Isolation and characterization of genetic expression and secretion signals from Enterococcus faecalis through the use of broad-host-range alpha-amylase probe vectors.
We have constructed two broad-host-range Gram+/Gram- probe vectors designed for the cloning of bacterial genetic expression and secretion signals. These vectors make use of a silent reporter gene encoding the mature alpha-amylase from Bacillus licheniformis whose reactivation can easily be monitored on iodine-stained starch plates. Shotgun cloning of Enterococcus faecalis DNA fragments allowed recovery of several cassettes directing transcription, translation of the reporter gene and secretion of alpha-amylase. Sequence analysis revealed, in each case, the presence of a putative promoter, ribosome-binding site and signal peptide similar to those described in other Gram+ bacteria.
Hols
P
P
Unité de Génétique, Université Catholique de Louvain, Louvain-La-Neuve, Belgium.
Baulard
A
A
Garmyn
D
D
Delplace
B
B
Hogan
S
S
Delcour
J
J
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular / Q000379:methods*; D013293:Enterococcus faecalis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics; D014176:Protein Biosynthesis; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000516:alpha-Amylases