RESUMEN
OBJECTIVE: To evaluate belimumab addition to the standard of care in patents with refractory idiopathic inflammatory myopathy (IIM). METHODS: We conducted a 40-week multicentre, randomized, double-blind, placebo-controlled trial with 1:1 IV belimumab 10 mg/kg or placebo randomization and a 24-week open-label extension. Clinical responses were measured by the definition of improvement (DOI) and total improvement score (TIS). Flow cytometry analyses were performed on available samples before randomization, at 24 and 60-64 weeks. Descriptive statistics, t-test, Fisher's exact test and analysis of variance tests were used. RESULTS: A total of 17 patients were randomized, 15 received five or more doses of belimumab or placebo and were included in the intention-to-treat analysis. More belimumab patients vs placebo attained a TIS ≥40 [55.5% vs 33.3%; P = non-significant (NS)] and achieved the DOI (33.3% vs 16.7%; P = NS) at weeks 40 and 64; the mean TIS was similar among groups. Two patients achieved major responses (TIS = 72.5) after week 40 in the belimumab arm and none in the placebo arm. No improvement in the placebo arm after switching to the open-label phase was observed. There was no steroid-sparing effect. No new safety signals were detected. Although total B cells were not reduced, belimumab induced naïve B cell depletion while enhancing the number and frequency memory B cells. CONCLUSION: The study did not meet the primary endpoint and no statistically significant differences were observed in clinical responses between arms. More patients achieved sustained TIS ≥40 and reached the DOI. Most patients who received belimumab for >40 weeks had clinical improvement. Phenotypic changes in B cell populations were not associated with clinical responses. CLINICAL TRIAL REGISTRATION NUMBER: Clinicaltrials.gov (https://clinicaltrials.gov/), NCT02347891.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Miositis , Adulto , Humanos , Anticuerpos Monoclonales Humanizados/uso terapéutico , Linfocitos B , Citometría de Flujo , Miositis/tratamiento farmacológicoRESUMEN
The Plasma Membrane Proteolipid 3 (PMP3, UPF0057 family in Uniprot) family consists of abundant small hydrophobic polypeptides with two predicted transmembrane helices. Plant homologues were upregulated in response to drought/salt-stresses and yeast deletion mutants exhibited conditional growth defects. We report here abundant expression of Group I PMP3 homologues (PMP3(i)hs) during normal vegetative growth in both prokaryotic and eukaryotic cells, at a level comparable to housekeeping genes, implicating the regular cellular functions. Expression of eukaryotic PMP3(i)hs was dramatically upregulated in response to membrane potential (Vm) variability (Vmvar ), whereas PMP3(i)hs deletion-knockdown led to Vm changes with conditional growth defects. Bacterial PMP3(i)h yqaE deletion led to a shift of salt sensitivity; Vmvar alternations with exogenous K+ addition downregulated prokaryotic PMP3(i)hs, suggesting [K+ ]-Vmvar axis being a significant feedback element in prokaryotic ionic homeostasis. Remarkably, the eukaryotic homologues functionally suppressed the conditional growth defects in bacterial deletion mutant, demonstrating the conserved cross-kingdom membrane functions by PMP3(i)hs. These data demonstrated a direct reciprocal relationship between PMP3(i)hs expression and Vm differentials in both prokaryotic and eukaryotic cells. Cumulative with PMP3(i)hs ubiquitous abundance, their lipid-binding selectivity and membrane protein colocalization, we propose [PMP3(i)hs]-Vmvar axis as a key element in membrane homeostasis.
