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R De Mot

Publications and source records attributed to R De Mot.

At least 37 records · Page 2Linked to original sources
PubMed1995

Channel-forming properties and structural homology of major outer membrane proteins from Pseudomonas fluorescens MFO and OE 28.3.

The major outer membrane proteins (OprF) from Pseudomonas fluorescens MFO and OE 28.3 were purified by a new method involving native electrophoresis in octyl-polyoxyethylene media. Both proteins, characterized by the same size, heat-modifiability and N-terminal sequence were re-incorporated in virtually solvent-free planar lipid bilayers. They displayed very similar channel-forming properties: the major conductance level was between 250 pS and 270 pS in 1 M NaCl. From experiments of zero-current potential, both porins were determined weakly cation selective. Amplification by PCR and sequencing of the oprF gene of strain MFO allowed to point out 94% identity between the amino acid sequences of these two OprFs isolated from ecological niches as different as milk (strain MFO) and soil (strain OE 28.3).

Amino Acid Sequence↗
PubMed1995

Characterization of the Rhodococcus sp. NI86/21 gene encoding alcohol: N,N'-dimethyl-4-nitrosoaniline oxidoreductase inducible by atrazine and thiocarbamate herbicides.

A protein with a mol.mass of 51,000 (ThcE) that was induced in Rhodococcus sp. NI86/21 during assimilation of thiocarbamate herbicides, atrazine, ethanol, propanol, glycerol, propionaldehyde or ethanolamine was identified by two-dimensional electrophoresis. The thcE gene was cloned and sequenced. The deduced amino acid sequence revealed ThcE as a member of group III alcohol dehydrogenases. ThcE displayed strong homology with sequenced subunit fragments of the homodecameric N,N'-dimethyl-4-nitrosoaniline-dependent alcohol oxidoreductases (MNO) of Amycolatopsis methanolica and Mycobacterium gastri. N-Terminal sequence analysis of purified MNO from Rhodococcus sp. NI86/21 confirmed the identity with ThcE. When overproduced in Escherichia coli, ThcE was insoluble and no MNO activity was detected.

Alcohol Oxidoreductases↗
PubMed1995

A single cytochrome P-450 system is involved in degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine by Rhodococcus sp. strain NI86/21.

During atrazine degradation by Rhodococcus sp. strain N186/21, N-dealkylated metabolites and an hydroxyisopropyl derivative are produced. The cytochrome P-450 system that is involved in degradation of thiocarbamate herbicides by strain N186/21 (I. Nagy, G. Schoofs, F. Compernolle, P. Proost, J. Vanderleyden, and R. De Mot, J. Bacteriol. 177:676-687, 1995) is also required for atrazine degradation. Atrazine-degrading activity was conferred on the atrazine-negative strains, mutant FAJ2027 of Rhodococcus sp. strain N186/21 and Rhodococcus erythropolis SQ1, upon transformation with the genes encoding the cytochrome P-450 system.

Alkylation↗
PubMed1994

Sequences of the cobalamin biosynthetic genes cobK, cobL and cobM from Rhodococcus sp. NI86/21.

Sequence analysis of a 5753-bp genomic fragment of Rhodococcus sp. NI86/21 revealed the presence of three homologues of known cobalamin biosynthetic genes. One of these genes encodes a protein showing strong homology with the precorrin-6x reductase (CobK) of Pseudomonas denitrificans. In addition, the rhodococcal homologue of the corrin methyltransferases, CobM from P. denitrificans and CbiF from Salmonella typhimurium, was identified. The protein deduced from a third Rhodococcus gene aligns well with CobL of P. denitrificans and with the protein pair CbiE/CbiT of S. typhimurium, which are involved in corrin methylation and decarboxylation.

Amino Acid Sequence↗
PubMed1994

Molecular characterization of the major outer-membrane protein OprF from plant root-colonizing Pseudomonas fluorescens.

