Biomedical subjects

E Galli

Publications and source records attributed to E Galli.

At least 37 records · Page 2Linked to original sources

PubMed1999

Cell envelope mutants of Pseudomonas putida: physiological characterization and analysis of their ability to survive in soil.

To generate mutants with altered lipopolysaccharides (LPS) of the wild-type Pseudomonas putida KT2442, we used the mini-Tn5luxAB-Km transposon. A mutant was found among luminescent colonies and selected as a negative clone in enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody (mAb) 7.3B, which recognizes the O-antigen of P. putida LPS. The DNA region of the LPS mutant interrupted by the minitransposon insertion was cloned and sequenced. Comparison of the deduced amino acid sequence with protein sequence databases showed similarity to the O-antigen polymerase (Wzy) of Salmonella enterica (muenchen). The wild-type gene was rescued by polymerase chain reaction (PCR), cloned into a broad-host-range plasmid and used to carry out complementation assays. The cloned gene was able to restore the wild-type phenotype of the P. putida wzy mutant. We constructed an isogenic mutant of the luminescent wzy mutant to which an oprL mutation was transferred by homologous recombination with an oprL::xylE cassette. The wzy mutants of P. putida were more sensitive to SDS, deoxycholate and EDTA than the corresponding parental strains. We analysed the ability of wzy, oprL and wzy oprL mutants of P. putida to colonize soil. In comparison with the wild-type strain, the ability of single mutants to colonize soil decreased; this characteristic was more evident for the double mutant, especially at high temperatures.

Amino Acid Sequence↗

PubMed1999

Activation and inactivation of Pseudomonas stutzeri methylbenzene catabolism pathways mediated by a transposable element.

The arrangement of the genes involved in o-xylene, m-xylene, and p-xylene catabolism was investigated in three Pseudomonas stutzeri strains: the wild-type strain OX1, which is able to grow on o-xylene but not on the meta and para isomers; the mutant M1, which grows on m-xylene and p-xylene but is unable to utilize the ortho isomer; and the revertant R1, which can utilize all the three isomers of xylene. A 3-kb insertion sequence (IS) termed ISPs1, which inactivates the m-xylene and p-xylene catabolic pathway in P. stutzeri OX1 and the o-xylene catabolic genes in P. stutzeri M1, was detected. No IS was detected in the corresponding catabolic regions of the P. stutzeri R1 genome. ISPs1 is present in several copies in the genomes of the three strains. It is flanked by 24-bp imperfect inverted repeats, causes the direct duplication of 8 bp in the target DNA, and seems to be related to the ISL3 family.

Base Sequence↗

PubMed1999

A new biocatalyst for production of optically pure aryl epoxides by styrene monooxygenase from Pseudomonas fluorescens ST.

We developed a biocatalyst by cloning the styrene monooxygenase genes (styA and styB) from Pseudomonas fluorescens ST responsible for the oxidation of styrene to its corresponding epoxide. Recombinant Escherichia coli was able to oxidize different aryl vinyl and aryl ethenyl compounds to their corresponding optically pure epoxides. The results of bioconversions indicate the broad substrate preference of styrene monooxygenase and its potential for the production of several fine chemicals.

Catalysis↗

PubMed1999

Identification of the Pseudomonas stutzeri OX1 toluene-o-xylene monooxygenase regulatory gene (touR) and of its cognate promoter.

Toluene-o-xylene monooxygenase is an enzymatic complex, encoded by the touABCDEF genes, responsible for the early stages of toluene and o-xylene degradation in Pseudomonas stutzeri OX1. In order to identify the loci involved in the transcriptional regulation of the tou gene cluster, deletion analysis and complementation studies were carried out with Pseudomonas putida PaW340 as a heterologous host harboring pFB1112, a plasmid that allowed regulated expression, inducible by toluene and o-xylene and their corresponding phenols, of the toluene-o-xylene monooxygenase. A locus encoding a positive regulator, designated touR, was mapped downstream from the tou gene cluster. TouR was found to be similar to transcriptional activators of aromatic compound catabolic pathways belonging to the NtrC family and, in particular, to DmpR (83% similarity), which controls phenol catabolism. By using a touA-C2,3O fusion reporter system and by primer extension analysis, a TouR cognate promoter (P(ToMO)) was mapped, which showed the typical -24 TGGC, -12 TTGC sequences characteristic of sigma(54)-dependent promoters and putative upstream activating sequences. By using the reporter system described, we found that TouR responds to mono- and dimethylphenols, but not the corresponding methylbenzenes. In this respect, the regulation of the P. stutzeri system differs from that of other toluene or xylene catabolic systems, in which the hydrocarbons themselves function as effectors. Northern analyses indicated low transcription levels of tou structural genes in the absence of inducers. Basal toluene-o-xylene monooxygenase activity may thus transform these compounds to phenols, which then trigger the TouR-mediated response.

