RESUMEN
A novel vacuum stable proton sponge, 4-maleicanhydridoproton sponge (MAPS), was prepared and applied as the matrix in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) of an aggressive brain tumor tissue (glioblastoma multiforme). Ionic maps of lactate, 2-hydroxyglutarate and chloride anions (m/z 89, 147, 35, respectively) were obtained using a routine MALDI ToF mass spectrometer.
Asunto(s)
Neoplasias Encefálicas/diagnóstico por imagen , Encéfalo/diagnóstico por imagen , Cloruros/análisis , Glioblastoma/diagnóstico por imagen , Glutaratos/análisis , Ácido Láctico/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Humanos , Anhídridos Maleicos/química , ProtonesRESUMEN
IscR (iron-sulfur cluster regulator) is encoded by an ORF located immediately upstream of genes coding for the Escherichia coli Fe-S cluster assembly proteins, IscS, IscU, and IscA. IscR shares amino acid similarity with MarA, a member of the MarA/SoxS/Rob family of transcription factors. In this study, we found that IscR functions as a repressor of the iscRSUA operon, because strains deleted for iscR have increased expression of this operon. In addition, in vitro transcription reactions established a direct role for IscR in repression of the iscR promoter. Analysis of IscR by electron paramagnetic resonance showed that the anaerobically isolated protein contains a [2Fe-2S](1+) cluster. The Fe-S cluster appears to be important for IscR function, because repression of iscR expression is significantly reduced in strains containing null mutations of the Fe-S cluster assembly genes iscS or hscA. The finding that IscR activity is decreased in strain backgrounds in which Fe-S cluster assembly is impaired suggests that this protein may be part of a novel autoregulatory mechanism that senses the Fe-S cluster assembly status of cells.
Asunto(s)
Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Genes Bacterianos , Proteínas Hierro-Azufre/genética , Proteínas Represoras/fisiología , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , ADN Bacteriano , Proteínas de Escherichia coli , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Proteínas Represoras/química , Proteínas Represoras/genética , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
DNA corresponding to two copies of the Rhizobium leguminosarum bv. viciae strain VF39 fixNOQP operon coding for a putative symbiotic terminal oxidase of the heme-copper oxidase superfamily was cloned, sequenced, and genetically analyzed. The first copy is located upstream of the fixK-fixL region on plasmid pRleVF39c, whereas the second copy resides on the nodulation plasmid pRleVF39d. Insertional mutagenesis with antibiotic resistance cassettes confirmed that both copies were functional, and that the presence of at least one functional copy was required for nitrogen fixation. The deduced amino acid sequences of both fixN genes are highly similar (95% identity) and contain 15 putative transmembrane helices, suggesting that the fixN gene products are integral membrane proteins. Furthermore, six histidine residues predicted to be the ligands for a heme-copper binuclear center and a low-spin heme b are conserved in both R. leguminosarum fixN proteins. The deduced fixO and fixP gene products show characteristics of membrane-bound monoheme and diheme cytochrome c, respectively. Upstream of both fixN copies putative Fnr-consensus binding sites (anaeroboxes) were found that differ in certain base pairs. As R. leguminosarum VF39 possesses two members of the Fnr/FixK regulator family, FnrN and FixK, the possible differential regulation of both fixN copies was analyzed with fixN-gusA reporter gene fusions. Both fixN fusions were induced under free-living microaerobic conditions and in the symbiotic zone of the root nodule. Induction of the expression of fixNc and fixNd was highly reduced in a fnrN mutant background and in a fixL mutant background, whereas fixK was only marginally involved in fixN regulation. Residual expression of fixN was observed in an fnrN/fixK double mutant.
Asunto(s)
Genes Bacterianos , Rhizobium leguminosarum/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN Bacteriano/genética , Fabaceae/microbiología , Expresión Génica , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Operón , Plantas Medicinales , Homología de Secuencia de Aminoácido , SimbiosisRESUMEN
Genes of Rhizobium leguminosarum bv. viciae VF39 coding for the regulatory elements NifA, FixL and FixK were isolated, sequenced and genetically analysed. The fixK-fixL region is located upstream of the fixNOQP operon on the non-nodulation plasmid pRleVF39c. The deduced amino acid sequence of FixL revealed an unusual structure in that it contains a receiver module (homologous to the N-terminal domain of response regulators) fused to its transmitter domain. An oxygen-sensing haem-binding domain, found in other FixL proteins, is conserved in R, leguminosarum bv. viciae FixL. R. leguminosarum bv. viciae possesses a second fnr-like gene, designated fixK, whose encoded gene product is very similar to Rhizobium meliloti and Azorhizobium caulinodans FixK. Individual R. leguminosarum bv. viciae fixK and fixL insertion mutants displayed a Fix+ phenotype. A reduced nitrogen-fixation activity was found for a R. leguminosarum bv. viciae fnrN-deletion mutant, whereas no nitrogen-fixation activity was detectable for a flxK/fnrN double mutant. The R. leguminosarum bv. viciae nifA gene is expressed independently of FixL and FixK under aerobic and microaerobic conditions, whereas fixL gene expression is induced under microaerobiosis. Another orf was identified down-stream of fixK-fixL and encodes a product which has homology to pseudoazurins from different species. Mutation of this azu gene showed that it is dispensable for nitrogen fixation.
Asunto(s)
Proteínas Bacterianas/genética , Genes Bacterianos , Hemoproteínas/genética , Rhizobium leguminosarum/genética , Secuencia de Aminoácidos , Mapeo Cromosómico , Expresión Génica , Histidina Quinasa , Datos de Secuencia Molecular , Mutagénesis , Homología de Secuencia de Aminoácido , Factores de Transcripción/genéticaRESUMEN
An open reading frame from Rhizobium leguminosarum bv. viciae strain VF39, previously identified and found to be similar to Escherichia coli fnr and Rhizobium meliloti fixK (orf240, thereafter called fnrN), was further analysed. Analysis of the expression of an fnrN-lacZ transcriptional fusion revealed that fnrN is preferentially expressed under oxygen limitation. Using R. meliloti fixN-lacZ fusions it was shown that the fnrN gene product only mediates transcriptional activation under microaerobiosis, indicating that the FnrN protein responds, directly or indirectly, to oxygen. Plasmids which expressed fnrN under the control of an E. coli promoter were able to complement an E. coli fnr mutant with respect to anaerobic growth on nitrate but not fumarate, and to promote anaerobic but not aerobic activation of the Fnr-dependent E. coli genes narGHJI, nirB and fdnGHI coding for nitrate reductase, NADH-dependent nitrite reductase and formate dehydrogenase-N, respectively. Fumarate and DMSO reductase activities were not induced by FnrN. The E. coli fnr gene substituted for fnrN in oxygen-regulated transcription of nirB- and fixN-lacZ fusions in R. leguminosarum. The results indicate that Fnr and FnrN are functionally very similar and share a common mode of oxygen-dependent transcriptional activation. From hybridization studies, it appeared that fnrN-like genes are present in a number of different R. leguminosarum strains.