N-terminal sequence analysis of peptides generated by proteolytic treatment of the Pseudomonas fluorescens OE 28.3 major outer-membrane protein OprF, embedded in outer membranes or present in whole cells, indicated a surface-exposed location for the proline-rich region of the protein. This region is absent from the P. aeruginosa and P. syringae OprFs. Evidence was obtained for the presence of additional exposed but less accessible regions in the carboxy half of OprF. Four OprF-specific monoclonal antibodies were all directed to the C-terminal part of the protein but did not recognize a surface-exposed epitope as shown by flow cytometry. Our data support the model previously proposed for P. aeruginosa OprF in which the entire protein is embedded in the outer membrane, unlike the topology proposed for the major outer-membrane protein from Escherichia coli, OmpA, whose carboxy half resides in the periplasmic space. For six other P. fluorescens strains producing OprF proteins with different isoelectric points, the primary structure was determined by sequence analysis of the PCR-amplified oprF genes. The proline-rich domain represented the most conserved region of the different P. fluorescens OprFs. Based on the sequence of its oprF gene, it was shown that the mushroom pathogen P. tolaasii is quite closely related to P. fluorescens. Comparative sequence analysis further showed that the carboxy half of OprF contains a sequence motif that is well conserved in the enterobacterial OmpA proteins but is also present in a number of other outer-membrane proteins, including peptidoglycan-associated lipoproteins.

Amino Acid Sequence↗
PubMed1994

Sequence of Rhodococcus gene cluster encoding the subunits of ethanolamine ammonia-lyase and an APC-like permease.

Sequence analysis of a 5173-bp genomic fragment from the nocardioform actinomycete Rhodococcus sp. strain NI86/21 revealed the presence of two genes, eutB and eutC, encoding the putative homologues of the large and small subunits of the ethanolamine ammonia-lyase, respectively, from Salmonella typhimurium. This is the first report of the characterization of these genes in a Gram-positive species. Immediately upstream of eutB, a gene encoding a putative permease of the APC (amino acids, polyamines, choline) transporter family was located. At present, no other Gram-positive members of this permease family are known. The translational coupling of these eut genes suggests an operon-like organization of the ethanolamine genes in Rhodococcus species. A truncated open reading frame downstream of eutC contained an N-terminal motif characteristic of membrane-anchored lipoproteins.

Amino Acid Sequence↗
PubMed1993

Characterization of the recA gene from Pseudomonas fluorescens OE 28.3 and construction of a recA mutant.

The recA gene of Pseudomonas fluorescens OE 28.3 was isolated by complementation of the Fec- phenotype of recombinant lambda EMBL3 phages in a RecA- Escherichia coli strain. The subcloned recA restored resistance to UV and methyl methanesulphonate (MMS) exposure in recA mutants of E. coli. DNA sequence analysis showed that the coding region of the P. fluorescens gene, specifying a protein of 352 amino acid residues, was preceded by an SOS box highly similar to those of Pseudomonas aeruginosa and Azotobacter vinelandii. The deduced amino acid sequence displayed highest homology to the RecA proteins from P. aeruginosa (87.8% identity) and A. vinelandii (84.3% identity). In both the regulatory region and the structural gene, a relatively high degree of sequence divergence from the Pseudomonas cepacia gene was observed. A mutant of P. fluorescens was constructed by inserting a kanamycin resistance cassette into its recA gene. This mutant exhibited an increased sensitivity to UV irradiation and MMS, and was strongly impaired in homologous recombinational activity.

Amino Acid Sequence↗
PubMed1992

Sequence of a Rhodococcus gene encoding a protein with extensive homology to the mammalian propionyl-CoA carboxylase beta chain.

