TY - JOUR
T1 - Septal and lateral wall localization of PBP5, the major D,D-carboxypeptidase of Escherichia coli, requires substrate recognition and membrane attachment
AU - Potluri, Lakshmiprasad
AU - Karczmarek, Aneta
AU - Verheul, Jolanda
AU - Piette, Andre
AU - Wilkin, Jean-Marc
AU - Werth, Nadine
AU - Banzhaf, Manuel
AU - Vollmer, Waldemar
AU - Young, Kevin D
AU - Nguyen-Distèche, Martine
AU - den Blaauwen, Tanneke
PY - 2010/7
Y1 - 2010/7
N2 - The distribution of PBP5, the major D,D-carboxypeptidase in Escherichia coli, was mapped by immunolabelling and by visualization of GFP fusion proteins in wild-type cells and in mutants lacking one or more D,D-carboxypeptidases. In addition to being scattered around the lateral envelope, PBP5 was also concentrated at nascent division sites prior to visible constriction. Inhibiting PBP2 activity (which eliminates wall elongation) shifted PBP5 to midcell, whereas inhibiting PBP3 (which aborts divisome invagination) led to the creation of PBP5 rings at positions of preseptal wall formation, implying that PBP5 localizes to areas of ongoing peptidoglycan synthesis. A PBP5(S44G) active site mutant was more evenly dispersed, indicating that localization required enzyme activity and the availability of pentapeptide substrates. Both the membrane bound and soluble forms of PBP5 converted pentapeptides to tetrapeptides in vitro and in vivo, and the enzymes accepted the same range of substrates, including sacculi, Lipid II, muropeptides and artificial substrates. However, only the membrane-bound form localized to the developing septum and restored wild-type rod morphology to shape defective mutants, suggesting that the two events are related. The results indicate that PBP5 localization to sites of ongoing peptidoglycan synthesis is substrate dependent and requires membrane attachment.
AB - The distribution of PBP5, the major D,D-carboxypeptidase in Escherichia coli, was mapped by immunolabelling and by visualization of GFP fusion proteins in wild-type cells and in mutants lacking one or more D,D-carboxypeptidases. In addition to being scattered around the lateral envelope, PBP5 was also concentrated at nascent division sites prior to visible constriction. Inhibiting PBP2 activity (which eliminates wall elongation) shifted PBP5 to midcell, whereas inhibiting PBP3 (which aborts divisome invagination) led to the creation of PBP5 rings at positions of preseptal wall formation, implying that PBP5 localizes to areas of ongoing peptidoglycan synthesis. A PBP5(S44G) active site mutant was more evenly dispersed, indicating that localization required enzyme activity and the availability of pentapeptide substrates. Both the membrane bound and soluble forms of PBP5 converted pentapeptides to tetrapeptides in vitro and in vivo, and the enzymes accepted the same range of substrates, including sacculi, Lipid II, muropeptides and artificial substrates. However, only the membrane-bound form localized to the developing septum and restored wild-type rod morphology to shape defective mutants, suggesting that the two events are related. The results indicate that PBP5 localization to sites of ongoing peptidoglycan synthesis is substrate dependent and requires membrane attachment.
KW - Carboxypeptidases
KW - Cell Division
KW - Escherichia coli
KW - Escherichia coli Proteins
KW - Mutation
KW - Peptidoglycan
KW - Protein Interaction Mapping
KW - Substrate Specificity
KW - Journal Article
KW - Research Support, N.I.H., Extramural
KW - Research Support, Non-U.S. Gov't
U2 - 10.1111/j.1365-2958.2010.07205.x
DO - 10.1111/j.1365-2958.2010.07205.x
M3 - Article
C2 - 20545860
SN - 0950-382X
VL - 77
SP - 300
EP - 323
JO - Molecular Microbiology
JF - Molecular Microbiology
IS - 2
ER -