Septal and lateral wall localization of PBP5, the major D,D-carboxypeptidase of Escherichia coli, requires substrate recognition and membrane attachment
[en] 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.
Disciplines :
Microbiology
Author, co-author :
Potluri, Lakshmiprasad; University of Arkansas for Medical Sciences > Department of Microbiology and Immunology
Karczmarek, Aneta; University of Amsterdam > Swammerdam Institute for Life Sciences > Molecular Cytology
Verheul, Jolanda; University of Amsterdam > Swammerdam Institute for Life Sciences > Molecular Cytology
Piette, André ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Wilkin, Jean-Marc; Université de Liège - ULiège > Centre d'Ingénierie des Protéines
Werth, Nadine; Eberhard Karls Universität Tübingen > Mikrobielle Genetik
Banzhaf, Manuel; Newcastle University > Centre for Bacterial Cell Biology
Vollmer, Waldemar; Newcastle University > Centre for Bacterial Cell Biology
Young, Kevin D; University of Arkansas for Medical Sciences > Department of Microbiology and Immunology
den Blaauwen, Tanneke; University of Amsterdam > Swammerdam Institute for Life Sciences > Molecular Cytology
Language :
English
Title :
Septal and lateral wall localization of PBP5, the major D,D-carboxypeptidase of Escherichia coli, requires substrate recognition and membrane attachment
Publication date :
July 2010
Journal title :
Molecular Microbiology
ISSN :
0950-382X
eISSN :
1365-2958
Publisher :
Blackwell Publishing, Oxford, United Kingdom
Volume :
77
Issue :
2
Pages :
300–323
Peer reviewed :
Peer Reviewed verified by ORBi
Funders :
UE - Union Européenne NIH - National Institutes of Health ABI - Arkansas Biosciences Institute
Funding text :
We thank Norbert Vischer for writing the Object-J macros.
We thank Rene van der Ploeg for the gift of LMC512 cells, Eefjan Breukink (University of Utrecht) for the gift of 14CGlcNAc-labelled lipid II and Mohammed Terrak (University of Liège) for the gift of 3H-lipid II. We thank Piet de Boer (Case Western Reserve University) for providing plasmid
pMLB1113. We are also very much in debt to Thomas Bernhardt (Harvard Medical School) for communicating unpublished information and for providing the Superfolder GFP dsbA-SS-sfgfp gene construct on plasmid pTB263. This work was supported by the European commission within the
‘EUR-INTAFAR’ LSHM-CT-2004-512138 network, by Grant R01-GM061019 from the US National Institutes of Health, and by the Arkansas Biosciences Institute, the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000.
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