Biomedical subjects

P Cornelis

Publications and source records attributed to P Cornelis.

At least 19 recordsLinked to original sources

PubMed1999

cumA, a gene encoding a multicopper oxidase, is involved in Mn2+ oxidation in Pseudomonas putida GB-1.

Pseudomonas putida GB-1-002 catalyzes the oxidation of Mn2+. Nucleotide sequence analysis of the transposon insertion site of a nonoxidizing mutant revealed a gene (designated cumA) encoding a protein homologous to multicopper oxidases. Addition of Cu2+ increased the Mn2+-oxidizing activity of the P. putida wild type by a factor of approximately 5. The growth rates of the wild type and the mutant were not affected by added Cu2+. A second open reading frame (designated cumB) is located downstream from cumA. Both cumA and cumB probably are part of a single operon. The translation product of cumB was homologous (level of identity, 45%) to that of orf74 of Bradyrhizobium japonicum. A mutation in orf74 resulted in an extended lag phase and lower cell densities. Similar growth-related observations were made for the cumA mutant, suggesting that the cumA mutation may have a polar effect on cumB. This was confirmed by site-specific gene replacement in cumB. The cumB mutation did not affect the Mn2+-oxidizing ability of the organism but resulted in decreased growth. In summary, our data indicate that the multicopper oxidase CumA is involved in the oxidation of Mn2+ and that CumB is required for optimal growth of P. putida GB-1-002.

Amino Acid Sequence↗

PubMed1999

Uptake of pyocin S3 occurs through the outer membrane ferripyoverdine type II receptor of Pseudomonas aeruginosa.

Pyocin S3 was found to kill exclusively Pseudomonas aeruginosa isolates producing type II pyoverdine (exemplified by strain ATCC 27853). Killing was specifically inhibited by addition of type II ferripyoverdine. All Tn5 mutants resistant to pyocin S3 were defective for pyoverdine-mediated iron uptake and failed to produce an 85-kDa iron-repressed outer membrane protein. We conclude that this protein is probably the type II ferripyoverdine receptor that is used by pyocin S3 to gain entry into the cell.

Bacterial Outer Membrane Proteins↗

PubMed1998

A new membrane-bound OprI lipoprotein expression vector. High production of heterologous fusion proteins in gram (-) bacteria and the implications for oral vaccination.

We have previously described the development of cloning vectors for the production of OprI-based outer membrane fusion proteins in E. coli (Cornelis et al., 1996) and now describe the construction of a new vector, containing a lacI(q) gene, resulting in tight repression of the promotor and allowing its use in other Gram (-) bacteria. The new pVUB3 expression vector encodes a truncated but active LacI(q)(341) repressor which binds to the single operator in the vector. A high repression of the trc promotor was observed, resulting in a very low basal leakage of expression and very high production levels of OprI or derivatives after IPTG induction in E. coli. Bacterial viability was not affected under uninduced conditions, but the number of viable cell counts decreased after production of large amounts of the outer membrane-bound OprI lipoprotein and its derivatives, both in E. coli and Salmonella typhimurium. This highly repressible system allows us to extend the use of OprI vectors in other Gram (-) bacteria, resulting in the production of outer membrane-bound lipid-modified molecules, opening the possibility for its application in the design of potential live Salmonella-based subunit vaccines.

Amino Acid Sequence↗

PubMed1998

Identification of a 25-aminoacid sequence from the major African swine fever virus structural protein VP72 recognised by porcine cytotoxic T lymphocytes using a lipoprotein based expression system.

Identification of African swine fever virus (ASFV) proteins recognised by cytotoxic T lymphocytes (CTL) from swine surviving ASFV/NH/P68 infection was assessed using expression vectors based on the Pseudomonas aeruginosa outer membrane lipoprotein I gene (oprI). Viral antigens expressed as fusion lipoproteins were shown to be taken efficiently by porcine blood-derived macrophages incubated with outer membrane protein preparations from transformed E. coli. To assess recognition by CTL the fusion lipoprotein-treated macrophages were used as targets in 51Cr release microcytotoxicity assays. Using this approach it was shown that the aminoacid sequence HKPHQSKPILTDENDTQRTCSHTNP from the major structural ASFV protein (VP72), encoded by a recombinant clone (pVUB72) is presented by macrophages, which are lysed under restriction of SLA class I antigens. Overall, the results demonstrate that the oprI based vectors are valuable tools to study ASFV-specific CTL activity.

