In many bacteria glucose is the preferred carbon source and represses the utilization of secondary substrates. In Bacillus subtilis, this carbon catabolite repression (CCR) is achieved by the global transcription regulator CcpA, whose activity is triggered by the availability of its phosphorylated cofactors, HPr(Ser46-P) and Crh(Ser46-P). Phosphorylation of these proteins is catalyzed by the metabolite-controlled kinase HPrK/P. Recent studies have focused on glucose as a repressing substrate. Here, we show that many carbohydrates cause CCR. The substrates form a hierarchy in their ability to exert repression via the CcpA-mediated CCR pathway. Of the two cofactors, HPr is sufficient for complete CCR. In contrast, Crh cannot substitute for HPr on substrates that cause a strong repression. Determination of the phosphorylation state of HPr in vivo revealed a correlation between the strength of repression and the degree of phosphorylation of HPr at Ser46. Sugars transported by the phosphotransferase system (PTS) cause the strongest repression. However, the phosphorylation state of HPr at its His15 residue and PTS transport activity have no impact on the global CCR mechanism, which is a major difference compared to the mechanism operative in Escherichia coli. Our data suggest that the hierarchy in CCR exerted by the different substrates is exclusively determined by the activity of HPrK/P.
SummaryThe histidine protein HPr has a key role in regulation of carbohydrate utilization in low-GC Gram-positive bacteria. Bacilli possess the paralogue Crh. Like HPr, Crh becomes phosphorylated by kinase HPrK/P in response to high fructose-1,6-bisphosphate concentrations. However, Crh can only partially substitute for the regulatory functions of HPr leaving its role mysterious. Using protein co-purification, we identified enzyme methylglyoxal synthase MgsA as interaction partner of Crh in Bacillus subtilis. MgsA converts dihydroxyacetone-phosphate to methylglyoxal and thereby initiates a glycolytic bypass that prevents the deleterious accumulation of phospho-sugars under carbon overflow conditions. However, methylgyloxal is toxic and its production requires control. We show here that exclusively the non-phosphorylated form of Crh interacts with MgsA in vivo and inhibits MgsA activity in vitro. Accordingly, Crh inhibits methylglyoxal formation in vivo under nutritional famine conditions that favour a low HPr kinase activity. Thus, Crh senses the metabolic state of the cell, as reflected by its phosphorylation state, and accordingly controls flux through the harmful methylglyoxal pathway. Interestingly, HPr is unable to bind and regulate MgsA, making this a bona fide function of Crh. Four residues that differ in the interaction surfaces of HPr and Crh may account for this difference.
Non-typeable Haemophilus influenzae (NTHi) is a common pathogen associated with diseases such as acute otitis media or exacerbations in patients with chronic obstructive pulmonary disease. The biofilm-forming capability substantially contributes to the persistence of NTHi. However, the regulation of biofilm formation is not completely understood. Quorum sensing regulated by autoinducer-2 produced by luxS is until now the only described regulatory mechanism. In this study, we show that the two-component signalling system QseB/C is involved in the biofilm formation of NTHi in vitro. An isogenic NTHi mutant of qseC (Hi3655KR2) showed a significant decrease in biofilm formation under static and semi-static conditions as assessed by crystal violet staining. In addition, under constant flow conditions, Hi3655KR2 formed less biofilm after 48 h. The biofilm defects were irrespective of autoinducer-2 levels. Hence, here we suggest for the first time a regulatory circuit in NTHi, which controls biofilm formation by mechanisms other than or in addition to luxS-dependent factors.
In the Gram-positive bacterium Bacillus subtilis as well as in other firmicutes, the HPr protein of the phosphotransferase system (PTS) has two distinct phosphorylation sites, His-15 and Ser-46. These sites are phosphorylated by the Enzyme I of the PTS and by the ATP-dependent HPr kinase/phosphorylase, respectively. As a result, the phosphorylation state of HPr reflects the nutrient supply of the cell and is in turn involved in several responses at the levels of transport activity and expression of catabolic genes. Most important, HPr(Ser-P) serves as a cofactor for the pleiotropic transcription regulator CcpA. In addition to the proteins that phosphorylate HPr, those that are involved in the dephosphorylation are important in controlling the overall HPr phosphorylation state and the resulting regulatory and physiological outputs. In this study, we found that in addition to the phosphorylase activity of the HPr kinase/phosphorylase, the serine/threonine protein phosphatase PrpC uses HPr(Ser-P) as a target.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.