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Stimulus Perception in Bacterial Signal-Transducing Histidine Kinases
No full textTwo-component signal-transducing systems are ubiquitously distributed communication interfaces in bacteria. They consist of a histidine kinase that senses a specific environmental stimulus and a cognate response regulator that mediates the cellular response, mostly through differential expression of target genes. Histidine kinases are typically transmembrane proteins harboring at least two domains: an input (or sensor) domain and a cytoplasmic transmitter (or kinase) domain. They can be identified and classified by virtue of their conserved cytoplasmic kinase domains. In contrast, the sensor domains are highly variable, reflecting the plethora of different signals and modes of sensing. In order to gain insight into the mechanisms of stimulus perception by bacterial histidine kinases, we here survey sensor domain architecture and topology within the bacterial membrane, functional aspects related to this topology, and sequence and phylogenetic conservation. Based on these criteria, three groups of histidine kinases can be differentiated. (i) Periplasmic-sensing histidine kinases detect their stimuli (often small solutes) through an extracellular input domain. (ii) Histidine kinases with sensing mechanisms linked to the transmembrane regions detect stimuli (usually membrane-associated stimuli, such as ionic strength, osmolarity, turgor, or functional state of the cell envelope) via their membrane-spanning segments and sometimes via additional short extracellular loops. (iii) Cytoplasmic-sensing histidine kinases (either membrane anchored or soluble) detect cellular or diffusible signals reporting the metabolic or developmental state of the cell. This review provides an overview of mechanisms of stimulus perception for members of all three groups of bacterial signal-transducing histidine kinases.NIGMS NIH HHS [GM047446, R01 GM047446
Transmembrane signaling and cytoplasmic signal conversion by dimeric transmembrane helix 2 and a linker domain of the DcuS sensor kinase
No full textAssignment of H-1, C-13 and N-15 resonances to the sensory domain of the membraneous two-component fumarate sensor (histidine protein kinase) DcuS of Escherichia coli
No full textSensing by the membrane-bound sensor kinase DcuS: Exogenous versus endogenous sensing of C-4-dicarboxylates in bacteria.
No full text Bacteria are able to grow at the expense of both common (succinate, L-malate, fumarate and aspartate) and uncommon (L-tartrate and D-malate) C4-dicarboxylates, which are components of central metabolism. Two types of sensors/regulators responding to the C4-dicarboxylates function in Escherichia coli, Bacillus, Lactobacillus and related bacteria. The first type represents membrane-integral two-component systems, while the second includes cytoplasmic LysR-type transcriptional regulators. The difference in location and substrate specificity allows the exogenous induction of metabolic genes by common C4-dicarboxylates, and endogenous induction by uncommon C4-dicarboxylates. The two-component sensors, DcuS and CitA, are composed of an extracellular Per-Arnt-Sim (PAS) domain, two transmembrane helices, a cytoplasmic PAS and the kinase domain. The structures of the extracellular PAS domains of DcuS and CitA have been determined in the ligand-bound and the apo form. Binding of the ligand results in closing and compaction of the binding site, and the structural change gives rise to piston-type movement of the adjacent membrane-spanning helix-2, and signal transmission to the cytoplasmic side. For DcuS, a membrane-embedded construct has been developed that suggests (by experimentation and modeling) that plasticity of the cytoplasmic PAS domain is central to signal transduction from the membrane to the kinase. Sensor kinase DcuS of E. coli requires the C4-dicarboxylate transporters DctA or DcuB as co-sensors for function under aerobic and anaerobic conditions, respectively. DcuB contains a regulatory site that controls the function of DcuS and is independent from the transport region. Therefore, DcuS senses C4-dicarboxylates in two independent modes, responding to the effector concentration and the metabolic flux of extracellular C4-dicarboxylates. </jats:p
