The role of protein phosphorylation in regulation of carbon catabolite repression in Bacillus subtilis

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CCR is one of the most thoroughly investigated signal transduction system in bacteria. It allows the bacteria to adapt to changes in the availability and supply of different carbon sources. In general, CCR is defined as the selective utilization of a preferred carbon source which represses the functions for the utilization of secondary carbon sources. At the molecular level, CCR is achieved by the global transcriptional regulator CcpA, in B. subtilis. CcpA forms a repressor complex with serine phosphorylated HPr and Crh proteins in the presence of a preferred carbon source like glucose. This repressor complex binds to the operator sites on the DNA called cre sites and thereby repressing a number of catabolic genes and operons involved in utilization of secondary carbon sources. The regulatory phosphorylation of HPr and Crh is achieved by a bifunctional enzyme HPrK/P.In this work, the repressing potential of various carbon sources besides glucose was analysed. A number of carbon sources could exert CcpA-mediated catabolite repression of the reporter system. Moreover, the substrates formed a hierarchy in their ability to exert repression. The different levels of repression by various carbon sources indicated the formation of the CcpA/co-repressor complex to different extents. CcpA and HPr levels were found to be similar in the cell, irrespective of the nature of the carbon source used. Thus, the phosphorylation pattern of the co-repressor was analysed. As a prerequisite for this experiment, it could be established that HPr and not Crh is the relevant co-repressor of CcpA. Thus, the focus was on analysing the level of HPr(Ser-P) in the cell. The presence of strong repressing carbon sources generated high intracellular HPr(Ser-P) as compared to the poor repressing carbon sources. Thus, it could be well established that the different repressing potential of various carbon sources is derived from the ability to generate different intracellular levels of HPr(Ser-P).In the presence of poor repressing carbon sources, besides low intracellular HPr(Ser-P), considerable amounts of histidine phosphorylated HPr were also present. HPr is also a part of the sugar PTS. The PTS is involved in the concomitant uptake and phosphorylation of various carbon sources. As a part of PTS, HPr is phosphorylated at its His-15 residue and receives phosphate from PEP via EI. Phosphorylation at histidine residue might decrease the available HPr as a substrate for HPrK/P. Therefore, the possibility that the presence of HPr(His-P) negatively regulates the phosphorylation of HPr by HPrK/P was addressed. In an EI mutant, HPr(His-P) is not formed. However, the repression potential of the non-PTS carbon sources remained unchanged in the EI mutant. This clearly established that the phosphorylation of HPr at Ser-46 is exclusively determined by HPrK/P activity and not by the PTS. Thus, in the presence of weaker repressing carbon sources, low HPr(Ser-P) levels are generated owing to the low kinase activity of HPrK/P. This hypothesis was confirmed by using an HPrK/P variant, which lost its phosphorylase activity. Such a variant exhibited a low but constitutive kinase activity and allowed repression even in the absence of a repressing carbon source.Modulation of the HPrK/P activity is suggested to be achieved by an allosteric regulation by metabolites like FBP and Pi. Therefore, we determined the FBP concentration in vivo. It turned out that on most sugars the intracellular FBP level is high enough to achieve theoretically a complete activation of the HPrK/P kinase activity. Therefore, further factors should exist that regulate HPrK/P activity in vivo. To probe into the possibility that HPr(Ser-P) levels can be affected by an enzyme other than HPrK/P, the role of PrpC was analysed. PrpC, a Ser/Thr phosphatase, has shown to dephosphorylate HPr(Ser-P) in M. pneumoniae. B. subtilis PrpC could dephosphorylate HPr(Ser-P) in vitro, but its absence or presence had no effect on CCR in vivo.Taken together, this work coherently demonstrates the central role of HPrK/P in CCR in B. subtilis. Moreover, the hierarchy of repression exerted by various carbon sources could be well explained with the levels of HPr(Ser-P) generated in the cell. This work further highlights the fundamental differences in the mechanism of catabolite repression in the two model organisms E. coli and B. subtilis. PTS transport activity is the decisive factor of both global and operon-specific CCR in E. coli. In the absence of HPr(His-P) in E. coli, EIIAglu is rendered unphosphorylated and exerts strong catabolite repression. In contrast, the global CCR mechanism in B. subtilis is not directly affected by the PTS activity.