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R De Mot

Publications and source records attributed to R De Mot.

At least 19 recordsLinked to original sources
PubMed2000

Characterization of an OprF-deficient mutant suggests that OprF is an essential protein for Pseudomonas fluorescens strain MF0.

A stable OprF-deficient mutant for Pseudomonas fluorescens strain MF0 was constructed using reverse genetics. This mutant, called MF372, showed a rounded morphology and grew more slowly in minimal medium, but not in rich medium. Contrary to other Pseudomonas strains, the loss of OprF for strain MF0 was accompanied by an altered outer membrane composition. At least three outer membrane proteins were overexpressed, apparently as a consequence of adaptive mutations. The N-terminal sequence of two of them revealed strong similarities with porins of the OprD family from P. aeruginosa. The data presented here shows that OprF may be an essential protein for this P. fluorescens strain.

Molecular Weight↗
PubMed2000

Identification and characterization of the omaA gene encoding the major outer membrane protein of Azospirillum brasilense.

The major outer membrane protein (MOMP) of Azospirillum brasilense was purified and degenerate oligonucleotides were constructed on the basis of partial internal amino acid sequences. PCR products were obtained using total DNA of A. brasilense as template. One of these, a 766-bp fragment, was DIG-labelled and used in Southern hybridization against A. brasilense DNA and a genomic library of A. brasilense in Escherichia coli. A clone containing a 20-kb EcoRI insert in pLAFR3 was identified by PCR screening. From this insert, an EcoRI-SalI fragment of approximately 3.5-kb was subcloned in pUC19. The gene encoding the A. brasilense MOMP was sequenced and analyzed. The deduced amino acid sequence contains a putative signal peptide of 23 residues, followed by 367 amino acids of the mature protein with a molecular mass of 38,753 Da. The deduced amino acid sequence shows similarity to certain bacterial porins.

Amino Acid Sequence↗
PubMed1999

Proteasomes and other self-compartmentalizing proteases in prokaryotes.

The proteasome represents the major non-lysosomal proteolytic system in eukaryotes. It confines proteolytic activity to an inner compartment that is accessible to unfolded proteins only. The strategy of controlling intracellular breakdown of proteins by self-compartmentalization is also used by different types of prokaryotic energy-dependent proteases. Genomic sequencing data reveal that various combinations of these energy-dependent proteases occur in prokaryotic cells from different lineages.

Adenosine Triphosphatases↗
PubMed1999

Degradation of pesticides by actinomycetes.

Actinomycetes have considerable potential for the biotransformation and biodegradation of pesticides. Members of this group of Gram-positive bacteria have been found to degrade pesticides with widely different chemical structures, including organochlorines, s-triazines, triazinones, carbamates, organophosphates, organophosphonates, acetanilides, and sulfonylureas. A limited number of these xenobiotic pesticides can be mineralized by single isolates, but often consortia of bacteria are required for complete degradation. Cometabolism of pesticides is frequently observed within this group of bacteria. When compared with pesticide degradation by Gram-negative bacteria, much less information about molecular mechanisms involved in biotransformations of pesticides by actinomycetes is available. Progress in this area has been seriously hampered by a lack of suitable molecular genetic tools for most representatives of this major group of soil bacteria. Overcoming this constraint would enable a better exploitation of the biodegradation and biotransformation abilities of actinomycetes for applications such as bioremediation and construction of transgenic herbicide-resistant crops.

Actinomyces↗
PubMed1999

Influence of a putative ECF sigma factor on expression of the major outer membrane protein, OprF, in Pseudomonas aeruginosa and Pseudomonas fluorescens.