African Swine Fever Virus↗

PubMed1998

Different residues in periplasmic domains of the CcmC inner membrane protein of Pseudomonas fluorescens ATCC 17400 are critical for cytochrome c biogenesis and pyoverdine-mediated iron uptake.

The inner membrane protein CcmC (CytA) of Pseudomonas fluorescens ATCC17400, which has homologues in several bacteria and plant mitochondria, is needed for the biogenesis of cytochrome c. A CcmC-deficient mutant is also compromised in the production and utilization of pyoverdine, the high-affinity fluorescent siderophore. A topological model for CcmC, based on the analysis of alkaline phosphatase fusions, predicts six membrane-spanning regions with three periplasmic loops. Site-directed mutagenesis was used in order to assess the importance of some periplasm-exposed residues, conserved in all CcmC homologues, for cytochrome c biogenesis, and pyoverdine production/utilization. Despite the conservation of the residues His-61, Val-62 and Pro-63 in the first periplasmic loop, and Leu-184, His-185 and Gln-186 in the third periplasmic loop, their simultaneous replacement with Ala only partially affected cytochrome c biogenesis and pyoverdine production/utilization. Simultaneous replacements of residues Trp-115 and Gly-116 in the second periplasmic loop substantially affected pyoverdine production/utilization but not cytochrome c production. An Ala substitution of Asp-127, in the second periplasmic loop, resulted in decreased production of cytochrome c, slower growth in conditions of anaerobiosis and reduced pyoverdine production. On the other hand, a mutation in Trp-126, also in the second periplasmic loop, totally suppressed the production of cytochrome c, whereas it had no effect on the production and utilization of pyoverdine. These results show a differential involvement of amino acid residues in periplasmic domains of CcmC in cytochrome c biogenesis and pyoverdine production/utilization.

ATP-Binding Cassette Transporters↗

PubMed1998

Sequence diversity of the oprI gene, coding for major outer membrane lipoprotein I, among rRNA group I pseudomonads.

The sequence of oprI, the gene coding for the major outer membrane lipoprotein I, was determined by PCR sequencing for representatives of 17 species of rRNA group I pseudomonads, with a special emphasis on Pseudomonas aeruginosa and Pseudomonas fluorescens. Within the P. aeruginosa species, oprI sequences for 25 independent isolates were found to be identical, except for one silent substitution at position 96. The oprI sequences diverged more for the other rRNA group I pseudomonads (85 to 91% similarity with P. aeruginosa oprI). An accumulation of silent and also (but to a much lesser extent) nonsilent substitutions in the different sequences was found. A clustering according to the respective presence and/or positions of the HaeIII, PvuII, and SphI sites could also be obtained. A sequence cluster analysis showed a rather widespread distribution of P. fluorescens isolates. All other rRNA group I pseudomonads clustered in a manner that was in agreement with other studies, showing that the oprI gene can be useful as a complementary phylogenetic marker for classification of rRNA group I pseudomonads.

Bacterial Proteins↗

PubMed1997

Analysis of epidemic Pseudomonas aeruginosa isolates by isoelectric focusing of pyoverdine and RAPD-PCR: modern tools for an integrated anti-nosocomial infection strategy in burn wound centres.

The Gram-negative bacterium Pseudomonas aeruginosa is an important life-threatening nosocomial pathogen in burn units. In this study we analysed epidemic P. aeruginosa isolates from patients and their hospital environment using two new molecular techniques in order to establish strain relatedness for epidemiological purposes. One technique was pyoverdine typing by isoelectric focusing (PVD-IEF) and the other was a genomic PCR-based fingerprinting technique called random amplification of polymorphic DNA actually referred to as RAPD-PCR. The described short epidemic (6 weeks) included 37 consecutive isolates from 9 different patients as well as two environmental isolates recovered, at the same time, from one of the hydrotherapy facilities. Only two of the three known pyoverdine types of P. aeruginosa could be found. Type I was absent while type II represented 49 per cent and type III, 51 per cent of the isolates. The two consecutive isolates from the environment were both of type III. The RAPD-PCR fingerprinting discriminated four patterns. Profile 1 represented 60 per cent; profile 2, 34 per cent; and profiles 3 and 4 only 3 per cent of the isolates respectively. The environmental isolates also had a RAPD-PCR 1 profile, arguing for the hydrotherapy facility as a possible contamination source. Prompt measures could prevent an outbreak. The study demonstrates the applicability of the techniques in a routine microbiology lab as well as their usefulness, in combination with other techniques, in the fight against nosocomial infections, which are so critical in burn units. Both techniques showed undoubtable evidence of the occurrence of polymicrobial infection of individual patients by P. aeruginosa species. Meanwhile pyoverdine typing by IEF seems suited to studying more profoundly the role of pyoverdines in burns.

