Fig. 1

Plasmids used in this study. All plasmids used in this study are based on the pMTL60000 modular shuttle vector series (A), defined by four modules (Gram-positive replicon (1), AscI/FseI, selectable marker (2), FseI/PmeI, gram-negative replicon (3), PmeI/SbfI, application-specific module (4), SbfI/AscI), with the corresponding components being S. aureus pUB110 replicon, thermostable kanamycin adenyltransferase gene (KanHT) conferring kanamycin resistance, ColEI origin of replication and the oriT-traJ mobilisation region, and LacZα MCS. The 5′ incompatible region of RepB (5′ IR) is reduced from 412 bp (a) to 362 bp (b) and to 189 bp (c) to give three variants of pMTL61110 (4809 bp), pMTL62110 (4591 bp) and pMTL63110 (4418 bp), respectively. Plasmids for pyrE-based allele-coupled exchange (ACE) are all derived from pMTL62110 (B). Insertion of a 299-bp internal pyrE sequence between SbfI and NotI (LHA) and a 1200-bp pyrE downstream sequence between HindIII and AscI gives the pyrE knockout vector pMTL-LS1. Insertion of a 1838-bp sequence consisting of the entire pyrE sequence lacking only the start codon and the same downstream RHA between SbfI and AscI gives the pyrE repair vector pMTL-LS2. Replacement of the LHA of pMTL-LS1 with the entire pyrE gene lacking only the start codon (639 bp) gives the pyrE integration vector, pMTL-LS3. DNA to be integrated chromosomally is inserted at the multiple cloning site. Fusion of KanHT with a heterologous pyrE (G. kaustophilus; G. thermoleovorans) gives deletion vectors pMTL-LS5 and pMTL-LS6. Homology arms of the desired knockout genes are cloned at the MCS