Fluorescent Staining Method Research Articles

Highly Selective and Fast Diagnosis of Alzheimer's Disease Hallmark Lesions using Congo Red in Isopropyl Alcoholic Solution

A highly selective, rapid, inexpensive, simple, and immunocytochemical compatible fluorescence staining method for Alzheimer's disease hallmark lesions applicable to sections of human specimens embedded in paraffin is described. Human necropsy material was fixed in buffered formalin, sectioned at 10 μm, mounted on slides, deparaffinized, and partially hydrated (70% ethanol). After partial hydration, sections were stained for 10 min in a solution of 0.2% Congo red in 70% isopropanol. After washing in 70% isopropanol and rehydration, auto-fluorescence of sections were quenched (optional) and processed for immunocytochemistry (optional). Finally, sections were mounted in an adequate mounting medium. Amyloid deposits appear pink at light microscopy and all Alzheimer's disease hallmark lesions appear orange or red under fluorescence microscopy using blue or green exciting light, respectively. The present method can be used in combination with all pre- or post-immunocytochemical techniques.

A novel, Golgi-Cox-based fluorescent staining method for visualizing full-length processes in primary rat neurons

The Golgi method, a well-known method used for staining whole dendrites and axonal trees of neurons, has been used widely for studying dendritic growth in vivo. Although detailed structural examination of neurons and their processes stained by the Golgi method has elucidated the complicated neuronal circuit, application of the method in cultured neurons has been unsuccessful to date.Here, we report the development of a stable, highly sensitive Golgi-Cox method that allows visualization of full-length processes, including the dendritic spines and the growth cones, of cultured rat neurons. This modified staining method requires: (1) rat cultured neurons fixed with a mixture containing 4% paraformaldehyde and 12.5% glutaraldehyde before impregnation with mercury; (2) rapid freezing of the fixed neurons using dry ice; and (3) immersion of the fixed neurons in Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulin-G antibody (Molecular Probes, Eugene, OR) for visualization after impregnation.

Alteration of T cell subtypes in spleen and antibodies of serum in mice infected with Angiostrongylus cantonensis

The immune responses of Angiostrongylus cantonensis infection are closely relevant to the host's self-protection and the nematode's pathogenesis. In the present study, BALB/c mice were randomly divided into uninfected control group, infection group 1, and infection group 2. The infection group 1 and infection group 2 were infected with 20 and 40 third-stage larvae of A. cantonensis per mouse, respectively. The splenocytes from the mice were collected and cultured on the 19th and 25th days post-infection; the subtypes of T cells in splenocytes were detected by flow cytometry with fluorescence staining method, and the cytokines in cultured supernatants of splenocytes were assayed by the method of ELISA. The specific IgG and IgE antibodies in sera of the mice were periodically detected by ELISA. The results showed that the percentages of CD4(+) and CD4(+) IL-4(+) T cells in splenocytes of infected mice were much higher (P < 0.05) than those in control mice; however, the percentages of CD4(+) IL-17(+) and CD4(+) IFN-γ(+) T cell were much lower(P < 0.01) after the infection. The levels of CD8(+) T cells in infected mice also rose, but differences between control mice and infected mice were not significant. In comparison with control mice, the concentration of IL-4 in the cultured supernatants of splenocytes in infected mice increased significantly (P < 0.05), but that of IL-17 decreased significantly (P < 0.01). In addition, the number of larvae infected and days after infection may influence levels of the T cell subtypes and the cytokines in spleen, too (P > 0.05). On humoral immunity, the levels of specific IgG antibodies in sera rose a bit at the fifth day post-infection, and reached a peak at the 20th day post-infection; the specific IgE antibodies gradually heightened during first 10days post-infection; then, it showed a downward trend during the 15th to 25th days post-infection. It is evident that the percentages of CD4(+) T lymphocytes of spleen in the mice infected with A. cantonensis markedly increase and polarize to Th2 phenotypes, and the function of Th17 cells is inhibited. In addition, the elevation of specific IgG antibodies in sera of the infected mice is more significant than that of specific IgE antibodies.