Asunto(s)
Potenciales de la Membrana/fisiología , Proteínas de la Membrana/metabolismo , Proteolípidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Estrés Fisiológico/fisiología , Archaea/metabolismo , Bacterias/metabolismo , Sequías , Canales Iónicos/fisiología , Proteínas de la Membrana/genética , Concentración Osmolar , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cloruro de Sodio/metabolismoRESUMEN
Haloacids are considered to be environmental pollutants, but some of them have also been tested in clinical research. The way that haloacids are transported across biological membranes is important for both biodegradation and drug delivery purposes. In this review, we will first summarize putative haloacids transporters and the information about haloacids transport when studying carboxylates transporters. We will then introduce MCT1 and SLC5A8, which are respective transporter for antitumor agent 3-bromopyruvic acid and dichloroacetic acid, and monochloroacetic acid transporters Deh4p and Dehp2 from a haloacids-degrading bacterium. Phylogenetic analysis of these haloacids transporters and other monocarboxylate transporters reveals their evolutionary relationships. Haloacids transporters are not studied to the extent that they deserve compared with their great application potentials, thus future inter-discipline research are desired to better characterize their transport mechanisms for potential applications in both environmental and clinical fields.
Asunto(s)
Ácido Dicloroacético/farmacocinética , Piruvatos/farmacocinética , Animales , Transporte Biológico , Membrana Celular/metabolismo , Humanos , Transportadores de Ácidos Monocarboxílicos/fisiología , Filogenia , Simportadores/fisiologíaRESUMEN
Bacterium Burkholderia sp. MBA4 can utilize haloacids as the sole carbon and energy source for growth. We have previously reported that a haloacid operon, encoding for a dehalogenase (Deh4a) and an associated permease (Deh4p), was responsible for the transformation and uptake of haloacids in MBA4. A disruption of deh4p in MBA4 caused a decrease in monochloroacetate (MCA) uptake, confirming its role as a haloacid transporter. However, this disruptant retained 68% of its MCA-uptake activity indicating the possibility of an alternative system. In this study, we report the identification of a second MCA-inducible haloacid transporter (Dehp2) in MBA4. Its function was confirmed by gene disruption and heterologous expression in Escherichia coli. A dehp2(-) mutant has 30% less, and an E. coli expressing Dehp2 has 40% more, of wildtype MCA-uptake activity. Quantitative RT-PCR illustrated that the minor loss of MCA-uptake activity in single disruptants of deh4p and dehp2 was partly due to a compensatory expression of the alternative gene. Competition assay and kinetics study revealed that Deh4p has a higher affinity for MCA while Dehp2 prefers chloropropionate. A deh4p(-)dehp2(-) double mutant retained 36% of MCA-uptake activity, indicating a robustness of the haloacid uptake systems. The MCA uptake activities mediated by Deh4p, Dehp2 and the uncharacterized system were completely abolished by protonophore carbonyl cyanide 3-chlorophenylhydrazone, suggesting that transmembrane electrochemical gradient is the driving force for MCA uptake.
Asunto(s)
Burkholderia/metabolismo , Regulación Bacteriana de la Expresión Génica , Hidrolasas/química , Unión Competitiva , Transporte Biológico , Carbono/química , Cloroacetatos/química , Relación Dosis-Respuesta a Droga , Electroquímica/métodos , Escherichia coli/metabolismo , Cinética , Modelos Biológicos , Modelos Genéticos , Mutación , Operón , Factores de TiempoRESUMEN
Haloacids are environmental pollutant and can be transformed to non-toxic alkanoic acids by microbial dehalogenase. Bacterium Burkholderia species MBA4 was enriched from soil for its ability to bioremediate haloacids such as mono-chloroacetate (MCA), mono-bromoacetate (MBA), 2-mono-chloropropionate, and 2-mono-bromopropionate. MBA4 produces an inducible dehalogenase Deh4a that catalyzes the dehalogenation process. The growth of MBA4 on haloacid also relies on the presence of a haloacid-uptake system. Similar dehalogenase genes can be found in the genome of many related species. However, wildtype Burkholderia caribensis MWAP64, Burkholderia phymatum STM815, and Burkholderia xenovorans LB400 were not able to grow on MCA. When a plasmid containing the regulatory and structural gene of Deh4a was transformed to these species, they were able to grow on haloacid. The specific enzyme activities in these recombinants ranges from 2- to 30-fold that of MBA4 in similar condition. Reverse transcription-quantitative real-time PCR showed that the relative transcript levels in these recombinant strains ranges from 9 to over 1,600 times that of MBA4 in similar condition. A recombinant has produced nearly five times of dehalogenase that MBA4 could ever achieve. While the expressions of Deh4a were more relaxed in these phylogenetically related species, an MCA-uptake activity was found to be inducible. These metabolically engineered strains are better degraders than the haloacid-enriched MBA4.
