Effect of in vitro DNA Rearrangement in the NH2-terminal Region of the Penicillinase Gene from Bacillus licheniformis on the Mode of Expression in Bacillus subtilis
Summary: We have constructed secretion vector plasmids that have unique Bg/II sites within or near the signal sequence of Bacillus licheniformis penicillinase, and have also constructed penicillinase cartridges that lack either one, two or three of the processing sites for the membrane-bound, exolarge and exo-small enzymes. Each of these penicillinase cartridges was cloned on secretion vectors in Bacillus subtilis, and enzyme production was examined. The presence of both the signal sequence and the three host-specific processing sites on the secretion vector was required for an effective expression of the enzyme in B. subtilis. The presence of any of the processing sites on the cartridge reduced the accumulation of penicillinase in the culture medium. When a vector plasmid lacking part of the hydrophobic region of the signal sequence and lacking the three processing sites was used, total penicillinase production decreased and enzyme accumulation in the medium was extremely low, despite the complete or incomplete presence of the processing sites on the cartridge. Molecular mass determination of these extracellular penicillinases suggested the existence of a new cleavage site for the enzyme.
BrammerW. J.,
MuirS.,
McMorrisA.1980; Molecular cloning of the gene for the β-lactamase of Bacillus licheniformis and its expression in Escherichia coli
. Molecular and General Genetics 178:217–224
ChangC. N.,
NielsenJ. B. K.,
IzuiK.,
BlobelG.,
LampenJ. O.1982; Identification of the signal peptidase cleavage site in Bacillus licheniformis prepenicillinase. Journal of Biological Chemistry 257:4340–4344
EmrS. D.,
SilhavyT. J.1983; Importance of secondary structure in the signal sequence for protein secretion. Proceedings of the National Academy of Sciences of the United States of America 80:4599–4603
FujiiM.,
ImanakaT.,
AibaS.1982; Molecular cloning and expression of penicillinase genes from Bacillus licheniformis in the thermophile Bacillus stearothermophilus
. Journal of General Microbiology 128:2997–3000
GrayO.,
ChangS.1981; Molecular cloning and expression of Bacillus licheniformis β-lactamase gene in Escherichia coli and Bacillus subtilis
. Journal of Bacteriology 145:422–428
ImanakaT.,
TsunekawaH.,
AibaS.1980; Phenotypic stability of trp operon recombinant plasmids in Escherichia coli
. Journal of General Microbiology 118:253–261
ImanakaT.,
FujiiM.,
AibaS.1981a; Isolation and characterization of antibiotic resistance plasmids from thermophilic bacilli and construction of deletion plasmids. Journal of Bacteriology 146:1091–1097
ImanakaT.,
TanakaT.,
TsunekawaH.,
AibaS.1981b; Cloning of the genes for penicillinase, penP and penI, of Bacillus licheniformis in some vector plasmids and their expression in Escherichia coli, Bacillus subtilis, and Bacillus licheniformis
. Journal of Bacteriology 147:776–786
ImanakaT.,
OshiharaW.,
HimenoT.,
AibaS.1983; Comparative studies on extracellular penicillinases of the same structural gene, penP, expressed in Bacillus licheniformis and Bacillus subtilis
. Journal of General Microbiology 129:2621–2628
ImanakaT.,
AnoT.,
FujiiM.,
AibaS.1984; Two replication determinants of an antibiotic-resistance plasmid, pTB19, from a thermophilic bacillus. Journal of General Microbiology 130:1399–1408
InouyeS.,
SoberonX.,
FranceschiniT.,
NakamuraK.,
ItakuraK.,
InouyeM.1982; Role of positive charge on the amino-terminal region of the signal peptide in protein secretion across the membrane. Proceedings of the National Academy of Sciences of the United States of America 79:3438–3441
IzuiK.,
NielsenJ. B. K.,
CaulfieldM. P.,
LampenJ. O.1980; Large exopenicillinase: initial extracellular form detected in cultures of Bacillus licheniformis
. Biochemistry 19:1882–1886
ManiatisT.,
FritschE. F.,
SambrookJ.1982; In Molecular Cloning, a Laboratory Manual,. pp 86–96136–139173–185 Cold Spring Harbor, New York: Cold Spring Harbor Laboratory;
MézesP. S. F.,
WangW.,
YehE. C. H.,
LampenJ. O.1983; Construction of penPᐃ1, Bacillus licheniformis 749/C β-lactamase lacking site for lipoprotein modification. Journal of Biological Chemistry 258:11211–11218
NeugebauerK.,
SprengelR.,
SchallerH.1981; Penicillinase from Bacillus licheniformis: nucleotide sequence of the gene and implications for the biosynthesis of a secretory protein in a Gram-positive bacterium. Nucleic Acids Research 9:2577–2588
NielsenJ. B. K.,
CaulfieldM. P.,
LampenJ. O.1981; Lipoprotein nature of Bacillus licheniformis membrane penicillinase. Proceedings of the National Academy of Sciences of the United States of America 78:3511–3515
PerlmanD.,
HalvorsonH. O.1983; A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides. Journal of Molecular Biology 167:391–409
SmithW. P.,
TaiP. C.,
DavisB. D.1981; Bacillus licheniformis penicillinase: cleavages and attachment of lipid during cotranslational secretion. Proceedings of the National Academy of Sciences of the United States of America 78:3501–3505
TalmadgeK.,
StahlS.,
GilbertW.1980; Eukaryotic signal sequence transports insulin antigen in Escherichia coli
. Proceedings of the National Academy of Sciences of the United States of America 77:3369–3373
Effect of in vitro DNA Rearrangement in the NH2-terminal Region of the Penicillinase Gene from Bacillus licheniformis on the Mode of Expression in Bacillus subtilis