To identify genes downstream of carB that are involved in phenotypic switching, the authors used a transposon mutagenesis screen and found that disruption of the genes involved in the biosynthesis of capsular colanic acid-like polymer (CAP) blocked colony switching, consistent with previous work that had linked colony switching in carB mutants to their capsulation state. Transcriptional analysis revealed that expression of the CAP biosynthetic locus was substantially higher in capsulated cells than in non-capsulated cells. These data suggest that the phenotypic switch occurs through bistable CAP expression, which is at least partly mediated at the transcriptional level.
To learn more about how the carB mutation influences CAP expression, the authors examined the pyrimidine biosynthesis pathway as it provides a molecular link between carB and CAP. carB encodes a subunit of the carbamoyl phosphate synthetase enzyme that catalyses a step at the start of the pyrimidine biosynthesis pathway, which concludes with the synthesis of UTP. UTP is itself a precursor for two pathways: DNA synthesis and CAP biosynthesis. The authors reasoned that bistable regulation could be mediated by the natural bifurcation point that exists when UTP is converted either into CTP (for DNA synthesis) or into UDP-glucose (for CAP biosynthesis). In agreement with this hypothesis, measurements of UTP showed that the carB mutation led to a decrease in the pool of available UTP at the bifurcation point. This decrease resulted in the upregulation of capsulation, whereas rescuing UTP availability by addition of its precursor, UMP, reduced capsulation. These findings suggest that a reduction in the cellular concentration of UMP and/or a downstream metabolite in the pyrimidine biosynthesis pathway is required for the switch to the capsulated phenotype.
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