The nature of the stimulus and of the fumarate binding site of the fumarate sensor DcuS of Escherichia coli
Full text linkDcuS is a membrane-associated sensory histidine kinase of Escherichia coli specific for C-4-dicarboxylates. The nature of the stimulus and its structural prerequisites were determined by measuring the induction of DcuS-dependent dcuB'-'lacZ gene expression. C4-dicarboxylates without or with substitutions at C-2/C-3 by hydrophilic (hydroxy, amino, or thiolate) groups stimulated gene expression in a similar way. When one carboxylate was replaced by sulfonate, methoxy, or nitro groups, only the latter (3-nitropropionate) was active. Thus, the ligand of DcuS has to carry two carboxylate or carboxylate/nitro groups 3.1-3.8 A apart from each other. The effector concentrations for half-maximal induction of dcuB'-'lacZ expression were 2-3 mM for the C-4-dicarboxylates and 0.5 mM for 3-nitropropionate or D-tartrate. The periplasmic domain of DcuS contains a conserved cluster of positively charged or polar amino acid residues (Arg(107)-X-2-His(110)-X-9-Phe(120)-X-26-Arg(147)-X-Phe(149)) that were essential for fumarate-dependent transcriptional regulation. The presence of fumarate or D-tartrate caused sharpening of peaks or chemical shift changes in HSQC NMR spectra of the isolated C-4-dicarboylate binding domain. The amino acid residues responding to fumarate or D-tartrate were in the region comprising residues 89-150 and including the supposed binding site. DcuS( R147A) mutant with an inactivated binding site was isolated and reconstituted in liposomes. The protein showed the same (activation-independent) kinase activity as DcuS, but autophosphorylation of DcuS was no longer stimulated by C-4-dicarboxylates. Therefore, the R147A mutation affected signal perception and transfer to the kinase but not the kinase activity per se
Intramolecular signal transduction of the sensor histidine kinase DcuS and the aerobic and anaerobic fumarate proteome in the regulation of the Escherichia coli C4-dicarboxylate metabolism
Full text linkThe aerobic and anaerobic utilization of C4-dicarboxylates in Escherichia coli is regulated by the DcuSR two-component system. Depending on oxygen availability, the transcription of the genes of fumarate respiration or the aerobic transporter DctA are induced. The C4-dicarboxylate transporters DctA and DcuB act as co-regulators of DcuS and convert DcuS to its responsive state in the DcuS-transporter sensor complex. C4-dicarboxylates bound at the periplasmic domain of DcuS and trigger a signal cascade across the membrane, emanating from the sensory PASP domain through TM2, a short Linker and the cytoplasmic PASC domain that results in cytoplasmic autophosphorylation at the C-terminal kinase domain of DcuS. TM2 was already shown to transduce the signal across the membrane via a piston type shift, but the entire mechanism in DcuS transmembrane signaling is unknown.
The structure and dynamics of the domains in DcuS intramolecular signal transduction were investigated by oxidative cysteine cross-linking. This revealed a membrane spanning dimeric continuous helix that is mostly stable in both DcuS signaling states. The continuous helix comprises the C-terminal α6 helix of PASP, TM2, the linker, and the N-terminal α1 helix of PASC and thus connects periplasmic signal input with the cytoplasmic signal output domains. The structural dynamics of selected DcuS residues in the DcuS signal transfer were tested by time-resolved cysteine cross-linking. TM2 was shown to be a rigid homo-dimer confirming the piston-type shift as major transmembrane signaling mechanism by TM2. In contrast, the linker represents a dynamic region in signal transduction. PASC also seems to undergo a restructuring in α1 and β1 upon DcuS activation. Furthermore, time-resolved cysteine crosslinking in the absence of the co-regulator DctA showed that DcuS adopts cross-linking reactivity resembling the fumarate activated state. It seems likely that DctA stabilizes homo-dimerization of the linker and α1 of PASC by direct interaction via the DctA helix 8b and thus converts DcuS in its responsive state in the DcuS-DctA sensor complex.
