An energetic profile of Corynebacterium glutamicum underpinned by measured biomass yield on ATP.

The supply and usage of energetic cofactors in metabolism is a central concern for systems metabolic engineering, particularly in case of energy intensive products. One of the most important parameters for systems wide balancing of energetic cofactors is the ATP requirement for biomass formation YATP/Biomass. Despite its fundamental importance, YATP/Biomass values for non-fermentative organisms are still rough estimates deduced from theoretical considerations. For the first time, we present an approach for the experimental determination of YATP/Biomass using comparative 13C metabolic flux analysis (13C MFA) of a wild type strain and an ATP synthase knockout mutant. We show that the energetic profile of a cell can then be deduced from a genome wide stoichiometric model and experimental maintenance data. Particularly, the contributions of substrate level phosphorylation (SLP) and electron transport phosphorylation (ETP) to ATP generation become available which enables the overall energetic efficiency of a cell to be characterized. As a model organism, the industrial platform organism Corynebacterium glutamicum is used. C. glutamicum uses a respiratory type of energy metabolism, implying that ATP can be synthesized either by SLP or by ETP with the membrane-bound F1FO-ATP synthase using the proton motive force (pmf) as driving force. The presence of two terminal oxidases, which differ in their proton translocation efficiency by a factor of three, further complicates energy balancing for this organism. By integration of experimental data and network models, we show that in the wild type SLP and ETP contribute equally to ATP generation. Thus, the role of ETP in respiring bacteria may have been overrated in the past. Remarkably, in the genome wide setting 65% of the pmf is actually not used for ATP synthesis. However, it turns out that, compared to other organisms C. glutamicum still uses its energy budget rather efficiently.

Isotropic-nematic transition of self-propelled rods in three dimensions.

Using overdamped Brownian dynamics simulations we investigate the isotropic-nematic (IN) transition of self-propelled rods in three spatial dimensions. For two well-known model systems (Gay-Berne potential and hard spherocylinders) we find that turning on activity moves to higher densities the phase boundary separating an isotropic phase from a (nonpolar) nematic phase. This active IN phase boundary is distinct from the boundary between isotropic and polar-cluster states previously reported in two-dimensional simulation studies and, unlike the latter, is not sensitive to the system size. We thus identify a generic feature of anisotropic active particles in three dimensions.

Phase separation on the sphere: Patchy particles and self-assembly.

Motivated by observations of heterogeneous domain structure on the surface of cells, we consider a minimal model to describe the dynamics of phase separation on the surface of a spherical particle. Finite-size effects on the curved particle surface lead to the formation of long-lived, metastable states for which the density is distributed in patches over the particle surface. We study the time evolution and stability of these states as a function of both the particle size and the thermodynamic parameters. Finally, by connecting our findings with studies of patchy particles, we consider the implications for self-assembly in many-particle systems.

Chalcone-based Selective Inhibitors of a C4 Plant Key Enzyme as Novel Potential Herbicides.

Weeds are a challenge for global food production due to their rapidly evolving resistance against herbicides. We have identified chalcones as selective inhibitors of phosphoenolpyruvate carboxylase (PEPC), a key enzyme for carbon fixation and biomass increase in the C4 photosynthetic pathway of many of the world's most damaging weeds. In contrast, many of the most important crop plants use C3 photosynthesis. Here, we show that 2',3',4',3,4-Pentahydroxychalcone (IC50 = 600 nM) and 2',3',4'-Trihydroxychalcone (IC50 = 4.2 µM) are potent inhibitors of C4 PEPC but do not affect C3 PEPC at a same concentration range (selectivity factor: 15-45). Binding and modeling studies indicate that the active compounds bind at the same site as malate/aspartate, the natural feedback inhibitors of the C4 pathway. At the whole plant level, both substances showed pronounced growth-inhibitory effects on the C4 weed Amaranthus retroflexus, while there were no measurable effects on oilseed rape, a C3 plant. Growth of selected soil bacteria was not affected by these substances. Our chalcone compounds are the most potent and selective C4 PEPC inhibitors known to date. They offer a novel approach to combat C4 weeds based on a hitherto unexplored mode of allosteric inhibition of a C4 plant key enzyme.

Neuregulin and laminin stimulate phosphorylation of the NF2 tumor suppressor in Schwann cells by distinct protein kinase A and p21-activated kinase-dependent pathways.

