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Description
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Fig. 1. Construction of suicide plasmids. (a) Arrangement of the pnpA, intB and orf379 genes in Dichelobacter nodosus strain 2483. The positions of thePH, C (core),KH and S1 domains of PNPase areindicated above the pnpA gene. E indicates the position of EcoRI sites. Plasmid pCF7 was constructed using primers BCH54 and 56 (Table 1) to amplify from the virulent D. nodosus strain A198 the segment of the pnpA gene from nt 518 to 891 (hatched) and primers BCH44 and 45 to amplify the segment from nt 1729 to 1988 (grey). These two segments of pnpA were cloned into pUC18, and the tetM cassette from plasmid pJIR1532 (Kennan et al., 1998) was introduced between them. Plasmid pCF5 was constructed in a similar manner, except that primers BCH42 and 43 were used instead of primers BCH54 and 56, so that pCF5 contains the segment of the pnpA gene from nt 1312 to 1718 (dotted). (b) Insertion of tetM into pnpA using pCF5 in strains 2483D1, D2 and D3. (c) Reconstruction of pnpA using pSK81 in strains 2483D1R, 2483D2R1 and 2483D2R2. Plasmid pSK81 was constructed using primers BCH337 and 338 to amplify from strain 2483 a segment containing nt 1198–2897 (bricks) and primers BCH339 and 340 to amplify a segment from nt 3091 to 3642 (checked). These were cloned into pUC18 using restriction sites and the kanR gene from pJIR537 (supplied by J. Rood, Monash University, Australia) was introduced between them. (d) Suicide plasmids pCF7, pCF5 and pSK81. |
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