SPEAKERS

Immune evasion by alphaherpesviruses

Herman Favoreel Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University

Thousands of years of co-evolution of animal viruses with the powerful immune defence mechanisms of host organisms have led to an awe-inspiring variety of viral mechanisms to avoid or delay elimination by the immune system. Herpesviruses, large DNA viruses that establish lifelong infections, have developed an exceptional diverse arsenal of such immune evasion tools. This lecture will focus on how members of an important subfamily of the herpesviruses, the alphaherpesviruses, avoid recognition by three important components of the innate and adaptive antiviral immune response: interferons, virus-specific antibodies, and cytotoxic T lymphocytes. Interferons

Interferons (IFNs) can be subdivided in type I and type II IFNs. Type I IFNs (IFN/) belong to the innate immunity and are produced in direct response of a cell to a virus infection. Type II IFN (IFN) makes part of both the innate and acquired immunity since it is synthesized by NK cells and T lymphocytes in response to the recognition of virus-infected cells. One of the important antiviral effects of IFN is the induction of a variety of proteins that interfere with different steps in virus replication. Alphaherpesvirus strategies to interfere with IFN-mediated suppression of replication that will be discussed in the lecture include degradation and inactivation of different IFN-induced antiviral proteins by the viral ICP0, ICP34.5, and US11 proteins. Virus-specific antibodies

Binding of virus-specific antibodies to viral proteins on the surface of virus particles may abolish infectivity of these particles, either by direct neutralization or by inducing opsonization and phagocytosis. Further, herpesvirus-infected cells express a variety of viral proteins on their cell surface, which can trigger antibody-dependent cell lysis via complement, phagocytes, and NK cells. Alphaherpesvirus tools to circumvent these effects of antibodies comprise disabling antibodies to activate antibody-dependent immune effectors by occupying their crucial Fc domain (gE-gI Fc receptor), binding and inactivating complement components by viral protein gC, and removal of antibody-antigen complexes from the surface of infected cells. Cytotoxic T lymphocytes (CTLs)

Some of the newly synthesized viral proteins in virus-infected cells are degraded to peptides, loaded on major histocompatibility complex class I (MHC I) molecules, and transported to the plasma membrane. Specific CTLs recognize these exposed MHC I-viral peptide complexes and subsequently eliminate the infected cell. Alphaherpesvirus methods to avoid CTL-mediated elimination of infected cells include prevention of peptide loading on MHC I molecules via the viral ICP47 protein and inhibition of CTL-induced apoptosis by the viral anti-apoptotic US3 protein. Reviews on different aspects of herpesvirus immune evasion Favoreel, H.W., Nauwynck, H.J., & Pensaert, M.B. (2000). Immunological hiding of herpesvirus-infected cells. Arch. Virol. 145:1269-1290. Johnson, D.C., & Hill, A.B. (1998). Herpesvirus evasion of the immune system. Curr. Top. Microbiol. Immunol. 232:149-177. Leib, D.A. (2002). Counteraction of interferon-induced antiviral responses by herpes simplex viruses. Curr. Top. Microbiol. Immunol. 269:171-185.

Microbial evasion of the plant immune system

Marcelle Holsters, Department Plant Systems Biology and Vakgroep Moleculaire Genetika VIB/Ugent

Technologiepark 927, 9000 Gent marcelle.holsters@psb.ugent.be

Plants have an immune system that allows them to perceive invaders (pathogenic or

potentially beneficial microbes) and to respond by production of antimicrobial compounds,

defense related proteins, and cell death to restrict intrusion. Several major components of the

plant surveillance system have been identified and share features with animal innate

immunity mechanisms. Pathogenic or symbiotic microbial partners have developed various

mechanism to avoid or counteract this immune system, e.g. by injection of effector proteins

via type III secretion systems, and by production of anti-oxidants and protective enzymes to

deal with reactive oxygen species (ROS) produced by the host. Here we shall discuss recent,

novel findings on the importance of surface polysaccharides as a protective device to shield

bacteria from toxic ROS during invasion of host tissues.

Genomics of Neisseria meningitidis to understand bacteria-host interactions

Xavier Nassif, Faculté de Médecine Necker-Enfants Malades, Paris.

Neisseria meningitidis is a commensal of the human nasopharynx which in some circumstances can invade the bloodstrean, cross the blood brain barrier and invade the meninges. The bacteria enter the central nervous system following a direct interaction with the luminal side of the cerebral endothelium, which constitutes the blood–brain barrier. To breach the barriers protecting the brain, N.meningitidis must cross a monolayer of tight junction-expressing endothelial or epithelial cells. The limited number of pathogens capable of crossing these tight barriers and invading the meninges suggests that they display very specific attributes. For Neisseria meningitidis, type IV pili have been identified as being essential for meningeal invasion by inducing the formation of microvilli like structures on the apical surface of the endothelial cells and thus promoting the internalisation of the bacteria. This lecture will focus on recent developpement obtained using a genomic approach to the study of meningococcal pathogenesis focusing on the interaction of N.meningitidis with the endothelial cells. References

Tinsley, C., X. Nassif. Meningococcal pathogenesis: at the boundary between the pre and post genomic eras Current Opinion in Microbiology,, 2001, 4, 47-52. Hoffmann I., E. Eugene, X. Nassif, P-O Couraud, S. Bourdoulous. Activation of ErbB2 receptor tyrosine kinase supports invasion of endothelial cells by Neisseria meningitidis. Journal of Cell Biology, 2001, 155, 133-143. Eugene E., I. Hoffmann, C Pujol, P-O. Couraud, S. Bourdoulous, X. Nassif. Microvilli-like structures are associated with the internalization of virulent cpsulated Neisseria meningitidis into vascular endothelial cells. Journal of Cell Science, 2002, 115, 1234-1241.

Nassif X., S. Bourdoulous, E. Eugene, P-O. Couraud. How do extracellular pathogens cross the blood-brain barrier ? Trends in Microbiology, 2002, 10, 227-232 Geoffroy M-C, S. Floquet, A. Metais, X. Nassif, V. Pelicic. Large-scale analysis of the meningococcus genome by gene disruption: resistance to complement-mediated lysis. Genome Research, 2003, 13, 391-398.

