Towards the development of Bacillus subtilis as a cell factory for membrane proteins and protein complexes

Background: The Gram-positive bacterium Bacillus subtilis is an important producer of high quality industrial enzymes and a few eukaryotic proteins. Most of these proteins are secreted into the growth medium, but successful examples of cytoplasmic protein production are also known. Therefore, one may anticipate that the high protein production potential of B. subtilis can be exploited for protein complexes and membrane proteins to facilitate their functional and structural analysis. The high quality of proteins produced with B. subtilis results from the action of cellular quality control systems that efficiently remove misfolded or incompletely synthesized proteins. Paradoxically, cellular quality control systems also represent bottlenecks for the production of various heterologous proteins at significant concentrations. Conclusion: While inactivation of quality control systems has the potential to improve protein production yields, this could be achieved at the expense of product quality. Mechanisms underlying degradation of secretory proteins are nowadays well understood and often controllable. It will therefore be a major challenge for future research to identify and modulate quality control systems of B. subtilis that limit the production of high quality protein complexes and membrane proteins, and to enhance those systems that facilitate assembly of these proteins.

Definition of the σW regulon of Bacillus subtilis in the absence of stress

Bacteria employ extracytoplasmic function (ECF) sigma factors for their responses to environmental stresses. Despite intensive research, the molecular dissection of ECF sigma factor regulons has remained a major challenge due to overlaps in the ECF sigma factor-regulated genes and the stimuli that activate the different ECF sigma factors. Here we have employed tiling arrays to single out the ECF σW regulon of the Gram-positive bacterium Bacillus subtilis from the overlapping ECF σX, σY, and σM regulons. For this purpose, we profiled the transcriptome of a B. subtilis sigW mutant under non-stress conditions to select candidate genes that are strictly σW-regulated. Under these conditions, σW exhibits a basal level of activity. Subsequently, we verified the σW-dependency of candidate genes by comparing their transcript profiles to transcriptome data obtained with the parental B. subtilis strain 168 grown under 104 different conditions, including relevant stress conditions, such as salt shock. In addition, we investigated the transcriptomes of rasP or prsW mutant strains that lack the proteases involved in the degradation of the σW anti-sigma factor RsiW and subsequent activation of the σW-regulon. Taken together, our studies identify 89 genes as being strictly σW-regulated, including several genes for non-coding RNAs. The effects of rasP or prsW mutations on the expression of σW-dependent genes were relatively mild, which implies that σW-dependent transcription under non-stress conditions is not strictly related to RasP and PrsW. Lastly, we show that the pleiotropic phenotype of rasP mutant cells, which have defects in competence development, protein secretion and membrane protein production, is not mirrored in the transcript profile of these cells. This implies that RasP is not only important for transcriptional regulation via σW, but that this membrane protease also exerts other important post-transcriptional regulatory functions

Stress-Responsive Systems Set Specific Limits to the Overproduction of Membrane Proteins in Bacillus subtilis▿†

Essential membrane proteins are generally recognized as relevant potential drug targets due to their exposed localization in the cell envelope. Unfortunately, high-level production of membrane proteins for functional and structural analyses is often problematic. This is mainly due to their high overall hydrophobicity. To develop new concepts for membrane protein overproduction, we investigated whether the biogenesis of overproduced membrane proteins is affected by stress response-related proteolytic systems in the membrane. For this purpose, the well-established expression host Bacillus subtilis was used to overproduce eight essential membrane proteins from B. subtilis and Staphylococcus aureus. The results show that the σW regulon (responding to cell envelope perturbations) and the CssRS two-component regulatory system (responding to unfolded exported proteins) set critical limits to membrane protein production in large quantities. The identified sigW or cssRS mutant B. subtilis strains with significantly improved capacity for membrane protein production are interesting candidate expression hosts for fundamental research and biotechnological applications. Importantly, our results pinpoint the interdependent expression and function of membrane-associated proteases as key parameters in bacterial membrane protein production

Protein transport across and into cell membranes in bacteria and archaea

In the three domains of life, the Sec, YidC/Oxa1, and Tat translocases play important roles in protein translocation across membranes and membrane protein insertion. While extensive studies have been performed on the endoplasmic reticular and Escherichia coli systems, far fewer studies have been done on archaea, other Gram-negative bacteria, and Gram-positive bacteria. Interestingly, work carried out to date has shown that there are differences in the protein transport systems in terms of the number of translocase components and, in some cases, the translocation mechanisms and energy sources that drive translocation. In this review, we will describe the different systems employed to translocate and insert proteins across or into the cytoplasmic membrane of archaea and bacteria

Assignment of clusters of genes with related transcript profiles across conditions to different groups of genes that are down-regulated or up-regulated in <i>sigW</i> mutant cells.

<p>The down-regulated genes are represented by groups 1 and 2 (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048471#pone-0048471-g001" target="_blank">Fig. 1</a>). Genes in group 3 were previously reported as σ^W-regulated, but our present studies provided no evidence for their proposed σ^W-dependency (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048471#pone-0048471-g001" target="_blank">Fig. 1</a>). The up-regulated genes are represented in a separate bar. Previously defined transcription clusters <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048471#pone.0048471-Nicolas1" target="_blank">[35]</a> are indicated in each bar by their C-number.</p

Up- and down-regulation of genes in <i>rasP</i>, <i>prsW</i> or <i>sigW</i> mutant strains compared to the wild-type.

<p>A, Venn diagram for down-regulated genes. B, Venn diagram for upregulated genes. Only genes with transcriptional changes that have p-values lower than 0.05 and effect values lower than −0.40 (A) or higher than 0.40 (B) are included. The genes that are considered to be σ^W-regulated are indicated between brackets.</p

Venn diagrams for the comparison of genes that were found to be downregulated in the <i>sigW</i> mutant strain with previously reported σ^W-regulated genes and genes that display similar condition-dependent transcription profiles as <i>sigW</i>.

<p>Diagram A includes only the so-called cluster C9 genes that have highly similar condition-dependent transcription profiles as defined by Nicolas <i>et al </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048471#pone.0048471-Nicolas1" target="_blank">[35]</a>. Notably, the <i>sigW</i> gene is included in cluster C9. Diagram B includes all genes that show condition-dependent expression profiles similar to that of <i>sigW</i>, including induction upon salt stress.</p

Organization of complex σ^W-regulated operons.

<p>The σ^W-regulated ORFs are indicated in black, and the σ^W-regulated ncRNAs are indicated in grey. Genes and an ncRNA on the opposite strand are indicated in white. A, The <i>yozO</i>-<i>yocM</i> operon. B, The <i>ydbST</i>-<i>acpS</i> operon.</p

Transcriptional changes of genes regulated by the CssRS two-component system.

<p>Transcriptional changes of genes regulated by the CssRS two-component system.</p

Expression profiles of <i>sigW</i>, <i>sigX</i>, <i>sigY</i> and <i>sigM</i> in <i>B. subtilis</i> 168 across 104 conditions.

<p>The 269 tiling array hybridizations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048471#pone.0048471-Nicolas1" target="_blank">[35]</a> are arranged along the x-axis. Of particular interest for discriminating the activities of the encoded sigma factors are the conditions heat stress (‘heat’), ethanol stress (‘etha’) and hypersaline stress (‘salt’), which are marked by pink shading.</p