Abstract
The metabolism of the cell surface during bacterial cell division involves synthesis and degradation of peptidoglycan (PGN), the major component of the bacterial cell wall. Bacteria have to ensure that their surface remains capable of withstanding high turgor pressures and, simultaneously, that the PGN at their surface is concealed from receptors produced by the host innate immune system. For cell separation to occur, and for PGN to be kept concealed, “old” PGN is degraded by specific PGN hydrolases, also known as autolysins, that are found at the bacterial cell surface or that are secreted into the growth medium. Bacterial PGN hydrolases are cell wall lytic enzymes that comprise a broad and diverse group of proteins. It is often difficult to assign a specific function to a PGN hydrolase mainly because an organism can have a large number of hydrolases with redundant activities and one hydrolase can have more than one enzymatic activity and participate in various cell processes (Vollmer et al. 2008). Bacillus subtilis has ca. 35 known or hypothetical PGN hydrolases, whereas Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) have, respectively, ca.16 and 19 PGN hydrolases (Vollmer, 2012; Heidrich et al., 2001; Singh et al., 2012). PGN hydrolases can be classified in three main classes: glycosidases, amidases and peptidases. Glycosidases cleave the glycan backbone and are divided into N-acetylglucosaminidases and N-acetylmuramidases. Amidases cleave the linkage between the peptide chain and the N-acetylmuramic residue of the glycan chain. Peptidases, such as endopeptidases and carboxypeptidases, are able to cleave peptide bonds between different amino acids of the PGN stem peptide. Here we describe a method to extract PGN hydrolases, which are non-covalently linked to the S. aureus cell wall (Vollmer, 2008). Analysis of extracts containing denatured PGN hydrolytic enzymes is performed by running a zymogram gel (a SDS-PAGE gel containing crude bacterial cell walls or substrate cells), which is then incubated in a non-denaturing buffer to allow renaturation of the PGN hydrolases. These renatured enzymes can then be identified through the production of clear bands that are observed where cell wall digestion has occurred. The protocol is divided into three steps: A) Preparation of the crude autolytic extracts from S. aureus cells; B) Preparation of substrate cells for gel zymograms; C) Analysis of crude autolytic extracts by gel zymography. We also show that this method can be used to determine the absence or altered activity of PGN hydrolases produced by different S. aureus mutant strains.
Keywords: Murein, Peptidoglycan, PGN hydrolases, Zymogram, Staphylococcus aureus
Materials and Reagents
Equipment
Procedure
Notes
Recipes
Acknowledgments
This protocol, which was adapted or modified from Grilo et al. (2014) and Yokoi et al. (2008), has been used to describe that autolysins can prevent detection of the PGN at the bacterial cell surface (Atilano et al., 2014). This work was supported by fellowship SFRH/BD/78748/2011 to FV and project PTDC/BIA-PLA/3432/2012 to SRF from Fundação para a Ciência e Tecnologia. We thank Inês Grilo for suggestions and Teresa Baptista da Silva for technical support.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.