Cloning and sequence analysis of the muramidase-2 gene from Enterococcus hirae.
Extracellular muramidase-2 of Enterococcus hirae ATCC 9790 was purified to homogeneity by substrate binding, guanidine-HCl extraction, and reversed-phase chromatography. A monoclonal antibody, 2F8, which specifically recognizes muramidase-2, was used to screen a genomic library of E. hirae ATCC 9790 DNA in bacteriophage lambda gt11. A positive phage clone containing a 4.5-kb DNA insert was isolated and analyzed. The EcoRI-digested 4.5-kb fragment was cut into 2.3-, 1.0-, and 1.5-kb pieces by using restriction enzymes KpnI, Sau3AI, and PstI, and each fragment was subcloned into plasmid pJDC9 or pUC19. The nucleotide sequence of each subclone was determined. The sequence data indicated an open reading frame encoding a polypeptide of 666 amino acid residues, with a calculated molecular mass of 70,678 Da. The first 24 N-terminal amino acids of purified extracellular muramidase-2 were in very good agreement with the deduced amino acid sequence after a 49-amino-acid putative signal sequence. Analysis of the deduced amino acid sequence showed the presence at the C-terminal region of the protein of six highly homologous repeat units separated by nonhomologous intervening sequences that are highly enriched in serine and threonine. The overall sequence showed a high degree of homology with a recently cloned Streptococcus faecalis autolysin.
Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
C P
CP
Chu
R
R
Kariyama
L
L
Daneo-Moore
G D
GD
Shockman
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D016983:Enterococcus / Q000235:genetics*; D013293:Enterococcus faecalis / Q000235:genetics; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics* / Q000378:metabolism; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid
D009113:Muramidase; D009238:N-Acetylmuramoyl-L-alanine Amidase