Biomedical subjects

M P Nuti

Publications and source records attributed to M P Nuti.

17 recordsLinked to original sources

PubMed2000

Nucleotide sequence and analysis of plasmid pMD136 from Pediococcus pentosaceus FBB61 (ATCC43200) involved in pediocin A production.

The complete sequence of the 19515-bp plasmid pMD136 from Pediococcus pentosaceus FBB61 (ATCC43200) has been determined. This plasmid is involved in Pediocin A production, a bacteriocin active against a wide range of gram-positive bacteria. It appears to replicate via a theta mechanism, with structures closely related to those of many lactococcal plasmids. Genes homologous to mobilization functions are also present, which are similar in sequence and arrangement to mobA, mobB, and mobC of some staphylococcal plasmids, although the last one contains a deletion in its central part. The region involved in bacteriocin activity has been limited to a 9.4-kb fragment, containing 10 open reading frames organized in a single operon. Since Pediocin A has a molecular weight of about 80 kDa (Piva and Headon, Microbiology, 140, 697-702, 1994), and a gene long enough to encode it is not present in pMD136, it is proposed that genes residing on the plasmid are responsible for the regulation of bacteriocinogenic activity. Gene arrangement and sequence homologies suggest the presence of a two-component-like regulatory mechanism.

Amino Acid Sequence↗

PubMed2000

Adhesion to hyphal matrix and antifungal activity of Pseudomonas strains isolated from Tuber borchii ascocarps.

Pseudomonas spp. isolates from Tuber borchii ascocarps, known to be able to produce phytoregulatory and biocontrol substances in pure culture, were used to perform studies on their possible physiological role in nature. Antimycotic activity was confirmed against fungal contaminants isolated from the ascocarps, suggesting that populations associated with Tuber borchii fruit bodies may play a role in the maintenance of ascocarp health. Fifty-five percent of strains tested were also able to release metabolites which affected T. borchii mycelial growth and morphogenesis in culture. On the contrary, growth of the arbuscular mycorrhizal fungus Glomus mosseae and the ectomycorrhizal fungus Laccaria bicolor, putative competitors of Tuber for mycorrhizal infection sites on roots, was not influenced by the presence of any bacterial strain. The possibility that these bacteria, which show antifungal activity and fungal growth modulation activities, might be incorporated in the developing ascocarp by means of their preferential adhesion to Tuber mycelium is discussed.

Ascomycota↗

PubMed1996

Identification, sequencing and mutagenesis of the gene for a D-carbamoylase from Agrobacterium radiobacter.

A clone positive for D-carbamoylase activity (2.7 kb HindIII-BamHI DNA fragment) was obtained by screening a genomic library of Agrobacterium radiobacter in Escherichia coli. This DNA fragment contains an open reading frame of 912 bp which is predicted to encode a peptide of 304 amino acids with a calculated molecular mass of 34247 Da. The D-carbamoylase gene, named cauA, was placed under the control of T7 RNA-dependent promoter and expressed in E. coli BL21(DE3). After induction with isopropyl-thio-beta-D-galactopyranoside, the synthesis of D-carbamoylase in E. coli reached about 40% of the total protein. The expressed protein was shown to possess a molecular mass, on SDS-PAGE, of 36 kDa and showed an enhanced stability with respect to that of the wild-type enzyme derived from A. radiobacter. Site-directed mutagenesis experiments allowed us to establish that a Pro14-->Leu14 exchange leads to an inactive enzyme species, while a Cys279-->Ser279 exchange did not impair the functional properties of the enzyme.

Amidohydrolases↗

PubMed1996

Fate of genetically modified Rhizobium leguminosarum biovar viciae during long-term storage of commercial inoculants.