In addition, the aerobic and anaerobic E. coli fumarate proteome was investigated in a ‘shotgun proteomics’ approach. The transcriptional regulation by DcuSR was displayed in the proteomic results. Upregulation of almost all TCA cycle enzymes, anaplerotic reactions, and gluconeogenesis under aerobic conditions was observed. This regulatory effect can be related to an EIIA-P/EIIA ratio-dependent indirect regulation by cAMP/CRP, but a previously unknown regulation by DcuSR is also possible in some cases. The most abundant category of proteins, which was upregulated by fumarate under anaerobic conditions, can be assigned to chemotaxis and motility.108 Seite
: role in signal transduction, dimer formation, and DctA interaction
Full text linkThe cytoplasmic PAS(C) domain of the fumarate responsive sensor kinase DcuS of Escherichia coli links the transmembrane to the kinase domain. PAS(C) is also required for interaction with the transporter DctA serving as a cosensor of DcuS. Earlier studies suggested that PAS(C) functions as a hinge and transmits the signal to the kinase. Reorganizing the PAS(C) dimer interaction and, independently, removal of DctA, converts DcuS to the constitutive ON state (active without fumarate stimulation). ON mutants were categorized with respect to these two biophysical interactions and the functional state of DcuS: type I-ON mutations grossly reorganize the homodimer, and decrease interaction with DctA. Type IIA-ON mutations create the ON state without grossly reorganizing the homodimer, whereas interaction with DctA is decreased. The type IIB-ON mutations were neither in PAS(C)/PAS(C), nor in DctA/DcuS interaction affected, similar to fumarate activated wild-typic DcuS. OFF mutations never affected dimer stability. The ON mutations provide novel mechanistic insight: PAS(C) dimerization is essential to silence the kinase. Reorganizing the homodimer and its interaction with DctA activate the kinase. The study suggests a novel ON homo-dimer conformation (type IIB) and an OFF conformation for PAS(C). Type IIB-ON corresponds to the fumarate induced wild-type conformation, representing an interesting target for structural biology
L-Aspartate is a high-quality nitrogen source of Escherichia coli: regulation and physiology
Full text linkEscherichia coli is a highly adaptable bacterium, which can utilize diverse carbon and nitrogen sources. In aerobic growth, the L-Asp transporter DcuA and the aspartate ammonia-lyase AspA catalyze a collaborative nitrogen shuttle in which L-Asp is transported into the bacterial cell and directly deaminated to form ammonium and fumarate. Ammonium is assimilated in the GS-GOGAT pathway, while fumarate is exported. In anaerobic growth, fumarate can be used to drive fumarate respiration, which transforms L-Asp to an elec-tron acceptor and nitrogen source, emphasizing the importance of L-Asp. AspA was stimulated by GlnB saturated with either 2-oxoglutarate and ATP, a bound UMP, or both. GlnB stimulates AspA deamination activity twofold, providing ammonium under nitrogen-limited conditions to supply the bacterial cell with a nitrogen source. The DcuA-AspA-GS-GOGAT pathway is able to produce L-Asp, L-Gln, L-Glu, and ammonium, which completely satisfies the nitrogen requirement of E. coli. L-Asp was found in millimolar concentration in the mouse intestine, which highlights its physiological relevance.
DctA is an aerobic C4-dicarboxylate transporter which has a high affinity for succinate.
Reporter gene assays of aspAp and dctAp expression, demonstrated a high sensitivity of the expression to the presence of different carbon sources, such as sugars, sugar alcohols, and C4-dicarboxylates. cAMP-CRP fine-tunes the expression of genes involved in the catabolism of substrates other than glucose, including aspA and dctA, in response to carbon availability. Bioinformatics revealed a mutation of the CRP-binding site in the dominant first half site of dctAp, explaining the higher repression in the presence of glycerol and D-xylose. In addition, DctA showed interaction with EIIAGlc of the glucose:phosphotransferase system, which is supposed to inhibit C4-dicarboxylate uptake in aerobic growth when upper and lower glycolytic substrates are available.
Enzymes that catalyze sequential reactions in metabolic pathways can be organized into
sequential complexes ('metabolons') capable of channeling metabolic intermediates and
increasing metabolic efficiency. Interaction between DcuA-AspA, DcuB-AspA, and DcuB-FumB was demonstrated and was suggested to drive nitrogen assimilation and fumarate
respiration.
The sensor histidine kinase DcuS was modeled using various bioinformatic tools to investigate the interaction between the DcuS linker region and DctA helix 8b. The interaction model postulated a salt bridge between two residues to stabilize the interaction. In addition, the DcuS model, multiple sequence alignment, and current research indicated an L-Proline hinge in the N-terminal α-helix of the cytoplasmic PAS domain.166 Seite
Citrate sensing by the C4-dicarboxylate/citrate sensor kinase DcuS of Escherichia coli: Binding site and conversion of DcuS to a C4-dicarboxylate- or citrate-specific sensor
No full textThe 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
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