The gene encoding OprF, a major outer membrane protein in Pseudomonas species (formerly known as type 1 pseudomonads), was thought to be constitutively transcribed from a single sigma 70 promoter immediately upstream of the gene. We now report the identification of a novel putative ECF (extracytoplasmic function) sigma factor gene, sigX, located immediately upstream of oprF in both Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens OE 28.3 and show that disruption of this gene significantly reduces OprF expression. In P. aeruginosa, Northern analysis demonstrated that this reduction was a result of an effect on transcription of monocistronic oprF combined with a polar effect due to termination of a transcript containing sigX and oprF. Comparison of sigX-disrupted and wild-type cell transcripts by primer extension indicated that monocistronic transcription of oprF occurs from two overlapping promoters, one that is SigX-dependent and resembles ECF sigma factor promoters in its minus-35 region and another promoter that is independent of SigX and is analogous to the sigma 70-type promoter previously reported. Complementation of the P. aeruginosa sigX-disrupted mutant with plasmid-encoded OprF did not resolve the phenotypes associated with this mutant, which included a markedly reduced logarithmic-phase growth rate in rich medium (compared to that in minimal medium), further reduction of the growth rate in a low-osmolarity environment, secretion of an unidentified pigment, and increased sensitivity to the antibiotic imipenem. This indicates that SigX is involved in the regulation of other genes in P. aeruginosa. Disruption of the sigX gene in P. fluorescens also had an effect on the logarithmic-phase growth rate in rich medium. A conserved sigX gene was also identified in a Pseudomonas syringae isolate and six P. aeruginosa clinical isolates. Collectively, these data indicate that an ECF sigma factor plays a role in the regulation and expression of OprF and also affects other genes.

Bacterial Proteins↗
PubMed1999

Sequence analysis of the oxidase/reductase genes upstream of the Rhodococcus erythropolis aldehyde dehydrogenase gene thcA reveals a gene organisation different from Mycobacterium tuberculosis.

The sequence of the DNA region upstream of the thiocarbamate-inducible aldehyde dehydrogenase gene thcA of Rhodococcus erythropolis NI86/21 was determined. Most of the predicted ORFs are related to various oxidases/reductases, including short-chain oxidases/reductases, GMC oxidoreductases, alpha-hydroxy acid oxidases (subfamily 1 flavin oxidases/dehydrogenases), and subfamily 2 flavin oxidases/dehydrogenases. One ORF is related to enzymes involved in biosynthesis of PQQ or molybdopterin cofactors. In addition, a putative member of the TetR family of regulatory proteins was identified. The substantial sequence divergence from functionally characterized enzymes precludes a reliable prediction about the probable function of these proteins at this stage. In Mycobacterium tuberculosis H37Rv, most of these ORFs have homologs that are also clustered in the genome, but some striking differences in gene organization were observed between Rhodococcus and Mycobacterium.

Aldehyde Dehydrogenase↗
PubMed1998

Characterization of ARC, a divergent member of the AAA ATPase family from Rhodococcus erythropolis.

A gene encoding a AAA ATPase was discovered in the 5' region of the second operon of 20 S proteasome subunits in the nocardioform actinomycete Rhodococcus erythropolis NI86/21. The gene was cloned and expressed in Escherichia coli. The protein, ARC (AAA ATPase forming Ring-shaped Complexes), is a divergent member of the AAA family. The deduced product of the arc gene is 591 residues long (66 kDa). The purified protein possesses a low, N-ethylmaleimide-sensitive ATPase activity and forms rings of six subunits, arranged symmetrically around a central opening or cavity. Two-dimensional crystals grown on lipid monolayers yielded images of the ATPase molecules in "end-on" orientation at 1.9 nm resolution.

Amino Acid Sequence↗
PubMed1998

The 20S proteasome of Streptomyces coelicolor.

20S proteasomes were purified from Streptomyces coelicolor A3(2) and shown to be built from one alpha-type subunit (PrcA) and one beta-type subunit (PrcB). The enzyme displayed chymotrypsin-like activity on synthetic substrates and was sensitive to peptide aldehyde and peptide vinyl sulfone inhibitors and to the Streptomyces metabolite lactacystin. Characterization of the structural genes revealed an operon-like gene organization (prcBA) similar to Rhodococcus and Mycobacterium spp. and showed that the beta subunit is encoded with a 53-amino-acid propeptide which is removed during proteasome assembly. The upstream DNA region contains the conserved orf7 and an AAA ATPase gene (arc).

Acetylcysteine↗
PubMed1998

A major outer membrane protein of Rahnella aquatilis functions as a porin and root adhesin.