Mutations in the neurofibromatosis type 2 (NF2) gene cause formation of schwannomas and other tumors in the nervous system. The NF2 protein, Schwannomin/Merlin, is a cytoskeleton-associated tumor suppressor regulated by phosphorylation at serine 518 (S518). Unphosphorylated Schwannomin restricts cell proliferation in part by inhibiting Rac- and p21-activated kinase (Pak). In a negative-feedback loop, Pak phosphorylates Schwannomin inactivating its ability to inhibit Pak. Little is known about receptor mechanisms that promote Pak activity and Schwannomin phosphorylation. Here we demonstrate in primary Schwann cells (SCs) that Schwannomin is rapidly phosphorylated on S518 by Pak following laminin-1 binding to beta1 integrin, and by protein kinase A following neuregulin-1beta (NRG1beta) binding to ErbB2/ErbB3 receptors. These receptors, together with phosphorylated Schwannomin, P-Pak, Cdc42 and paxillin are enriched at the distal tips of SC processes, and can be isolated as a complex using beta1 integrin antibody. Dual stimulation with laminin-1 and NRG1beta does not synergistically increase Schwannomin phosphorylation because ErbB2 kinase partially antagonizes integrin-dependent activation of Pak. These results identify two parallel, but interactive pathways that inactivate the tumor suppressor activity of Schwannomin to allow proliferation of subconfluent SCs. Moreover, they identify ErbB2, ErbB3 and beta1 integrins as potential therapeutic targets for NF2.

Global expression profiling and physiological characterization of Corynebacterium glutamicum grown in the presence of L-valine.

Addition of L-valine (50 to 200 mM) to glucose minimal medium had no effect on the growth of wild-type Corynebacterium glutamicum ATCC 13032 but inhibited the growth of the derived valine production strain VAL1 [13032 DeltailvA DeltapanBC(pJC1ilvBNCD)] in a concentration-dependent manner. In order to explore this strain-specific valine effect, genomewide expression profiling was performed using DNA microarrays, which showed that valine caused an increased ilvBN mRNA level in VAL1 but not in the wild type. This unexpected result was confirmed by an increased cellular level of the ilvB protein product, i.e., the large subunit of acetohydroxyacid synthase (AHAS), and by an increased AHAS activity of valine-treated VAL1 cells. The conclusion that valine caused the limitation of another branched-chain amino acid was confirmed by showing that high concentrations of L-isoleucine could relieve the valine effect on VAL1 whereas L-leucine had the same effect as valine. The valine-caused isoleucine limitation was supported by the finding that the inhibitory valine effect was linked to the ilvA deletion that results in isoleucine auxotrophy. Taken together, these results implied that the valine effect is caused by competition for uptake of isoleucine by the carrier BrnQ, which transports all branched-chained amino acids. Indeed, valine inhibition could also be relieved by supplementing VAL1 with the dipeptide isoleucyl-isoleucine, which is taken up by a dipeptide transport system rather than by BrnQ. Interestingly, addition of external valine stimulated valine production by VAL1. This effect is most probably due to a reduced carbon usage for biomass production and to the increased expression of ilvBN, indicating that AHAS activity may still be a limiting factor for valine production in the VAL1 strain.

Historadioautographic localization of oxytocin and V1a vasopressin binding sites in the kidney of developing and adult rabbit, mouse and merione and of adult human.

The localization of oxytocin (OT) binding sites and vasopressin (VP) binding sites of the V1a subtype was investigated by radioautography in kidneys of rabbits, mice and meriones during postnatal development and in the adult, and in the human kidney. Kidney sections were incubated in the presence of selective radioiodinated OT and V1a antagonists, respectively. The localizations were compared with those previously described in the rat. The main finding of the study was the almost constant presence in the cortex of V1a binding sites in the connecting tubule, the cortical collecting duct and in the juxtaglomerular apparatus (on the intra- and extraglomerular mesangium and the afferent arteriole). This distribution suggests an interaction of VP via V1a receptors and the kallikrein-kinin system in the kidney. OT binding sites, in comparison with V1a binding sites, were fewer and less constantly detectable in the kidney of the different species. In the mouse, their presence on the limbs of Henle's loop in the medulla points to the possibility of their involvement in the medullary concentrating process. In the kidneys of the various species, OT and V1a binding sites occurred always in differential structures. In contrast, in the human kidney cortex, a colocalization of OT and V1a binding sites was almost constantly observed. This raises the question as to the specificity of the neurohypophysial hormone receptors in the human kidney.

