Characterization of Listeria monocytogenes protein Lmo0327 with murein hydrolase activity.
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.
Popowska
Magdalena
M
Department of General Microbiology, Institute of Microbiology, Warsaw University, Miecznikowa 1, 02-096 Warsaw, Poland. magdapop@biol.uw.edu.pl
Markiewicz
Zdzislaw
Z
D000595:Amino Acid Sequence; D001483:Base Sequence; D002473:Cell Wall / Q000201:enzymology; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D008089:Listeria monocytogenes / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D050436:Regulatory Elements, Transcriptional; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D009238:N-Acetylmuramoyl-L-alanine Amidase