ABSTRACT
Herbaspirillum seropedicae GlnB (GlnB-Hs) is a signal transduction protein involved in the control of nitrogen, carbon and energetic metabolism. The adsorption of GlnB-Hs deposited by spin coating on hydrophilic and hydrophobic silicon forms a thin layer that was characterized using atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared attenuated total reflectance spectroscopy (FTIR-ATR). AFM allowed the identification of globular, face-up donut like array of protein on hydrophilic silicon substrate, favoring deprotonated residues to contact the silicon oxide surface. Over hydrophobic silicon, GlnB-Hs adopts a side-on conformation forming a filament network, avoiding the contact of protonated residues with silicon surface. XPS allowed us to determine the protonated and non-protonated states of nitrogen 1s (N 1s). The FTIR-ATR measurements provided information about protein secondary structure and its conservation, after surface adsorption.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Microscopy, Atomic Force/methods , Photoelectron Spectroscopy/methods , Silicon/chemistry , Spectroscopy, Fourier Transform Infrared/methods , Herbaspirillum/metabolism , Hydrophobic and Hydrophilic Interactions , Protein Binding , Protein Structure, Secondary , Static ElectricityABSTRACT
The control of nitrogen metabolism in pathogenic Gram-positive bacteria has been studied in a variety of species and is involved with the expression of virulence factors. To date, no data have been reported regarding nitrogen metabolism in the odontopathogenic species Streptococcus mutans. GlnR, which controls nitrogen assimilation in the related bacterial species, Bacillus subtilis, was assessed in S. mutans for its DNA and protein binding activity. Electrophoretic mobility shift assay of the S. mutans GlnR protein indicated that GlnR binds to promoter regions of the glnRA and amtB-glnK operons. Cross-linking and pull-down assays demonstrated that GlnR interacts with GlnK, a signal transduction protein that coordinates the regulation of nitrogen metabolism. Upon formation of this stable complex, GlnK enhances the affinity of GlnR for the glnRA operon promoter. These results support an involvement of GlnR in transcriptional regulation of nitrogen metabolism-related genes and indicate that GlnK relays information regarding ammonium availability to GlnR.
Subject(s)
Animals , Rats , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial/genetics , Nitrogen/metabolism , Operon/genetics , Promoter Regions, Genetic/genetics , Streptococcus mutans/metabolism , Base Sequence , Bacterial Proteins/genetics , DNA, Bacterial/genetics , Molecular Sequence Data , Rats, Wistar , Streptococcus mutans/geneticsABSTRACT
The control of nitrogen metabolism in pathogenic Gram-positive bacteria has been studied in a variety of species and is involved with the expression of virulence factors. To date, no data have been reported regarding nitrogen metabolism in the odontopathogenic species Streptococcus mutans. GlnR, which controls nitrogen assimilation in the related bacterial species, Bacillus subtilis, was assessed in S. mutans for its DNA and protein binding activity. Electrophoretic mobility shift assay of the S. mutans GlnR protein indicated that GlnR binds to promoter regions of the glnRA and amtB-glnK operons. Cross-linking and pull-down assays demonstrated that GlnR interacts with GlnK, a signal transduction protein that coordinates the regulation of nitrogen metabolism. Upon formation of this stable complex, GlnK enhances the affinity of GlnR for the glnRA operon promoter. These results support an involvement of GlnR in transcriptional regulation of nitrogen metabolism-related genes and indicate that GlnK relays information regarding ammonium availability to GlnR.
Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial/genetics , Nitrogen/metabolism , Operon/genetics , Promoter Regions, Genetic/genetics , Streptococcus mutans/metabolism , Animals , Bacterial Proteins/genetics , Base Sequence , DNA, Bacterial/genetics , Molecular Sequence Data , Rats , Rats, Wistar , Streptococcus mutans/geneticsABSTRACT
Streptococcus mutans is a Gram-positive bacterium present in the oral cavity, and is considered to be one of the leading causes of dental caries. S. mutans has a glnK gene, which codes for a PII-like protein that is possibly involved in the integration of carbon, nitrogen and energy metabolism in several organisms. To characterize the GlnK protein of S. mutans, the glnK gene was amplified by PCR, and cloned into the expression vectors pET29a(+) and pET28b(+). The native GlnK-Sm was purified by anion exchange (Q-Sepharose) and affinity (Hi-Trap Heparin) chromatography. The GlnK-His-Sm protein was purified using a Hi-Trap Chelating-Ni2+ column. The molecular mass of the GlnK-His-Sm proteins was 85 kDa as determined by gel filtration, indicating that this protein is a hexamer in solution. The GlnK-His-Sm protein is not uridylylated by the Escherichia coli GlnD protein. The activities of the GlnK-Sm and GlnK-His-Sm proteins were assayed in E. coli constitutively expressing the Klebsiella pneumoniae nifLA operon. In K. pneumoniae, NifL inhibits NifA activity in the presence of high ammonium levels and the GlnK protein is required to reduce the inhibition of NifL in the presence of low ammonium levels. The GlnK-Sm protein was unable to reduce NifL inhibition of NifA protein. Surprisingly, the GlnK-His-Sm protein was able to partially reduce NifL inhibition of the NifA protein under nitrogen-limiting conditions, in a manner similar to the GlnK protein of E. coli. These results suggested that S. mutans GlnK is functionally different from E. coli PII proteins.