Base Sequence↗

PubMed1998

Two aberrant mercury resistance transposons in the Pseudomonas stutzeri plasmid pPB.

The two mer operons of the Pseudomonas stutzeri OX plasmid pPB and their flanking regions have been sequenced and found to be part of two aberrant transposons. The narrow spectrum mer operon is almost identical to that of Tn501, but is associated with the remnants of Tn5053 tni genes rather than the Tn501 transposition module. The broad spectrum mer operon shows an overall homology with that of Tn5053, but differs from it in the presence of a merB gene, absent in Tn5053, and a merC gene instead of a merF. The pPB broad spectrum mer operon is associated with an incomplete Tn5053-like transposition module and with the Tn501 tnp genes, which are proximal, respectively, to the end and to the beginning of the mer operon. A hypothesis about pPB evolution is presented.

DNA Transposable Elements↗

PubMed1998

Analysis of the gene cluster encoding toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1.

The toluene/o-xylene monooxygenase cloned from Pseudomonas stutzeri OX1 displays a very broad range of substrates and a very peculiar regioselectivity, because it is able to hydroxylate more than one position on the aromatic ring of several hydrocarbons and phenols. The nucleotide sequence of the gene cluster coding for this enzymatic system has been determined. The sequence analysis revealed the presence of six open reading frames (ORFs) homologous to other genes clustered in operons coding for multicomponent monooxygenases found in benzene- and toluene-degradative pathways cloned from Pseudomonas strains. Significant similarities were also found with multicomponent monooxygenase systems for phenol, methane, alkene, and dimethyl sulfide cloned from different bacterial strains. The knockout of each ORF and complementation with the wild-type allele indicated that all six ORFs are essential for the full activity of the toluene/o-xylene monooxygenase in Escherichia coli. This analysis also shows that despite its activity on both hydrocarbons and phenols, toluene/ o-xylene monooxygenase belongs to a toluene multicomponent monooxygenase subfamily rather than to the monooxygenases active on phenols.

Cloning, Molecular↗

PubMed1997

Production of substituted naphthalene dihydrodiols by engineered Escherichia coli containing the cloned naphthalene 1,2-dioxygenase gene from Pseudomonas fluorescens N3.

Naphthalene dioxygenase, a key enzyme in the dihydroxylation of naphthalene, is encoded by the plasmid pN3, responsible for naphthalene metabolism in Pseudomonas fluorescens N3. The naphthalene dioxygenase, including all the sequences for its expression and the regulatory region, has been localized on the 4.3-kb HindIII-ClaI fragment and on the 3.5-kb HindIII fragment of the plasmid pN3, by Southern analysis using as probes nahA and nahR genes, the homologous genes of the plasmid NAH7 from Pseudomonas putida G7. We cloned in Escherichia coli JM109 the dioxygenase gene and its regulatory region and developed an efficient bacterial system inducible by salicylic acid, able to produce dihydrodiols. E. coli containing recombinant plasmids carrying the dioxygenase gene were analysed for their potential as a biocatalytic tool to produce dihydrodiols from different naphthalenes with the substituent on the aromatic ring at the alpha or beta position. The dihydrodiols, identified by HPLC (high-performance liquid chromatography) and 1H-NMR (nuclear magnetic resonance) were produced with yields ranging from 50 to 94%. The degree of bioconversion efficiency depends on the nature and the position of the substituent and indicates the broad substrate specificity of this dioxygenase and its potential for the production of a wide variety of fine chemicals.

