109 research outputs found
Cryptic operon for beta-glucoside metabolism in E. coli K-12: genetic evidence for a regulatory protein. Genetics : 11-25 (1981).
No full text
Regulation of glutamine synthetase isoenzymes in Rhizobium leguminosarum biovar viciae.
No full textAmmonia assimilation in Rhizobium leguminosarum biovar viceae strain RCRlOO 1 (hereafter
called R. leguminosarum) appears to take place only through the glutamine synthetase/glutamate
synthase pathway since (a) no glutamate dehydrogenase was detected in crude extracts of
bacteria grown in different nitrogen sources, and (b) the growth rate on glutamine as a nitrogen
source was faster than that observed on NH4Cl. In contrast to reports for other Rhizobium
species, R. leguminosarum can definitely utilize NH4C1 for growth. R. Ieguminosarum contains
two glutamine synthetase isoenzymes, GSI and GSII, which can be detected in the presence of
each other by differential heat stability, or separated by affinity chromatography or
immunoabsorption with an antiserum raised against pure GSI. GSII does not cross-react with
an anti-GSI antiserum. GSI was shown to be reversibly adenylylated and it was also shown that
adenylylation inhibits the biosynthetic activity of this enzyme, in a similar way to that reported
for Escherichia coli glutamine synthetase and in contrast to that observed for glutamine
synthetase of Rhizobium sp. strain ANU289. The apparent adenylylation level in different
growth conditions changes from 21% to 99%, indicating a physiological role of this posttranslational
modification in the in vivo regulation of GSI activity. The intracellular
concentration of GSI varies very little when R . leguminosarum is grown on different nitrogen
sources (twofold when measured by the transferase assay, or fourfold when measured by
ELISA). In addition, the concentration of mRNA specific for GSI in different nitrogen sources
does not show appreciable differences. The intracellular concentration of GSII varies from a
specific activity value higher than 1000 when R. leguminosarum is grown on glutamate or nitrate,
to an undetectable level when grown on NH4C1. When NH4C1 is added to a culture growing in
glutamate, GSII activity is rapidly diluted out, suggesting a post-translational mechanism of
enzyme inhibition or inactivation. Chloramphenicol prevents the disappe
Studio sulla relazione tra la resistenza alla kanamicina ed il gene “Putative aminoglycoside 3’-phosphotranferase (PAPH) in Sinorhizobium meliloti ceppo 1021
No full textIl ceppo selvatico di Sinorhizobium meliloti 1021 è sensibile alla kanamicina (30 mg/l) anche se Capela et al. (Proc. Natl. Acad. Sci. U.S.A.(2001), 98: 9877-9882), analizzando l’intero genoma, hanno trovato un gene codificante per una “putative aminoglycoside 3'-phosphotransferase” (PAPH), coinvolta nella resistenza ad antibiotici aminoglicosidici quali la kanamicina. Un mutante spontaneo (GM42) kanamicina resistente (200 mg/l) di Sinorhizobium meliloti 1021 è stato caratterizzato da un punto di vista molecolare e fenotipico per analizzare l’attività del gene PAPH.
Il ceppo parentale e GM42 sono stati caratterizzati da un punto di vista microbiologico tramite il MicroLog System Biolog. I risultati mostrano pattern differenti tra il ceppo parentale ed il mutante sulla base della loro capacità di utilizzare o meno fonti di carbonio quali mannitolo o maltosio.
L'amplificazione del gene PAPH tramite PCR ha prodotto un'unica banda sia nel ceppo wild-type 1021 che in GM42 e dal loro sequenziamento non è emersa alcuna differenza nucleotidica. Tuttavia, l’analisi trascrizionale mediante RT-PCR del gene PAPH ha evidenziato differenze tra i due ceppi. Infatti il wild-type non ha prodotto alcun amplicone, mentre GM42 esprime il gene PAPH in diverse condizioni di crescita saggiate.
Sono state condotte ulteriori analisi mediante Western blotting sulle proteine totali del parentale e del ceppo mutante utilizzando l’anticorpo anti-neomicina fosfotransferasi II. GM42 contiene un peptide che immunoreagisce con l’anticorpo utilizzato, tale reattività è assente nel ceppo selvatico. Inoltre tale gene è stato clonato nel vettore di espressione pKK223-3 in E. coli TOP10 e i cloni ottenuti hanno acquisito resistenza alla kanamicina.
Dal confronto dell’espressione genica effettuata mediante DNA-microarrays si evidenzia un’overespressione del gene PAPH e della regione a monte di tale gene nel mutante kanamicina resistente.
Saranno condotte ulteriori indagini per la comprensione dei meccanismi genetici alla base del fenotipo del ceppo mutante GM42. Ciò rappresenterà un considerevole passo in avanti nello studio dei fattori di resistenza delle popolazioni naturali di S. meliloti 1021, aprendo la strada ad un razionale impiego in agricoltura sostenibile
The spliceosomal intron of rolA gene of Agrobacterium rhizogenes is a prokaryotic promoter.