Subject(s)
Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial/genetics , Nitrogen/metabolism , PII Nitrogen Regulatory Proteins/genetics , Streptococcus mutans/genetics , Bacterial Proteins/metabolism , Chromatography, Affinity , Escherichia coli/genetics , Klebsiella pneumoniae/genetics , Nitrogen Fixation , PII Nitrogen Regulatory Proteins/metabolism , Polymerase Chain Reaction , Streptococcus mutans/metabolismABSTRACT
Streptococcus mutans is a Gram-positive bacterium present in the oral cavity, and is considered to be one of the leading causes of dental caries. S. mutans has a glnK gene, which codes for a PII-like protein that is possibly involved in the integration of carbon, nitrogen and energy metabolism in several organisms. To characterize the GlnK protein of S. mutans, the glnK gene was amplified by PCR, and cloned into the expression vectors pET29a(+) and pET28b(+). The native GlnK-Sm was purified by anion exchange (Q-Sepharose) and affinity (Hi-Trap Heparin) chromatography. The GlnK-His-Sm protein was purified using a Hi-Trap Chelating-Ni2+ column. The molecular mass of the GlnK-His-Sm proteins was 85 kDa as determined by gel filtration, indicating that this protein is a hexamer in solution. The GlnK-His-Sm protein is not uridylylated by the Escherichia coli GlnD protein. The activities of the GlnK-Sm and GlnK-His-Sm proteins were assayed in E. coli constitutively expressing the Klebsiella pneumoniae nifLA operon. In K. pneumoniae, NifL inhibits NifA activity in the presence of high ammonium levels and the GlnK protein is required to reduce the inhibition of NifL in the presence of low ammonium levels. The GlnK-Sm protein was unable to reduce NifL inhibition of NifA protein. Surprisingly, the GlnK-His-Sm protein was able to partially reduce NifL inhibition of the NifA protein under nitrogen-limiting conditions, in a manner similar to the GlnK protein of E. coli. These results suggested that S. mutans GlnK is functionally different from E. coli PII proteins.
Subject(s)
Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial/genetics , Nitrogen/metabolism , PII Nitrogen Regulatory Proteins/genetics , Streptococcus mutans/genetics , Bacterial Proteins/metabolism , Chromatography, Affinity , Escherichia coli/genetics , Klebsiella pneumoniae/genetics , Nitrogen Fixation , PII Nitrogen Regulatory Proteins/metabolism , Polymerase Chain Reaction , Streptococcus mutans/metabolismABSTRACT
The protein GlnB-Hs (GlnB of Herbaspirillum seropedicae) in diazotroph micro-organisms signalizes levels of nitrogen, carbon, and energy for a series of proteins involved in the regulation of expression and control of the activity of nitrogenase complex that converts atmospheric nitrogen in ammonia, resulting in biological nitrogen fixation. Its structure has already been determined by X-ray diffraction, revealing a trimer of (36 kDa) with lateral cavities having hydrophilic boundaries. The interactions of GlnB-Hs with the well-known Si(111) surface were investigated for different incubation times, protein concentrations in initial solution, deposition conditions, and substrate initial state. The protein solution was deposited on Si(111) and dried under controlled conditions. An atomic force microscope operating in dynamic mode shows images of circular, linear, and more complex donut-shaped protein arrangement, and also filament types of organization, which vary from a few nanometers to micrometers. Apparently, the filament formation was favored because of protein surface polarity when in contact with the silicon surface, following some specific orientation. The spin-coating technique was successfully used to obtain more uniform surface covering.