Bacterial Proteins↗

PubMed1997

Sequencing and functional analysis of styrene catabolism genes from Pseudomonas fluorescens ST.

The nucleotide sequence of the 4,377-bp chromosomal region of Pseudomonas fluorescens ST that codes for the oxidation of styrene to phenylacetic acid was determined. Four open reading frames, named styA, styB, styC, and styD, were identified in this region. Sequence analysis and biotransformation assays, performed with batch and continuous cultures, allowed us to identify the functions of the sequenced genes. styA and styB encode a styrene monooxygenase responsible for the transformation of styrene to epoxystyrene; styC codes for the second enzyme of the pathway, an epoxystyrene isomerase that converts epoxystyrene to phenylacetaldehyde; and the styD gene produces a phenylacetaldehyde dehydrogenase that oxidizes phenylacetaldehyde to phenylacetic acid. StyA, 415-amino-acids long, was found to be weakly homologous to p-hydroxybenzoate hydroxylase from both P. fluorescens and P. aeruginosa and to salicylate hydroxylase from P. putida, suggesting that it might be a flavin adenine dinucleotide-binding monooxygenase. StyB was found to be partially homologous to the carboxyterminal part of the 2,4-dichlorophenol-6-monooxygenase encoded by plasmid pJP4, while the styC product did not share significant homology with any known proteins. The fourth open reading frame, styD, could encode a protein of 502 amino acids and was strongly homologous to several eukaryotic and prokaryotic aldehyde dehydrogenases. The order of the genes corresponds to that of the catabolic steps. The previously suggested presence of the gene for epoxystyrene reductase, which directly converts epoxystyrene to 2-phenylethanol (A.M. Marconi, F. Beltrametti, G. Bestetti, F. Solinas, M. Ruzzi, E. Galli, and E. Zennaro, Appl. Environ. Microbiol. 61:121-127, 1996), has not been confirmed by sequencing and by biotransformation assays performed in continuous cultures. A copy of the insertion sequence ISI162, belonging to the IS21-like family of elements, was identified immediately downstream of the styrene catabolic genes.

Base Sequence↗

PubMed1997

Isolation and Metabolic Characterization of a Pseudomonas stutzeri Mutant Able To Grow on the Three Isomers of Xylene.

From an o-xylene-degrading Pseudomonas stutzeri strain (OX1), we previously isolated mutant M1, which had acquired the ability to grow on m-xylene and p-xylene but lost the ability to utilize the ortho isomer. From M1 cultures we have now isolated a revertant strain (R1) which grows on o-xylene and retains the ability to grow with the meta and para isomers regardless of the selective pressure applied. In P. stutzeri R1, o-xylene is degraded through two successive monooxygenations of the aromatic ring, while m-xylene and p-xylene catabolism proceeds through the progressive oxidation of a methyl substituent, although unquantifiable amounts of these two substrates are transformed into the corresponding dimethylphenols, which are not utilized for further growth. The two catabolic pathways are inducible by all three xylene isomers.

Journal Article↗

PubMed1996

Organisation of the tmb catabolic operons of Pseudomonas putida TMB and evolutionary relationship with the xyl operons of the TOL plasmid pWW0.

In Pseudomonas putida (Pp) TMB the genes involved in the catabolism of methyl-substituted aromatic hydrocarbons 1,2,4-trimethylbenzene, m- and p-xylene (tmb operon), are functionally and genetically homologous to the xyl genes of the plasmid pWW0, but are chromosomally encoded. We have analysed by cloning. Southern blotting and sequencing of selected regions the organisation of the tmb cluster. This analysis shows that the structural and regulatory genes of the tmb and xyl systems exhibit a high degree of homology and are similarly organised in operons. However the operons are differently arranged on the Pp TMB chromosome and on the pWW0 plasmid. Comparison of the two systems suggests that the operon arrangement found in pWW0 may have originated from that found in Pp TMB via cointegration mediated by replicative transposition or by intermolecular recombination between two copies of the insertion element IS1246.