Sequence analysis of a 2-kilobase DNA fragment from Rhodococcus sp. NI86/21 revealed an open reading frame encoding a 476-amino-acid protein with striking homology to the rat and human propionyl-CoA carboxylase beta subunits. The nucleotide sequence of a corresponding prokaryotic gene has not yet been reported. Upstream, the C-terminal part of a putative beta-ketoacyl synthase was identified.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed1992

Homology of the root adhesin of Pseudomonas fluorescens OE 28.3 with porin F of P. aeruginosa and P. syringae.

The gene encoding the root adhesin from the outer membrane of Pseudomonas fluorescens OE 28.3 was isolated from a genomic lambda EMBL3 library and sequenced. The deduced protein (32104 daltons) displayed strong homology with the amino- and carboxyterminal parts of porin F (OprF) from P. aeruginosa and P. syringae. Significant homology was also found within the C-terminal domain of the OmpA proteins from Enterobacteria and major outer membrane proteins from Neisseria species. However, a cysteine-rich domain present in the OprFs of P. aeruginosa and P. syringae is absent from the adhesin of P. fluorescens. Instead, it contains a shorter sequence with eight alternating proline residues.

Adhesins, Bacterial↗
PubMed1990

Isolation of behavioral mutants of Azospirillum brasilense by using Tn5 lacZ.

Tn5 lacZ mutants were generated with Azospirillum brasilense 7030 by mating that strain with Escherichia coli strains carrying suicide plasmid pCIB100 or pCIB110. Kanamycin-resistant Azospirillum colonies were obtained with a maximum frequency of 10(-6) per recipient cell. The potential of Tn5 lacZ for random transposon mutagenesis coupled to transcription analysis in A. brasilense 7030 was demonstrated. Sixty percent of all Kmr A. brasilense 7030 mutants expressed beta-galactosidase activity. Mutants affected in motility (Fla-) and general chemotaxis (Che-) were identified. Chromosomal insertions of Tn5 lacZ are involved, except for two Che- mutants. The latter che loci reside on a 90-megadalton plasmid. Expression of an acidic protein (Mr, 110,000) was abolished in these mutants.

Azospirillum brasilense↗
PubMed1987

Purification and characterization of extracellular alpha-amylase and glucoamylase from the yeast Candida antarctica CBS 6678.

An alpha-amylase and a glucoamylase were purified to homogeneity from the culture fluid of beta-cyclodextrin-grown Candida antarctica CBS 6678 by protamine sulfate treatment, ammonium sulfate precipitation, gel filtration (Sephadex G-75 sf, Ultrogel AcA 54), DEAE-Sephacel chromatography, hydroxyapatite chromatography and affinity chromatography on acarbose--AH-Sepharose 4B. Both enzymes were monomeric glycoproteins with fairly different amino acid compositions. Their apparent relative molecular mass, sedimentation coefficient (Szero20,w), isoelectric point, absorption coefficient (280 nm), pH and temperature optima were estimated as 48,500, 4.7 S, 10.1, 1.74 cm2 mg-1, 4.2 and 57 degrees C, respectively, for glucoamylase and as 50,000, 4.9 S, 10.3, 1.53 cm2 mg-1, 4.2 and 62 degrees C, respectively, for alpha-amylase. Kinetic analyses indicated that both enzymes preferentially hydrolyzed high-molecular-mass substrates, including some raw starches. alpha-Amylase was active on cyclodextrins, whereas debranching activity was demonstrated for glucoamylase. Trestatins were potent inhibitors of both alpha-amylase (Ki less than 1 microM) and glucoamylase (Ki less than 0.1 microM), being more effective than Bay e 4609 (Ki less than 10 microM). Glucoamylase was selectivity and strongly inhibited by acarbose (Ki less than 0.1 microM). Activity of the latter enzyme was also affected by 1-deoxynojirimycin (Ki less than 1 mM), maltitol and amino alcohols (Ki less than 10 mM). Unlike alpha-amylase, glucoamylase adsorbed strongly onto raw starch, the adsorption site being non-identical with the active site.

Adsorption↗
PubMed1986

Secretion of alpha-amylase and multiple forms of glucoamylase by the yeast Trichosporon pullulans.