Subject(s)
Bacterial Proteins/chemistry , Models, Chemical , PII Nitrogen Regulatory Proteins/chemistry , Silicon/chemistry , Adsorption , Bacterial Proteins/ultrastructure , Gene Expression Regulation, Bacterial , Genes, Bacterial/physiology , Gram-Negative Bacteria/chemistry , Herbaspirillum , Microscopy, Atomic Force/methods , Nitrogen/chemistry , Nitrogenase/chemistry , PII Nitrogen Regulatory Proteins/ultrastructure , Rhizobiaceae , Solutions , Surface Properties , X-Ray Diffraction/methodsABSTRACT
Two Azospirillum brasilense open reading frames (ORFs) exhibited homology with the two-component NtrY/NtrX regulatory system from Azorhizobium caulinodans. These A. brasilense ORFs, located downstream to the nifR3ntrBC operon, were isolated, sequenced and characterized. The present study suggests that ORF1 and ORF2 correspond to the A. brasilense ntrY and ntrX genes, respectively. The amino acid sequences of A. brasilense NtrY and NtrX proteins showed high similarity to sensor/kinase and regulatory proteins, respectively. Analysis of lacZ transcriptional fusions by the beta-galactosidase assay in Escherichia coli ntrC mutants showed that the NtrY/NtrX proteins failed to activate transcription of the nifA promoter of A. brasilense. The ntrYX operon complemented a nifR3ntrBC deletion mutant of A. brasilense for nitrate-dependent growth, suggesting a possible cross-talk between the NtrY/X and NtrB/C sensor/regulator pairs. Our data support the existence of another two-component regulatory system in A. brasilense, the NtrY/NtrX system, probably involved in the regulation of nitrate assimilation.
Subject(s)
Azospirillum brasilense/genetics , Bacterial Proteins/isolation & purification , Gene Expression Regulation, Bacterial/genetics , Genes, Bacterial , Amino Acid Sequence , Azospirillum brasilense/growth & development , Azospirillum brasilense/isolation & purification , Base Sequence , DNA, Bacterial/genetics , Escherichia coli/chemistry , Genetic Complementation Test , Molecular Sequence Data , Mutation , Nitrogen Fixation/geneticsABSTRACT
Two Azospirillum brasilense open reading frames (ORFs) exhibited homology with the two-component NtrY/NtrX regulatory system from Azorhizobium caulinodans. These A. brasilense ORFs, located downstream to the nifR3ntrBC operon, were isolated, sequenced and characterized. The present study suggests that ORF1 and ORF2 correspond to the A. brasilense ntrY and ntrX genes, respectively. The amino acid sequences of A. brasilense NtrY and NtrX proteins showed high similarity to sensor/kinase and regulatory proteins, respectively. Analysis of lacZ transcriptional fusions by the ß-galactosidase assay in Escherichia coli ntrC mutants showed that the NtrY/NtrX proteins failed to activate transcription of the nifA promoter of A. brasilense. The ntrYX operon complemented a nifR3ntrBC deletion mutant of A. brasilense for nitrate-dependent growth, suggesting a possible cross-talk between the NtrY/X and NtrB/C sensor/regulator pairs. Our data support the existence of another two-component regulatory system in A. brasilense, the NtrY/NtrX system, probably involved in the regulation of nitrate assimilation
Subject(s)
Azospirillum brasilense , Bacterial Proteins , Gene Expression Regulation, Bacterial , Nitrogen Fixation , Amino Acid Sequence , Azospirillum brasilense , Base Sequence , DNA, Bacterial , Escherichia coli , Genetic Complementation Test , MutationABSTRACT
Herbaspirillum seropedicae is a nitrogen-fixing bacterium found in association with economically important gramineae. Regulation of nitrogen fixation involves the transcriptional activator NifA protein. The regulation of NifA protein and its truncated mutant proteins is described and compared with that of other nitrogen fixation bacteria. Nitrogen fixation control in H. seropedicae, of the beta-subgroup of Proteobacteria, has regulatory features in common with Klebsiella pneumoniae, of the gamma-subgroup, at the level of nifA expression and with rhizobia and Azospirillum brasilense, of the alpha-subgroup, at the level of control of NifA by oxygen.
Subject(s)
Betaproteobacteria/genetics , Genes, Bacterial , Nitrogen Fixation/genetics , Bacterial Proteins/genetics , Carrier Proteins/genetics , Gene Expression Regulation, Bacterial , Gene Order , PII Nitrogen Regulatory Proteins , Transcription Factors/geneticsABSTRACT
The PII protein is apparently involved in the control of NifA activity in Herbaspirillum seropedicae. To evaluate the probable role of PII in signal transduction, uridylylation assays were conducted with purified H. seropedicae PII and Escherichia coli GlnD, or a cell-free extract of H. seropedicae as sources of uridylylating activity. The results showed that alpha-ketoglutarate and ATP stimulate uridylylation whereas glutamine inhibits uridylylation. Deuridylylation of PII-UMP was dependent on glutamine and inhibited by ATP and alpha-ketoglutarate. PII uridylylation and (or) deuridylylation in response to these effectors suggests that PII is a nitrogen level signal transducer in H. seropedicae.
