Development of in vitro transposon assisted signal sequence trapping and its use in screening Bacillus halodurans C125 and Sulfolobus solfataricus P2 gene libraries Fiona Becker a, * , Kirk Schnorr a , Reinhard Wilting a , Niels Tolstrup b , Jannick Dyrløv Bendtsen c , Peter Bjarke Olsen a a Novozymes A/S, Krogshoejvej 36, 2880 Bagsværd, Denmark b Exiqon, Bygstubben 9, 2950 Vedbaek, Denmark c Center for Biological Sequence Analysis, The Technical University of Denmark, 2800 Lyngby, Denmark Received 3 December 2003; received in revised form 5 December 2003; accepted 6 December 2003 Abstract To identify genes encoding extracytosolic proteins, a minitransposon, TnSig, containing a signal-less h-lactamase (V bla) as reporter gene, was constructed and used for in vitro transposition of genomic libraries made in Escherichia coli. The V bla gene was cloned into a bacteriophage Mu minitransposon enabling translational fusions between V 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 BA000004 and EMBL AE006641), 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. D 2004 Elsevier B.V. All rights reserved. Keywords: MuA transposon; Library screening; Signal trapping; Secreted; Industrial enzymes 1. Introduction The discovery of enzymes for industrial applica- tions traditionally involves screening donor isolates on specific substrates under the desired application conditions. Once an enzyme activity is found the corresponding gene is cloned, sequenced and investi- gated for novelty. Recent advances in sequencing 0167-7012/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2003.12.002 Abbreviations: aa, amino acids(s); Ap, ampicillin; hLac, beta- lactamase; B., Bacillus; bp, base pair; CAT, chloramphenicol acetyl transferase; Cm, chloramphenicol; E., Escherichia; Km, Kanamy- cin; LSP, lipoprotein signal peptide; ORF, open reading frame; RBS, ribosome binding site; SD, Shine-Dalgarno sequence; SP(s), signal peptide(s), signal sequence or secretion signal; Tn, transposon. * Corresponding author. Tel.: +45-44426640; fax: +45- 44427828. E-mail address: fiod@novozymes.com (F. Becker). www.elsevier.com/locate/jmicmeth Journal of Microbiological Methods 57 (2004) 123 – 133