Industrial production processes often involve the use of complex and cheap nutrient sources. This results in carbon catabolite repression of genes that are in the focus of production. To overcome catabolite repression (see Fig. C), the use of strains lacking the central regulator, CcpA, is of interest. However, these strains exhibit severe growth defects, among them an auxotrophy for glutamate. We have demonstrated that mutations in DNA topoisomerase A (TopA) that result in hyperactivity of the protein and thus in enhanced DNA relaxation can overcome the growth defect of the ccpA mutant (Fig. A, B, C). Reduced supercoiling results in a global re-organization of gene expression and metabolism in B. subtilis. DNA topoisomerases are essential enzymes in all domains of life, and TopA of B. subtilis has long been regarded essential as well. Recent evidence suggests that the protein becomes available if a distinct topoisomerases, encoded by the genes parE and parC, is overexpressed (Fig. D). It is therefore tempting to speculate that the topoisomerase IV (ParEC) can take over the essential function of topoisomerase A (TopA) in removing negative supercoils imposed by the transcription and replication machineries. This knowledge is important for the construction of a genome-minimized strain.

Control of DNA metabolism

Figures. (A) A mutation in topA allows growth of a ccpA mutant on minimal medium. (B) Mutations in distinct domains of TopA result in hyperactivity. (C) The hyperactive TopA2 variant rapidly relexes supecoiled DNA. (D) The deletion of the essential topA gene provokes an amplification of the parEC locus. (E) The amplification can be visualized by placing a gfp gene behind the parEC operon. Eventually, the amplification geets lost when the bacteria acquire a mutation resulting in higher parEC promoter activity.



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Reuß et al., 2018 Metab. Eng. 45: 171-179.