A Comparison of Ziehl-Neelsen Staining and Fluorescent Microscopy for Diagnosis of Pulmonary Tuberculosis

According to WHO one third of the world population have tuberculosis. The present study was undertaken to compare the efficacy of flurochrome(FI) stain with conventional Z-N stain in the diagnosis of pulmonary & extra- pulmonary tuberculosis. 388 cases of suspected pulmonary tuberculosis were included in the study. All samples were screen for Acid Fast Bacilli (AFB) by Z-N & Fluorescent staining methods. Positive samples detected by fluorescent stain were 57(14.69%) when compared to Zn stain 29(7.47%). Conclusion: Compared to Z-N stain (7.47%) flurochrome staining was found to be more efficient (14.69%) in AFB detection of AFB from cases of Pulmonary Tuberculosis.

A fluorescent staining method for marking the cuttlefish(Sepiella japonica)

It is necessary to establish a highly efficient method of marking,which will provide technical guarantee for a more accurate assessment of stock-enhancement effect of the cuttlefish(Sepiella japonica).The cuttlefish were chosen as the experimental material in this study.The larval cuttlefish were immersed in alizarin complexone(ALC)to dye the cuttlebone.The experiment was divided into 5 groups with concentration gradient and 2 time periods,and doral mantle length,body weight and survival rate of cuttlefish were measured and observed.The trial group and control group(50 ind/group)were reared in 2.5 m diameter circular concrete ponds,respectively.The cuttlefish were sampled randomly in order to examine the dye-retention of the tagged cuttlebone after being tagged 5,12,19,26 d.Moreover,the tagged cuttlefish were examined at high temperature treatment for 5 min.The results showed that the optimised dye concentration and immerged time were 90 mg/L and 24 h,respectively.The doral mantle length of cuttlefish had a significant positive correlation with body weight(P0.01),and there was no significant difference in growth between trial group and control group(P0.05).The tagged oval pink ring was still clear in the cuttlebone even after being tagged 26 d.The range and color of tagging parts were not changed before and after high temperature treatment.Additionally,the proposed method has effect on both internal signs and external signs,and the survival rate and dye-retention rate of the tagged cuttlefish are 100%.It is concluded that this method can be successfully applied to marking cuttlefish,and offer a number of benefits,including simple,efficient,high sample throughput,and a wide range of application prospects.

Genotoxic and Cytotoxic Evaluation of Pyrethroid Insecticides λ-Cyhalothrin and α-Cypermethrin on Human Blood Lymphocyte Culture

The present study aimed to investigate the genotoxic, cytotoxic and aneugenic effects of 1, 2, 3.75, 7.5, 15, 30 μM concentrations of the insecticides λ-cyhalothrin (LCT) and α-cypermethrin (CYP) on human peripheral blood lymphocyte culture using micronucleus (MN) and fluorescence in situ hybridisation (FISH) methods. All the concentrations were tested to assess the MN and apoptosis effects, and 1 and 2 μM LCT and 7.5 and 15 μM CYP concentrations were tested for FISH analysis. The cytotoxic effect was also observed using trypan blue and the acridine orange/ethidium bromide fluorescence staining method to measure the apoptotic effect. It was observed that both of the insecticides had a cytotoxic effect at all the concentrations (p ≤ 0.001) and apoptotic effect for LCT at 15-30 μM (p ≤ 0.05; p ≤ 0.01) for CYP between 2 and 30 μM concentrations (p ≤ 0.05; p ≤ 0.01). The micronuclei that developed after exposure were induced because of an aneugenic effect (p ≤ 0.001). LCT and CYP might be spindle poisons or caused damaged to centromere/kinetochore function.

Anti-bacterial performance of Zirconia coatings on Titanium implants

Bacterial adhesion and colonization are considered to play key roles in the pathogenesis of peri-implant disease, which is an inflammatory process leading the destruction of both soft and hard tissue around a Titanium (Ti) implant. The osseointegration of Ti implants is influenced by their composition and surface treatment. Zirconia (ZrO2) coatings have been shown to improve implant osseointegration in various biomedical fields, including orthopedic devices and dental implants. This study used a twin-gun magnetron sputtering system to deposit ZrO2–Sliver (Ag) and ZrO2–Copper (Cu) coatings on biograde pure-Ti implant materials. The effects of Ag and Cu on the antibacterial properties of the coated Ti samples were investigated using Staphylococcus aureus (S. aureus) and Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans), which are found frequently in implant-associated infections. The in vitro antibacterial analyses employed a fluorescence staining method using SYTO9 and bacterial-viability tests on agar plates. Incorporating Ag and Cu into ZrO2 changed the structure of crystalline ZrO2, and the nanostructures of the resulting ZrO2–Ag and ZrO2–Cu coatings were found to be correlated with the antibacterial performance. The adherence of S. aureus and A. actinomycetemcomitans was less for ZrO2 samples with a small doping of Cu than for uncoated Ti. The ZrO2–Ag coating with an Ag content of 10.6at.%, which showed hydrophobicity, exhibited the lowest bacterial retention and viability of S. aureus and A. actinomycetemcomitans. The results suggest that ZrO2–Ag and ZrO2–Cu coatings improve the antibacterial performance relative to pure-Ti implant materials.

