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P Cornelis

Publications and source records attributed to P Cornelis.

17 recordsLinked to original sources
PubMed1994

Detection and differentiation of microbial siderophores by isoelectric focusing and chrome azurol S overlay.

Siderophores are microbial, low molecular weight iron-chelating compounds. Fluorescent Pseudomonads produce different, strain-specific fluorescent siderophores (pyoverdines) as well as non-fluorescent siderophores in response to low iron conditions. We present an isoelectric focusing method applicable to unpurified as well as to purified pyoverdine samples where the fluorescent siderophores are visualized under UV illumination. Siderophores from different Pseudomonas sp., amongst which are P. aeruginosa, P. fluorescens and P. putida, including egg yolk, rhizospheric and clinical isolates as well as some derived Tn5 mutants were separated by this technique. Different patterns could be observed for strains known to produce different siderophores. The application of the chrome azurol S assay as a gel overlay further allows immediate detection of non-fluorescent siderophores or possibly degradation products with residual siderophore activity. The method was also applied to other microbial siderophores such as deferrioxamine B.

Culture Media↗
PubMed1993

Detection of the outer membrane lipoprotein I and its gene in fluorescent and non-fluorescent pseudomonads: implications for taxonomy and diagnosis.

The open reading frame of the OprI lipoprotein gene from Pseudomonas aeruginosa was amplified by polymerase chain reaction (PCR) starting from purified DNA or colony lysates. A fragment of the expected size (249 bp) was detected in all P. aeruginosa strains from various clinical and geographical origins. The gene could only be amplified in pseudomonads of rRNA group I which are considered to be the authentic genus Pseudomonas. Digestions with HaeIII, PvuII and SphI of the amplified fragments demonstrated a sequence variation in the oprI gene. Colony, dot and Western blots with two monoclonal antibodies (mAbs) against the lipoprotein I confirmed our PCR results. These findings open interesting perspectives for the molecular taxonomy of the genus Pseudomonas and the development of diagnostic tools.

Antibodies, Monoclonal↗
PubMed1992

Molecular typing of Brucella with cloned DNA probes.

Brucella constitutes a single genomic species (B. melitensis); however, for epidemiological studies, methods are needed for discriminating strains within this genomic species. DNA samples from 112 Brucella strains were cleaved by restriction endonucleases and the fragments separated by agarose gel electrophoresis and transferred to nylon membranes. When the DNA fragments on the membranes were probed with 32P-labelled 16 + 23 S rRNA from Escherichia coli, a single rRNA gene restriction pattern was obtained after cleavage with all endonucleases tested (HindIII, EcoRI, SmaI, and XhoI) except BamHI. This indicated high genomic homogeneity within the single Brucella species. Of 30 probes consisting of random Brucella DNA fragments cloned into lambda EMBL3, 20 yielded a single BamHI restriction pattern per probe when applied to 112 Brucella DNA tested. However, 7 probes yielded 3 to 12 different patterns among DNA tested. These patterns more-or-less correlated with the classification of strains into biogroups (Melitensis, Abortus, Suis, Neotomae, Ovis and Canis) and biovars (18 biovars represented). Probe A was capable of separating biogroup Melitensis from the other biogroups. Probe C separated the set of biogroups Melitensis-Abortus-Ovis from the other biogroups. By reference to the patterns obtained using 1 to 7 probes, the most frequently occurring biovars (Melitensis 1, Melitensis 3, Abortus 1, Abortus 3, Suis 2 and Ovis) could be distinguished from each other. Eight biovars showed more than one pattern with 1 to 7 probes. The proposed typing system should be useful for epidemiological subtyping and does not pose safety problems once the DNA has been extracted.

Autoradiography↗
PubMed1992

Stability, frequency and multiplicity of transposon insertions in the pyoverdine region in the chromosomes of different fluorescent pseudomonads.

Tn5 mutagenesis of different fluorescent pseudomonads was achieved by conjugational transfer of the suicide vector pSUP 10141. Pyoverdine negative (Pvd-) mutants were detected by the absence of fluorescence on King's B medium and by their inability to grow in the presence of the iron chelator EDDHA [ethylenediamine di(o-hydroxyphenylacetic acid)]. In P. fluorescens ATCC 17400 and three rhizosphere isolates (one P. putida and two P. fluorescens), the percentage of Pvd- mutants ranged between 0 and 0.54%. In a P. chlororaphis rhizosphere isolate, this percentage was higher (4%). In these mutants both of the Tn5 antibiotic resistances (Km and Tc) were stable and the transposon could be detected by hybridization. In Pvd- mutants of P. fluorescens ATCC 17400, the transposon was found to be inserted twice in the chromosome while single insertions were detected in the DNA of other, randomly tested mutants. In P. aeruginosa PAO1, where 13.1% of the mutants were Pvd-, both antibiotic resistances were rapidly lost and accordingly no transposon insertion could be detected by hybridization. However, the Pvd- phenotype was generally stable in these mutants. The plasmid pNK862 containing a mini-Tn10 transposon was introduced by electroporation into P. aeruginosa PAO1 and Kmr mutants were recovered, 89% of which were Pvd- and confirmed to be P. aeruginosa by PCR amplification of the P. aeruginosa lipoprotein gene. The mini-Tn10 insertions were also found to be unstable in PAO1.