Catabolite repression of the citrate fermentation genes in Klebsiella pneumoniae: evidence for involvement of the cyclic AMP receptor protein.

Klebsiella pneumoniae is able to grow anaerobically with citrate as a sole carbon and energy source by a fermentative pathway involving the Na(+)-dependent citrate carrier CitS, citrate lyase, and oxaloacetate decarboxylase. The corresponding genes are organized in the divergent citC and citS operons, whose expression is strictly dependent on the citrate-sensing CitA-CitB two-component system. Evidence is provided here that the citrate fermentation genes are subject to catabolite repression, since anaerobic cultivation with a mixture of citrate and glucose or citrate and gluconate resulted in diauxic growth. Glucose, gluconate, and also glycerol decreased the expression of a chromosomal citS-lacZ fusion by 60 to 75%, whereas a direct inhibition of the citrate fermentation enzymes was not observed. The purified cyclic AMP (cAMP) receptor protein (CRP) of K. pneumoniae bound to two sites in the citC-citS intergenic region, which were centered at position -41.5 upstream of the citC and citS transcriptional start sites. Binding was apparently stimulated by the response regulator CitB. These data indicate that catabolite repression of the citrate fermentation genes is exerted by CRP and that in the absence of repressing carbon sources the cAMP-CRP complex serves to enhance the basal, CitB-dependent transcription level.

In vivo and in vitro development of the protist Helicosporidium sp.

We describe the discovery and developmental features of a Helicosporidium sp. isolated from the black fly Simulium jonesi. Morphologically, the helicosporidia are characterized by a distinct cyst stage that encloses three ovoid cells and a single elongate filamentous cell. Bioassays have demonstrated that the cysts of this isolate infect various insect species, including the lepidopterans, Helicoverpa zea, Galleria mellonella, and Manduca sexta, and the dipterans, Musca domestica, Aedes taeniorhynchus, Anopheles albimanus, and An. quadrimaculatus. The cysts attach to the insect peritrophic matrix prior to dehiscence, which releases the filamentous cell and the three ovoid cells. The ovoid cells are short-lived in the insect gut with infection mediated by the penetration of the filamentous cell into the host. Furthermore, these filamentous cells are covered with projections that anchor them to the midgut lining. Unlike most entomopathogenic protozoa, this Helicosporidium sp. can be propagated in simple nutritional media under defined in vitro conditions, providing a system to conduct detailed analysis of the developmental biology of this poorly known taxon. The morphology and development of the in vitro produced cells are similar to that reported for the achorophyllic algae belonging to the genus Prototheca.

Proteome analysis of Corynebacterium glutamicum.

By the use of different Corynebacterium glutamicum strains more than 1.4 million tons of amino acids, mainly L-glutamate and L-lysine, are produced per year. A project was started recently to elucidate the complete DNA sequence of this bacterium. In this communication we describe an approach to analyze the C. glutamicum proteome, based on this genetic information, by a combination of two-dimensional (2-D) gel electrophoresis and protein identification via microsequencing or mass spectrometry. We used these techniques to resolve proteins of C. glutamicum with the aim to establish 2-D protein maps as a tool for basic microbiology and for strain improvement. In order to analyze the C. glutamicum proteome, methods were established to fractionate the C. glutamicum proteins according to functional entities, i.e., cytoplasm, membranes, and cell wall. Protein spots of the cytoplasmic and membrane fraction were identified by N-terminal sequencing, immunodetection, matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electrospray ionization-mass spectrometry (ESI-MS). Additionally, a protocol to analyze proteins secreted by C. glutamicum was established. Approximately 40 protein spots were observed on silver-stained 2-D gels, 12 of which were identified.

Molecular analysis of the cytochrome bc1-aa3 branch of the Corynebacterium glutamicum respiratory chain containing an unusual diheme cytochrome c1.