Incorporated nematocysts in Aeolidiella stephanieae (Gastropoda, Opisthobranchia, Aeolidoidea) mature by acidification shown by the pH sensitive fluorescing alkaloid Ageladine A

The sequestration of nematocysts (a special group of cnidocysts) from cnidarian prey with subsequent use in defence is described for few metazoan phyla. Members of the taxon Aeolidoidea (Nudibranchia, Gastropoda) are well-known for this. Questions regarding the reasons some nematocysts do not discharge when the gastropod feeds and how these same nematocysts can be transported along the digestive tract into specialized morphological structures called cnidosacs, remain unanswered. Within the cnidosac, nematocysts are incorporated in cells and finally be used for defence against predators.The most plausible explanation for this phenomenon suggests there are immature and therefore non-functional nematocysts in the food. A recent study by Berking and Herrmann (2005) on cnidarians suggested that the nematocysts mature by acidification via proton transfer into the nematocyst capsule. According to this hypothesis only immature nematocysts are transported into the cnidosac where they are then made functional through an accumulation of protons. In this study we present a fluorescence staining method that tests the hypothesis by Berking and Herrmann (2005) and detects changes in the pH values of incorporated nematocysts, interpreted as changes in maturation stages. This marker, the fluorescent dye Ageladine A, stains nematocyst capsules according to their pH values. With Ageladine A we were able to show that kleptocnides indeed change their pH value after incorporation into the aeolidoidean cnidosac.

Rapid Image-based Cytometry for Comparison of Fluorescent Viability Staining Methods

The ability to accurately measure cell viability is important for any cell-based research. Traditionally, viability measurements have been performed using trypan blue exclusion method on hemacytometer, which allowed researchers to visually distinguish viable from nonviable cells. However, the trypan blue method is often limited to only cell lines or primary cells that have been rigorously purified. In the recent years, small desktop image-based cell counters have been developed for rapid cell concentration and viability measurement due to advances in imaging and optics technologies as well as novel fluorescent stains. In this work, we employed the Cellometer image-based cytometer to demonstrate the ability to simplify viability detection compared to the current methods. We compared various fluorescence viability detection methods using single- or dual-staining technique. Single-staining method using nucleic acid stains including ethidium bromide, propidium iodide, 7AAD, DAPI, Sytox Green and Sytox Red, and enzymatic stains including CFDA and Calcein AM were performed. All stains produced comparable results to trypan blue exclusion method for cell line samples. Dual-staining method using AO/PI, CFDA/PI, Calcein AM/PI and Hoechst 33342/PI that enumerates viable and non-viable cells was tested on primary cell samples with high debris contents. This method allowed exclusion of cellular debris and non-nucleated cells from analysis, which can eliminate the need to perform purification step during sample preparation, and improves the efficiency of viability detection method. Overall, these image-based fluorescent cell counters can simplify assay procedures as well as capture images for visual confirmation.

Effects of salvianolic acid B on in vitro growth inhibition and apoptosis induction of retinoblastoma cells.

To observe the effects of salvianolic acid B (SalB) on in vitro growth inhibition and apoptosis induction of retinoblastoma HXO-RB44 cells. The effects of SalB on the HXO-RB44 cells proliferation in vitro were observed by MTT colorimetric method. The morphological changes of apoptosis before and after the treatment of SalB were observed by Hoechst 33258 fluorescent staining method. Apoptosis rate and cell cycle changes of HXO-RB44 cells were detected by flow cytometer at 48 hours after treated by SalB. The expression changes of Caspase-3 protein in HXO-RB44 cells were detected by Western Blot. SalB significantly inhibited the growth of HXO-RB44 cells, while the inhibition was in a concentration-and time-dependent manner. The results of fluorescent staining method indicated that HXO-RB44 cells showed significant phenomenon of apoptosis including karyorrhexis, fragmentation and the formation of apoptotic bodies, etc. after 24, 48 and 72 hours co-culturing of SalB and HXO-RB44 cells. The results of flow cytometer showed that the apoptosis rate and the proportion of cells in S phase were gradually increased at 48 hours and 72 hours after treated by different concentrations of SalB. Western Blot strip showed that the expression of Caspase-3 protein in HXO-RB44 cells was gradually increased with the increase of the concentration of SalB. SalB can significantly affect on HXO-RB44 cells growth inhibition and apoptosis induction which may be achieved through the up-regulation of Caspase-3 expression and the induction of cell cycle arrest.

