130,395 research outputs found
In vitro activity of beta-lactam antibiotics against beta-lactamase producing enterobacteria.
No full textComparative activity of Ceftizoxime and four other Cephalosporins against Gram negative bacteria and their sensitivity to Beta Lactamases. Microbiologica:
No full textA peroxiredoxin of Thermus thermophilus HB27: Biochemical characterization of a new player in the antioxidant defence.
No full textTo fight oxidative damage due to reactive oxygen species (ROS), cells are equipped of different enzymes, among which Peroxiredoxins (Prxs) (EC 1.11.1.15) play a key role. Prxs are thiol-based enzymes containing one (1-Cys Prx) or two (2-Cys Prx) catalytic cysteine residues. In 2-Cys Prxs the cysteine residues form a disulfide bridge following reduction of peroxide which is in turn reduced by Thioredoxin reductase (Tr) /Thioredoxin (Trx) disulfide reducing system to regenerate the enzyme. In this paper we investigated on Prxs of Thermus thermophilus whose genome contains an ORF TT_C0933 encoding a putative Prx, belonging to the subfamily of Bacterioferritin comigratory protein (Bcp): the synthetic gene was produced and expressed in E. coli and the recombinant protein, TtBcp, was biochemically characterized. TtBcp was active on both organic and inorganic peroxides and showed stability at high temperatures. To get insight into disulfide reducing system involved in the recycling of the enzyme we showed that TtBcp catalically eliminates hydrogen peroxide using an unusual partner, the Protein Disulfide Oxidoreductase (TtPDO) that could replace regeneration of the enzyme. Altogether these results highlight not only a new anti-oxidative pathway but also a promising molecule for possible future biotechnological applications
A consensus motif common to all Rho-dependent prokaryotic transcription terminators
No full textWe have characterized at the molecular level several polar mutations in four different cistrons of the his operon of S. typhimurium. An analysis of the his-specific transcripts produced in vivo in the mutant strains, together with in vitro transcription assays, led to the identification of several cryptic Rho-dependent transcription termination elements within the his operon that are activated by the uncoupling of transcription and translation. Common features of these elements were sought and found with a computer program. We have identified a consensus motif, consisting of a cytosine-rich and guanosine-poor region, that is located upstream of the heterogeneous 3' endpoints of the prematurely terminated in vivo transcripts and that is present in all the Rho-dependent transcription terminators described thus far
Biochemical characterization of a novel thermostable β-glucosidase from Dictyoglomus turgidum
No full textDtur_0462 gene from the hypertermophilic bacterium Dictyoglomus turgidum, encoding a β-glucosidase, was synthetically produced and expressed in Escherichia coli BL21(DE3)-RIL. DturβGlu was purified to homogeneity by affinity chromatography and its homotetrameric structure was determined by gel filtration. The monomer is composed by 418 amino acidic residues and showed high sequence similarity with Glycoside Hydrolases (GHs) belonging to GH1 family. The maximum activity of DturβGlu was observed at 80 °C and at pH 5.4. DturβGlu was stable in the range of pH 5–8 and retained 70% of its activity after 2 h of incubation at 70 °C. Metal ions and chemical reagents differently influenced the β-glucosidase activity; furthermore, DturβGlu displays a good ethanol and glucose tolerance (Ki 750 mM). The enzyme is active on p-nitrophenyl-β-D-glucopyranoside (pNPGlu) (Km 0.84 mM) and p-nitrophenyl-β-D-galactopyranoside (pNPGal) (Km 1.36 mM) and shows a broad substrate specificity towards natural compounds as salicin, cellobiose and genistin. The ability to hydrolyze different substrates, the activation in the presence of surfactants, the good thermal resistance, and finally the high glucose and ethanol tolerance make this enzyme a good candidate for industrial applications
The Histidinol Phosphate Phosphatase Involved in Histidine Biosynthetic Pathway Is Encoded by SCO5208 (hisN) in Streptomyces coelicolor A3(2)
No full textThrough the screening of a Streptomyces coelicolor
genomic library, carried out in a histidinol
phosphate phosphatase (HolPase) deficient strain,
SCO5208 was identified as the last unknown gene involved
in histidine biosynthesis. SCO5208 is a phosphatase, and it
can restore the growth in minimal medium in this HolPase
deficient strain when cloned in a high or low copy number
vector. Moreover, it shares sequence homology with other
HolPases recently identified in Actinobacteria. During this
work a second phosphatase, SCO2771, sharing no homologies
with SCO5208 and all so far described phosphatases
was identified. It can complement HolPase activity mutation
only at high copy number. Sequence analysis of
SCO5208 and SCO2771, amplified from the HolPase
mutant strain, revealed that SCO5208 shows a mutation in
a conserved amino acid, whereas SCO2771 does not show
any mutation. All these results show that S. coelicolor
SCO5208, recently renamed hisN, is the HolPase involved
in histidine biosynthesi
Comparative in vitro activity of Norfloxacin and four other chemotherapeutics against urinary Gram negative isolates.
