Characterization of the pneumococcal bacteriophage HB-3 amidase: cloning and expression in Escherichia coli.
HB-3, a temperate bacteriophage of Streptococcus pneumoniae, synthesizes its own murein hydrolase activity when multiplying on cultures of pneumococcus. The enzyme (HBL) was purified and biochemically characterized as an N-acetylmuramoyl-L-alanine amidase of 36,000 daltons, and a 2.1-kilobase DraI fragment containing the lysin gene (hbl) was cloned and expressed in Escherichia coli. Our results demonstrated that the primary product of the hbl gene is a form with low enzyme activity that can be converted to a more active form under conditions similar to those previously described for the major pneumococcal autolysin (E. García, J.L. García, C. Ronda, P. García, and R. López, Mol. Gen. Genet. 201:225-230, 1985). The phage-encoded amidase requires the presence of choline in the teichoic acids of the pneumococcal cell walls for in vivo and in vitro activity. Comparative biochemical and immunological tests of the phage-encoded and host amidases revealed a remarkable similarity between these enzymes, although analyses of their N-terminal amino acid sequences allowed us to conclude that the amidases are similar but not identical. This appears to be the first description of the cloning of a phage-encoded amidase in gram-positive bacteria.
Romero
A
A
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Lopez
R
R
Garcia
P
P
D000581:Amidohydrolases / Q000235:genetics*; D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005814:Genes, Viral; D007700:Kinetics; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid; D013296:Streptococcus pneumoniae / Q000201:enzymology / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics*
D004279:DNA, Viral; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; D000581:Amidohydrolases; D009238:N-Acetylmuramoyl-L-alanine Amidase