How trypanosomes evade immune responses and become infective

E. Pays Université Libre de Bruxelles

Human sleeping sickness — African trypanosomiasis — is caused by the protozoan parasite Trypanosoma brucei rhodesiense and is transmitted to humans through the bite of infected tsetse flies. Human serum contains a trypanosome lytic factor that can prevent infection by susceptible trypanosomes. Most T. b. rhodesiense strains exhibit resistance to this factor through antigenic variation-linked expression of a gene encoding a truncated form of the variant surface glycoprotein, termed serum resistance associated protein (SRA) (ref. 1). We will detail recent results (ref. 2) showing that SRA mediates resistance to lysis by interaction of its N-terminal alpha-helix with the C-terminal alpha-helix of an apolipoprotein associated with high density lipoproteins, termed apolipoprotein L-I (apoL-I). Direct evidence indicated that apoL-I is indeed the trypanosome lytic factor of human serum. This protein was observed to enter parasite cells via the endocytic pathway and to co-localize with SRA in the lysosome, where the two proteins subsequently interacted. Blocking the SRA-apoL-I interaction may help to prevent or treat the disease.

References. 1. H.V. Xong et al., "A VSG expression site-associated gene confers resistance to human serum in Trypanosoma rhodesiense," Cell, 95:839-846, 1998.

2. L. Vanhamme et al., "Apolipoprotein L-I is the trypanosome lytic factor of human serum," Nature, 422:83-87, 2003.

Vaccinia virus-mediated suppression of the host response to infection

Geoffrey L. Smith Department of Virology, Faculty of Medicine, Imperial College London, St. Mary’s

Campus, Norfolk Place, London W2 1PG, UK Vaccinia virus is the live vaccine that was used to eradicate smallpox and is a member of the orthopoxvirus genus. These large DNA viruses replicate in the cell cytoplasm and encode about 200 genes. Bioinformatic analyses have shown that 90 of these are conserved in all chordopoxviruses and these are clustered in the centre of the virus genome. Genes near the genome termini are more variable and are non-essential for virus replication. Instead these genes affect virus host range, virulence and interactions with the immune system. The latter genes are the subject of this lecture. The variety of immunomodulators encoded by vaccinia virus is quite remarkable. Within the infected cell the virus makes proteins that block apoptosis, signalling pathways or the anti-viral effects of interferons, and even an enzyme that synthesizes steroid hormones. There are also a group of proteins that are released from the infected cell and that bind complement factors, cytokines, chemokines or interferons. The lecture will give an overview of immune evasion by vaccinia virus and then consider recent work with specific genes that influence the immune response to infection. These include a secreted protein that binds IL-18 in solution and an intracellular protein that synthesizes steroid hormones.

POSTER ABSTRACTS

Cytotoxicity of spirulina extracts on human cancer cell lines.

Sarah Baatout (1,*), Matthias Deruelle (2), Hanane Derradji (1,2), Max Mergeay (1), PatrickVan Oostveldt (2), Sofie Bekaert (2,*).

(1) Laboratory of Radiobiology, Belgian Nuclear Research Centre, SCK-CEN, Mol, Belgium; (2) Laboratory for Biochemistry and Molecular Cytology, Department for

Molecular Biotechnology, FLTBW – Ghent University, Belgium. The cyanobacteria A. platensis (also named spirulina) is known for its potential health effects and is reported in the literature to diminish cholesterol level, to be active against diabetes, hypertension, anemia and iron bioavailability, to reduce kidney toxicity from drugs and heavy metals, to help to loose weight, to inhibit HIV replication and to reduce the effects induced after Chernobyl radiation. Some of the beneficial effects are attributed to its natural amount of antioxidants (spirulina is the richest beta-carotene (main source of vitamin A) food known (10x > carrots) as well as iron (58 x > raw spinach and 28 x > raw beef liver). However, spirulina also contains toxins that could be responsible for its cytotoxicity. In this study, we were interested in studying the threshold of intrinsic cytotoxic effect of spirulina on human cancer cells and its cell type dependency. For that purpose, we used flow cytometry to estimate apoptosis and necrosis in three human leukaemic cell lines (HELA : cervix carcinoma; IM-9 : multiple myeloma; K562 : chronic myelogenous leukaemia). Cells were cultured in the presence of spirulina (concentrations ranging from 0 to 500 µg/ml) between 15 to 40 hours. Apoptosis and necrosis were evaluated by the annexin-V-PI staining, cell size and granularity. Early apoptosis was checked by analysing the maintenance of mitochondrial membrane potential (DioC(6)3) and the production of superoxides (hydoethidine) whereas advanced apoptosis was studied by measuring the propensity of the sub-G1 peak, esterase activity (fluorescein diacetate) and intracellular pH (carboxyfluorescein diacetate). The three cell lines had a different sensitivity to spirulina extracts (HELA most resistant > K562 > IM-9 most sensitive). Indeed, concerning the HELA cell line, a significant effect on enzymatic activity was observed only at 500 µg/ml spirulina whilst for K562 cells, spirulina extracts showed a significant effect on K562 enzymatic activity, cell size, cell granularity and intracellular pH from 250 µg/ml. IM-9 showed a significant induction of apoptosis and necrosis when cultured in the presence of 20 µg/ml onwards. These results suggest that, depending on the concentration, spirulina extracts could enhance apoptosis in leukaemic cell lines and may open a perspective for cancer therapy at the condition to also address the intrinsic toxicity of spirulina on normal cells. (*) These authors equally contributed to the work. This work is partially supported by ESA/ESTEC (contract number n°15680/01/NL/ND).

Physiological changes induced in bacteria following oxidative stress.