Assessment of Salmonella enteritidis Viability in Egg White during Early Incubation Stages by Fluorescent Staining Method

Assessment of Salmonella enteritidis Viability in Egg White during Early Incubation Stages by Fluorescent Staining Method

Characterization and antibacterial performance of ZrNO–Ag coatings

Composite metal nitride–metal (MeN–Me) physical vapor deposited (PVD) films (e.g. TaN–Cu, TiN–Cu, ZrN–Ag, etc.) are often considered for applications such as tribological and antibacterial coatings. In this study, ZrN, ZrO2–Ag and ZrNO–Ag coatings with different Ag contents were deposited by reactive magnetron sputtering. Monoclinic ZrO2 and fcc Ag composite structures were obtained when Zr and Ag were cosputtered to react with oxygen. ZrNO–Ag showed a porous structure containing monoclinic ZrO2, fcc Ag and Zr2ON2. Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) and Staphylococcus aureus (S. aureus), major pathogens frequently found in the implant-associated infections, were cultured on the coated samples. The antibacterial effects were determined using in vitro anti-bacterial analyses by a fluorescence staining method employing Syto9 and bacterial viability agar tests. In addition, the biocompatibility of human gingival fibroblast (HGF) cells on coatings was also evaluated by a MTT test assay. It showed that the nanostructure and Ag content of the ZrO2–Ag and ZrNO–Ag coatings were correlated with the biocidal property. The ZrN coating deposited by magnetron sputtering did not improve the bacterial reduction of A. actinomycetemcomitans and S. aureus. The ZrO2–Ag and ZrNO–Ag coated samples had lower bacterial retention, and the ZrNO–Ag coatings with silver content of 11.8at.% possessed the best antibacterial performance with excellent HGF cell compatibility as well.

Enhancement of amygdalin activated with β-d-glucosidase on HepG2 cells proliferation and apoptosis

The growth inhibition and induction of apoptosis brought by amygdalin and activated with β-d-glucosidase were tested for cytoactivity in HepG2 cells. The MTT viability assay showed that all samples had effects on HepG2 proliferation in dose and time response manners. IC50 of stand-alone amygdalin and activation with β-d-glucosidase on the proliferation of HepG2 cells for 48h were 458.10mg/mL and 3.2mg/mL, respectively. Moreover, apoptotic cells were determined by AO/EB (acridine orange/ethidium bromide) fluorescent staining method and Annexin V-FITC/PI staining flow cytometry cell cycle analysis. With increasing of amygdalin concentration and the incubation time, the apoptotic rate was heightened. Compared with the control, there was significant difference (p<0.01). Together, these findings indicate that amygdalin had no strong anti-HepG2 activity; however the ingredients of amygdalin activated with β-d-glucosidase had a higher and efficient anti-HepG2 activity. It was therefore suggested that this combination strategy may be applicable for treating tumors with a higher activity.

The effects of thermal stimuli on intracellular calcium change and histamine releases in rat basophilic leukemia mast cells

The effects of thermal stimuli on rat basophilic leukemia mast cells were studied. The cells in calcium-contained or calcium-free buffers were thermally stimulated in the temperature range of 25–60 °C. The corresponding calcium ion concentration in cells [Ca2+]i as well as the released histamine from cells was measured with fluorescence staining methods. The ruthenium red (RR), a block of membrane calcium channels (transient receptor potential family V (TRPV)), was used in experiments. Under the stimulus of 25–50 °C, no significant difference on [Ca2+]i was found between these three groups of the cells in calcium-contained buffer without or with RR and cells in calcium-free saline, indicating that the increased calcium in cytosol did not result from the extracellular buffer but came from the intracellular calcium stores. The [Ca2+]i continuously increased under the temperature of 50–60 °C, but the RR and calcium-free saline can obviously diminish the [Ca2+]i increase at these high temperatures, reflecting that the opening of the TRPV2 channels leads to a calcium influx resulting in the [Ca2+]i increment. The histamine release also became significant in these cases. Since the released histamine is a well-known mediator for the microcirculation promotion, the histamine release from mast cells could be one of the mechanisms of thermal therapy.

