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Journal of Bacteriology, November 2008, p. 7079-7086, Vol. 190, No. 21
0021-9193/08/$08.00+0     doi:10.1128/JB.00519-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Detection and Localization of Single LysM-Peptidoglycan Interactions{triangledown}

Guillaume Andre,1 Kees Leenhouts,2 Pascal Hols,3 and Yves F. Dufrêne1*

Unité de Chimie des Interfaces, Université catholique de Louvain, Croix du Sud 2/18, B-1348 Louvain-la-Neuve, Belgium,1 Mucosis BV, Nijenborgh 4, NL-9747 AG Groningen, The Netherlands,2 Unité de Génétique, Institut des Sciences de la Vie, Université catholique de Louvain, Croix du Sud 5/6, B-1348 Louvain-la-Neuve, Belgium3

Received 16 April 2008/ Accepted 21 August 2008

The lysin motif (LysM) is a ubiquitous protein module that binds peptidoglycan and structurally related molecules. Here, we used single-molecule force spectroscopy (SMFS) to measure and localize individual LysM-peptidoglycan interactions on both model and cellular surfaces. LysM modules of the major autolysin AcmA of Lactococcus lactis were bound to gold-coated atomic force microscopy tips, while peptidoglycan was covalently attached onto model supports. Multiple force curves recorded between the LysM tips and peptidoglycan surfaces yielded a bimodal distribution of binding forces, presumably reflecting the occurrence of one and two LysM-peptidoglycan interactions, respectively. The specificity of the measured interaction was confirmed by performing blocking experiments with free peptidoglycan. Next, the LysM tips were used to map single LysM interactions on the surfaces of L. lactis cells. Strikingly, native cells showed very poor binding, suggesting that peptidoglycan was hindered by other cell wall constituents. Consistent with this notion, treatment of the cells with trichloroacetic acid, which removes peptidoglycan-associated polymers, resulted in substantial and homogeneous binding of the LysM tip. These results provide novel insight into the binding forces of bacterial LysMs and show that SMFS is a promising tool for studying the heterologous display of proteins or peptides on bacterial surfaces.

* Corresponding author. Mailing address: Unité de Chimie des Interfaces, Université catholique de Louvain, Croix du Sud 2/18, B-1348 Louvain-la-Neuve, Belgium. Phone: (32) 10 47 36 00. Fax: (32) 10 47 2005. E-mail: yves.dufrene{at}uclouvain.be

{triangledown} Published ahead of print on 29 August 2008.

Journal of Bacteriology, November 2008, p. 7079-7086, Vol. 190, No. 21
0021-9193/08/$08.00+0     doi:10.1128/JB.00519-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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