Asunto(s)
Proteínas Bacterianas/metabolismo , Burkholderia , Hidrolasas/metabolismo , Ingeniería Metabólica/métodos , Acetatos/metabolismo , Proteínas Bacterianas/genética , Biodegradación Ambiental , Burkholderia/enzimología , Burkholderia/genética , Burkholderia/metabolismo , Hidrolasas/genética , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad por SustratoRESUMEN
BACKGROUND: Acetate is a commonly used substrate for biosynthesis while monochloroacetate is a structurally similar compound but toxic and inhibits cell metabolism by blocking the citric acid cycle. In Burkholderia species MBA4 haloacetate was utilized as a carbon and energy source for growth. The degradation of haloacid was mediated by the production of an inducible dehalogenase. Recent studies have identified the presence of a concomitantly induced haloacetate-uptake activity in MBA4. This uptake activity has also been found to transport acetate. Since acetate transporters are commonly found in bacteria it is likely that haloacetate was transported by such a system in MBA4. RESULTS: The haloacetate-uptake activity of MBA4 was found to be induced by monochloroacetate (MCA) and monobromoacetate (MBA). While the acetate-uptake activity was also induced by MCA and MBA, other alkanoates: acetate, propionate and 2-monochloropropionate (2MCPA) were also inducers. Competing solute analysis showed that acetate and propionate interrupted the acetate- and MCA- induced acetate-uptake activities. While MCA, MBA, 2MCPA, and butyrate have no effect on acetate uptake they could significantly quenched the MCA-induced MCA-uptake activity. Transmembrane electrochemical potential was shown to be a driving force for both acetate- and MCA- transport systems. CONCLUSIONS: Here we showed that acetate- and MCA- uptake in Burkholderia species MBA4 are two transport systems that have different induction patterns and substrate specificities. It is envisaged that the shapes and the three dimensional structures of the solutes determine their recognition or exclusion by the two transport systems.
Asunto(s)
Acetatos/metabolismo , Ácido Acético/metabolismo , Burkholderia/metabolismo , Redes y Vías Metabólicas/genética , Transporte Biológico , Burkholderia/genética , Membrana Celular/metabolismo , Activación TranscripcionalRESUMEN
BACKGROUND: 2-Haloacids can be found in the natural environment as degradative products of natural and synthetic halogenated compounds. They can also be generated by disinfection of water and have been shown to be mutagenic and to inhibit glyceraldehyde-3-phosphate dehydrogenase activity. We have recently identified a novel haloacid permease Deh4p from a bromoacetate-degrading bacterium Burkholderia sp. MBA4. Comparative analyses suggested that Deh4p is a member of the Major Facilitator Superfamily (MFS), which includes thousands of membrane transporter proteins. Members of the MFS usually possess twelve putative transmembrane segments (TMS). Deh4p was predicted to have twelve TMS. In this study we characterized the topology of Deh4p with a PhoA-LacZ dual reporters system. RESULTS: Thirty-six Deh4p-reporter recombinants were constructed and expressed in E. coli. Both PhoA and LacZ activities were determined in these cells. Strength indices were calculated to determine the locations of the reporters. The results mainly agree with the predicted model. However, two of the TMS were not verified. This lack of confirmation of the TMS, using a reporter, has been reported previously. Further comparative analysis of Deh4p has assigned it to the Metabolite:H+ Symporter (MHS) 2.A.1.6 family with twelve TMS. Deh4p exhibits many common features of the MHS family proteins. Deh4p is apparently a member of the MFS but with some atypical features. CONCLUSION: The PhoA-LacZ reporter system is convenient for analysis of the topology of membrane proteins. However, due to the limitation of the biological system, verification of some of the TMS of the protein was not successful. The present study also makes use of bioinformatic analysis to verify that the haloacid permease Deh4p of Burkholderia sp. MBA4 is a MFS protein but with atypical features.