Analysis of oxidative stress-induced protein carbonylation using fluorescent hydrazides

Protein carbonyl detection has been commonly used to analyze the degree of damage to proteins under oxidative stress conditions. Most laboratories rely on derivatization of carbonyl groups with dinitrophenylhydrazine followed by Western blot analysis using antibodies against the dinitrophenyl moiety. This paper describes a protein carbonyl detection method based on fluorescent Bodipy, Cy3 and Cy5 hydrazides. Using this approach, Western blot and immunodetection are no longer needed, shortening the procedure and increasing accuracy. Combination of Cy3 and Cy5 hydrazides allows multiplexing analyses in a single two-dimensional gel. Derivatization with Bodipy hydrazide allows easy matching of the spots of interest and those obtained by general fluorescent protein staining methods, which facilitates excising target proteins from the gels and identifying them. This method is effective for detecting protein carbonylation in samples of proteins submitted to metal-catalyzed oxidation "in vitro" and assessing the effect of hydrogen peroxide and chronological aging on protein oxidative damage in yeast cells.

92P Tff1 Trefoil Protein Involves in Resistance to Doxorubicin-Induced Apoptosis in Breast Cancer Cell

ABSTRACT TFF1 trefoil protein is a small secretory protein expressed in a various type of carcinomas including breast cancer. TFF1 gene contained estrogen responsive element and its expression could be regulated by estrogen. In addition, estrogen has been demonstrated its ability to promote resistance to doxorubicin in estrogen receptor positive MCF-7 breast cancer cell line. Moreover, it has been reported that TFF1 can protect conjunctival cells from UV-induced apoptosis. In this study, we demonstrated the role of TFF1 in estrogen-promoted resistance to doxorubicin-induced apoptosis using MCF-7 model. Permanent knockdown of TFF1 gene in MCF-7 cell had been generated and used to test the sensitivity to doxorubicin treatment compared to parental cell in the conditions present or absent of 17b-estradiol, a potent estrogenic agent. The apoptosis cells were measured by fluorescence staining and flow cytometry method. The results showed that among the stimulation of apoptosis by doxorubicin, 17b-estradiol could suppress this process in parental MCF-7 cell but not in TFF1 knockdown MCF-7 cell. Moreover, by trypan blue staining method, it has been shown that anti-TFF1 antibody could reverse the anti-apoptotic effect of estrogen in parental MCF-7 cell and recombinant TFF1 could recover TFF1 knockdown MCF-7 cell death induced by doxorubicin. However, this process could not be inhibited by fulvestrant, an estrogen antagonist. Apoptosis protein array experiment reflected the role of the anti-oxidative enzymes catalase and heme oxygenase 1 in estrogen and TFF1 modulated apoptosis. These phenomena determine the role of TFF1 in estrogen-promoted resistance to apoptosis induced by doxorubicin in MCF-7 breast cancer cell. TFF1 gene may be a target for enhancing of sensitivity to chemotherapy in breast cancer treatment. Disclosure All authors have declared no conflicts of interest.

Rapid flow cytometric method for viability determination of solventogenic clostridia

We endeavored to develop a method for viability determination of solventogenic clostridia and to apply it for monitoring acetone-butanol-ethanol (ABE) fermentation. Six fluorescent probes (propidium iodide [PI], ethidium bromide, fluorescein diacetate, carboxyfluorescein diacetate [cFDA], rhodamine 123, bis-(1,3-dibutylbarbituric acid)trimethine oxonol [BOX]) were tested in order to distinguish two subpopulations of live and dead clostridial cells in suspension. Three of them were found to be appropriate (PI, BOX and cFDA) for this purpose. Developed fluorescent staining methods were applied to batch fermentation processes of Clostridium pasteurianum and C. beijerinckii carried out in a laboratory bioreactor under anaerobic conditions. Whereas PI was found to be applicable to both strains, BOX was convenient only for viability determination of C. pasteurianum. Although cFDA can distinguish two cell subpopulations in suspension, it was found to be unsuitable for viability determination under tested conditions, since it reflected more variable esterase activity during sporulation cell cycle than viability. Flow cytometry in combination with convenient fluorescent probe has been proved to be a valuable tool for viability determination. We assume this rapid and simple method can help to obtain more complex and precise information about ABE fermentation.