Patrick De Boever (1,*), Sarah Baatout (2,*), Max Mergeay (1,2). (1) Laboratory for Microbiology and (2) Laboratory of Radiobiology,

Belgian Nuclear Research Centre, SCK-CEN, Mol, Belgium. Flow cytometric tests were performed to estimate physiological changes associated with oxidative stress. Maintenance of membrane integrity and potential as well as the esterase activity, the intracellular pH and the production of superoxide anions was determined in four bacterial strains (Ralstonia metallidurans, Escherichia coli, Shewanella oneidensis and Deinococcus radiodurans). The physiology of the bacterial strains is being studied in order to understand their behaviour and resistance under extreme conditions (such as oxidative stress). This knowledge is of importance in the light of the potential use of these strains for bioremediation. Suspensions of R. metallidurans, E. coli, S. oneidensis and D. radiodurans were submitted to a 1-hour oxidative stress induced by H2O2 concentrations varying from 0 to 880 mM. Fluorochromes, including propidium iodide, rhodamine-123, 3,3'-dihexyloxacarbocyanine iodide, fluorescein diacetate, carboxy-fluorescein diacetate or hydroethidine were chosen as analytical parameters for identifying the physiological state and the overall fitness of individual cells. An individual cell's physiologic state was assessed with a Coulter EPICS XL analyser based on the differential uptakes of these fluorescent stains. The four bacterial strains exhibited varying staining intensities. For all bacterial strains, moderate physiological damage was observed from 13.25 mM H2O2 onwards. Membrane permeability and potential, esterase activity, intracellular pH and production of superoxide anion production were significantly modified at high H2O2 concentrations. In conclusion, it is apparent that a range of significant physiological alterations occurs after oxidative stress. Fluorescent staining methods coupled with flow cytometry are useful for monitoring physiological changes induced by different stress situations. The impact of other stress conditions (radiation, pressure, pH, etc.) is currently being investigated in our laboratories. (*) These authors have equally contributed to the work. This work is partly supported by ESA/ESTEC (contract number n°15680/01/NL/ND).

Fishing genes in lactic acid bacteria to become resistant to the

vancomycin

Marie Deghorain*1, Laetitia Fontaine1, Jean-Luc Mainardi2, Bernard Hallet1, Jean Delcour1and Pascal Hols1

1Unité de Génétique, I.S.V., Université Catholique de Louvain, Belgique, 2Laboratoire de Recherche Moléculaire sur les Antibiotiques, Université Paris VI,

France 1 5/bte6 Place Croix du Sud, B-1348 Louvain-La-Neuve, Belgique.

*Deghorain@gene.ucl.ca.be The Gram-positive bacterial cell wall is mainly composed of peptidoglycan, a polymer of glycan strands linked together via a peptide cross-bridge. Vancomycin inhibits cell wall biosynthesis through binding to the dipeptide D-ala-D-ala located at the end of the peptidoglycan precursor after his transport through the membrane and before cross-bridge formation. Most vancomycin resistant strains contain a modified peptidoglycan precursor in which the terminal D-alanine residue is replaced by a D-lactate, resulting in 1000 fold weaker affinity of the antibiotic to its target. Specific Ddl ligases are responsible of the formation of the dipeptide D-ala-D-ala or the depsipetide D-ala-D-lac. In enterococci, vancomycin resistance requires the acquisition of a set of proteins including a D-lactate dehydrogenase (VanH) and a D-ala-D-ala dipeptidase (VanX), in addition to an exogenous D-ala-D-lac ligase. The lactic acid bacterium Lactobacillus plantarum is intrinsically vancomycin resistant and produces exclusively D-lactate ending peptidoglycan precursors. In contrast, Lactococcus lactis is vancomycin sensitive and produces only D-alanine ending precursors. The heterologous expression of the ddl gene from L. lactis in L. plantarum resulted in vancomycin sensitivity while the reciprocal was unsufficient to make L. lactis vancomycin resistant. A L. lactis ddl mutant strain expressing solely the L. plantarum Ddl ligase was still sensitive and 40% of the precursors were ending by D-ala-D-ala. This result indicates the "in vivo" bispecificity of the L. plantarum Ddl ligase. A resistant L. lactis strain could only be obtained by coexpressing the L. plantarum ddl gene and the vanX gene from Enterococcus faecium. Interestingly, a vanX-like gene was found in the genome of L. plantarum and a D-ala-D-ala dipeptidase activity was detected when this gene was overexpressed. Coproduction of this dipeptidase with the L. plantarum Ddl ligase resulted in a L. lactis ddl mutant strain, which was vancomycin resistant as observed with the enterococcal vanX gene. This finding suggests that the vanX-like gene of L. plantarum may contribute to the intrinsic vancomycin resistance by removing the D-ala-D-ala dipeptides arising from the bispecificity of the L. plantarum Ddl ligase.

The TatA component of the Streptomyces lividans twin-arginine translocation transporter.

De Keersmaeker S., Geukens N., Van Mellaert L., Schaerlaekens K., Lammertyn E.

and J. Anné. Laboratory of Bacteriology, Rega Institute, Katholieke Universiteit Leuven, B-3000

Leuven, Belgium The twin-arginine translocation (Tat) transporter is an unusual protein translocase that is present in the cytoplasmic membrane of many bacteria. This translocase has the unique property to transport folded proteins across the plasma membrane. Protein substrates recognised by the Tat system have a cleavable N-terminal signal peptide that contains an essential S/T-R-R-x-Φ-Φ-“twin-arginine” motif. The bacterial Tat pathway is best characterised in Escherichia coli. Four genes were identified on the chromosome (tatA, B, C and E) of which TatA, TatB and TatC are the major components of the translocase. In Streptomyces lividans, homologues of TatA, TatB and TatC were identified. Recently, the functionality of the Tat pathway has been demonstrated in S. lividans (1). Because of the high number of predicted Tat substrates (230 precursor proteins), the Tat export system is proposed to be more important for protein secretion in Streptomyces than in other bacteria (2). Here, we studied the role of S. lividans TatA in the translocation transporter. TatA is an integral membrane protein of 96 amino acids with a predicted N-terminal α-helical transmembrane domain followed by an amphipatic -α-helix at the cytoplasmic side of the membrane. Hexahistidine-tagged S. lividans TatA was overproduced in E. coli and affinity purified. Gel filtration chromatography demonstrated that S. lividans TatA is present in E. coli as a large homooligomeric complex with an apparent molecular mass of approximately 460 kDa. In addition, a TatA complex with a similar molecular mass could be detected in the membranes of wildtype S. lividans. Furthermore, Western blotting experiments showed that in S. lividans TatA was found to be stable in the absence of TatB or TatC and may therefore participate in a separate complex lacking TatB and TatC. Finally, TatA was found both in the cytoplasmic membrane as well as in the cytoplasm. The latter may be linked to a possible role in recruiting the Tat substrates to the Tat translocase. References : (1) Schaerlaekens, K. et al. 2001. Twin-arginine translocation pathway in Streptomyces lividans. J. Bacteriol. 183: 6727-6732. (2) Schaerlaekens, K. et al. 2003. The importance of the Tat-dependent protein secretion pathway in Streptomyces as revealed by phenotypic changes in tat deletion mutants and genome analysis. Microbiology, in press.