A study was carried out to assess the behaviour, in terms of strain survival and genetic stability, of genetically modified micro-organisms (GEMs) during their storage in commercial-type agricultural inoculants. Three genetically modified Rhizobium leguminosarum biovar viciae strains were constructed, using a gene cassette containing an inducible lacZ gene from Escherichia coli and mercury resistance determinants from transposon Tn 1831. In the first case the genes have been integrated into the chromosome, the second strain contains the inducible cassette on a plasmid, in the third case the cassette is carried by the same plasmid, but the lacZ is constitutively expressed at high levels, due to the removal of the regulatory structure (lac operator) between the gene and its promoter. Three inoculum formulations, based on liquid, vermiculite and peat carriers, were prepared using the genetically modified strains, and were monitored during a period of up to 16 months. Results indicate a high stability of the chromosomally integrated markers. The plasmid-borne modification also was very stable, though the presence of the plasmid affected the strain growth kinetics. In contrast, the strain containing the highly expressed lacZ showed dramatic marker instability. Strain behaviour in stored inoculant packages reflected that observed in batch cultures; moreover, prolonged storage appeared to magnify differences found in in vitro cultures.

Cell Survival↗

PubMed1995

Poly-beta-hydroxybutyrate (PHB) biosynthetic genes in Rhizobium meliloti 41.

Genes encoding beta-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHB-synthase (phaC) from R. meliloti 41, together with a fourth gene, referred to as ORF1, presumed to be involved in PHB biosynthesis, have been cloned and sequenced. phaA, phaB and ORF1 were identified by heterologous hybridization on a cosmid library, while phaC was isolated by cloning the transposon-tagged fragment from a R. meliloti PHB- Tn5 mutant. phaA and phaB were functionally expressed in Escherichia coli while phaC was able to complement a PHB- strain of R. meliloti 41. The three genes were sufficient to direct the production of polyhydroxyalkanoate in E. coli. The homology of ORF1 with an ORF located near the PHB genes in two phototrophic bacteria suggests its involvement in PHB synthesis.

Acetyl-CoA C-Acyltransferase↗

PubMed1994

Construction of multipurpose gene cartridges based on a novel synthetic promoter for high-level gene expression in gram-negative bacteria.

A series of gene cartridges containing a novel synthetic promoter (Psyn) was constructed. The Psyn sequence is based on the consensus of a number of naturally occurring promoters and displays strong activity in Escherichia coli and Rhizobium leguminosarum. In a direct comparison, Psyn proved to be about twice as strong as the tac promoter in E. coli, while the difference in Rhizobium was about tenfold. A small Psyn cartridge was constructed by adding a Shine-Dalgarno sequence, an ATG codon, and a removable lac operator, whose excision can convert the regulated cartridge into a constitutively expressed unit. A second cassette was obtained by the addition of a lacIq gene in order to provide autonomous regulation also in hosts lacking lacI functions, such as R. leguminosarum. A promoterless lacZ gene was inserted to monitor the activity. This gene can be either replaced with genes of interest, or used for gene fusions by means of conveniently positioned restriction sites. A third cassette was generated by adding a mercury-resistance determinant as a selectable marker, suitable for monitoring tagged bacteria released into environments. In such cases, where a non-antibiotic-resistant marker is preferable, the use of mercury chloride adds the advantage of inhibiting fungal growth when plating soil suspensions. The presence of the second marker, lacZ driven by the strong Psyn, facilitates the selection. Furthermore, the Psyn fragment can be used as a specific probe for the detection of released bacteria engineered with any of the above constructs.

Base Sequence↗

PubMed1992

Experimental conditions may affect reproducibility of the beta-galactosidase assay.

Several experimental conditions and parameters contributing to the determination of beta-galactosidase activity, as proposed in Miller's assay, were studied. Use of the absorbance correction factor and the nature and concentration of permeabilizing agents were taken into account as different experimental conditions. Reaction time, culture volume, and growth stage were investigated as equation parameters. From a quantitative point of view the results, in terms of Miller units, are markedly affected by variation in these conditions. Therefore, to ensure reproducibility it is advisable to use constant values for all the parameters.

Chloroform↗

PubMed1990

Lignin-solubilizing ability of actinomycetes isolated from termite (Termitidae) gut.