No full textComparative activity of Ceftazidime and four other Cephalosporins against Gram negative bacteria and their sensitivity to Beta lactamases
No full textIdentification and molecular characterisation of thermophilic endoglucanase from Sulfolobus solfataricus.
No full textINTRODUCTION: Microbial degradation of cellulose has enormous economic potential for the conversion of plant biomass into fuel and chemicals. Cellulose is a linear polymer composed of D-glucose units linked by 1,4--D-glucosidic bonds. Its enzymatic hydrolysis requires the action of both endoglucanases (1,4--D-glucan glucanohydrolase [EC 3.2.1.4]) and exoglucanases (1,4--D-glucan cellobiohydrolase [EC 3.2.1.91]). A synergic interaction of these enzymes is necessary for the complete hydrolysis of crystalline cellulose. Thermophilic microrganisms have received considerable attention as source of highly active and thermostable cellulolytic enzymes; genes encoding endoglucanases are widely distributed among fungi and Bacteria; recently one was identified in the Archaeon Pyrococcus furiosus suggesting the occurrence of polysaccharides in hydrothermal vent environments (1).
Our results report the identification of cellulasic activity in the Archaeon Sulfolobus solfataricus and molecular characterisation of celS, encoding a putative cellulase homologous to thermophilic endoglucanases.
MATERIALS AND METHODS: S. solfataricus MT4 strain, kindly provided by Prof. Mario De Rosa, was grown at 82°C in a rotary shaker.
A gene bank of S solfataricus MT4 strain was constructed as described previously (2). pGEM1.1 was the recombinant plasmid screened with adh gene as probe and containing celS . It was sequenced using primers constructed ad hoc. Analysis of the putative open reading frames (ORFs), was performed using Blast program.
Total RNA was extracted in the late exponentially growth phase and primer extension was performed (3).
Detectection of cellulasic activity was determined on CMC-cellulose plates and after SDS-PAGE (4).
RESULTS: Cellulase activitiy was detected in the supernatant of S. solfataricus MT4 cultures and specific enzyme staining after SDS-PAGE revealed the presence of two proteins with apparent molecular mass of about 49 kDa and 40 kDa. At the same time we have cloned a DNA fragment from S. solfataricus MT4 containing an ORF (CelS) of 322 aminoacids (molecular weight 36703 Da) with significant homology to the P. furiosus (1), Thermotoga maritima (4) and T. neapolitana (5) endo-1,4--glucanases. The gene was demonstrated to be transcribed in vivo.
In order to optimise the expression of celS we tried to grow MT4 on different -glucans, namely in minimal media containing Avicel, lichenan, carboxylmethylcellulose, but they were unable to support growth. Therefore enzymatic and transcriptional analysis were performed on cells cultured in more unspecific minimal and rich media. The results obtained suggested a catabolite repression by glucose and in general a down regulation by complex nutrients.
The transcription start site was unambiguously identified by primer extension and revealed coincident with translational initiation.
In order to demonstrate the relationship between structure and function of celS, the gene was fused with gst (glutathione-S- tranferase) in the expression vector pGEX-2tk and expressed in E.coli. The purification and the characterisation of the recombinant protein are underway
An extracellular endoglucanase from Sulfolobus solfataricus.
No full textCellulose, is an unbranched glucose polymer composed of D-glucose units linked by 1,4--D-glucosidic bonds. It is one of the most abundant biopolymers on Earth and represents an important renewable energy source.
Genes encoding endoglucanases are widely distributed among fungi and Bacteria; recently one was identified in the Archaeon Pyrococcus furiosus suggesting the occurrence of polysaccharides in hydrothermal vent environments (1).
We have identified a DNA fragment from Sulfolobus solfataricus Gstrain containing an ORF encoding a putative endoglucanase (CelSs) of 322 aminoacids (about 36 kDa) with significant homology to the Thermotoga maritima and T. neapolitana endo-1,4--glucanase. The gene was demonstrated to be transcribed in vivo and the transcription start site was identified by primer extension.
The cellulase activity was detected in the supernatant of S. solfataricus G cultures and specific enzyme staining after SDS-PAGE revealed the presence of two proteins with apparent molecular mass of about 43 kDa and 36 kDa.
The insertion into suitable vectors for the high level expression of the celSs open reading frame in E.coli and the characterisation of the recombinant protein are underway
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