Cloning and expression of a gene encoding Moraxella sp. CK-1 autolysin.
A gene of Moraxella sp. CK-1 encoding cell wall lytic activity has been cloned and expressed in E. coli. A genomic library of Moraxella sp. CK-1 was constructed in the multifunctional phagemid vector pT7T3 19u, with partial Sau3A digests of Moraxella sp. CK-1 genomic DNA inserted at the BamHI restriction site. Screening of about 5,000 transformants for cell lysis activity in LB agar plates containing Micrococcus Iuteus cells gave one positive clone harboring the 3.7 kb insert (pMXA282). Restriction mapping and deletion analysis of the recombinant plasmid carrying a 3.7 kb insert suggested that the autolysin gene was located within a 1.1 kb BamHI-PstI fragment. Analysis of extracts of E. coli clone harboring recombinant plasmids on renaturing SDS-polyacrylamide gels containing heat-killed Micrococcus luteus cells showed a clear zone around a polypeptide of about 32 kDa. Lytic activity against Micrococcus luteus cell walls by the cloned autolysin was maximum at pH 9.0. Even in conditions of over pH 10.0, this cloned autolysin showed a vigrous lytic activity. Southern blot analysis suggested the existence of other homologous regions in Moraxella sp. CK-1 genome.
Ohn
T B
TB
Department of Biology, Hanyang University, Seoul, Korea.
Kim
C H
CH
Min
B R
BR
Choi
Y K
YK
D001433:Bacteriolysis / Q000187:drug effects; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D015698:Genomic Library; D006863:Hydrogen-Ion Concentration; D016982:Micrococcus luteus / Q000187:drug effects; D009016:Moraxella / Q000201:enzymology* / Q000235:genetics*; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology
D009238:N-Acetylmuramoyl-L-alanine Amidase