Multimer recognition and secretion by the non-classical secretion pathway in Bacillus subtilis

Abstract

Non-classical protein secretion in bacteria is a common phenomenon. However, the selection principle for non-classical secretion pathways remains unclear. Here, our experimental data, to our knowledge, are the first to show that folded multimeric proteins can be recognized and excreted by a non-classical secretion pathway in Bacillus subtilis. We explored the secretion pattern of a typical cytoplasmic protein D-psicose 3-epimerase from Ruminococcus sp. 5_1_39BFAA (RDPE), and showed that its non-classical secretion is not simply due to cell lysis. Analysis of truncation variants revealed that the C- and N-terminus, and two hydrophobic domains, are required for structural stability and non-classical secretion of RDPE. Alanine scanning mutagenesis of the hydrophobic segments of RDPE revealed that hydrophobic residues mediated the equilibrium between its folded and unfolded forms. Reporter mCherry and GFP fusions with RDPE regions show that its secretion requires an intact tetrameric protein complex. Using cross-linked tetramers, we show that folded tetrameric RDPE can be secreted as a single unit. Finally, we provide evidence that the non-classical secretion pathway has a strong preference for multimeric substrates, which accumulate at the poles and septum region. Altogether, these data show that a multimer recognition mechanism is likely applicable across the non-classical secretion pathway.

Engineering Biology to Construct Microbial Chassis for the Production of Difficult-to-Express Proteins

“…Alternatively, several reports have shed light on nonclassical protein secretion systems that mediate translocation of proteins that lack signal peptides as a more straightforward means to direct protein secretion [85]. Specifically, all routes by which the cytoplasmic proteins without apparent secretion motifs travel out into the extracellular space are termed as nonclassical secretion pathway due to their largely elusive mechanism of transport [86]. Notably, in analogy to fusion tags, physically linking such nonclassically secreted proteins to heterologous expression target was shown to functionally mimic the role of signal peptides, enhancing the secretion rates of fusion proteins without compromising their biological functions [85].…”

EngineeringBacillusfor efficient production of heterologous protein: current progress, challenge and prospect

“…; Zhao et al . ). Among these strategies, the genetic modification of host strains is regarded as an efficient tactic due to its universality and efficiency, and many studies for developing effective hosts for heterologous protein production have been reported.…”

“…The low yield of heterologous proteins from Bacillus protein expression systems hindered its application, and (Zhou et al 2017;Huang et al 2018), signal peptides (Cai et al 2016), transportation elements (Chen et al 2015c), signal peptidases (Cai et al 2016;Song et al 2017), signal peptide peptidases , chaperones (Chen et al 2015d), proteases (Pummi et al 2002;Wei et al 2015), transcriptional factors ), etc, have been genetically modified for enhanced production of target proteins (a-amylase, protease, pullulanase, etc. ), also, the nonclassical secretion pathways have been developed and applied for protein production in B. subtilis Zhao et al 2017). Among these strategies, the genetic modification of host strains is regarded as an efficient tactic due to its universality and efficiency, and many studies for developing effective hosts for heterologous protein production have been reported.…”

The Native Monomer of Bacillus Pumilus Ribonuclease Does Not Exist Extracellularly

“…Proteins translocated through the SecYEG pore to the cell surface immediately acquire a native conformation to avoid proteolytic degradation. Secretion of proteins directly in a dimeric state is also possible [ 12 , 13 ]. Most of them are secreted through nonclassical pathways and require an unknown three-dimensional recognition signal for secretion.…”