A novel screening system for secretion of heterologous proteins in Bacillus subtilis

Abstract

SummaryHigh‐level production of secretory proteins in Bacillus subtilis leads to a stress response involving the two‐component system CssRS and its target genes htrA and htrB. Here, we used this sensing system in a reporter strain in which gfp is under control of PhtrA, the secretion stress responsive promoter of htrA. Overexpression of heterologous secretory proteins in this strain results in green fluorescent cells, which can be separated from non‐secreting, low fluorescent cells using a fluorescence‐activated cell sorter (FACS). Using this principle, genomic libraries of uncharacterized prokaryotic organisms, expressed in the reporter strain, can be screened for genes encoding secretory proteins.

Comparative transcriptional analysis of Bacillus subtilis cells overproducing either secreted proteins, lipoproteins or membrane proteins

“…In this study, overproduction of the membrane proteins LmrA and XylP did not significantly induce htrA or htrB . This is in agreement with previous results from an analysis of the activation of the htrA promoter in response to overproduction of secretory proteins, including MntA, XynA, TEM-1 β-lactamase, Usp45 and LmrA, showing that the stress signal is sensed on the outside of the cell and not from within the membrane [48]. Surprisingly, NprE overproduction did not induce the CssRS response.…”

“…These encode membrane-anchored HtrA and HtrB proteins, which have their active site on the trans side of the membrane and are thought to have proteolytic as well as chaperone activity for removal of misfolded protein or for assisting in folding of newly secreted proteins, respectively [47]. The CssRS two component system is activated by accumulation of mis- or unfolded secreted protein at the membrane - cell wall interface, as a result of, e.g., overexpression of these proteins or heat stress [48,49]. In this study, overproduction of the membrane proteins LmrA and XylP did not significantly induce htrA or htrB .…”

Discovery and Therapeutic Targeting of Differentiated Biofilm Subpopulations

“…However, it has been recently proposed that this technology could be exploited by biofilm researchers to enable the analysis of subpopulations of bacterial cells with different physiological states such as those arising within biofilms (Ambriz-Avina et al, 2014). Once these populations have been labeled and separated out via FACS, they can be independently characterized by high-throughput molecular techniques (Trip et al, 2011) and studied using proteomic or transcriptomic analyses (Ambriz-Avina et al, 2014). Recently, FCM has been used for studying the distribution of different subpopulations present in the biofilm strata under different nutrient availability and at different stages of biofilm formation (Wojciech et al, 2018).…”

A salt, detergent, and solvent tolerant protease from <i>Bacillus</i> sp. B001: Low-cost, easy-purified, and enhanced production by raw material based culture strategy

“…Biosynthesis of these enzymes is controlled by various regulation systems in microorganisms, such as quorum-sensing and two-component systems [7,8]. In this case cells can regulate gene expression and activation of proteins in response to a large variety of environmental signals including nutrients and cell density [9].…”