In this work, the genes for cytochrome aa3 oxidase and the cytochrome bc1 complex in the gram-positive soil bacterium Corynebacterium glutamicum were identified. The monocistronic ctaD gene encoded a 65-kDa protein with all features typical for subunit I of cytochrome aa3 oxidases. A ctaD deletion mutant lacked the characteristic 600 nm peak in redox difference spectra, and growth in glucose minimal medium was strongly impaired. The genes encoding subunit III of cytochrome aa3 (ctaE) and the three characteristic subunits of the cytochrome bc1 complex (qcrABC) were clustered in the order ctaE-qcrCAB. Analysis of the deduced primary structures revealed a number of unusual features: (1) cytochrome c1 (QcrC, 30 kDa) contained two Cys-X-X-Cys-His motifs for covalent heme attachment, indicating that it is a diheme c-type cytochrome; (2) the 'Rieske' iron-sulphur protein (QcrA, 45 kDa) contained three putative transmembrane helices in the N-terminal region rather than only one; and (3) cytochrome b (QcrB, 60 kDa) contained, in addition to the conserved part with eight transmembrane helices, a C-terminal extension of about 120 amino acids, which presumably is located in the cytoplasm. Staining of C. glutamicum proteins for covalently bound heme indicated the presence of a single, membrane-bound c-type cytochrome with an apparent molecular mass of about 31 kDa. Since this protein was missing in a qcrCAB deletion mutant, it most likely corresponds to cytochrome c1. Similar to the deltactaD mutant, the deltaqcrCAB mutant showed strongly impaired growth in glucose minimal medium, which indicates that the bc1-aa3 pathway is the main route of respiration under these conditions.

A high-resolution reference map for cytoplasmic and membrane-associated proteins of Corynebacterium glutamicum.

We present a high-resolution reference map for soluble proteins obtained from Corynebacterium glutamicum cells grown in glucose minimal medium. The analysis window covers the pl range from 4-6 and the molecular mass range from 5-100 kDa. Using overlapping narrow immobilized pH gradients for isoelectric focusing, 970 protein spots were detected after second-dimensional separation on SDS-polyacrylamide gels and colloidal Coomassie-staining. By tryptic peptide mass fingerprinting 169 protein spots were identified, representing 152 different proteins including many enzymes involved in central metabolism (18), amino acid biosynthesis (24) and nucleotide biosynthesis (11). Thirty-five of the identified proteins have no known function. A comparison of the observed and the expected physicochemical properties of the identified proteins indicated that nine proteins were covalently modified, since variants with apparently identical molecular mass, but differing pl were detected. The N-termini of eight proteins were determined by post-source decay (PSD) analysis of selected peptides. In addition to the soluble proteins, a map of the membrane-bound proteins within the pl range 4-7 is presented, which contains 660 protein spots, 22 of which were identified, representing 13 different proteins.

Identification of triphosphoribosyl-dephospho-CoA as precursor of the citrate lyase prosthetic group.

The gamma-subunit of citrate lyase (EC 4.1.3.6) contains the prosthetic group 2'-(5"-phosphoribosyl)-3'-dephospho-CoA and serves as an acyl carrier protein (ACP). We recently showed that in Escherichia coli the proteins CitG and CitX are essential for holo-ACP synthesis and provided evidence that CitG catalyzes the formation of a prosthetic group precursor from ATP and dephospho-CoA, which is subsequently attached via phosphodiester linkage to apo-ACP by CitX. Here we prove that CitG indeed catalyzes the conversion of ATP and dephospho-CoA to adenine and 2'-(5"-triphosphoribosyl)-3'-dephospho-CoA, the predicted precursor of the prosthetic group. Furthermore, this precursor was transferred by CitX to apo-ACP, yielding holo-ACP. Thus, our proposed mechanism for holo-ACP synthesis could be verified.

Biosynthesis of the prosthetic group of citrate lyase.

