Septal and lateral wall localization of PBP5, the major D,D‐carboxypeptidase of Escherichia coli, requires substrate recognition and membrane attachment

Abstract

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.

Peptidoglycan Hydrolases of Escherichia coli

“…DD-Carboxypeptidase PBP5. As the major PBP of E. coli (60,243), PBP5 has been extensively studied (72,205,280,301). It was identified with D-alanine carboxypeptidase IA activity encoded by the dacA gene (30,166,191,242).…”

“…Various N-and C-terminally truncated forms were produced (68,205,280). The soluble and membrane-bound forms of PBP5 recognize the same range of substrates.…”

“…2) in many soluble substrates (UDP-MurNAcpentapeptide, lipid II, muropentapeptides from different sources, pentapeptides, and synthetic compounds) and in uncross-linked and cross-linked PG (1,188,205,246,281). In cross-linked PG, PBP5 recognizes D-Ala3D-Ala bonds in both the monomer and dimer subunits (205).…”

“…The overproduction of PBP5 is lethal, causing E. coli to grow as spherical cells before lysing (162,247). The proposed function of PBP5 is to regulate the availability of pentapeptide subunits for the formation of the cross-linkages by transpeptidation (48,205).…”

In Vivo Bacterial Morphogenetic Protein Interactions

“…These are the membrane proteins FtsK, FtsQ, FtsB, FtsL, FtsW, FtsI, FtsN and periplasmic protein AmiC (Aarsman et al, 2005). Four penicillin-binding proteins localize to the constriction site namely PBP1B, PBP2, PBP3 also known as FtsI and PBP5 (Bertsche et al, 2006;Den Blaauwen et al, 2003;Potluri et al, 2010;Weiss et al, 1997).…”