High-Precision, Whole-Genome Sequencing of Laboratory Strains Facilitates Genetic Studies
A) Verification of the Solexa-identified yjbM* and ywaC* mutations by Sanger sequencing. The reference sequence for yjbM or ywaC is shown first, followed by the Solexa results for these genes in the strain relA-, in which the relA gene is deleted, and the strain relA-* which has the identical relA deletion as well as second site suppressor mutations. Chromatograms of the Sanger sequencing results for each gene are shown last. B) Partial suppression of the relA- growth defect by introduction of either the yjbM* or ywaC* allele from relA-*. The strains shown in clockwise order are: i) JH642 (wt), ii) relA- with the yjbM* mutation from relA-* (relA-yjbM*), iii) relA- with the ywaC* mutation from relA-* (relA- ywaC*), iv) relA- with both suppressor mutations yjbM* and ywaC* (relA-*), v) yjbM* (in the JH642 background), vi) ywaC* (in the JH642 background) and vii) relA- (in the JH642 background, with no suppressors). The strains were plated on LB. relA- has a significant growth defect, which is partially relieved by introduction of either yjbM* or ywaC*, and is fully rescued by both mutations together. C) Re-introduction of a growth defect in the relA-* suppressor strain by expression of either yjbM or ywaC. The strains shown in clockwise order are: i) JH642 (wt), ii) relA-*, with suppressor mutations in both yjbM and ywaC, expressing wild type yjbM (relA- p_yjbM), iii) relA-* expressing wild type ywaC (relA-* p_ywaC), iv) relA- with suppressor mutations in yjbM and ywaC (relA-*), v) JH642 with ectopic expression of wild type yjbM (p_yjbM), vi) JH642 with ectopic expression of wild type ywaC (p_ywaC), and vii) relA- (in the JH642 background, with no suppressors). Full length wild type yjbM and ywaC were expressed from the IPTG-inducible pHyperSpac promoter and strains were plated on LB-IPTG. Expression of either yjbM or ywaC abolishes the effect of the suppressors in relA-*, and introduces a significant growth defect that is comparable to relA-.