Development of in vitro transposon assisted signal sequence trapping and its use in screening Bacillus halodurans C125 and Sulfolobus solfataricus P2 gene libraries.
To identify genes encoding extracytosolic proteins, a minitransposon, TnSig, containing a signal-less beta-lactamase ('bla) as reporter gene, was constructed and used for in vitro transposition of genomic libraries made in Escherichia coli. The 'bla gene was cloned into a bacteriophage Mu minitransposon enabling translational fusions between 'bla and target genes. Fusion of TnSig in the correct reading frame to a protein carrying transmembrane domains or signal peptides resulted in ampicillin resistance of the corresponding clone. Prokaryotic gene libraries from the alkaliphilic bacterium Bacillus halodurans C125 and the hyperthermophilic archaeon Sulfolobus solfataricus P2 were tagged with TnSig. The genomic sequences, which are publicly available (EMBL and EMBL ), were used for rapid open reading frame (ORF) identification and prediction of protein localisation in the cell. Genes for secreted proteins, transmembrane proteins and lipoproteins were successfully identified by this method. In contrast to previous transposon based identification strategies, the method described here is fast and versatile and essentially enables any selectable marker compatible library to be tagged. It is suited for identifying genes encoding extracytosolic proteins in gene libraries of a wide range of prokaryotic organisms.
Novozymes A/S, Krogshoejvej 36, 2880 Bagsvaerd, Denmark. fiod@novozymes.com
Fiona
F
Becker
Kirk
K
Schnorr
Reinhard
R
Wilting
Niels
N
Tolstrup
Jannick Dyrløv
JD
Bendtsen
Peter Bjarke
PB
Olsen
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics; D010583:Bacteriophage mu / Q000235:genetics; D001483:Base Sequence; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D015723:Gene Library; D019766:Genes, Archaeal / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D006026:Glycoside Hydrolases / Q000235:genetics; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics; D016944:Sulfolobus / Q000201:enzymology / Q000235:genetics*
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D006026:Glycoside Hydrolases; C100181:pullulanase