Citrate lyase (EC 4.1.3.6) catalyzes the cleavage of citrate to acetate and oxaloacetate and is composed of three subunits (alpha, beta, and gamma). The gamma-subunit serves as an acyl carrier protein (ACP) and contains the prosthetic group 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA, which is attached via a phosphodiester linkage to serine-14 in the enzyme from Klebsiella pneumoniae. In this work, we demonstrate by genetic and biochemical studies with citrate lyase of Escherichia coli and K. pneumoniae that the conversion of apo-ACP into holo-ACP is dependent on the two proteins, CitX (20 kDa) and CitG (33 kDa). In the absence of CitX, only apo-ACP was synthesized in vivo, whereas in the absence of CitG, an adenylylated ACP was produced, with the AMP residue attached to serine-14. The adenylyltransferase activity of CitX could be verified in vitro with purified CitX and apo-ACP plus ATP as substrates. Besides ATP, CTP, GTP, and UTP also served as nucleotidyl donors in vitro, showing that CitX functions as a nucleotidyltransferase. The conversion of apo-ACP into holo-ACP was achieved in vitro by incubation of apo-ACP with CitX, CitG, ATP, and dephospho-CoA. ATP could not be substituted with GTP, CTP, UTP, ADP, or AMP. In the absence of CitG or dephospho-CoA, AMP-ACP was formed. Remarkably, it was not possible to further convert AMP-ACP to holo-ACP by subsequent incubation with CitG and dephospho-CoA. This demonstrates that AMP-ACP is not an intermediate during the conversion of apo- into holo-ACP, but results from a side activity of CitX that becomes effective in the absence of its natural substrate. Our results indicate that holo-ACP formation proceeds as follows. First, a prosthetic group precursor [presumably 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA] is formed from ATP and dephospho-CoA in a reaction catalyzed by CitG. Second, holo-ACP is formed from apo-ACP and the prosthetic group precursor in a reaction catalyzed by CitX.

Evaluation of performance of the Gen-Probe human immunodeficiency virus type 1 viral load assay using primary subtype A, C, and D isolates from Kenya.

Accurate and sensitive quantification of human immunodeficiency virus type 1 (HIV-1) RNA has been invaluable as a marker for disease prognosis and for clinical monitoring of HIV-1 disease. The first generation of commercially available HIV-1 RNA tests were optimized to detect the predominant HIV-1 subtype found in North America and Europe, subtype B. However, these tests are frequently suboptimal in detecting HIV-1 genetic forms or subtypes found in other parts of the world. The goal of the present study was to evaluate the performance of a new viral load assay with non-subtype B viruses. A transcription-mediated amplification method for detection and quantitation of diverse HIV-1 subtypes, called the Gen-Probe HIV-1 viral load assay, is under development. In this study we examined the performance of the Gen-Probe HIV-1 viral load assay relative to that of the commonly used commercial HIV-1 RNA assays using a panel of primary isolates from Kenya. For comparison, we included several subtype B cloned viruses, and we quantified each virus using an in-house quantitative-competitive reverse transcriptase PCR (QC-RT-PCR) method and gag(p24) antigen capture. The Gen-Probe HIV-1 viral load assay and a version of the Roche AMPLICOR HIV-1 MONITOR test (version 1.5) that was designed to detect a broader range of subtypes were both sensitive for the quantification of Kenyan primary isolates, which represented subtype A, C, and D viruses. The Gen-Probe HIV-1 viral load assay was more sensitive for the majority of viruses than the Roche AMPLICOR HIV-1 MONITOR test version 1.0, the Bayer Quantiplex HIV RNA 3.0 assay, or a QC-RT-PCR method in use in our laboratory, suggesting that it provides a useful method for quantifying HIV-1 RNAs from diverse parts of the world, including Africa.

The impact of cultural values on patient education for the elderly.

Little literature has examined the interaction of culture and patient education with specific reference to the elderly. It is necessary for nurses to develop a respectful understanding of their patient's culture in order to provide meaningful and relevant patient education. Understanding of cultural differences is especially important when educating elderly immigrants, as many elderly retain significant aspects of their native cultural traditions and may be slower to acculturate to Western cultural values. This paper will outline a number of assessment parameters and educational strategies necessary for nurses to begin to provide culturally sensitive patient education for elderly patients within the nephrology setting.

Reading research: a review of a research critical appraisal workshop.

Evidence-based practice requires that nurses possess the skills to critically evaluate and apply research findings. Unfortunately, many nurses have had no formal training in research appraisal. A five-hour workshop was offered to nurses working within nephrology settings, to assist nurses to develop research appraisal skills. This article provides an outline of the workshop agenda and reviews the session evaluations made by participants and group facilitators. Based upon the feedback from the five-hour workshop, a model for a future research appraisal workshop is presented.

Managers' leadership and critical care nurses' intent to stay.

