Date Published: July 21, 2011
Author(s): Hoang Duc Nguyen, Trang Thi Phuong Phan, Wolfgang Schumann.
Bacillus subtilis codes for two putative sortases, YhcS and YwpE, and two surface proteins, YhcR and YfkN, harboring sorting motifs supposed to be recognized by the putative sortase(s). However, there is no experimental evidence to show a direct link between these sortases and sorting sequences. To study the role of these two putative sortases on displaying YhcR and YfkN on the cell wall, expression of yhcS and ywpE was analyzed by transcriptional fusions and by Northern blot. It turned out that yhcS gene is expressed at a higher level during the late stationary phase from both experiments, while ywpE expression is not confirmed in the Northern blot analysis. Next, we constructed yhcS and ywpE single and double knockout strains and plasmids that express one or both genes to restore the functions of the knockout strains. It could be shown that display of YhcR and YfkN on the surface depended on the presence of YhcS while YwpE seems not to play a major role if any as a sortase. Finally, the putative sorting motif together with a 123-amino-acid spacer derived from YhcR and YfkN designated YhcR123 and YfkN123, respectively, were fused to an α-amylase reporter enzyme. The fusion protein YhcR123-AmyQ could be displayed on the surface at high amounts, while YfkN123-AmyQ could be hardly detected. We conclude that the sortase YhcS can recognize and anchor YhcR on the cell wall. This result further indicates that the YhcR sorting sequence can be used to display recombinant proteins on the surface of B. subtilis cells.
Cell surface display of recombinant proteins is usually achieved through a translational fusion of the target protein to one of the naturally occurring surface proteins of the host cell. Display of proteins on the surface of microorganisms, enabled by means of recombinant DNA technology, has become an increasingly used strategy in various applications in microbiology, biotechnology and vaccination (Samuelson et al. 2002; Wernerus and Stahl 2004; Daugherty 2007).
Using bioinformatics tools, two sortase-like genes and two substrate proteins have been identified (Comfort and Clubb 2004; Pallen et al. 2001; Boekhorst et al. 2005). We could show here that the putative sortase genes ywpE and yhcS are preferentially expressed in the late stationary phase. This finding suggests that these enzymes fulfill their task mainly during that growth phase. Furthermore, we could demonstrate that the two putative sortase-dependent substrate proteins, YfkN and YhcR, can be anchored on the cell wall in the presence of YhcS. In terms of application, this work demonstrated that the YhcR sorting sequence can be specifically used to display heterologous proteins on the cell-wall of B. subtilis cells. The B. subtilis cell wall contains peptide crosslinks identical to those present in the L. monocytogenes cell walls. This suggests that the crosslink of potential surface proteins to the peptidoglycan is formed by the nucleophilic attack of the amino group of m-diaminopimelic acid cross-bridge within the lipid II precursor as in the case of L. monocytogenes (Dhar et al. 2000).
The authors declare that they have no competing interests.