Construction of penP delta 1, Bacillus licheniformis 749/C beta-lactamase lacking site for lipoprotein modification. Expression in Escherichia coli and Bacillus subtilis.
Membrane-bound penicillinases in Gram-positive bacteria are glyceride-cysteine lipoproteins (Nielsen, J. B. K., and Lampen, J. O. (1982) J. Biol. Chem. 257, 4490-4495) that can be, but are not necessarily, intermediates in formation of exocellular enzymes. We have now deleted from the signal region of the Bacillus licheniformis 749/C beta-lactamase gene (penP) 15 base pairs that code for Ala-Leu-Ala-Gly-Cys. This sequence includes the Cys residue that undergoes lipophilic modification and the site of cleavage by signal peptidase and is well conserved in diverse prokaryotic lipoproteins. In the deletion gene, penP delta 1, the remaining Cys is preceded by 8 hydrophobic residues instead of 14 for the original modification site. PenP delta 1 has been cloned in Escherichia coli and Bacillus subtilis and its expression and products compared with penP. Penicillinase synthesis by penP delta 1 clones was greater than or equal to amounts formed by penP clones or B. licheniformis. Lipoprotein production from penP delta 1 was very low in either host and appears physiologically insignificant. In E. coli carrying penP delta 1, 25% of the penicillinase was released into the periplasm as a processed form. The remainder was a membrane-associated form of translation product size and was oriented to the periplasm. In mid-log cultures of B. subtilis carrying penP delta 1, the translation product and 2 protease-shortened species were present both in the cytoplasm and on the outer surface of the membrane. Release began at stationary phase and was dependent on the presence of enzyme processing to exo-small and a pH value greater than 7.5. We conclude that the shortened lipophilic sequence of the penP delta 1 prepenicillinase is adequate for transfer of the nascent chain through the membrane.
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005786:Gene Expression Regulation; D008074:Lipoproteins / Q000378:metabolism*; D010405:Penicillinase / Q000096:biosynthesis; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D008074:Lipoproteins; D010405:Penicillinase; D001618:beta-Lactamases