The leader protein of Theiler's virus interferes with nucleo-cytoplasmic trafficking of cellular proteins.

Sophie Delhaye, Vincent van Pesch and Thomas Michiels

Christian de Duve Institute of Cellular Pathology, Université Catholique de Louvain, MIPA-VIRO 74-49, 74, avenue Hippocrate, B-1200, Brussels, Belgium

Theiler's virus is a picornavirus responsible for infections of the central nervous system (CNS) of the mouse. The DA and BeAn strains of the virus provoke a chronic demyelinating disease considered as a model for multiple sclerosis. These viruses have a striking ability to persist and multiply lifelong in the CNS, in the face of a strong specific immune response. We showed previously that the leader (L) protein of Theiler's virus inhibits the production of type-I interferons by infected cells. A L-mutant virus was dramatically impaired in its ability to persist in the CNS of SJL/J mice 1. Here, we observed that the L protein can trigger the subcellular redistribution of cellular proteins. It can promote the redistribution of a NLS-eGFP protein from the nucleus to the cytoplasm and, conversely, of a cytoplasmic protein to the nucleus. It can notably interfere with trafficking of interferon regulatory factor-3 (IRF-3), a cellular factor crucial for early transcriptional activation of interferon genes. Perturbation of nucleo-cytoplasmic trafficking can be viewed as a broader strategy to escape immune responses since the expression of many immune effectors depends on the nuclear translocation of transcription factors. Other picornaviruses were reported to affect the nucleo-cytoplasmic traffic of host proteins. However, none of them expresses a protein related to the leader protein of Theiler's virus. 1. van Pesch, V., O. van Eyll, and T. Michiels, The leader protein of Theiler's virus inhibits immediate-early alpha/beta interferon production. J Virol 75:7811-7817

Entry process of porcine reproductive and respiratory syndrome virus in porcine alveolar macrophages

P. Delputte and H.J. Nauwynck

Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke Belgium

Introduction

Porcine reproductive and respiratory syndrome virus (PRRSV) replicates almost exclusively in cells of the monocyte-macrophage lineage both in vitro and in vivo (2,3). New insights in the early stages of PRRSV entry in macrophages have been gained in the author’s laboratory and are summarized in this overview.

Role of macrophage receptors in PRRSV attachment and internalization

The first step in the replication cycle of a virus is its binding to cell surface receptor(s). At present, two receptors were identified for PRRSV on macrophages: heparan sulphate glycosaminoglycans and sialoadhesin. Another candidate receptor is under investigation (7).

I. Heparan sulphate glycosaminoglycans (1) – The involvement of heparan sulphate glycosaminoglycans in the binding of PRRSV to alveolar macrophages has been shown by (i) reducing both binding and infection of alveolar macrophages by heparin and heparan sulphate in a dose dependent way and (ii) reducing infection of alveolar macrophages by heparinase treatment. By heparin-precipitation, the matrix protein alone, or in a combination with GP5 was identified as a viral ligand for this receptor. II. Sialoadhesin (6) – A monoclonal antibody (mAb 41D3) was produced that reduces binding and blocks PRRSV infection of macrophages (4). A 210 kDa protein (p210) was purified by immunoaffinity with mAb 41D3 and submitted for internal peptide sequencing after tryptic digestion. Amino acid sequence identities ranging from 56% to 91% with mouse sialoadhesin, a macrophage-restricted membrane-anchored glycoprotein, were obtained with four p210 derived peptides. Using these peptide data, the full p210 cDNA sequence (5193 bp) was subsequently determined. It shares 69% and 78% amino acid identity respectively with mouse and human sialoadhesins. Swine (PK15) cells, resistant to viral entry, were transfected with the cloned porcine sialoadhesin cDNA, and inoculated PRRSV. Bound and internalized virus particles were detected only in PK15 cells expressing the recombinant sialoadhesin, demonstrating that this protein mediates uptake of PRRSV. However, nucleocapsid desintegration, like observed in Marc-145 cells as a result of virus uncoating after fusion of the virus with the endocytic vesicle membrane, was not observed, suggesting a block in the fusion process.

The viral ligand that interacts with sialoadhesin is not yet identified. III. A 150 kDa protein doublet - Wissink et al. (7) used a similar protocol as Duan et al. (4) in order to obtain monoclonal antibodies that inhibit PRRSV infection in macrophages. Two mAbs were directed against a 220 kDa protein, most likely the sialoadhesin, and two mAbs directed against a 150 kDa glycoprotein doublet. Necessity of a pH-drop in the PRRSV replication cycle

After uptake in the endosomes, a pH drop is necessary to cause disintegration of the virion and release of the viral genome in the cytoplasm, and to start the replication cycle of PRRSV in macrophages (5). The sequence of changes that allows the PRRSV genome to become released from the endosome into the cytoplasm has still to be determined. References

1. Delputte P., Vanderheijden N. Nauwynck H.J., and Pensaert M.B. 2002. J.Virol. 76, 4312-4320.

2. Duan X., Nauwynck H.J., and Pensaert M.B. 1997a. Vet. Microb. 56, 9-19. 3. Duan X., Nauwynck H.J., and Pensaert M.B. 1997b. Arch. Virol. 142, 2483-2487.

4. Duan X., Nauwynck H.J., Favoreel H., and Pensaert M.B. 1998. J.Virol. 72, 4520-4523. 5. Nauwynck H.J., Duan X, Favoreel H., Van Oostveldt P., and Pensaert M.B. 1999. J. Gen.

Virol. 80, 297-305. 6. Vanderheijden N., Delputte P.L., Favoreel H.W., Vandekerckhove J., Van Damme J., van

Woensel P.A., and Nauwynck H.J. 2003. J.Virol. 77:8207-8215. 7. Wissink, E., Van Wijk H., Pol J., Godeke G., Van Rijn P., Rottier P., and Meulenberg J. 2003.

Arch. Virol. 148, 177-187.

IS231-MIC231, a modular family of transposable elements from Bacillus cereus senso lato

De Palmenaer D., Vermeiren C., Chen Y. and Mahillon J.