Asunto(s)
Proteínas Bacterianas/genética , Burkholderia/enzimología , Proteínas de Transporte de Membrana/genética , Fosfatasa Alcalina/genética , Secuencia de Aminoácidos , Burkholderia/genética , Biología Computacional , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Genes Reporteros , Halogenación , Operón Lac , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
DehIVa is a haloacid dehalogenase (EC 3.8.1.2) from the soil and water borne bacterium Burkholderia cepacia MBA4, which belongs to the functionally variable haloacid dehalogenase (HAD) superfamily of enzymes. The haloacid dehalogenases catalyse the removal of halides from haloacids resulting in a hydroxlated product. These enzymes are of interest for their potential to degrade recalcitrant halogenated environmental pollutants and their use in the synthesis of industrial chemicals. The haloacid dehalogenases utilise a nucleophilic attack on the substrate by an aspartic acid residue to form an enzyme-substrate ester bond and concomitantly cleaving of the carbon-halide bond and release of a hydroxylated product following ester hydrolysis. We present the crystal structures of both the substrate-free DehIVa refined to 1.93 A resolution and DehIVa covalently bound to l-2-monochloropropanoate trapped as a reaction intermediate, refined to 2.7 A resolution. Electron density consistent with a previously unidentified yet anticipated water molecule in the active site poised to donate its hydroxyl group to the product and its proton to the catalytic Asp11 is evident. It has been unclear how substrate enters the active site of this and related enzymes. The results of normal mode analysis (NMA) are presented and suggest a means whereby the predicted global dynamics of the enzyme allow for entry of the substrate into the active site. In the context of these results, the possible role of Arg42 and Asn178 in a "lock down" mechanism affecting active site access is discussed. In silico substrate docking of enantiomeric substrates has been examined in order to evaluate the enzymes enantioselectivity.
Asunto(s)
Proteínas Bacterianas/química , Burkholderia cepacia/enzimología , Hidrolasas/química , Modelos Moleculares , Secuencia de Aminoácidos , Sitios de Unión , Hidrocarburos Clorados , Hidrólisis , Ligandos , Datos de Secuencia Molecular , Propionatos/química , Conformación Proteica , Especificidad por Sustrato , Agua/químicaRESUMEN
BACKGROUND: S-Adenosylmethionine synthetase (AdoMetS) catalyzes the formation of S-Adenosylmethionine (AdoMet), the major methyl group donor in cells. AdoMet-mediated methylation of DNA is known to have regulatory effects on DNA transcription and chromosome structure. Transcription of environmental-responsive genes was demonstrated to be mediated via DNA methylation in dinoflagellates. RESULTS: A full-length cDNA encoding AdoMetS was cloned from the dinoflagellate Crypthecodinium cohnii. Phylogenetic analysis suggests that the CcAdoMetS gene, is associated with the clade of higher plant orthrologues, and not to the clade of the animal orthrologues. Surprisingly, three extra stretches of residues (8 to 19 amino acids) were found on CcAdoMetS, when compared to other members of this usually conserved protein family. Modeled on the bacterial AdeMetS, two of the extra loops are located close to the methionine binding site. Despite this, the CcAdoMetS was able to rescue the corresponding mutant of budding yeast. Southern analysis, coupled with methylation-sensitive and insensitive enzyme digestion of C. cohnii genomic DNA, demonstrated that the AdoMetS gene is itself methylated. The increase in digestibility of methylation-sensitive enzymes on AdoMet synthetase gene observed following the addition of DNA methylation inhibitors L-ethionine and 5-azacytidine suggests the presence of cytosine methylation sites within CcAdoMetS gene. During the cell cycle, both the transcript and protein levels of CcAdoMetS peaked at the G1 phase. L-ethionine was able to delay the cell cycle at the entry of S phase. A cell cycle delay at the exit of G2/M phase was induced by 5-azacytidine. CONCLUSION: The present study demonstrates a major role of AdoMet-mediated DNA methylation in the regulation of cell proliferation and that the CcAdoMetS gene is itself methylated.