Bacterial Typing Techniques↗

PubMed1997

Trehalose induces antagonism towards Pythium debaryanum in Pseudomonas fluorescens ATCC 17400.

Pseudomonas fluorescens ATCC 17400 shows in vitro activity against Pythium debaryanum under conditions of iron limitation. A lacZ reporter gene introduced by transposon mutagenesis into the P. fluorescens ATCC 17400 trehalase gene (treA) was induced by a factor released by the phytopathogen Pythium debaryanum. The induction of the lacZ gene was lost upon treatment of the Pythium supernatant with commercial trehalase. A trehalose concentration as low as 1 microM could induce the expression of treA. The mutation did not affect the wild-type potential for fungus antagonism but drastically decreased the osmotolerance of the mutant in liquid culture and suppressed the ability of P. fluorescens ATCC 17400 to utilize trehalose as a carbon source. A subsequent transposon insertion in treP, one of the trehalose phosphotransferase genes upstream of treA, silenced the lacZ gene. This double mutant restricted fungal growth only under conditions of high osmolarity, which probably results in internal trehalose accumulation. These data confirm the role of the disaccharide trehalose in osmotolerance, and they indicate its additional role as an initiator of or a signal for fungal antagonism.

Amino Acid Sequence↗

PubMed1997

Direct detection and identification of Pseudomonas aeruginosa in clinical samples such as skin biopsy specimens and expectorations by multiplex PCR based on two outer membrane lipoprotein genes, oprI and oprL.

A multiplex PCR test based on the simultaneous amplification of two lipoprotein genes, oprI and oprL, was designed and evaluated for its ability to directly detect fluorescent pseudomonads (amplification of oprI open reading frame, 249 bp) and Pseudomonas aeruginosa (amplification of oprL open reading frame, 504 bp) in clinical material. A collection of reference strains including 20 different species of fluorescent pseudomonads was tested. Positive PCR results for both genes were observed only for P. aeruginosa isolates (n = 150), including strains of clinical and environmental origin, while only one gene, oprI, was amplified from the other fluorescent pseudomonads. All other bacteria tested (n = 15) were negative by the amplification test. The lower detection level for P. aeruginosa was estimated to be 10(2) cells/ml. Preliminary evaluation on testing skin biopsy specimens from patients with burns (n = 14) and sputum samples from cystic fibrosis patients (n = 49) and other patients (n = 19) showed 100% sensitivity and 74% specificity in comparison with culture. This multiplex PCR assay appears promising for the rapid and sensitive detection of P. aeruginosa in clinical specimens. Further evaluation of its specificity in longitudinal clinical studies is warranted.

Bacterial Outer Membrane Proteins↗

PubMed1996

Development of new cloning vectors for the production of immunogenic outer membrane fusion proteins in Escherichia coli.

The Pseudomonas aeruginosa lipoprotein gene (oprI) was modified by cloning an in-frame polylinker in both orientations at the end of oprI. The resulting plasmids pVUB1 and pVUB2 allow high lipoprotein production in E. coli after IPTG induction. The modified lipoproteins are present in the outer membrane and surface-exposed. Outer membrane-bound fusion proteins of different sizes were produced and used to generate antibodies without use of adjuvant. An 87 bp DNA fragment from the vp72 capsid protein gene of African Swine Fever virus (ASFV) and the entire Leishmania major glycoprotein gp63 gene were expressed in this system. Finally, a fusion lipoprotein containing a 16 amino acid epitope from the pre-S2b region of Hepatitis B virus (HBV) was presented by an antigen-presenting cell line to a T-cell hybridoma while the corresponding cross-linked S2b peptide was not. The results suggest that OprI-based fusion proteins can be used to generate both humoral and cellular immune responses.

African Swine Fever Virus↗

PubMed1996

A cytochrome c biogenesis gene involved in pyoverdine production in Pseudomonas fluorescens ATCC 17400.

