A lysine cluster in domain II of Bacillus subtilis PBP4a plays a role in the membrane attachment of this C1 PBP A lysine cluster in domain II of Bacillus subtilis PBP4a plays a role in the membrane attachment of this C1 PBP Arnaud Vanden Broeck, Marjorie Dauvin, Eric Sauvage and Colette Duez Centre d’Ingénierie des Protéines, Institut de Chimie, B6a. Université de Liège. ULg Introduction Bacillus subtilis PBP4a belongs to the class-C1 PBPs characterized by two internal additional domains of unknown function. Seven lysine residues (K) are protruding from domain II. Four of them: K86, K114, K119 and K265 have been mutated in glutamine residues (Q). Both proteins (WT and Mut4KQ PBP4a) have been produced without signal peptide in E. coli and their sub-cellular localizations determined by measuring the DD-carboxypeptidase activities in the different compartments (cytoplasmic vs membrane attached proteins). After purification, their binding to B. subtilis membranes has been compared: WT PBP4a interacts in vitro with membranes isolated from this organism in contrast to Mut4KQ PBP4a that remains entirely unbound. In absence of any amphiphilic peptide in PBP4a, the crown of positive charges on the surface of domain II is likely responsible for the PBP cellular localization in interaction with the cytoplasmic membrane. Fig.1: Electrostatic potentials of Bacillus subtilis PBP4a showing the basic surface in domain II. The negatively charged residues are coloured red, the positively charged residues are coloured blue. Site-directed mutagenesis of K86, K114, K119 and K265 Cloning of dacC into pBAD/Myc-HisA (Kan R ) was performed to produce B. subtilis PBP4a without its signal peptide in E. coli LMG194 cells. Rolling circle mutagenesis allowed to exchange lysine residues K86, K114, K119 and K265 (see Figure 1) into glutamine residues. Production of WT or Mut 4KQ PBP4a and study of their cellular localisations Production of WT PBP4a or mutated PBP4a in E. coli LMG194 cells were induced by addition of 0.2% L-arabinose. Figure 2 presents the percentages of DD-carboxypeptidase activities in the soluble fraction or in a 1M NaCl membrane extract from each transformed strain. Purified WT PBP4a was mainly attached to membranes isolated from B. subtilis in opposition to the mutated protein. Fig. 3: Immunodetection of PBP4a in extracts of B. subtilis membranes. - Lane 1: Prestained PageRuler Protein Ladder. -Lane 2: aliquot of proteins extracted with 1 M NaCl form B. subtilis membranes. - Lane 3: Purified PBP4a produced in E. coli without signal peptide Conclusions  After replacement of four lysine residues protruding from the B. subtilis PBP4a domain II, WT and Mut4KQ PBP4a have been produced in E. coli.  The WT PBP4a was mainly associated with the E. coli cytoplasmic membrane in opposition to the Mut4KQ protein.  Purified WT PBP4a binds in vitro to B. subtilis cytoplasmic membranes in contrast to purified Mut4KQ PBP4a.  Western blotting of proteins extracted from B. subtilis membranes revealed the presence of PBP4a with a size actually indicating processing and secretion of PBP4a in B. subtilis. FUNDING: IAP n° P6/19 and P7/44. C. Duez is a Research Associate of the FRS-FNRS. Multiple sequence alignment between canonical conserved SxxK and SxN motifs from eight C1-PBPs Alignment of B. subtilis PBP4a sequence with Actinomadura R39 DD-peptidase, E. coli PBP4, Haemophilus influenzae PBP4, Clostridium sp. DD-peptidase, Neisseria gonorrhoeae PBP3, Pseudomonas aeruginosa PBP4 and Haladaptatus paucihalophilus PBP. Lysine residues protruding from domain II in B. subtilis PBP4a are generally not conserved among these PBPs. A positively charged surface at the tip of domain II also exists in the DD-peptidase of the A. R39 Gram positive bacterium but not in the E. coli or H. influenzae C1 PBPs suggesting a different mechanism of membrane association for the Gram negative class-C1 PBPs. Processed PBP4a is present in B. subtilis membranes. Rabbit polyclonal antibodies directed against purified PBP4a were used to detect PBP4a in B. subtilis membrane extracts. A band, identical in size to PBP4a lacking the predicted 29- residue signal peptide is visible on a Western blot (Figure 3). This result indicates for the first time that PBP4a is actually processed in B. subtilis and translocated through the cytoplasmic membrane Fig. 2: Percentage of DD-carboxypeptidase activities in the cytoplasmic fraction or associated to the E. coli inner membrane 70 kDa 55 kDa 40 kDa 1 2 3