Person:

DeLoughery, Aaron

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

Bacteria are capable of sensing changes in their environment and responding to those changes by altering patterns of gene expression. There are many mechanisms by which bacteria can sense internal and external signals. The most well studied systems for this, two-component systems, are composed of a histidine kinase that can act as a detector and a response regulator that when activated by the histidine kinase can carry out a function. In the case of regulating gene expression, once a signal is detected, the response regulator can directly act as a transcription factor or control the activity of other transcription factors to determine the genes that are transcribed and the genes that are not. Historically, much of gene regulation has been thought to be regulated at the level of transcription. There are, however, several processes that take place after a gene is transcribed that ultimately determine the level of a gene product These include mRNA stability, translation initiation, translation efficiency, and protein degradation, all of which the cell could potentially target to fine-tune protein levels. Many such post-transcriptional mechanisms of regulation have been identified, such as controlled proteolysis and the regulation of translation by riboswitches. Here I report on mechanisms operating at the level of translational efficiency and mRNA stability that control entry into a multicellular state in the bacterium Bacillus subtilis.

Like many bacteria, B. subtilis can form architecturally complex communities known as biofilms. Biofilm formation by B. subtilis is largely governed by a dedicated repressor for matrix production, SinR. At the transcriptional level, biofilm formation is regulated by the response regulator Spo0A, which turns on the gene for the anti-repressor SinI. SinI, in turn, binds to and inactivates SinR. The work that I have been a part of and that is presented here has revealed two additional mechanisms in which SinR is regulated post-transcriptionally. The first is a simple mechanism in which B. subtilis can sense changes in nutrient availability without a dedicated protein or RNA. Instead certain synonymous serine codons cause pausing when serine levels are reduced, and enrichment for these codons in sinR decreases its rate of translation under biofilm-inducing conditions, contributing to derepression of matrix genes.

The second, which is the main focus of this thesis, acts at the level of the stability of the mRNA for sinR. This mechanism was unexpectedly uncovered while determining the function of three mysterious proteins, YlbF, YmcA, and YaaT, which are required for biofilm formation. I have found that YlbF, YmcA, and YaaT form a three-protein complex that interacts with and is required for the full activity of the ribonuclease RNase Y. This complex is required for biofilm formation because it destabilizes the mRNA for sinR. In addition RNA-sequencing experiments reveal a global role for the complex in RNA turnover. Turnover of many known targets of RNase Y, including mRNA and non-coding RNAs, are dependent on the complex.

In light of these findings, the decision to form a multicellular community can be seen as the convergence of three regulatory pathways operating at three different levels of the expression of the gene for the master regulatory protein for biofilm formation SinR.