The lignocellulose-degrading abilities of 11 novel actinomycete strains isolated from termite gut were determined and compared with that of the well-characterized actinomycete, Streptomyces viridosporus T7A. Lignocellulose bioconversion was followed by (i) monitoring the degradation of [14C]lignin- and [14C]cellulose-labeled phloem of Abies concolor to 14CO2 and 14C-labeled water-soluble products, (ii) determining lignocellulose, lignin, and carbohydrate losses resulting from growth on a lignocellulose substrate prepared from corn stalks (Zea mays), and (iii) quantifying production of a water-soluble lignin degradation intermediate (acid-precipitable polymeric lignin). The actinomycetes were all Streptomyces strains and could be placed into three groups, including a group of five strains that appear superior to S. viridosporus T7A in lignocellulose-degrading ability, three strains of approximately equal ability, and three strains of lesser ability. Strain A2 was clearly the superior and most effective lignocellulose decomposer of those tested. Of the assays used, total lignocellulose weight loss was most useful in determining overall bioconversion ability but not in identifying the best lignin-solubilizing strains. A screening procedure based on 14CO2 evolution from [14C-lignin]lignocellulose combined with measurement of acid-precipitable polymeric lignin yield was the most effective in identifying lignin-solubilizing strains. For the termite gut strains, the pH of the medium showed no increase after 3 weeks of growth on lignocellulose. This is markedly different from the pattern observed with S. viridosporus T7A, which raises the medium pH considerably. Production of extracellular peroxidases by the 11 strains and S. viridosporus T7A was followed for 5 days in liquid cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinomycetales↗

PubMed1988

Purification of cellulases from Streptomyces strain A20 by electroendosmotic preparative electrophoresis.

Five cellulase components were identified and purified in one step from Streptomyces strain A20 using electroendosmotic preparative electrophoresis. By this procedure up to 18 mg of protein mixture could be loaded on the column, with an estimated recovery of 60-70% of total activity; activity and protein recovery could be estimated 32% and 47% respectively, if only activity peaks were considered. In comparison to other purification methods, this technique results in high protein recovery and resolution of applied samples.

Bacterial Proteins↗

PubMed1988

Identification and characterization of the nodD gene in Rhizobium leguminosarum strain 1001.

A gene library of the symbiotic 240-kb plasmid of Rhizobium leguminosarum strain 1001 was constructed in pUC18. The clones showing homology with a 6.6-kb fragment containing nodEFDABC from the Sym plasmid pRLlJI were detected by colony hybridization. Additional probes from the symbiotic region of pRLlJI were used to localize the corresponding genes on the map of pRle1001a. The relative positions of nod and nif gene clusters are different than those of pRLlJI. A comparison of the amino acid sequence for NodD from pRle1001a with NodD proteins from other Rhizobium species showed a high degree of sequence conservation at the amino terminus of the protein.

Amino Acid Sequence↗

PubMed1981

Large plasmids of fast-growing rhizobia: homology studies and location of structural nitrogen fixation (nif) genes.

A single large plasmid was isolated from multiplasmid-harboring strains Rhizobium leguminosarum 1001 and R. trifolii 5. These single plasmids, as well as the largest plasmid detectable in R. phaseoli 3622, hybridized with part of the nif structural genes of Klebsiella pneumoniae. In contrast, the plasmids of R. meliloti strains V7 and L5-30 did not show hybridization with the nif genes of K. pneumoniae, indicating that these genes might be located either on the chromosome or on a much larger plasmid which as yet has not been isolated. Studies of the homology between plasmids of fast-growing Rhizobium species showed that a specific deoxyribonucleic acid sequence, which carries the structural genes for nitrogenase, is highly conserved on a plasmid in R. leguminosarum, R. trifolii, and R. phaseoli. Furthermore, it was found that this type of plasmid in the different species shares extensive deoxyribonucleic acid homology, suggesting that strains in the R. leguminosarum cluster have preserved a nif plasmid.

Base Sequence↗

PubMed1978

Interrelationships between soil microorganisms and polystyrene.

Transmission and scanning electron microscopy along with autoradiographic procedures were used for evaluating the interrelationship between soil microorganisms and polystyrene. Two hypotheses, allowing to explain the properties of polystyrene as a soil conditioner, were investigated: the first is concerned with a possible action of soil microorganisms on the compound; the second, reciprocally, with the polystyrene interference on microorganisms. Radioactivity translocation of 14C-Ecolyte-polystyrene along fungal hyphae and asexual fructification of strains, isolated from soil, as well as cytological modification at the cell wall level of the same microfungi, cultivated in the presence of polystyrene have been ascertained.

Cladosporium↗

PubMed1973