A 38-kDa major outer membrane protein (OMP) was isolated from the nitrogen-fixing enterobacterium Rahnella aquatilis CF3. This protein exists as a stable trimer in the presence of 2% sodium dodecyl sulfate at temperatures below 60 degrees C. Single channel experiments showed that this major OMP of R. aquatilis CF3 is able to form pores in the planar lipid membrane. Two oligonucleotides encoding the N-terminal portion of the 38-kDa OMP and C-terminal portion of OmpC were used to amplify the 38-kDa gene by PCR. The deduced amino acid sequence showed a strong homology with Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, and Serratia marcescens OmpC sequences, except loops L6 and L7, which are postulated to be cell surface exposed. On the basis of the OmpF-PhoE three-dimensional structure, it seems likely that this 38-kDa organizes three 16-strand beta-barrel subunits. The relationship between the structure and the double functionality of this protein as porin and as a root adhesin is discussed.

Amino Acid Sequence↗
PubMed1997

Subunit topology of the Rhodococcus proteasome.

The 20S proteasome, isolated from the nocardioform actinomycete Rhodococcus erythropolis strain NI86/21, is built from two alpha-type and two beta-type subunits. In order to probe the subunit topology, we have set up an expression system which allows coexpression of the genes encoding the alpha- and beta-subunits in all possible combinations. The four respective constructs obtained yielded fully assembled and proteolytically active proteasomes. Biochemical, kinetic and electron microscopy analysis allow us to rule out several of the models which were originally envisaged for the subunit topology of the Rhodococcus proteasome. The experiments further indicate that the assembly pathways of the Rhodococcus and of the Thermoplasma proteasome differ in some important details.

Cloning, Molecular↗
PubMed1997

Eubacterial proteasomes.

Proteasomes are large, multisubunit proteases with highly conserved structures. The 26S proteasome of eukaryotes is an ATP-dependent enzyme of about 2 MDa, which acts as the central protease of the ubiquitin-dependent pathway of protein degradation. The core of the 26S complex is formed by the 20S proteasome, an ATP-independent, barrel-shaped protease of about 700 kDa, which has also been detected in archaebacteria and, more recently, in eubacteria. Currently, the distribution of 20S proteasomes in eubacteria appears limited to the actinomycetes, while most other eubacteria contain a related complex of simpler structure.

Amino Acid Sequence↗
PubMed1997

New method for RNA isolation from actinomycetes: application to the transcriptional analysis of the alcohol oxidoreductase gene thcE in Rhodococcus and Mycobacterium.

A new protocol for the isolation of RNA from Rhodococcus and other actinomycetes such as Mycobacterium and Amycolatopsis was developed. The method is based on rapid lysis of cells in a high-speed reciprocal shaker using small abrasive particles followed by spin column purification of the lysate. This quick procedure yields RNA preparations suitable for functional studies. This was shown for the thcE gene of R. erythropolis NI86/21, which encodes a N,N'-dimethyl-4-nitrosoaniline-dependent alcohol oxidoreductase. The thcE transcript was detected by Northern hybridization in R. erythropolis, R. fascians, Mycobacterium chlorophenolicum and Mycobacterium smegmatis. The transcriptional start point of the gene was determined by primer extension of the R. erythropolis mRNA.

Alcohol Oxidoreductases↗
PubMed1997

Thiocarbamate herbicide-inducible nonheme haloperoxidase of Rhodococcus erythropolis NI86/21.

During biodegradation of thiocarbamate herbicides by Rhodococcus erythropolis NI86/21, a protein with an M(r) of 30,000 is induced (I. Nagy, G. Schoofs, F. Compernolle, P. Proost, J. Vanderleyden, and R.De Mot, J. Bacteriol. 177:676-687, 1995). Based on N-terminal sequence data for the protein purified by two-dimensional electrophoresis, the corresponding structural gene, thcF, was cloned and sequenced. The deduced protein sequence of ThcF is homologous to those of nonheme haloperoxidases. A particularly high level of sequence identity (72.6%) was observed for the chloroperoxidase from Pseudomonas pyrrocinia. A polyclonal antibody against the latter enzyme cross-reacted with ThcF either produced by the original Rhodococcus cells or overexpressed heterologously in Escherichia coli. In both thiocarbamate-grown Rhodococcus cells and E. coli cells expressing thcF, the haloperoxidase activity of ThcF was demonstrated. The thiocarbamate-inducible R. erythropolis ThcF protein represents the first (nonheme) haloperoxidase to be identified in a nocardioform actinomycete.

