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.
Site-directed mutagenesis of K86, K114, K119 and K265
Cloning of dacC into
pBAD/Myc-HisA (KanR)
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.
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.
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.
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. 2: Percentage of DD-carboxypeptidase
activities in the cytoplasmic fraction or
associated to the E. coli inner membrane
Multiple sequence alignment between canonical conserved
SxxK and SxN motifs from eight C1-PBPs
A band, identical in size to PBP4a lacking the predicted 29residue 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. 3: Immunodetection of PBP4a
in extracts of B. subtilis
membranes.
70 kDa
- Lane 1: Prestained PageRuler
55 kDa
Protein Ladder.
-Lane 2: aliquot of proteins
extracted with 1 M NaCl form B. 40 kDa
subtilis membranes.
- Lane 3: Purified PBP4a produced
in E. coli without signal peptide
1
2
3
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.
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.