Laboratory of Food and Environmental Microbiology, UCL, B-1348 Louvain-La-Neuve, Belgium and Department of Microbiology and Molecular Genetics, University

of Texas HMS, Houston, USA.

Mobile elements are DNA entities able to move about in bacterial genomes. Due to their mobility, they modulate gene expression and translocate “passenger genes”. Combined with horizontal gene transfer mechanisms, they also participate to intercellular fluxes of important features such as virulence determinants or antibiotic resistance genes. IS231A is an Insertion Sequence originally isolated from B. thuringiensis in association with delta-endotoxin genes. Several IS231A-related sequences have since been detected in most members of the B. cereus group, including B. anthracis and B. cereus. Based on recent data, these sequences can now be classified in two groups according to their structural organisation. The first group includes 16 typical iso-IS231 sequences, while the second contains larger entities called Mobile Insertion Cassettes (MIC). MIC231A, from B. cereus, is composed of a D-stereospecific endopeptidase (adp) gene flanked by extremities similar to those of IS231A. Its mobility has been shown both in Bacillus and Escherichia coli. A total of 8 copies of MIC231A were found in almost all members of the B. cereus s.l. group. MIC231V/D, also from B. cereus, displays a unique organisation of inter-digitised cassettes. The first one, named MIC231D, contains an adp gene and is included into a larger cassette, MIC231V, carrying a fosfomycin resistance gene. MIC231E is located upstream of a B. thuringiensis delta-endotoxin gene and seems to be an empty MIC. B. anthracis also hosts several interesting elements. MIC231C, S and T contain, in addition to their transposases, ORFs sharing similarities with antibiotic resistance determinants or transcriptional regulators. The mobility and putative biological functions of these elements will be reported.

The RSH gene of Brucella Melitensis 16M is essential for full virulence and controls VIRB expression in vitro.

M. Dozot, R-M. Delrue, R. Hallez, J.J. Letesson and X. De Bolle.

Unité de Recherche en Biologie Moléculaire (URBM), Laboratoire d’Immunologie et de Microbiologie, Facultés Universitaires Notre-Dame de la Paix, Namur (Belgique).

Several studies suggest that Brucella has to cope with nutritional deprivation during its trafficking inside professional and non professional phagocytes. Moreover, data from Kohler et al. (2002) and Kim et al. (2003) suggest that rsh (for relA spoT homolog) is important for the virulence of Brucella. This gene encodes the main factor of starvation response (also called stringent response) in bacteria. In E. coli, the RelA and SpoT proteins synthesize the nucleotides (p)ppGpp which mediate the adaptation to starvation. In pathogenic or symbiotic bacteria like Legionella and Rhizobium, the Rsh homologs are involved in the adaptation to the intracellular environment. In order to understand the role of rsh in the virulence of Brucella, we created a strain of B. melitensis 16M deleted for rsh using a non polar cassette called aphA4. It is expected that rsh deletion will have pleiotropic effects, as demonstrated in E. coli. In agreement with this, we observed that the rsh mutant presents a particular morphology : after growth in a rich medium, the rsh mutant is two to three-fold bigger than the wild-type strain when observed with electron microscopy. This phenotype was very similar to the one described for the double relA spoT mutant of E. coli. The non polar rsh mutant fail to replicate in ovine macrophages and HeLa cells. The virulence of the rsh mutant in a murine model is currently under investigation. Western blot analysis revealed that some VirB proteins are less abundant in the rsh mutant compared to the wild-type strain when they are grown until mid-exponential phase in rich medium. These data suggest that Rsh may indirectly regulate the expression of the virB operon. KOHLER, S., FOULONGNE, V., OUAHRANI-BETTACHE, S., BOURG, G., TEYSSIER, J., RAMUZ, M., AND LIAUTARD, J-P. (2002). Proc Natl Acad Sci USA 99(24):15711-6. KIM, S., WATARAI, M., KONDO, Y., ERDENEBAATAR, J., MAKINO, S, AND SHIRAHATA, T. (2003). Infect Immun 71(6): 3020-3027

Identification and characterization of a locus involved in bacteriocin biosynthesis in the lactic acid bacterium Streptococcus thermophilus

Laetitia Fontaine1, Benoît Grossiord2, Jean Delcour1 and pascal Hols1 1: Unité de Génétique, Université Catholique de Louvain (UCL), B-1348, Louvain-la-

Neuve, Belgique. 2: Laboratoire de Microbiologie et de Biochimie Appliquée (LMBA), ENITA

Bordeaux, F-33170, Gradignan, France. Streptococcus thermophilus is one of the main economically important lactic acid bacteria

used in the dairy industry. Increasing knowledge of its metabolic properties and

physiology is a prerequisite for the improvement of its industrial properties.

Determination of the entire genome sequence of S. thermophilus LMG18311 was

used in this study to investigate the presence of two-component systems (TCSs) and

their respective functions. Attention was especially focussed on TCSs possibly

involved in the regulation of the production of antimicrobial compounds such as

bacteriocins. Among the ten TCSs identified, two of them showed homology with

TCSs related to bacteriocin biosynthesis loci. The aim of the present study was to

characterize one of the gene clusters containing a TCS showing strong homology with

the previously identified blp gene cluster (bacteriocin like peptide) of Streptococcus

pneumoniae. Such a gene cluster was also present in the two related S. thermophilus

strains, LMD9 and CNRZ1066, for which the full genome sequence is also available.

However, some differences were present when comparing the organization of the blp

gene clusters of the three strains. That allowed us to look for a functional cluster and

to characterize the antimicrobial activity related to those dedicated gene clusters. The

comparison of those gene clusters together with the identification of the bacteriocin

encoding genes will be presented.

