Controlled expression and structural organization of a Lactococcus lactis bacteriophage lysin encoded by two overlapping genes.
The phi vML3 bacteriophage lysin is specific for lactococci and could be used to promote enzyme release during cheese manufacture. The level of lysin expression from the cloned gene using its own upstream sequences is very low. Expression in Escherichia coli by using a synthetic hybrid lysin gene and a series of BAL 31 deletions of the original cloned DNA fragment suggested that the start of the gene had previously been incorrectly assigned. Reevaluation of homology between the lysin and Bacillus subtilis PZA protein 15 led to the identification of a new potential ribosome binding site (RBS). A 0.72-kb PCR-generated fragment including this RBS and the complete lysin gene was expressed and inducibly controlled. The translational start of the lysin gene was identified as an isoleucine codon, and this may lead to a low translation rate. During the analysis of the BAL 31 deletion fragments, two proteins of 20 and 8 kDa were shown to be expressed from the originally defined lysin gene. The DNA sequence has a second open reading frame with a good RBS and two potential start methionines. The smaller lysin protein was isolated, and the N terminus was sequenced, confirming that one methionine codon acted as the start of a second gene. The larger lysin protein has homology with lysozymes. The smaller lysin protein has some features resembling those of a holin. The possible roles of these two proteins in lysis of lactococci are discussed.
Shearman
C A
CA
Institute of Food Research, Norwich, United Kingdom.
Jury
K L
KL
Gasson
M J
MJ
D000595:Amino Acid Sequence; D001433:Bacteriolysis / Q000235:genetics; D001435:Bacteriophages / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004798:Enzymes / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005807:Genes, Overlapping; D005814:Genes, Viral; D013294:Lactococcus lactis / Q000821:virology*; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D015678:Viral Structural Proteins / Q000235:genetics
D017931:DNA Primers; D004279:DNA, Viral; D004798:Enzymes; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; D009113:Muramidase