Asunto(s)
División Celular/fisiología , Metilación de ADN , ADN Protozoario/genética , Dinoflagelados/genética , Fase G2/fisiología , Metionina Adenosiltransferasa/genética , Animales , Antimetabolitos/farmacología , Bacterias/enzimología , Bacterias/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , División Celular/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , ADN Complementario/genética , ADN Complementario/metabolismo , ADN Protozoario/metabolismo , Dinoflagelados/enzimología , Etionina/farmacología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fase G2/efectos de los fármacos , Metionina Adenosiltransferasa/metabolismo , Modelos Moleculares , Filogenia , Desarrollo de la Planta , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/enzimología , Plantas/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Saccharomycetales/enzimología , Saccharomycetales/genéticaRESUMEN
We report the complete genome sequence of Burkholderia caribensis MWAP64 (LMG 18531), which was isolated from soil for its proficiency in producing large amounts of exopolysaccharide that help form microaggregates in a vertisol. There are four replicons with a total size of 9,032,119 bp.
RESUMEN
Biodegradation is an effective way to remove environmental pollutants haloacids, and haloacids uptake is an important step besides cytoplasmic dehalogenation. Previous study has identified a robust haloacids transport system in Burkholderia caribensis MBA4 with two homologous genes deh4p and dehp2 as major players. Both genes are inducible by monochloroacetate (MCA), and dehp2 is conserved among the Burkholderia genus with a two component system upstream. Here we show that dehp2 is not in the same operon with the upstream two component system, and fusion with lacZ confirmed the presence of MCA-inducible promoter activity in the 228bp upstream non-coding region of dehp2. Serial deletion confirmed 112bp upstream is enough for basic promoter activity, but sequence further upstream is useful for enhanced promoter activity. Electrophoretic mobility shift assay of the 228bp region showed a retardation complex with stronger hybridization in the induced condition, suggesting a positive regulation pattern. Regulator(s) binding region was found to lie between -228 to -113bp of dehp2. Quantitative real-time PCR showed that the expressions of dehp2 orthologs in three other Burkholderia species were also MCA-inducible, similar as dehp2. The 5' non-coding regions of these dehp2 orthologs have high sequence similarity with dehp2 promoter, and 100bp upstream of dehp2 orthologs is especially conserved. Our study identified a promoter of haloacids transporter gene that is conserved in the Burkholderia genus, which will benefit future exploitation of them for effective biodegradation of haloacids.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Proteínas Bacterianas/metabolismo , Burkholderia/efectos de los fármacos , Burkholderia/genética , Proteínas Portadoras/metabolismo , Cloroacetatos/farmacología , Secuencia ConservadaRESUMEN
DehIVa is one of two dehalogenases produced by the soil- and water-borne bacterium Burkholderia cepacia MBA4. It acts to break down short-chain halogenated aliphatic acids through a nucleophilic attack and subsequent hydrolysis of an enzyme-substrate intermediate to remove the halide ions from L-enantiomers substituted at the C2 position (e.g L-2-monochloropropionic acid). Dehalogenases are an important group of enzymes that are responsible for breaking down a diverse range of halogenated environmental pollutants. The dhlIVa gene coding for DehIVa was expressed in Escherichia coli and the protein was purified and crystallized using the hanging-drop method. Crystals grown in PEG 4000 and ammonium sulfate diffracted to 3.1 A. The crystals had a primitive hexagonal unit cell, with unit-cell parameters a = b = 104.2, c = 135.8 A, alpha = beta = 90, gamma = 120 degrees. Determining this structure will provide valuable insights into the characterization of the catalytic mechanisms of this group of enzymes.