Pseudomonas fluorescens ATCC 17400 produces pyoverdine under iron-limiting conditions. A Tn5 mutant, 2G11, produced lower amounts of different pyoverdine forms and was unable to grow under iron limitation caused by ethylenediamine-di(o-hydroxy-phenylacetic acid) (EDDHA) or zinc. This mutant was complemented by a 9.6 kb HindIII-BamHI DNA fragment that contained eight contiguous open reading frames (ORFs cytA to cytH). The proteins possibly encoded by this polycistronic gene cluster were all similar to the products of cytochrome c biogenesis genes from, amongst others, Rhodobacter capsulatus and Bradyrhizobium japonicum, not only in terms of amino acid sequence, but also in the overall hydropathy index of these proteins. By TnphoA mutagenesis and site-specific gene replacement it was found that the first three ORFs (cytA to cytC) were essential for cytochrome c production while only the product of cytA was needed for normal pyoverdine production. The presence of a putative haem-binding site in the CytA protein (WGSWWVWD) was confirmed. From analysis of a constructed phoA fusion, a periplasmic location was found for this motif. The ability of the cytA gene to restore both cytochrome c and pyoverdine production suggests the involvement of this particular gene both in haem and in pyoverdine transport in P. fluorescens.

Alkaline Phosphatase↗

PubMed1996

Siderophore-mediated iron uptake in Alcaligenes eutrophus CH34 and identification of aleB encoding the ferric iron-alcaligin E receptor.

Siderophore production in response to iron limitation was observed in Alcaligenes eutrophus CH34, and the corresponding siderophore was named alcaligin E. Alcaligin E was characterized as a phenolate-type siderophore containing neither catecholate nor hydroxamate groups. Alcaligin E promoted the growth of siderophore-deficient A. eutrophus mutants under iron-restricted conditions and promoted 59Fe uptake by iron-limited cells. However, the growth of the Sid- mutant AE1152, which was obtained from CH34 by Tn5-Tc mutagenesis, was completely inhibited by the addition of alcaligin E. AE1152 also showed strongly reduced 59Fe uptake in the presence of alcaligin E. This indicates that a gene, designated aleB, which is involved in transport of ferric iron-alcaligin E across the membrane is inactivated. The aleB gene was cloned, and its putative amino acid sequence showed strong similarity to those of ferric iron-siderophore receptor proteins. Both wild-type strain CH34 and aleB mutant AE1152 were able to use the same heterologous siderophores, indicating that AleB is involved only in ferric iron-alcaligin E uptake. Interestingly, no utilization of pyochelin, which is also a phenolate-type siderophore, was observed for A. eutrophus CH34. Genetic studies of different Sid- mutants, obtained after transposon mutagenesis, showed that the genes involved in alcaligin E and ferric iron-alcaligin E receptor biosynthesis are clustered in a 20-kb region on the A. eutrophus CH34 chromosome in the proximity of the cys-232 locus.

Alcaligenes↗

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

Zinc affects siderophore-mediated high affinity iron uptake systems in the rhizosphere Pseudomonas aeruginosa 7NSK2.

Zinc concentrations ranging between 0.1 and 1 mM only slightly reduced maximal growth of wild-type Pseudomonas aeruginosa 7NSK2 in iron-limiting casamino acid medium, but had a clear negative effect on the growth of mutant MPFM1 (pyoverdin negative) and especially mutant KMPCH (pyoverdin and pyochelin negative). Production of pyoverdin by wild-type strain 7NSK2 was significantly increased in the presence of 0.5 mM zinc and could not be repressed by iron even at a concentration of 100 microM. Siderophore detection via isoelectrofocusing revealed that mutant KMPCH did not produce any siderophores, while mutant MPFM1 overproduced a siderophore with an acidic isoelectric point, most likely pyochelin. Pyochelin production by MPFM1 was stimulated by the presence of zinc in a similar way as pyoverdin for the wild-type. Analysis of outer membrane proteins revealed that three iron regulated outer membrane proteins (IROMPs) (90, 85 and 75 kDa) were induced by iron deficiency in the wild-type, while mutants were found to have altered IROMP profiles. Zinc specifically enhanced the production of a 85 kDa IROMP in 7NSK2, a 75 kDa IROMP in MPFM1 and a 90 kDa IROMP in KMPCH.

Bacterial Outer Membrane Proteins↗

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↗