Changes of DNA topology affect the global transcription landscape and allow rapid growth of a Bacillus subtilis mutant lacking carbon catabolite repression.

Bacteria are able to prioritize preferred carbon sources from complex mixtures. This is achieved by the regulatory phenomenon of carbon catabolite repression. To allow the simultaneous utilization of multiple carbon sources and to prevent the time-consuming adaptation to each individual nutrient in biotechnological applications, mutants lacking carbon catabolite repression can be used. However, such mutants often exhibit pleiotropic growth defects. We have isolated and characterized mutations that overcome the growth defect of Bacillus subtilis ccpA mutants lacking the major regulator of catabolite repression, in particular their glutamate auxotrophy. Here we show, that distinct mutations affecting the essential DNA topoisomerase I (TopA) cause glutamate prototrophy of the ccpA mutant. These suppressing variants of the TopA enzyme exhibit increased activity resulting in enhanced relaxation of the DNA. Reduced DNA supercoiling results in enhanced expression of the gltAB operon encoding the biosynthetic glutamate synthase. This is achieved by a significant re-organization of the global transcription network accompanied by re-routing of metabolism, which results in inactivation of the glutamate dehydrogenase. Our results provide a link between DNA topology, the global transcriptional network, and glutamate metabolism and suggest that specific topA mutants may be well suited for biotechnological purposes.

Overexpression of the Rv0805 phosphodiesterase elicits a cAMP-independent transcriptional response.

The Rv0805 gene in Mycobacterium tuberculosis encodes a metallophosphoesterase which shows cAMP-hydrolytic activity. Overexpression of Rv0805 has been used as a tool to lower intracellular cAMP levels and thereby elucidate the roles of cAMP in mycobacteria. Here we show that levels of cAMP in M. tuberculosis were lowered by only ∼30% following overexpression of Rv0805, and transcript levels of a number of genes, which include those associated with virulence and the methyl citrate cycle, were altered. The genes that showed altered expression were distinct from those differentially regulated in a strain deleted for the cAMP-receptor protein (CRP(Mt)), consistent with the relatively low dependence on cAMP of CRP(Mt) binding to DNA. Using mutants of Rv0805 we show that the transcriptional signature of Rv0805 overexpression is a combination of catalysis-dependent and independent effects, and that the structurally flexible C-terminus of Rv0805 is crucial for the catalysis-independent effects of the protein. Our study demonstrates the dissociation of Rv0805 and cAMP-regulated gene expression, and reveals alternate functions for this phosphodiesterase from M. tuberculosis.

The regulation of Enzyme IIA(Glc) expression controls adenylate cyclase activity in Escherichia coli.

During the last few years, several genes, such as pap, bgl and flhDC, have been shown to be coregulated by the histone-like nucleoid-structuring (H-NS) protein and the cyclic AMP-catabolite activator protein (cAMP/CAP) complex, suggesting an interaction between both systems in the control of some cellular functions. In this study, the possible effect of H-NS on the cAMP level was investigated. In a CAP-deficient strain, the presence of an hns mutation results in a strong reduction in the amount of cAMP, due to a decrease in adenylate cyclase activity. This is caused by the reduced expression of crr, which encodes the Enzyme IIA(Glc) of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), from its specific P2 promoter. This leads to a twofold reduction in the global amount of Enzyme IIA(Glc), the adenylate cyclase activator, responsible for the decrease in adenylate cyclase activity observed in the hns crp strain.