Amino Acid Sequence↗
PubMed1997

Transposition of the IS21-related element IS1415 in Rhodococcus erythropolis.

Three copies of the IS21-related transposable element IS1415 were identified in Rhodococcus erythropolis NI86/21. Adjacent to one of the IS1415 copies, a 47-bp sequence nearly identical to the conserved 5' end of integrons was found. Accurate transposition of IS1415 carrying a chloramphenicol resistance gene (Tn5561) was demonstrated following delivery from a suicide vector to R. erythropolis SQ1.

Amino Acid Sequence↗
PubMed1997

Further sequence analysis of the DNA regions with the Rhodococcus 20S proteasome structural genes reveals extensive homology with Mycobacterium leprae.

The sequence of the respective DNA regions downstream of the 20S proteasome structural genes prcB1A1 (6 kb) and prcB2A2 (3.3 kb) of Rhodococcus erythropolis NI86/21 were determined. A highly conserved gene organization was observed between the two clusters which differed significantly in G + C content (68.8% versus 62.6%). Several ORFs were homologues of putative genes previously identified by genomic sequencing of the equivalent DNA in the related nocardioform actinomycete, Mycobacterium leprae, and thought to be specific for this pathogen. Three ORFs (ORF8(1), ORF8(2), ORF12[1]) without a counterpart in M. leprae were found. No significant homology to known sequences including proteasome-related gene products was detected, except for ORF9(1) and ORF9(2) which display a high level of sequence identity with a partially sequenced ORF in Streptomyces chrysomallus. These downstream ORFs also show a significant level of sequence homology with the ORF6(1) and ORF6(2) which are located upstream of the proteasome structural genes in the respective clusters.

Amino Acid Sequence↗
PubMed1996

Identification of a Rhodococcus gene cluster encoding a homolog of the 17-kDa antigen of Brucella and a putative regulatory protein of the AsnC-Lrp family.

By sequence analysis, a gene encoding a homolog of the 17-kDa protein antigen of the Gram-negative pathogen Brucella abortus (Hemmen et al., Clin Diagn Lab Immunol 2: 263, 1995) was identified in the nocardioform actinomycete Rhodococcus sp. NI86/21. Database searching also revealed a partial human cDNA sequence for a putative eukaryotic homolog of this presumptive Brucella-specific protein. These proteins display a low but significant level of similarity with lumazine synthases involved in bacterial riboflavin biosynthesis. In the upstream region, a Rhodococcus gene for a putative regulatory protein of the AsnC family is located.

Amino Acid Sequence↗
PubMed1995

The first characterization of a eubacterial proteasome: the 20S complex of Rhodococcus.

BACKGROUND: The 26S proteasome is the central protease of the ubiquitin-dependent pathway of protein degradation. The proteolytic core of the complex is formed by the 20S proteasome, a cylinder-shaped particle that in archaebacteria contains two different subunits (alpha and beta) and in eukaryotes contains fourteen different subunits (seven of the alpha-type and seven of the beta-type). RESULTS: We have purified a 20S proteasome complex from the nocardioform actinomycete Rhodococcus sp. strain NI86/21. The complex has an apparent relative molecular mass of 690 kD, and efficiently degrades the chymotryptic substrate Suc-Leu-Leu-Val-Tyr-AMC in the presence or absence of 0.05% SDS. Purified preparations reveal the existence of four subunits, two of the alpha-type and two of the beta-type, the genes for which we have cloned and sequenced. Electron micrographs show that the complex has the four-ringed, cylinder-shaped appearance typical of proteasomes. CONCLUSIONS: The recent description of the first eubacterial ubiquitin, and our discovery of a eubacterial proteasome show that the ubiquitin pathway of protein degradation is ancestral and common to all forms of life.

Amino Acid Sequence↗