Subject(s)
Bacterial Proteins/metabolism , Betaproteobacteria/metabolism , Signal Transduction , Uridine Monophosphate/metabolism , Adenosine Triphosphate/metabolism , Escherichia coli/metabolism , Glutamine/metabolism , Ketoglutaric Acids/metabolism , Nucleotidyltransferases/metabolism , PII Nitrogen Regulatory ProteinsABSTRACT
Two glnB-like genes have been isolated from Herbaspirillum seropedicae by complementation of the Klebsiella pneumoniae glnB502 mutant for growth on nitrate. One of these glnB-like genes has been sequenced and shows strong identity with GlnB proteins derived from other organisms. A Tn5-20 mutation of this glnB was Nif negative.
Subject(s)
Bacterial Proteins/genetics , Genes, Bacterial , Glutamate-Ammonia Ligase/metabolism , Gram-Negative Aerobic Bacteria/genetics , Ammonium Chloride/metabolism , Conjugation, Genetic , DNA Transposable Elements , Gene Expression Regulation, Bacterial , Genetic Complementation Test , Gram-Negative Aerobic Bacteria/metabolism , Klebsiella pneumoniae/genetics , Molecular Sequence Data , Mutagenesis, Insertional , Nitrates/metabolism , Nitrogen Fixation , PII Nitrogen Regulatory Proteins , Phenotype , Potassium Compounds/metabolismABSTRACT
The purpose of this study was to compare the amount of fluoride in plaque formed on glass ionomer cement or composite and to evaluate the effects of fluoride released on growth of cariogenic microflora, fluoride uptake, and secondary caries formation under in situ conditions of a high cariogenic challenge. Ten adult volunteers took part in this crossover study performed in two phases of 28 days. Eighty enamel blocks were randomly restored with glass ionomer cement (Chelon-Fil-Espe) or composite (Silux). During each phase of the study, an acrylic resin appliance, containing four enamel blocks restored with the same material, was constructed for each of the volunteers. During the experimental period, all subjects used fluoride-free dentifrice, refrained from brushing the restored enamel blocks, and immersed the appliances into 20 por cento sucrose solution eight times a day. Fluoride levels, mutans streptococci, and lactobacilli were assessed in dental plaque. Fluoride uptake and microhardness profiles were determined in enamel around the restorations. Statistical analyses indicated a significantly higher level of fluoride (p<0.05) and a lower level of mutans streptococci plaque formed on glass ionomer cement. Analysis of variance in a split-plot model indicated that in the enamel around the glass ionomer restoration the fluoride uptake was significantly greater (p<0.025) and the mineral loss significantly lower (p<0.01). The results show that glass ionomer cement presents a broad anticariogenic effect and may be of value in preventing secondary caries,even under conditions of a high caries risk
Subject(s)
Humans , Dental Caries/classification , Glass Ionomer Cements/therapeutic use , Dental Enamel/anatomy & histology , Fluorine/therapeutic use , In Vitro TechniquesABSTRACT
The purpose of this study was to compare the amount of fluoride in plaque formed on glass ionomer cement or composite and to evaluate the effects of fluoride released on growth of cariogenic microflora, fluoride uptake, and secondary caries formation under in situ conditions of a high cariogenic challenge. Ten adult volunteers took part in this crossover study performed in two phases of 28 days. Eighty enamel blocks were randomly restored with glass ionomer cement (Chelon-Fil-Espe) or composite (Silux). During each phase of the study, an acrylic resin appliance, containing four enamel blocks restored with the same material, was constructed for each of the volunteers. During the experimental period, all subjects used fluoride-free dentifrice, refrained from brushing the restored enamel blocks, and immersed the appliances into 20% sucrose solution eight times a day. Fluoride levels, mutans streptococci, and lactobacilli were assessed in dental plaque. Fluoride uptake and microhardness profiles were determined in enamel around the restorations. Statistical analyses indicated a significantly higher level of fluoride (p < 0.05) and a lower level of mutans streptococci plaque formed on glass ionomer cement. Analysis of variance in a split-plot model indicated that in the enamel around the glass ionomer restoration the fluoride uptake was significantly greater (p < 0.025) and the mineral loss significantly lower (p < 0.01). The results show that glass ionomer cement presents a broad anticariogenic effect and may be of value in preventing secondary caries, even under conditions of a high caries risk.