Base Sequence↗

PubMed1996

Cloning and characterization of styrene catabolism genes from Pseudomonas fluorescens ST.

A gene bank from Pseudomonas fluorescens ST was constructed in the broad-host-range cosmid pLAFR3 and mobilized into Pseudomonas putida PaW340. Identification of recombinant cosmids containing the styrene catabolism genes was performed by screening transconjugants for growth on styrene and epoxystyrene. Transposon mutagenesis and subcloning of one of the selected genome fragments have led to the identification of three enzymatic activities: a monooxygenase activity encoded by a 3-kb PstI-EcoRI fragment and an epoxystyrene isomerase activity and an epoxystyrene reductase activity encoded by a 2.3-kb BamHI fragment. Escherichia coli clones containing the 3-kb PstI-EcoRI fragment were able to transform styrene into epoxystyrene, and those containing the 2.3-kb BamHI fragment converted epoxystyrene into phenylacetaldehyde or, only in the presence of glucose, into 2-phenylethanol. The three genes appear to be clustered and are probably encoded by the same DNA strand. In E. coli, expression of the epoxystyrene reductase gene was under the control of its own promoter, whereas the expression of the other two genes was dependent on the presence of an external vector promoter.

Acetaldehyde↗

PubMed1996

Cloning of the genes for and characterization of the early stages of toluene and o-xylene catabolism in Pseudomonas stutzeri OX1.

In order to study the toluene and o-xylene catabolic genes of Pseudomonas stutzeri OX1, a genomic library was constructed. A 28-kb EcoRI restriction endonuclease DNA fragment, cloned into the vector plasmid pLAFR1 and designated pFB3401, permitted Pseudomonas putida PaW340 to convert toluene and o-xylene into the corresponding meta-ring fission products. Physical and functional endonuclease restriction maps have been derived from the cloned DNA fragment. Further subcloning into and deletion analysis in the Escherichia coli vector pGEM-3Z allowed the genes for the conversion of toluene or o-xylene into the corresponding catechols to be mapped within a 6-kb region of the pFB3401 insert and their direction of transcription to be determined. Following exposure to toluene, E. coli cells carrying this 6-kb region produce a mixture of o-cresol, m-cresol, and p-cresol, which are further converted to 3-methylcatechol and 4-methylcatechol. Similarly, a mixture of 2,3-dimethylphenol and 3,4-dimethylphenol, further converted into dimethylcatechols, was detected after exposure to o-xylene. The enzyme involved in the first step of toluene and o-xylene degradation exhibited a broad substrate specificity, being able to oxidize also benzene, ethylbenzene, m-xylene, p-xylene, styrene, and naphthalene. Deletions of the 6-kb region which affect the ability to convert toluene or o-xylene into the corresponding methylphenols compromise also their further oxidation to methylcatechols. This suggests that a single enzyme system could be involved in both steps of the early stages of toluene and o-xylene catabolism.

Benzene↗

PubMed1996

[Clinical comparison of different replacement milk formulas in children with allergies to cow's milk proteins. 24-month follow-up study].

The elective treatment for allergy to cow's milk protein is the elimination of these proteins from the diet. The present study with a follow-up of over two years took the form of a comparison between different replacement formulas based on soya (group A), hydrolysate of soya and bovine collagen (group B), and hydrolysate of casein (group C), randomly administered to 55 children (30 males and 25 females, aged between 2-48 months) with documented allergy to cow's milk proteins, but with different clinical symptoms. Tests to evaluate the acquisition of clinical tolerance to cow's milk proteins were performed using a day-hospital regime every 6 months. Sensitivity reactions were observed in 22% of cases in group A, 8% in group B and 37.5% in group C. It is worth underlining that 5 of the 6 children with reactions to soya protein then showed an excellent tolerance to hydrolysate of soya when it was administered subsequently until tolerance was achieved. Weight and statutory growth was uniformly good in all three groups. A high percentage of children achieved tolerance after 24 months (72%); the mean time taken to acquire clinical tolerance was 11.6 +/- 4.8 in group A, 11.6 +/- 6.02 in group B, and 14 +/- 5.6 in group C. No correlation was found between the type of initial symptoms, age at onset, method of response to first challenge and the time taken to acquire tolerance.