Swapping control mechanisms between two site-specific recombination systems

C. Galloy, V. Vanhooff, J. Delcour and B. Hallet

Unité de Génétique, Université Catholique de Louvain-La-Neuve, Place Croix du Sud, 5 bte 6, B-1348 Louvain-La-Neuve

Site-specific recombination is a reaction in which DNA strands are broken and exchanged at specific positions of two distant sites on a DNA molecule. Different forms of this reaction are used in many organisms to bring about programmed and tightly controlled genetic rearrangements. Each site-specific recombination system functions with its own recombinase protein that recognises and acts on determined recombination sites. The recombination reaction is catalysed within a nucleoprotein complex containing 4 recombinase molecules bound as dimers onto short sequences (termed the "core" site, ~ 30 pb) of both recombination sites. In most natural recombination systems, formation of the recombination complex requires additional DNA sequences and proteins to control the outcome and/or the frequency or recombination. Transposon Tn4430 from Bacillus thuringiensis encodes a DNA site-specific recombination system which functions to resolve cointegrate intermediates arising form its replicative mode of transposition. This reaction is catalysed by the TnpI protein in the internal resolution site (irs) of Tn4430. In addition of the plalindromic recombination core region, IR1-IR2, this site contains two accessory TnpI-binding motifs, DR1-DR2. In the Xer system of Escherichia coli, recombination at natural plasmid sites (such as the psi site of pSC101) requires specific host proteins (PepA and ArcA), in addition to the site-specific recombinase XerC and XerD. In both cases, the function of the accessory sequences and proteins is to restrict recombination between sites that are present on a same DNA molecule, and to prevent intermolecular recombination events. However, the topology of the product are different. Recombination by the TnpI/irs system gives a 2-noded catenane, whereas the organisation of the recombination complex of Xer system generates a 4-noded catenane. In order to determine whether the control mechanisms of the two recombination systems can be interchanged, hybrid recombination sites were constructed by joining the core site of one recombination system to the accessory sequences of the other system. These different sites were studied in vivo in E.coli and in vitro with purified recombinases. In vivo analysis of the hybrid sites show that the Xer accessory sequences and protein are proficient in controlling the directionality of the recombination reaction catalysed by the TnpI protein; whereas the converse is not true, the accessory TnpI binding sequences of the irs recombination site have no effect on Xer recombination activity. This indicates that communication between the catalytic and regulatory parts of the recombination complex of these two system is different. In vitro analysis show that PepA can effect the same architectural role in the hybrid site as in the natural Xer site by stimulating the reaction and imposing a specific geometry to the recombination complex.

Higher resistance of porcine trigeminal ganglion neurons towards pseudorabies virus-induced cell death compared to other porcine cell

types in vitro

K. Geenen, H.W. Favoreel, H.J. Nauwynck, and M.B. Pensaert

Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Belgium

Trigeminal ganglion (TG) neurons are the major sites for latency for many

alphaherpesviruses, including the human herpes simplex virus, the porcine pseudorabies virus, and the bovine herpesvirus type 1. Latency is a quiescent form of infection that allows herpesviruses to establish lifelong infections in their host. Virus reactivation from latently infected TG neurons can occur from time to time and may lead to virus replication, virus spread and infection of new hosts. To date little is known about the kinetic time course of alphaherpesvirus replication in TG neurons, and how long TG neurons may survive ongoing intracellular alphaherpesvirus replication.

Therefore, we determined the kinetics of viral antigen expression in porcine TG neurons infected with the swine alphaherpesvirus pseudorabies virus (PRV) in vitro and compared these with other porcine cell types (superior cervical ganglion neurons, primary and continuous swine kidney cells, nonneuronal TG cells, endothelial cells, and blood monocytes). By confocal microscopy, we showed that all TG neurons were infected and expressed early and late viral genes normally. Viral envelope glycoproteins, a subpolulation of the proteins encoded by viral late genes, showed normal processing through the Golgi and reached the cell surface, all as expected. However, expression and cell surface transport of viral proteins was slightly, but significantly, delayed (>3h) in TG neurons versus the other porcine cell types. We next examined the time course of PRV-induced cell death in the different cell types using the live/dead marker ethidium-monoazide-bromide. We showed that 51.1% of the PRV-infected TG neurons survived up to 96 hpi (end of experiment), whereas all other cell types died within 48 hpi. This remarkable difference in time course of PRV-induced cell death in TG neurons versus other porcine cell types cannot solely be attributed to the slightly delayed kinetics in virus replication, and implicates that specific viral and/or cellular factors allow TG neurons to survive a PRV infection for unusual long times. We are currently investigating which viral factors may contribute to survival of PRV-infected TG neurons.

In conclusion, porcine TG neurons can be infected with PRV in vitro and are more resistant to PRV-induced cell death compared with other PRV-infected porcine cell types. High resistance of TG neurons towards PRV-induced cell death may lead to longer time of virus production and spread during virus reactivation and perhaps even allow re-establishment of latency in TG neurons that have reactivated.

High prevalence of Brucella spP. in tissues from apparently healthy GREENLAND SEA hooded seals (Cystophora cristata)

M.Tryland1,2, K.K.Sørensen3, and J.Godfroid4. 1Department of Arctic Veterinary

Medicine, The Norwegian School of Veterinary Science, Tromsø, Norway, 2Department of Microbiology and Virology, University of Tromsø, Norway, 3National

Veterinary Institute, Regional laboratory, Tromsø, Norway, 4Veterinary and Agrochemical Research Centre, Brussels, Belgium

We have previously reported high prevalence (35%) of anti-Brucella antibodies in sera from hooded seals. The aim of the present study was to look further into Brucella-infections in hooded seals by bacteriological and and serological investigations in apparently healthy animals caught in their natural habitat. The animals were caught during a scientific hunt between Spitsbergen (Svalbard) and Greenland in September 2002. Serum and samples (tonsils, lung, mediastinal lymph nodes, spleen, liver, kidney and testes) from 29 seals were obtained. The organ samples (n=174) showed no gross lesions. Tissues were plated on blood agar and modified Farrell’s medium (without nalidixic acid and bacitracin). Suspected colonies were identified as Brucella spp. based on morphological and biochemical characteristics and IS711/IS6501-PCR tests. Marine Brucella spp. were recovered from 37% (11/29) of the animals. The highest prevalence was in spleen (10/29) and mediastinal lymph node (10/24). PCR assays based on the IS711 element found downstream of the bp26 gene that is specifific for marine mammal isolates, gave consistently positive results. All isolates were classified as Brucella pinnipediae according to their omp2 gene locus organisation. Three new insertion sequences were identified and named ISBm1, ISBm2, and ISBm3, in course of sequencing of the wbk locus of B. melitensis 16M. ISBm1 EcoRI RFLP allows the differentiation between pinniped and cetacean Brucella isolates. All isolates were classified as Brucella cetaceae. Our results suggest that at least two different types of Brucella spp. infect the different seal species in the Northern Atlantic. The sera were tested for anti-Brucella antibodies by SAW (with and without EDTA), RB, CFT and a protein-A iELISA, and considered positive only if found sero-positive in all tests. Anti-Brucella antibodies were detected in the serum of 8/11 bacteriologically positive animals. Two additional animals also were serologically positive, giving a total sero-prevalence of 33% (10/29), confirming our previous results. In course of sequencing of the wbk locus of B. melitensis 16M, three new insertion sequences were identified and named ISBm1, ISBm2, and ISBm3. As diagnostic tool, ISBm1 EcoRI RFLP allows the discrimination between terrestrial and marine mammal Brucella strains as does the PCR assay based on an IS711 element found downstream of the bp26 gene only in marine mammal Brucella isolates. Moreover, ISBm1 EcoRI RFLP allows the differentiation between pinniped and cetacean Brucella isolates. Recently specific PCR identification tests have been described [Cloeckaert et al., Microbes Infect. 5(2003) 593-602]: PCR I was specific for B. pinnipediae strains, whereas PCRs II, III, and IV were specific of B. cetaceae strains. It has been suggested that, with respect to the current classification of brucellae, isolates from pinnipeds and cetaceans could actually comprise two new species, B. pinnipediae and B. cetaceae. Our results based on the aforementioned typing methods, suggest that all the hooded seal isolates belong to the B. pinnipediae species.

