Reconstruction and expression of the autolytic gene from Clostridium acetobutylicum ATCC 824 in Escherichia coli.
The complete lyc gene encoding the autolytic lysozyme of Clostridium acetobutylicum ATCC 824 was reconstructed from two overlapping DNA fragments and cloned into a suitable plasmid enabling Escherichia coli to produce this lytic enzyme under the control of the lac promoter. A polypeptide with an apparent M(r) of 35,000, corresponding to that predicted from the nucleotide sequence, was observed by maxicell analysis of whole-cell extracts of E. coli harboring the clostridial gene. The enzyme yield was shown to depend on the pH of the culture medium, since the protein was unstable at alkaline pH. The expression of the lyc gene was not increased by using the E. coli strong promoter, lpp-lac, probably due to the limit imposed by the extreme differences in codon usage. Although the LYC lysozyme does not contain a cleavable signal peptide, most of the protein was found in the periplasmic fraction of E. coli suggesting that this enzyme was secreted through a specific mechanism, as already observed for other autolysins.
Département de Génie Biochimique et Alimentaire, Institut National des Sciences Appliquées, Toulouse, France.
C
C
Croux
J L
JL
García
D001433:Bacteriolysis; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology / Q000235:genetics*; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids
D009238:N-Acetylmuramoyl-L-alanine Amidase