology and division.

Inferring the contribution of small RNAs to changes in gene expression in response to stress.

A main strategy of bacteria adapting to environmental changes is the remodeling of their transcriptome. Changes in the transcript levels of specific genes are due to combined effects of various regulators, including small RNAs (sRNAs). sRNAs are post-transcriptional regulators of gene expression that mainly control translation, but also directly and indirectly affect the levels of their target transcripts. Yet, the relative contribution of an sRNA to the total change in the transcript level of a gene upon an environmental change has not been assessed. We present a design of differential gene expression analysis by RNA-seq that allows extracting the contribution of an sRNA to the total change in the transcript level of each gene in response to an environmental change by fitting a linear model to the data. We exemplify this for the sRNA RyhB in cells growing under iron limitation and show a variation among genes in the relative contribution of RyhB to the change in their transcript level upon iron limitation, from subtle to very substantial. Extracting the relative contribution of an sRNA to the total change in expression of genes is important for understanding the integration of regulation by sRNAs with other regulatory mechanisms in the cell.

Hierarchy in Hfq Chaperon Occupancy of Small RNA Targets Plays a Major Role in Their Regulation.

Bacterial small RNAs (sRNAs) are posttranscriptional regulators of gene expression that base pair with complementary sequences on target mRNAs, often in association with the chaperone Hfq. Here, using experimentally identified sRNA-target pairs, along with gene expression measurements, we assess basic principles of regulation by sRNAs. We show that the sRNA sequence dictates the target repertoire, as point mutations in the sRNA shift the target set correspondingly. We distinguish two subsets of targets: targets showing changes in expression levels under overexpression of their sRNA regulator and unaffected targets that interact more sporadically with the sRNA. These differences among targets are associated with their Hfq occupancy, rather than with the sRNA-target base-pairing potential. Our results suggest that competition among targets over Hfq binding plays a major role in the regulatory outcome, possibly awarding targets with higher Hfq binding efficiency an advantage in the competition over binding to the sRNA.