Citrate Sensing by the C ₄ -Dicarboxylate/Citrate Sensor Kinase DcuS of Escherichia coli : Binding Site and Conversion of DcuS to a C ₄ -Dicarboxylate- or Citrate-Specific Sensor

Abstract

The histidine protein kinase DcuS of Escherichia coli senses C 4 -dicarboxylates and citrate by a periplasmic domain. The closely related sensor kinase CitA binds citrate, but no C 4 -dicarboxylates, by a homologous periplasmic domain. CitA is known to bind the three carboxylate and the hydroxyl groups of citrate by sites C1, C2, C3, and H. DcuS requires the same sites for C 4 -dicarboxylate sensing, but only C2 and C3 are highly conserved. It is shown here that sensing of citrate by DcuS required the same sites. Binding of citrate to DcuS, therefore, was similar to binding of C 4 -dicarboxylates but different from that of citrate binding in CitA. DcuS could be converted to a C 4 -dicarboxylate-specific sensor (DcuS DC ) by mutating residues of sites C1 and C3 or of some DcuS-subtype specific residues. Mutations around site C1 aimed at increasing the size and accessibility of the site converted DcuS to a citrate-specific sensor (DcuS Cit ). DcuS DC and DcuS Cit had complementary effector specificities and responded either to C 4 -dicarboxylates or to citrate and mesaconate. The results imply that DcuS binds citrate (similar to the C 4 -dicarboxylates) via the C 4 -dicarboxylate part of the molecule. Sites C2 and C3 are essential for binding of two carboxylic groups of citrate or of C 4 -dicarboxylates; sites C1 and H are required for other essential purposes.Escherichia coli is able to use C 4 -dicarboxylates as substrates for anaerobic growth by fumarate respiration, which requires the synthesis of fumarate reductase (frdABCD genes) and the fumarate/succinate antiporter DcuB (dcuB gene) (for reviews, see references 6, 15, and 28). Expression of the frdABCD and dcuB genes is stimulated by the DcuSR two-component system (12,14,15,29). DcuS responds to C 4 -dicarboxylates and related compounds through a periplasmic sensing domain (19). The two carboxylic groups of the C 4 -dicarboxylates are crucial for stimulus perception, whereas other parts of the C 4 -dicarboxylates such as ligands at position C2 or C3 are of minor significance. The apparent K D values of C 4 -dicarboxylates for stimulating the expression of DcuS-regulated genes are in the range of 0.45 to 3 mM.DcuS is a member of the CitA/DcuS family of sensory histidine kinases and shares significant sequence similarities with CitA (4,5,15,17,18). CitA is the highly specific and highaffinity citrate sensor kinase of the CitAB two-component system that controls expression of the citrate fermentation genes in Klebsiella pneumoniae and E. coli. The CitA and DcuS sensors have similar membrane topologies, with a periplasmic sensory domain and a cytoplasmic kinase domain (15,18,29). CitA and DcuS are typical members of the periplasmic sensing histidine kinases (20). The structure of the periplasmic domain of DcuS has been solved by nuclear magnetic resonance (NMR) spectroscopy, and that of CitA from K. pneumoniae has been solved by crystallography and X-ray analysis (23, 25).The overall structures for the two domains are similar and resemble the PAS (Per-Arnt-Sim)...

CitA/CitB Two-Component System Regulating Citrate Fermentation in Escherichia coli and Its Relation to the DcuS/DcuR System In Vivo

“…Figure 4 summarizes the physical and functional interaction between DcuS/DcuR and CitA/CitB systems. First, the expression studies demonstrate linear signal transfer, CitA¡Cit-B¡citC and DcuS¡DcuR¡dcuB, and exclude branched signal transfer between both systems in accordance with earlier suggestions (25). The linear signal transfer is confirmed by the interaction studies by BACTH that show interaction between DcuS and DcuR but the lack of interaction between CitA and DcuR.…”

“…A similar result was obtained when the bacteria were grown anaerobically in eM9 medium with gluconate as the carbon source and citrate as the effector, where the citrate induction of dcuB-lacZ decreased by a factor of 1.7 in the CitA deletion strain (data not shown). An even stronger decrease (factor of 3.3) was observed earlier for the citA mutant after the anaerobic growth of the bacteria in eM9 medium with glucose and citrate (25). The decrease in dcuB-lacZ expression in the CitA mutant was not observed with fumarate and is citrate specific (Table 4).…”

“…Additionally, it was tested whether CitA requires, like DcuS, the presence of transporters (CitT and DcuB) of the pathway or of related pathways (the succinate efflux carrier DcuC) to function. Expression studies (25) showed that the induction of dcuB-lacZ expression is diminished slightly in a citA-negative strain but not in a citB-negative strain, suggesting an interaction between the DcuS/DcuR and the CitA/CitB systems in the transcriptional regulation of target genes. The interaction between both systems was studied here in more detail.…”

“…The sensor kinase DcuS responds to all types of C 4 -dicarboxylates and citrate with apparent K d values in the range of 2 to 13 mM (23). Fumarase and FrdABCD also are required for citrate fermentation, and the respective genes then are induced by the response of DcuS/DcuR to citrate (23,25). …”

Probing bacterial transmembrane histidine kinase receptor–ligand interactions with natural and synthetic molecules

“…Furthermore, like CqsS, many two-component sensors have complicated membrane-spanning domains, which has made structural analyses particularly difficult. For these reasons, analyses of ligandreceptor interactions using structural approaches have been limited to a few histidine kinases with well-defined periplasmic sensing domains and known ligands (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). Thus, despite intensive effort, it remains unclear how ligand binding affects signaling activity in this important family of receptors.…”