Genetic and biochemical characterization of the Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H lysin.
LL-H, a virulent phage of Lactobacillus delbrueckii subsp. lactis, produces a peptidoglycan-degrading enzyme, Mur, that is effective on L. delbrueckii, Lactobacillus acidophilus, Lactobacillus helveticus, and Pediococcus damnosus cell walls. In this study, the LL-H gene mur was cloned into Escherichia coli, its nucleotide sequence was determined, and the enzyme produced in E. coli was purified and biochemically characterized. Mur was purified 112-fold by means of ammonium sulfate precipitation and cation-exchange chromatography. The cell wall-hydrolyzing activity was found to be associated with a 34-kDa protein. The C-terminal domain of Mur is not essential for catalytic activity since it can be removed without destroying the lytic activity. The N-terminal sequence of the purified lysin was identical to that deduced from the nucleotide sequence, but the first methionine is absent from the mature protein. The N-terminal part of this 297-amino-acid protein had homology with several Chalaropsis-type lysozymes. Reduction of purified and Mur-digested L. delbrueckii cell wall material with labeled NaB3H4 indicated that the enzyme is a muramidase. The temperature optimum of purified Mur is between 30 and 40 degrees C, and the pH optimum is around 5.0. The LL-H lysin Mur is stable at temperatures below 60 degrees C.
Vasala
A
A
Department of Genetics, University of Oulu, Finland.
Välkkilä
M
M
Caldentey
J
J
Alatossava
T
T
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics* / Q000472:pathogenicity; D001483:Base Sequence; D002473:Cell Wall / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D005814:Genes, Viral; D007778:Lactobacillus / Q000821:virology*; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014764:Viral Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D004279:DNA, Viral; D014764:Viral Proteins; D009113:Muramidase