Blotting, Southern↗
PubMed1990

Pyoverdin-facilitated iron uptake in Pseudomonas aeruginosa: immunological characterization of the ferripyoverdin receptor.

A purified polyclonal antiserum directed against the isolated main 80 kD IROMP (iron-regulated outer-membrane protein) from Pseudomonas aeruginosa PAO1 detected only the 80 kD polypeptide of outer-membrane proteins from PAO1 cells grown in iron deficiency in Western blots. It was also shown to inhibit the uptake of 59Fe pyoverdin by PAO1 cells as well as its binding to purified outer membranes. Immunofluorescence experiments with intact PAO1 cells confirmed that the receptor is present only at the surface of cells grown under conditions of iron deficiency. All these data allow us to conclude that the 80 kD main IROMP of P. aeruginosa is indeed the receptor for the siderophore ferripyoverdin.

Bacterial Outer Membrane Proteins↗
PubMed1989

Cloning and analysis of the gene for the major outer membrane lipoprotein from Pseudomonas aeruginosa.

The gene for the Pseudomonas aeruginosa outer membrane lipoprotein I was isolated from a genomic library in the phage lambda EMBL3 vector and subsequently subcloned in the low copy-number, wide host-range plasmid vector, pKT240. The cloned gene was highly expressed, resulting in the production of a low molecular-weight protein (8 kD) that was found to be associated with the outer membrane. Sequence analysis showed an open reading frame of 83 amino acids with a putative N-terminal hydrophobic signal peptide of 19 residues immediately followed by the lipoprotein consensus sequence, GLY-CYS-SER-SER (residues 19-22). The predicted amino acid composition of the mature polypeptide and that of the purified lipoprotein I of P. aeruginosa (Mizuno and Kageyama, 1979) were identical. In contrast with other Gram-negative outer membrane lipoproteins, conformation predictions suggested that the mature protein was a single alpha helix.

Amino Acid Sequence↗
PubMed1989

Evidence for different pyoverdine-mediated iron uptake systems among Pseudomonas aeruginosa strains.

Fourteen strains of Pseudomonas aeruginosa (P. aeruginosa ATCC 15692, P. aeruginosa ATCC 27853, and 12 clinical isolates) were checked for the production of pyoverdine and for pyoverdine-mediated iron uptake. Under iron restriction, two isolates produced undetectable amounts of pyoverdine, but all the other strains produced a compound with physicochemical properties identical or close to those of the pyoverdine of P. aeruginosa ATCC 15692 (strain PAO1). The pyoverdines were purified and tested for their growth-promoting activity and for their ability to facilitate 59Fe uptake in homologous experiments involving each pyoverdine and its producing strain, as well as in heterologous systems involving all the other strains. The results of both types of experiments suggested the existence of three specificity groups. This was confirmed by analysis of the amino acid composition of the pyoverdines, which differed for each group. A partially purified polyclonal antiserum raised against a major 80-kilodalton (kDa) iron-regulated outer membrane protein (IROMP) of P. aeruginosa PAO1 recognized the 80-kDa IROMPs from P. aeruginosa PAO1 and the clinical isolates belonging to the same group, whereas the IROMPs from the strains belonging to the two other groups were not detected. A second antiserum raised against the P. aeruginosa ATCC 27853 80-kDa IROMP gave similar results by reacting specifically with the 80-kDa IROMP from the strains belonging to this group. Thus, together with the already known pyoverdine from P. aeruginosa PAO1, two new types of pyoverdines produced by strains belonging to this species were characterized.

Amino Acids↗
PubMed1989

[Parry-Romberg progressive facial hemiatrophy and localized scleroderma. Nosologic and pathogenic problems].

A case of a 65-year-old caucasian woman with progressive hemifacial atrophy (Parry-Romberg syndrome) is reported. The atrophy had begun ten years before. At time of presentation, she was suffering from facial pain and diplopia. She presented with severe enophthalmos associated with eyelid atrophy, loss of cilia and mild blepharoptosis. There was a patch of alopecia and atropic areas of skin, recognised as scleroderma "en coup de sabre". Ophthalmic examination showed miosis. Horner's syndrome and ocular motility disturbance. Biomicroscopic examination of anterior and posterior segments was normal; there was no heterochromia and no disc atrophy. Aesthetic surgical treatment was proposed. The etiology of hemifacial atrophy remains controversial, and its relations with scleroderma and autonomic nervous system disorders are discussed.