Asunto(s)
Proteínas Bacterianas/química , Burkholderia cepacia/enzimología , Hidrolasas/química , Proteínas Bacterianas/aislamiento & purificación , Cristalización , Hidrolasas/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Difracción de Rayos XRESUMEN
Burkholderia caribensis MBA4 was isolated from soil for its capability to grow on haloacids. This bacterium has a genome size of 9,482,704 bp. Here we report the genome sequences and annotation, together with characteristics of the genome. The complete genome sequence consists of three replicons, comprising 9056 protein-coding genes and 80 RNA genes. Genes responsible for dehalogenation and uptake of haloacids were arranged as an operon. While dehalogenation of haloacetate would produce glycolate, three glycolate operons were identified. Two of these operons contain an upstream glcC regulator gene. It is likely that the expression of one of these operons is responsive to haloacetate. Genes responsible for the metabolism of dehalogenation product of halopropionate were also identified.
RESUMEN
2-Haloacid dehalogenases are hydrolytic enzymes that cleave the halogen-carbon bond(s) in haloalkanoic acids. We have previously isolated a cryptic haloacid dehalogenase gene from Burkholderia cepacia MBA4 and expressed it in Escherichia coli. This recombinant protein is unusual in having a long leader sequence, a property of periplasmic enzymes. In this paper, we report the functional role of this leader sequence. Western blot analyses showed that Chd1 is translocated to the periplasm. The results on the expression of Chd1 in the presence of sodium azide suggested the cleavage of the leader to be Sec-dependent. Chimeras of Chd1 and green fluorescent protein demonstrated that the leader sequence is fully functional in translocating the fusion protein to the periplasm. The expression of the chimeras in Sec mutants supported the Sec-dependent translocation. Surprisingly, recombinant Chd1 and a chimera with no leader sequence were also found in the periplasm.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Proteínas Bacterianas/metabolismo , Burkholderia cepacia/enzimología , Proteínas de Escherichia coli/fisiología , Escherichia coli/genética , Hidrolasas/metabolismo , Proteínas de Transporte de Membrana/fisiología , Adenosina Trifosfatasas/genética , Proteínas Bacterianas/genética , Burkholderia cepacia/genética , Clonación Molecular , ADN Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Hidrolasas/genética , Proteínas de Transporte de Membrana/genética , Plásmidos , Transporte de Proteínas , Proteínas Recombinantes/biosíntesis , Canales de Translocación SEC , Proteína SecARESUMEN
Burkholderia caribensis MBA4 was isolated from soil for its ability to utilize 2-haloacid. An inducible haloacid operon, encoding a dehalogenase and a permease, is mainly responsible for the biotransformation. Here, we report the draft genome sequence of this strain.
RESUMEN
Purple acid phosphatases (PAP) are a group of dimetallic phosphohydrolase first identified in eukaryotes. Bioinformatics analysis revealed 57 prokaryotic PAP-like sequences in the genomes of 43 bacteria and 4 cyanobacteria species. A putative PAP gene (BcPAP) from the bacteria Burkholderia cenocepacia J2315 was chosen for further studies. Synteny analysis showed that this gene is present as an independent gene in most of the members of the genus Burkholderia. The predicted 561 a.a. polypeptide of BcPAP was found to harbour all the conserved motifs of the eukaryotic PAPs and an N-terminal twin-arginine translocation signal. Expression and biochemical characterization of BcPAP in Escherichia coli revealed that this enzyme has a relatively narrow substrate spectrum, preferably towards phosphotyrosine, phosphoserine and phosphoenolpyruvate. Interestingly, this enzyme was found to have a pH optimum at 8.5, rather than an acidic optima exhibited by eukaryotic PAPs. BcPAP contains a dimetallic ion centre composed of Fe and Zn, and site-directed mutagenesis confirmed that BcPAP utilizes the invariant residues for metal-ligation and catalysis. The enzyme is secreted by the wild type bacteria and its expression is regulated by the availability of orthophosphate. Our findings suggest that not all members in the PAP family have acidic pH optimum and broad substrate specificity.