Glycol chitosan improves the efficacy of intranasally administrated replication defective human adenovirus type 5 expressing

glycoprotein D of bovine herpesvirus 1

Sacha Gogev1, Katalin de Fays 1, Marie-France Versali2, Sandrine Gautier3, Etienne Thiry1

1 Virology-Immunology, Department of Infectious and Parasitic diseases, Faculty of Veterinary Medicine, University of Liège, Belgium

2 Chitin-Chitosan Research Group, Department of Life Sciences, University of Liège, Belgium

3 KitoZyme S.A., Liège, Belgium The ability of two soluble formulations, namely chitosan and glycol chitosan, when used as

an intranasal adjuvant, to improve the immunogenicity of an intranasal human adenovirus

type 5 replication defective expressing bovine herpesvirus 1 (BoHV-1) glycoprotein D based

vaccine, was investigated in cattle. Their adjuvant effects on immune response by increasing

clinical and especially virological protection against an intranasal BoHV-1 challenge were

then evaluated. The best virological protection was obtained in calves immunized with the

vaccine vector adjuvanted with glycol chitosan which decreased the challenge BoHV-1 virus

excretion titres by 0.5-1.5 log when compared to those obtained in calves immunized with the

vaccine vector alone or adjuvanted with chitosan. A slight difference in clinical scores was

observed in calves immunized with the adjuvanted vaccine vector compared to calves

immunized with the vaccine vector alone. The obtained data suggest that the tested soluble

formulation of glycol chitosan has promising potential use as an intranasal adjuvant for

recombinant viral vector vaccines in cattle.

Characterization of the CtrA regulon in Alpha Proteobacteria.

R.Hallez, A. F. Bellefontaine, C. Lambert, J-J. Letesson, J. Vandenhaute and X. De Bolle.

(Unité de Recherche en Biologie Moléculaire (URBM), Laboratoire de Microbiologie et de Génétique Moléculaire, Facultés Universitaires Notre-Dame de la Paix, B-5000

Namur, Belgium). CtrA is a transcriptional regulator found in all alpha proteobacteria analysed to date. In this

group, we find notably the free living bacterium Caulobacter crescentus, the plant’s

symbionts Sinorhizobium meliloti and Mesorhizobium loti, the plant’s pathogen

Agrobacterium tumefaciens, or yet the facultative or obligate animal’s pathogens Brucella sp.

or Rickettsia prowazekii, respectively. In C. crescentus, CtrA is controlled by a complex

multicomponent signal transduction system that regulates and synchronises multiple cell

cycle events (Quon et al., 1996). All the promoters directly regulated by CtrA contain a

characteristic DNA motif (TTAA-N7-TTAAC or TTAACCAT), defined as the CtrA binding

sites since CtrA is able to bind it in vitro and in vivo (Laub et al, 2002). We analysed the CtrA

regulon in other alpha proteobacteria to get a better understanding of the CtrA role in these

bacteria. The release of the alpha proteobacterial genomes has permitted us to characterize in

silico both the upstream and downstream pathways of CtrA regulons. Our results indicate first

that the upstream regulating pathway is partially conserved between these bacterial species.

Indeed, in all genomes except for R. prowazekii, we find not only one homolog for each

factor potentially involved in the control of a CtrA activity in C. crescentus, but also other

potential CtrA activating factor(s) in some species. For identification of CtrA targets, we have

searched all promoter regions containing the CtrA binding site consensus in the seven

genomes. Our observations suggest that similar processes are regulated by CtrA through the

control of distinct target genes.

In conclusion, our results indicate that the alpha proteobacteria are adapted to their lifestyles

while keeping an essential transcriptional regulator with a view to controlling generic cellular

processes.

Laub, M. T., Chen, S. L., Shapiro, L. and McAdams, H. H. (2002) Proc. Natl. Acad. Sci. 99 : 4632-4637. Quon, K. C., Marczynski, G. T., and Shapiro, L. (1996) Cell 84 : 83-93.

Arginine deiminase from Giardia intestinalis, study of a possible drug target.

