Complete nucleotide sequence of the bacteriophage K1F tail gene encoding endo-N-acylneuraminidase (endo-N) and comparison to an endo-N homolog in bacteriophage PK1E.
Endo-N-acylneuraminidase (endo-N) is a phage-encoded depolymerase that degrades the alpha (2-8)-linked polysialic acid chains of K1 serotypes of Escherichia coli and vertebrate neural cell adhesion molecules. We have determined the DNA sequence of the bacteriophage K1F tail protein structural gene, which codes for a polypeptide of 920 residues. Purification of the tail protein yields a 102-kDa species upon denaturing gel electrophoresis and detection by Western immunoblot analysis. An identical polypeptide was detected by Western blot analysis of K1F virions. Peptide sequencing confirmed that the open reading frame determined by nucleotide sequencing encodes endo-N. Immunoelectron microscopy with neutralizing antibodies raised against the depolymerase confirmed that endo-N is a component of the K1F tail apparatus. Antibodies in the serum cross-reacted with endo-N from another K1-specific phage, PK1E, demonstrating the presence of shared epitopes. Homology between K1F and PK1E endo-N was confirmed by Southern, Northern (RNA), and Western blot analyses. The endo-N amino-terminal domain is homologous to the amino termini of phage T7 and T3 tail proteins, indicating by analogy that this domain functions in attachment of endo-N to the K1F virion's head. A central domain of 495 residues has weak similarity to sea urchin aryl sulfatase, suggesting that this region may contain the endo-N catalytic site. Failure to detect homology between the PK1E homolog and the carboxy-terminal domain of K1F endo-N is consistent with the central domain's involvement in binding and catalysis of polysialic acid. These results provide the initial molecular and genetic description of polysialic acid depolymerase, which has so far been detected only in K1-specific phage.
Petter
J G
JG
Department of Veterinary Pathobiology, University of Illinois, Urbana-Champaign 61801.
Vimr
E R
ER
D000595:Amino Acid Sequence; D001483:Base Sequence; D003090:Coliphages / Q000201:enzymology* / Q000235:genetics* / Q000648:ultrastructure; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005798:Genes, Bacterial; D007700:Kinetics; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; C035355:endo-N-acetylneuraminidase; D009439:Neuraminidase