BACKGROUND: Cyclical fluctuations in turnover of critical care nurses are a large and complex problem. Managers' leadership characteristics may be a determinant of critical care nurses' intent to stay in the job. OBJECTIVE: To examine the direct and indirect effects of nurse-managers' characteristics of power, influence, and leadership style on critical care nurses' intent to stay in the nurses' employment positions. METHODS: The sample was 255 staff nurses in intensive care units at 4 urban hospitals. Established instruments with sound reliability and validity were used to assess the predictor, intervening, and outcome variables. Path analysis was used to examine the relationships in a conceptual model of intent to stay. RESULTS: The model explained 52% of the variance in intent to stay, and managers' characteristics were significant at each stage. Managers' position power and influence over work coordination had a direct link to intent to stay; structuring expectations and consideration contributed indirectly through the variables of instrumental communication, autonomy, and group cohesion. Instrumental communication, autonomy, and group cohesion decreased job stress and thus increased job satisfaction. Job satisfaction was directly linked with intent to stay. CONCLUSIONS: Inclusion of nurse-managers' characteristics explained more variance in intent to stay than did previous models. Managers with leadership styles that seek and value contributions from staff, promote a climate in which information is shared effectively, promote decision making at the staff nurse level, exert position power, and influence coordination of work to provide a milieu that maintains a stable cadre of nurses.

Historadioautographic localization, pharmacology and ontogeny of V(1a) vasopressin binding sites in the rat kidney.

The localization and pharmacological characteristics of vasopressin (VP) binding sites of the V(1a) subtype in developing and adult rat kidney were investigated by radioautography on kidney sections incubated in the presence of a radioiodinated selective V(1a) antagonist. Their localization after in vivo systemic infusion of the radioligand was also investigated. V(1a) binding sites first appear at embryonic day 16 on vascular elements. In the adult, they were localized in the cortex (vascular and tubular structures, juxtaglomerular apparatus), the outer medulla outer stripe (vasa recta) and inner stripe (thin descending limbs of short looped nephrons) and the inner medulla (collecting ducts). Data obtained in vitro were confirmed by in vivo binding at postnatal day 30 (PN30). Whatever their localizations, the V(1a) binding sites exhibited full V(1a) pharmacological profile in postnatal stages rats and in adult rats: a high affinity (nM range) for VP and for the V(1a) agonist, a lower affinity (microM range) for oxytocin and no affinity for the oxytocin agonist. The presence of V(1a) binding sites in these different structures raises the question of the putative roles of VP in modulating renal functions. A striking finding is the presence of V(1a) binding sites in the outer medullary thin descending limbs of short looped nephrons suggesting their colocalization with urea transporters.

The periplasmic domain of the histidine autokinase CitA functions as a highly specific citrate receptor.

The two-component regulatory system CitA/CitB is essential for induction of the citrate fermentation genes in Klebsiella pneumoniae. CitA represents a membrane-bound sensor kinase consisting of a periplasmic domain flanked by two transmembrane helices, a linker domain and the conserved kinase or transmitter domain. A fusion protein (MalE-CitAC) composed of the maltose-binding protein and the CitA kinase domain (amino acids 327-547) showed constitutive autokinase activity and transferred the gamma-phosphate group of ATP to its cognate response regulator CitB. The autokinase activity of CitA was abolished by an H350L exchange, and phosphorylation of CitB was inhibited by a D56N exchange, indicating that H-350 and D-56 represent the phosphorylation sites of CitA and CitB respectively. In the presence of ATP, CitB-D56N formed a stable complex with MalE-CitAC. To analyse the sensory properties of CitA, the periplasmic domain (amino acids 45-176) was overproduced as a soluble, cytoplasmic protein with a C-terminally attached histidine tag (CitAPHis). Purified CitAPHis bound citrate, but none of the other tri- and dicarboxylates tested, with high affinity (KD approximately 5 microM at pH 7) in a 1:1 stoichiometry. As shown by isothermal titration calorimetry, the binding reaction was driven by the enthalpy change (DeltaH = -76.3 kJ mol-1), whereas the entropy change was opposed (-TDeltaS = + 46.3 kJ mol-1). The pH dependency of the binding reaction indicated that the dianionic form H-citrate2- is the citrate species recognized by CitAPHis. In the presence of Mg2+ ions, the dissociation constant increased significantly, suggesting that the Mg-citrate complex is not bound by CitAPHis. This work defines the periplasmic domain of CitA as a highly specific citrate receptor and elucidates the binding characteristics of CitAPHis.