Animals↗

PubMed1995

Cloning and comparison of mercury- and organomercurial-resistance determinants from a Pseudomonas stutzeri plasmid.

Plasmid pPB confers broad-spectrum mercury resistance (HgR) to a Pseudomonas stutzeri strain. Two pPB regions, separated by 25-30 kb and sharing homology with Tn501 mer (Hg detoxification) genes, were cloned separately and each was shown to carry a cluster of functional and independently regulated mer genes. One of the two gene clusters conferred resistance only to inorganic mercury, and had a structure identical to the classical model of narrow-spectrum mer operons. In the other cluster a novel merB gene, not homologous to the other known merB, but with the same function, was mapped upstream from merA, interposed between an organomercurial-responsive regulatory element and transport genes. Evidence suggests that merB and the other structural mer genes might be transcribed from two distinct promoters. The presence of two inverted repeat-like elements, identical to those of Tn5053, upstream from merR suggests that the pPB broad-spectrum-gene cluster could be part of a transposon-like element.

Amino Acid Sequence↗

PubMed1994

Analysis of polyunsaturated fatty acids in newborn sera: a screening tool for atopic disease?

It has been demonstrated that patients with atopic disease have anomalies of fatty acid composition, as a result of altered metabolism or abnormal incorporation of fatty acids into the tissues. In the present study, in 57 newborns 'at risk' for atopic disease, the polyunsaturated fatty acid (PUFA) levels were found to be lower in cord blood in infants who subsequently developed atopic disease than in non-atopics. In all babies, levels of arachidonic acid and dihomo-gamma-linolenic acid in sera at 1 and 3 months of age were lower than those in cord blood. These changes were more marked in children who subsequently developed atopic disease, and in those who, independently of signs and/or symptoms of atopic disease, were formula-fed. A comparison between IgE and PUFA levels revealed no significant differences at any tested time interval. In conclusion, our data suggest that in children 'at risk' for atopy, PUFA levels may be predictive of atopic disease.

8,11,14-Eicosatrienoic Acid↗

PubMed1994

Methylprednisolone bolus: a novel therapy for severe atopic dermatitis.

Seven children suffering from severe atopic dermatitis, unresponsive to standard therapy, received an iv bolus dose of methylprednisolone (20 mg/kg/day) for three days. Immunological parameters were evaluated before and after treatment. At the end of bolus therapy both skin lesions and itching improved for several months in five of seven patients. No side effects were observed, but a significant and transient lymphopenic response occurred, with lower CD4+ than CD8+ lymphocyte counts. Our data suggest that this therapy may be a novel and safe therapeutic approach in severe atopic dermatitis.

Adolescent↗

PubMed1993

Effect on wheat root development of inoculation with an Azospirillum brasilense mutant with altered indole-3-acetic acid production.

To evaluate the involvement of indole-3-acetic acid (IAA) in promotion of root development, the effects of inoculation of wheat seedlings with Azospirillum brasilense SpM7918, a very low-IAA producer, were estimated. Compared with the wild-type strain Sp6, SpM7918 showed a reduced ability to promote root system development in terms both of number and length of lateral roots and of distribution of root hairs.

Azospirillum brasilense↗

PubMed1991

Intrathecal synthesis of free immunoglobulin light chains and IgM in initial multiple sclerosis.

We studied the intrathecal synthesis of free kappa, free lambda immunoglobulin light chains and of IgM in 33 consecutive patients with possible or probable MS at the time of their first diagnosis. Nineteen patients presented free kappa or lambda light chain bands in CSF after agarose isoelectric focusing, protein transfer to nitrocellulose and immunostaining with specific antisera. Nineteen patients had increased CSF levels of free kappa light chains as measured with a competitive ELISA. Fourteen had an increased IgM index, as evaluated with a sandwich ELISA. Twenty-six patients displayed CSF oligoclonal IgG bands in CSF and 25 had cerebral magnetic resonance imaging lesions suggestive of MS. The local production of free immunoglobulin light chains and IgM is often detected in the CSF of patients with early MS.

Adolescent↗