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Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes
Published on: September 19, 2013
Characterization of Listeria monocytogenes protein Lmo0327 with murein hydrolase activity
Magdalena Popowska1, Zdzislaw Markiewicz
1Department of General Microbiology, Institute of Microbiology, Warsaw University, Miecznikowa 1, 02-096 Warsaw, Poland. magdapop@biol.uw.edu.pl
Abstract:
Listeria monocytogenes is an ubiquitous gram-positive, opportunistic food-borne human and animal pathogen. To date, five L. monocytogenes autolysins have been characterized: p60, p45, Ami, MurA and Auto and the preliminary results of our studies show that FlaA, a flagellar protein of L. monocytogenes, also has murein-degrading activity. In this study, a gene coding a 144 kDa protein (Lmo0327) with murein hydrolase activity was identified from a lambda Zap expression library of L. monocytogenes EGD genomic DNA, using a direct screening protocol involving the plating of infected Escherichia coli XL1-blue MRF' cells onto medium containing Bacillus subtilis murein, a substrate for autolytic proteins. Protein Lmo0327 has a signal sequence, a N-terminal LRR domain and a C-terminal wall-anchoring LPXTG motif. In order to examine the roles of this enzyme and the putative transcription regulator coded by gene lmo0326 located upstream of lmo0327, both structural genes were insertionally inactivated by site-specific integration of a temperature-sensitive plasmid. We show that Lmo0327 is a surface protein covalently linked to murein and that the putative transcription regulator Lmo0326 can be assumed to positively regulate the expression of gene lmo0327. The enzyme, which we have shown to have murein-hydrolysing activity, plays a role in cell separation and murein turnover.
Insights
This study identifies a novel murein hydrolase, Lmo0327, in Listeria monocytogenes. This surface protein, regulated by Lmo0326, is crucial for bacterial cell separation and murein turnover.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Enzymology
Background:
- Listeria monocytogenes is a significant food-borne pathogen.
- Autolysins play key roles in bacterial cell wall metabolism.
- Previous studies identified several L. monocytogenes autolysins, with preliminary data suggesting flagellar protein FlaA also possesses murein-degrading activity.
Purpose of the Study:
- To identify and characterize novel murein hydrolases in Listeria monocytogenes.
- To investigate the function of the identified enzyme and its regulatory elements.
Main Methods:
- Screening a L. monocytogenes genomic DNA library using Bacillus subtilis murein as a substrate.
- Identification and characterization of the murein hydrolase gene (lmo0327) and its upstream regulator (lmo0326).
- Gene inactivation studies using site-specific integration of a temperature-sensitive plasmid.
Main Results:
- A 144 kDa protein, Lmo0327, with murein hydrolase activity was identified.
- Lmo0327 is a surface protein covalently linked to the murein.
- The upstream gene lmo0326 encodes a putative transcription regulator that positively regulates lmo0327 expression.
- Lmo0327 is involved in cell separation and murein turnover.
Conclusions:
- Lmo0327 is a novel, surface-anchored murein hydrolase in Listeria monocytogenes.
- The expression of Lmo0327 is positively regulated by the putative transcription regulator Lmo0326.
- This enzyme plays a significant role in Listeria monocytogenes cell division and peptidoglycan remodeling.