Asunto(s)
Fosfatasa Ácida/química , Proteínas Bacterianas/química , Burkholderia cepacia/enzimología , Genoma Bacteriano/genética , Glicoproteínas/química , Fosfatasa Ácida/clasificación , Fosfatasa Ácida/genética , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Burkholderia cepacia/genética , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Glicoproteínas/clasificación , Glicoproteínas/genética , Concentración de Iones de Hidrógeno , Modelos Genéticos , Filogenia , Especificidad por SustratoRESUMEN
We have developed a method for rapid screening of genes that affected the expression of dehalogenase IVa of Burkholderia cepacia MBA4. The promoter region of the dehalogenase gene was used to drive the expression of a beta-galactosidase gene. A plasmid containing this reporter was first electroporated into MBA4, and a Tn5 containing suicidal plasmid was introduced subsequently. The use of electroporation was necessary because Escherichia coli mediated transconjugation was ineffective in plasmid-carrying MBA4. The number of integrants generated was directly proportional to the amount of plasmid DNA used. Integrants with an elevated beta-galactosidase activity were isolated. Mutants with a disruption in a putative iron-transporter gene and in a putative response regulator receiver gene were identified. The basal dehalogenase transcript levels of these mutants were higher than the wild type. These mutants also grow faster than the wild type in chloroacetate-containing medium. This methodology of isolating regulatory mutants is theoretically feasible and convenient for any kinds of bacteria.
Asunto(s)
Burkholderia cepacia/enzimología , Genes Reguladores , Hidrolasas/química , Hidrolasas/genética , Técnicas Bacteriológicas , Burkholderia cepacia/genética , Burkholderia cepacia/crecimiento & desarrollo , Elementos Transponibles de ADN , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Hidrolasas/metabolismo , Mutagénesis , Transformación GenéticaRESUMEN
Burkholderia cepacia MBA4 is a bacterium that degrades 2-haloacids by removing the halogen and subsequent metabolism of the product for energy. In this study, 2-DE, MS/MS, and N-terminal amino acid sequencing were used to investigate the protein expression profiles of MBA4 grown in a 2-haloacid (monochloroacetate, MCA) and in the corresponding metabolic product (glycolate). Glycolate was used as a control to eliminate the proteins induced by it. Five proteins were found to be up-regulated and five proteins were down-regulated in response to MCA. The differentially expressed proteins were examined, seven of them were identified by MS/MS and two of them were sequenced by Edman degradation. Our results definitely provide an insight for understanding the physiology of B. cepacia MBA4 in response to organohalide contaminated site.
Asunto(s)
Acetatos/metabolismo , Proteínas Bacterianas/química , Burkholderia cepacia/química , Burkholderia cepacia/metabolismo , Proteómica , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Burkholderia cepacia/genética , Electroforesis en Gel Bidimensional , Regulación Bacteriana de la Expresión Génica , Glicolatos/metabolismo , Datos de Secuencia Molecular , Espectrometría de Masas en TándemRESUMEN
Burkholderia cepacia MBA4 is a bacterium that can utilize 2-haloacids as carbon and energy sources for growth. It has been proposed that dehalogenase-associated permease mediates the uptake of haloacid. In this paper, we report the first cloning and characterization of such a haloacid permease. The structural gene, designated deh4p, was found 353 bases downstream of the dehalogenase gene deh4a. Quantitative analysis of the expression of deh4p showed that it was induced by monochloroacetate (MCA), to a level similar to the MCA-induced level of deh4a. The nucleotide sequence of deh4p was determined, and an open reading frame of 1,656 bp encoding a putative peptide of 552 amino acids was identified. Deh4p has a putative molecular weight of 59,414 and an isoelectric point of 9.88. Deh4p has the signatures of sugar transport proteins and integral membrane proteins of the major facilitator superfamily. Uptake of [(14)C]MCA into the cell was Deh4p dependent. Deh4p has apparent K(m)s of 5.5 and 8.9 muM and V(max)s of 9.1 and 23.1 nmol mg(-1) min(-1) for acetate and MCA, respectively. A mutant with a transposon-inactivated haloacid operon failed to grow on MCA even when deh4a was provided in trans.