No full textAgrobacterium rhizogenes transfers DNA (T-DNA)from its Ri plasmid to plant cells. All T-DNA genesare expressed in plant cells. The rolA gene is the onlyT-DNA gene that contains an intron in the untranslatedleader region of its mRNA. This paper showsthat (i) the rolA gene is also transcribed in bacteria;(ii) the 85 bp corresponding to the spliceosomalintron drives prokaryotic gene expression in agrobacteria,in free-living rhizobia and in bacteroidswithin root nodules; and (iii) promoter activity isabolished by the deletion of 63 bp from its 50 end andis reduced by mutations changing its sequence nearthe putative 210 region. The expression pattern of achimeric reporter gene shows that, in free-livingbacteria, gene expression takes place during theexponential phase of growth and increases at theonset of the stationary phase. Within root nodules,reporter gene expression occurs in the invasion,nitrogen fixing and senescent zones
Use of nodulation pattern, stress tolerance, nodC gene amplification, RAPD-PCR and RFLP-16S rDNA analysis to discriminate genotypes of Rhizobium leguminosarum biovar viciae
No full textTwenty-seven new Rhizobium isolates were obtained from root nodules of wild and crop legumes belonging to the genera Vicia, Lathyrus and Pisum from different agroecological areas in central and southern Italy. A polyphasic approach including phenotypic and genotypic techniques was used to study their diversity and their relationships with other biovars and species of rhizobia. Analysis of symbiotic properties and stress tolerance tests revealed that wild isolates showed a wide spectrum of nodulation and a marked variation in stress tolerance compared with reference strains tested in this study. All rhizobial isolates (except for the isolate CG4 from Galega officinalis) were presumptively identified as Rhizobium leguminosarum biovar viciae both by their symbiotic properties and the specific amplification of the nodC gene. In particular, we found that the nodC gene could be used as a diagnostic molecular marker for strains belonging to the bv. viciae. RFLP-PCR 16S rDNA analysis confirms these results, with the exception of two strains that showed different RFLP-genotypes from those of the reference strains of R. leguminosarum bv. viciae. Analysis of intraspecies relationship among strains by using the RAPD-PCR technique showed a high level of genetic polymorphism, grouping our isolates and reference strains into six different major clusters with a similarity level of 20%. Data from seven parameters of phenotypic and genotypic analyses were evaluated by using principal component analysis which indicated the differences among strains and allowed them to be divided into seven different groups
Use of nodulation pattern, stress tolerance, nodC gene amplification, RAPD-PCR and RFLP-16S rDNA analysis to discriminate genotypes of Rhizobium leguminosarum biovar viciae
No full textTwenty-seven new Rhizobium isolates were obtained from root nodules of wild and crop legumes belonging to the genera Vicia, Lathyrus and Pisum from different agroecological areas in central and southern Italy. A polyphasic approach including phenotypic and genotypic techniques was used to study their diversity and their relationships with other biovars and species of rhizobia. Analysis of symbiotic properties and stress tolerance tests revealed that wild isolates, showed a wide spectrum of nodulation and a marked variation in stress tolerance compared with reference strains tested in this study. All rhizobial isolates (except for the isolate CG4 from Galega officinalis) were presumptively identified as Rhizobium leguminosarum biovar viciae both by their symbiotic properties and the specific amplification of the nodC gene. In particular, we found that the nodC gene could be used as a diagnostic molecular marker for strains belonging to the bv. viciae. RFLP-PCR 16S rDNA analysis confirms these results, with the exception of two strains that showed different RFLP-genotypes from those of the reference strains of R. leguminosarum bv. viciae. Analysis of intraspecies relationship among strains by using the RAPD-PCR technique showed a high level of genetic polymorphism, grouping our isolates and reference strains into six different major clusters with a similarity level of 20%. Data from seven parameters of phenotypic and genotypic analyses were evaluated by using principal component analysis which indicated the differences among strains and allowed them to be divided into seven different groups. (c) 2005 Elsevier GmbH. All rights reserved
Indole-3-acetic acid regulates the central metabolic pathways in Escherichia coli.
No full textThe physiological changes induced by indoleacetic acid (IAA) treatment were investigated in the totally sequenced Escherichia coli K-12 MG1655. DNA macroarrays were used to measure the mRNA levels for all the 4290 E. coli protein-coding genes; 50 genes (1-1 %) exhibited significantly different expression profiles. In particular, genes involved in the tricarboxylic acid cycle, the glyoxylate shunt and amino acid biosynthesis (leucine, isoleucine, valine and proline) were up-regulated, whereas the fermentative adhE gene was down-regulated. To confirm the indications obtained from the macroarray analysis the activity of 34 enzymes involved in central metabolism was measured; this showed an activation of the tricarboxylic acid cycle and the glyoxylate shunt. The malic enzyme, involved in the production of pyruvate, and pyruvate dehydrogenase, required for the channelling of pyruvate into acetyl-CoA, were also induced in IAA-treated cells. Moreover, it was shown that the enhanced production of acetyl-CoA and the decrease of NADH/NAD+ ratio are connected with the molecular process of the IAA response. The results demonstrate that IAA treatment is a stimulus capable of inducing changes in gene expression, enzyme activity and metabolite level involved in central metabolic pathways in E. col
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