Trichosporon pullulans IGC 3488 produced extracellular alpha-amylase and glucoamylase activities when grown in batches in a medium containing corn steep liquor and soluble starch or corn starch. alpha-Amylase, unlike glucoamylase activity, was secreted biphasically. For both amylases the maximum concentration was found in stationary phase cultures. The amylolytic enzymes, previously concentrated by ammonium sulfate precipitation, were separated into a glucoamylase fraction and an alpha-amylase fraction by Ultrogel AcA 54 gel filtration. Pullulanase activity was located in the glucoamylase fraction, whereas cyclodextrinase activity was restricted to the alpha-amylase fraction. Isoamylase and alpha-glucosidase were not detected. Electrophoretic analysis showed that alpha-amylase activity was due to a single protein. Glucoamylase, however, occurred in multiple forms. The four glucoamylases and the alpha-amylase were glycoproteins.

Chromatography, Gel↗
PubMed1985

Purification and characterization of an extracellular glucoamylase from the yeast Candida tsukubaensis CBS 6389.

The starch-degrading yeast Candida tsukubaensis CBS 6389 secreted amylase at high activity when grown in a medium containing soluble starch. The extracellular alpha-amylase activity was very low. The major amylase component was purified by DEAE-Sephadex A-50 chromatography and Ultrogel AcA 44 gel filtration and characterized as a glucoamylase. The enzyme proved to be a glycoprotein with a molecular weight of 56 000. The glucoamylase had a temperature optimum at 55 degrees C and displayed highest activity in a pH range of 2.4-4.8. Acarbose strongly inhibited the purified glucoamylase. Debranching activity was present as demonstrated by the release of glucose from pullulan.

Candida↗
PubMed1985

Purification and Characterization of Extracellular Amylolytic Enzymes from the Yeast Filobasidium capsuligenum.

The extracellular amylolytic system of Filobasidium capsuligenum consisted of an alpha-amylase (1,4-alpha-d-glucan glucanhydrolase, EC 3.2.1.1) and two forms of glucoamylase (1,4-alpha-d-glucan glucohydrolase, EC 3.2.1.3). The enzymes were purified by ammonium sulfate fractionation, repeated ion-exchange chromatography (DEAE-Sephadex A-50), and gel filtration (Sephadex G-25, Sephadex G-100 sf). alpha-Amylase had an optimum pH of 5.6 and an optimum temperature of 50 degrees C but was rapidly inactivated at higher temperature. The molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 64,000. An acarbose concentration of 20 mug/ml was required for 50% inhibition of the alpha-amylase. Both glucoamylases are glycoproteins of identical molecular weight (60,000) and produce only glucose by exohydrolysis. The debranching activity of the glucoamylases was evidenced with substrates containing alpha-1,6 linkages. The pH optima were 5.0 to 5.6 for glucoamylase I and 4.8 to 5.3 for glucoamylase II. Glucoamylase I had a higher optimum temperature (55 degrees C) than glucoamylase II (50 degrees C) and was also more resistant to thermal inactivation. Only low acarbose concentrations (<0.1 mug/ml) were required to reduce the activity of the glucoamylases by 50%.

Journal Article↗
PubMedDate not supplied

A self-compartmentalizing protease in Rhodococcus: the 20S proteasome.

The 26S proteasome represents a major, energy-dependent and self-compartmentalizing protease system in eukaryotes. The proteolytic core of this complex, the 20S proteasome, is also ubiquitous in archaea. Although absent from most eubacteria, this multi-subunit protease was recently discovered in Rhodococcus and appears to be confined to actinomycetes. The eubacterial 20S proteasome represents an attractive complementary system to study proteasome assembly, quaternary structure, and catalytic mechanism. In addition, it is likely to contribute substantially to our understanding of the role of various self-compartmentalizing proteases in bacterial cells.

Adenosine Triphosphatases↗