Cellular attachment and osteoblast differentiation of mesenchymal stem cells on natural cuttlefish bone

The purpose of this study was to describe an approach that aims to provide fundamental information for the application of natural cuttlefish bone. Before applying cuttlefish bone as a bone defect filling material, we evaluated proliferation, adhesion, and cell viability of human mesenchymal stem cells (hMSCs) cultured on cuttlefish bone. Cuttlefish bone was separated into two parts (dorsal shield and lamellar region) and each part was used. Cell proliferation and viability were assessed using the MTS assay and live/dead fluorescence staining method. The morphology was observed using scanning electron microscopy (SEM). hMSCs were stimulated with osteogenic medium and osteoblast differentiation was evaluated. The fluorescence images showed that the seeded cells grew well and that cell distribution was in accordance with the surface morphology of the cuttlefish bone. Compared with the dorsal shield, cells penetrated deeper into the three-dimensional inner space of the lamellar part. Furthermore, under osteogenic differentiation conditions, alkaline phosphatase activity increased and the mRNA expression of ALP, runt-related transcription factor 2, and collagen type I α1 was increased in hMSCs cultured on both the dorsal shield and lamellar block. These results indicate the potential of cuttlefish bone as an ideal scaffold for bone regenerative materials. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

A new fluorescent staining method for callose of microspore mother cells during meiosis

To observe the dynamic behavior of callose of microspore mother cells during meiosis, we developed a convenient, rapid and efficient staining method using an improved carbol fuchsin/aniline blue solution. The stained microspore mother cells during meiosis showed yellowish green callose, red cytoplasm and dark red chromosomes when excited with blue light, which produced a contrasting image with a three-dimensional effect. When stained with only improved carbol fuchsin solution, the cells had red cytoplasm and chromosomes when excited with green light. The improved carbol fuchsin solution can be used to replace other more expensive DNA-specific dyes, such as DAPI and H33258, to reduce experimental costs.

The ChrA Response Regulator in Corynebacterium diphtheriae Controls Hemin-Regulated Gene Expression through Binding to the hmuO and hrtAB Promoter Regions

Corynebacterium diphtheriae, the etiologic agent of diphtheria, utilizes heme and hemoglobin (Hb) as iron sources for growth. Heme-iron utilization involves HmuO, a heme oxygenase that degrades cytosolic heme, resulting in the release of heme-associated iron. Expression of the hmuO promoter is under dual regulation, in which transcription is repressed by DtxR and iron and activated by a heme source, such as hemin or Hb. Hemin-dependent activation is mediated primarily by the ChrAS two-component system, in which ChrS is a putative heme-responsive sensor kinase while ChrA is proposed to serve as a response regulator that activates transcription. It was recently shown that the ChrAS system similarly regulates the hrtAB genes, which encode an ABC transporter involved in the protection of C. diphtheriae from hemin toxicity. In this study, we characterized the phosphorelay mechanism in the ChrAS system and provide evidence for the direct regulation of the hmuO and hrtAB promoters by ChrA. A fluorescence staining method was used to show that ChrS undergoes autophosphorylation and that the phosphate moiety is subsequently transferred to ChrA. Promoter fusion studies identified regions upstream of the hmuO and hrtAB promoters that are critical for the heme-dependent regulation by ChrA. Electrophoretic mobility shift assays revealed that ChrA specifically binds at the hmuO and hrtAB promoter regions and that binding is phosphorylation dependent. A phosphorylation-defective mutant of ChrA [ChrA(D50A)] exhibited significantly diminished binding to the hmuO promoter region relative to that of wild-type ChrA. DNase I footprint analysis further defined the sequences in the hmuO and hrtAB promoters that are involved in ChrA binding, and this analysis revealed that the DtxR binding site at the hmuO promoter partially overlaps the binding site for ChrA. DNase I protection studies as well as promoter fusion analysis suggest that ChrA and DtxR compete for binding at the hmuO promoter. Collectively, these data demonstrate that the ChrA response regulator directly controls the expression of hmuO and the hrtAB genes and the binding activity of ChrA is dependent on phosphorylation by its cognate sensor kinase ChrS.