Aronson, A. be produced in large quantity. A surface display system using CotB, a component of the spore coat, has been developed and used for producing heterologous antigens (14). During sporulation, cells form protein crystals of insecticidal protein toxins (the delta-endotoxins), which have been used as biological pesticides. Many studies have shown that the 130-kDa protoxin from the Cry1Ac subgroup is also a major component of the spore coat. Protein extracts from spores react with antiprotoxin antibody and have insecticidal activity like that of the crystal protein (9). An electron microscopy study showed that the protoxin exists in the spore coat layer (23) but only in strains (which produce the toxins), not in mutant strains, which have lost the toxin-encoding plasmids and do not produce the crystals (3). We previously proposed a model in which the N-terminal business end of the protoxin is exposed on the spore surface and CH-223191 the C-terminal region anchors the protoxin inside the spore coat (10). Plasmids. The toxin sporulation-specific promoter region from strain HD73 (GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”M11068″,”term_id”:”142721″M11068; hereinafter, nucleotide numbers refer to this sequence) has been determined (1). It contains two individual promoter sites, Bt1 and Bt2. A 241-bp NsiI fragment containing both promoters was inserted into the PstI site of CH-223191 the shuttle vector pHT3101 (16). The resulting plasmid, pCD2, also encodes the first 11 amino acids of Cry1Ac toxin (Fig. ?(Fig.1A).1A). Various C-terminal regions of the protoxin were amplified by PCR, fused with full-length green fluorescent protein (GFP) from plasmid pGreen Lantern-1 (Invitrogen), and inserted in frame into pCD2. The following individual constructs, shown in Fig. ?Fig.1C,1C, contain the indicated protoxin CH-223191 C-terminal regions: (i) Bt3, GFP alone, no fusion partner; (ii) Bt5, nucleotides (nt) 2202 to 3217; (iii) Bt6, nt 2565 to 2980; (iv) Bt7, nt 3217 to 3925 (stop codon); (v) Bt8, nt 2278 to 3925; (vi) Bt9, nt 2278 to 2980; (vii) Bt10, nt 2278 to 3685; and (viii) Bt12, nt 2566 to 3685. Detailed maps for these constructs are available on request. Plasmid pCD4 was made by inserting the fragment of nt 2565 to 2980 (415 bp) into the KpnI and EcoRI sites of pCD2 (Fig. ?(Fig.1B1B). Open in a separate window FIG. 1. Display of GFP on the spore surface. (A) Vector pCD2 (6.6 kb) was derived from pHT3101 by inserting the toxin promoter region into CH-223191 the PstI site. This plasmid can grow in both (with ampicillin selection [Amp-R]) and (with erythromycin selection [Ery-R]). The inserted promoters (P) are sporulation specific. (B) pCD4, derived from pCD2, is a spore surface display vector. A 415-bp fragment from the gene was inserted downstream of multiple cloning sites. Fusion proteins can be displayed on the spore surface anchored by the portion derived from the protoxin. (C) GFP fusion proteins are shown schematically. The approximate nucleotide numbers and restriction sites are shown. The positions of cysteine residues are also shown. (D) Expression of various GFP fusion proteins on the surfaces of spores. The white arrows indicate mother cells. Electroporation. electroporation was performed as described by Macaluso and Mettus (17). mutant strains were grown in brain heart infusion medium plus 0.5% glycerol overnight at 30C. The culture was diluted 1:20 into prewarmed fresh medium and grown for 1 h. The culture was pelleted and washed twice with ice-cold electroporation buffer (0.625 M sucrose, 1 mM MgCl2). The cells were resuspended in electroporation buffer at half the volume of the original culture. Eight-tenths milliliter of cells and 2 l of plasmid (about 0.1 to 0.2 g DNA, purified with a QIAGEN Qiaprep mini column) were mixed in a 4-mm cuvette and incubated on ice for Hes2 5 min. Electroporation was carried out in a Bio-Rad GenePulse II with settings of 1 1.3 kV, 25 F, and 50 . The cells were then mixed with 1.6 ml of brain heart infusion medium plus 0.5% glycerol and incubated at 30C for 1 h, and 5 to 100 l was plated on LB plates.