Identification of suppressor mutantsĀ
To screen for spontaneous mutations, the lpdA mutant was streaked
from frozen stocks on TSA plate supplemented with a mixture of the
branched-chain carboxylic acids a C5, i C5, and i C4.
Independent colonies were then grown overnight in borosilicate glass
tubes containing TSB supplemented with the mixture of the branched-chain
carboxylic acids. A double-back dilution was performed in unsupplemented
medium, and once cells reached an OD600 of 0.1 (growth
arrest) they were serial diluted, plated on TSA without supplementation,
and incubated at 37oC. Colonies (one per liquid
culture) were picked, restreaked, and growth in the absence of
carboxylic acids was confirmed. Following confirmation, gDNAs were
purified and submitted for whole genome sequencing as a fee for service
at SeqCenter. Analysis of spontaneous mutations was performed using
BRESEQ software (Barrick et al., 2009).