Aged↗
PubMed1987

Microbial amylases.

Amylases are of tremendous importance in the food industry. Different enzymes used separately or together convert starch into glucose, maltose, dextrins or cyclodextrins. Most enzymes of industrial importance are of microbial origin, and the list of amylases produced by molecular cloning is rapidly expanding.

Amylases↗
PubMed1983

Growth defects of Escherichia coli cells which contain the gene of an alpha-amylase from Bacillus coagulans on a multicopy plasmid.

An alpha-amylase gene from Bacillus coagulans has previously been cloned in Escherichia coli and shown to direct the synthesis of an enzymically active protein of 60,000 Dal (Cornelis et al., 1982). In one particular E. coli host, strain HB101, amylase was found to accumulate in the periplasmic space. To study the processing and the location of the amylase, plasmid pAMY2 was introduced into E. coli 188 which is a strain constitutive for alkaline phosphatase, a periplasmic marker, and for beta-galactosidase, a cytoplasmic marker. Abnormally large amounts of both alpha-amylase and beta-galactosidase were found in the culture fluid of cells grown in rich medium. Furthermore a severe growth defect was found when cells containing pAMY2 were grown in maltose and glycerol media, while the ability to grow on glucose remained normal. This defect could be reversed by two types of spontaneous mutations. Mutations in the first class are located on the plasmid and correspond to the insertional inactivation of the amylase gene by IS1. Mutations in the second class are located on the host chromosome. These results suggest that the synthesis and export of B. coagulans alpha-amylase is deleterious to E. coli, especially in media containing maltose or glycerol as sole carbon source.

Alkaline Phosphatase↗
PubMed1982

Cloning and expression of a Bacillus coagulans amylase gene in Escherichia coli.

A partial EcoRI fragment of Bacillus coagulans DNA cloned in an Escherichia coli K12 bacteriophage lambda host-vector system was shown to direct the synthesis of a thermostable alpha-amylase whose activity could be detected in situ on petri plates using the iodine staining method. A 3.31 kb EcoRI fragment containing the active gene with its own promoter was subcloned in pBR322; in the new clone, called pAMY2, the amylase was shown to accumulate in the periplasmic space. The molecular weight of the enzyme, confirmed by in vivo labelling of plasmid products in minicells, was estimated to be 60000. The restriction map of the plasmid was determined for five restriction enzymes and two new plasmids with smaller DNA inserts were constructed, both directing the synthesis of amylase; one of them with a 2.2 kb PstI insert was shown to be responsible for the synthesis of a fused beta-lactamase-alpha-amylase protein with amylase activity.

Amylases↗
PubMed1979

Functionally impaired tRNA from ethionine treated rats as detected in injected Xenopus oocytes.

Treatment of rats with ethionine was found to cause severe impairment in the aminoacylation capacity of tRNA. This effect was only observed when assayed in injected oocytes, while invitro assays of aminoacylation failed to detect differences between normal tRNA and tRNA from ethionine treated animals. The effect of ethionine on the tRNA population was not uniform and differed for various amino acid specific tRNAs. Thus liver tRNA from ethionine treated rats showed a decreased capacity for phenylalanine aminoacylation, while no change was found in the case of leucine. On the other hand, the level of histidine aminoacylation was higher for tRNA from ethionine treated animals. An even more complex response was observed with methionine aminoacylation where tRNA from ethionine treated animals showed an initially faster rate than control tRNA. With more prolonged incubation periods, the methionyl-tRNA from ethionine treated animals was deacylated at an accelerated rate while the level of normal methionyl-tRNA remained almost constant. In addition to the aminoacylation reaction, the participation of aminoacyl-tRNA in protein synthesis was severely impaired. In this case, both the injected oocyte system and the cell-free wheat germ assay revealed these differences which were manifested with various mRNA and viral RNA preparations.

Amino Acyl-tRNA Synthetases↗
PubMed1975

Reversed phase chromatography of isoaccepting tRNA's from healthy and crown gall tissues from Nicotiana tabacum.

RPC 5 (Reversed Phase Chromatography) of aminoacyl-tRNA's from healthy and crown gall (induced by Agrobacterium tume-faciens strain B6) tobacco tissues were compared for eleven amino acids. For ten amino acids: alanine, arginine, glutamic acid, glycine, isoleucine, leucine, lysine, methionine, tyrosine, and valine, no qualitative or quantitative differences could be detected between aminoacyl-tRNA's from both sources. Phenylalanyl-tRNA's from crown gall tissues gave two peaks on RPC 5; the minor early eluting species (peak 1) was always absent in elution profiles of phenylalanyl-tRNA's from healthy tissues or from tobacco leaves. After the "Y" base was removed by pH 2.9 treatment, peak 2 of phenylalanine tRNA was shifted to the position of peak 1.

Amino Acyl-tRNA Synthetases↗