A. Koaik 1, F. Van Vliet 2, C. Vander Wauven 2, V. Stalon 1,2 & J. Wouters 2

1 Laboratoire de Microbiologie, Université Libre de Bruxelles, Av. Emile Gryson, 1 - B-1070 Bruxelles.

2 Institut de Recherche Microbiologiques Jean-Marie WIAME, Av. Emile Gryson, 1 - B-1070 Bruxelles.

The amitochondriate protozoon Giardia intestinalis is a human parasite: it causes diarrhoea, nausea and it is a major cause of infantile death in developing countries. A number of different classes of drugs have been successful in the therapy of giardiosis. However, treatment fealures in conjunction with drug resistance and the induction of drug-resistant parasites in vitro justifies the search for alternative antigiardials. Arginine fermentation through the arginine deiminase (ADI) pathway is the major source of energy in Giardia (Edwards et al ; 1992). Because it is absent in the host , the ADI enzyme appears as a suitable drug target. ADI from G. intestinalis was produced from a recombinant strain of Escherichia coli and its kinetic properties (Km, Vm, optimum pH) were studied. The effect on the activity of substrate analogues and monovalent or bivalent metallic ions was also tested. Bacterial ADIs are highly conserved proteins. They form a superfamily with the amidinotransferases (Shirai et al ; 2001). The conserved domains can be recognized in the Giardia enzyme primary sequence. Conserved residues, which are part of the catalytic site in the amidinotransferase structure (Humm et al ; 2001), were substituted in the Giardia enzyme. They were all inactive, in accordance with their supposed implication in the catalytic reaction. Compared to the bacterial enzymes, ADI from Giardia intestinalis shows a 145 amino acids long C-terminal extension. Deletion of this extension results in the loss of enzymatic activity, although it is not expected to be part of the catalytic site. A recombinant protein with a N-terminal 6 His-tag was purified to homogeneity. Crystals were obtained and a XR diffraction analysis is planned. The determination of the three dimensional structure will give more insights into the role of the C-terminal extension. It will allow an integration of the kinetic data and the characteristics of the catalytic site, with the ultimate aim to design an efficient inhibitor. References:

1. Edwards MR, Schofield PJ, O'Sullivan WJ, Costello M. Arginine metabolism during culture of Giardia intestinalis. Mol Biochem Parasitol. (1992) ; 53 : 97-103.

2. Shirai H, Blundell TL, Mizuguchi K.A novel superfamily of enzymes that catalyze

the modification of guanidino groups. Trends Biochem Sci. (2001) ; 26 : 465-8.

3. Humm A, Fritsche E, Steinbacher S, Huber R. Crystal structure and mechanism of human L-arginine:glycine amidinotransferase: a mitochondrial enzyme involved in creatine biosynthesis. EMBO J. (1997) ; 16 : 3373-85.

Interspecific recombination between ruminant alphaherpesviruses in vitro

Meurens F.1a, Keil G.2, Muylkens B.1, Gogev S.1, Negro S.1, Thiry E1.

1Department of Infectious and Parasitic Diseases, Laboratory of Virology, Faculty of Veterinary Medicine, University of Liège, Belgium

2Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany

aAspirant FNRS Recombination between different strains of a same alphaherpesvirus after co-infection has

been already described both in vitro and in vivo. Recombination between different

alphaherpesviruses was described between herpes virus simplex 1 and 2 but has not yet been

observed between other alphaherpesviruses. In order to evaluate interspecific recombination,

epithelial bovine kidney cells (Madin Darby Bovine Kidney, MDBK) were infected with a

double deleted mutant of bovine herpesvirus 1 (BoHV-1) (enhanced Green Fluorescent

Protein replaces glycoprotein I and enhanced Red Fluorescent Protein replaces glycoprotein

C, “green and red” phenotype) and different ruminant alphaherpesviruses (“wild type”

phenotype). After 24 h, supernatants were collected and progeny viruses were characterized.

Four phenotypes can be described: “red and green”, “wild type”, and the recombinant

phenotypes “red”, and “green”. While numerous recombinant viruses were isolated (up to 30

%) after BoHV-1 co-infections, only two recombinant viruses were isolated from the three

intertypic co-infections (BoHV-1/caprine herpesvirus 1, BoHV-1/cervid herpesvirus 2,

BoHV-1/BoHV-5). Both recombinant viruses resulted from BoHV-1/BoHV-5 co-infection.

Restriction analysis of recombinant virus genomes showed different genetic backgrounds.

One possessed a restriction pattern more similar to BoHV-1 than to BoHV-5 while the other

possessed a restriction pattern more similar to BoHV-5. Analysis of phenotype percentages

obtained after co-infections show very frequent events of recombination between same or

different strains of BoHV-1, rare events between BoHV-1 and BoHV-5 and no recombination

between BoHV-1 and caprine and cervid herpesviruses. A possible explanation of this result

could be given by parental virus kinetics and genomic characteristics. Indeed, recombination

could only occur with viruses having a very high genome similarity as well as similar kinetic

properties. Although unfrequent, the intertypic recombination occurred in vitro and therefore

should be considered in the control of infections caused by ruminant alphaherpesviruses.

Investigation of the plasmid-borne copper resistance cluster of genes in Ralstonia metallidurans CH34

S. van Aelst1,2, S. Monchy1, A. Benotmane1, R.Wattiez3, T. Vallaeys4 and 1,2M.

Mergeay 1Laboratory for Microbiology, Division Radioactive Waste & Clean-up, Center of

Studies for Nuclear Energy, SCK/CEN, Mol, Belgium. 2Laboratory for Microbiology, Université Libre de Bruxelles, Belgium.

³Laboratoire de Chimie biologique, Université de Mons-Hainaut, Belgium. 4Departement de Microbiologie, INRA, Thiverval Grignon 78850, France.

Ralstonia metallidurans CH34, was isolated from sediments of a zinc factory in Belgium. A facultative hydrogenotroph, it displays multiple resistances to a variety of heavy metals: Ag+, Bi3+, Cd2+, Co2+.CrO4

2-, Cu2+, Hg2+, Ni2+, Pb2+, Tl+ and Zn2+... Strain CH34 carries two megaplasmids, pMOL28 (171kb) and pMOL30 (234kb) that are especially rich in heavy metal resistance genes. Nevertheless, chromosomal material contains also much more metal resistance genes than most of the genomes that have been sequenced up to now (1). In the present communication, we focus on the genes that are involved in the resistance to copper. Chromosomal material contains a cluster copSRcopABCD that is reminiscent of cop genes found in P.syringae or pco genes found on an E.coli plasmid. Equivalents of these six genes are also present in a 15 genes plasmid-bourne copper resistance cluster copVTKSRABCDIJGFLH carried by the plasmid pMOL30. In this study, the function of the plasmid-borne copper resistance genes will be investigated by in-silico analysis (annotation, phylogeny...), by quantitative RT-PCR (transcriptomic) and by insertional mutagenesis. The cluster copV to H has been cloned in the broad host range vector pLAFR3 that was introduced in the plasmid free derivative of CH34 (strain AE104) to give rise to the strain AE1744 (pLAFR3::cop). The observed MICs show that the cloned fragment containing the whole region copV to copH confers a resistance to copper (in the strain AE1744) quite similar to the resistance conferred by the plasmid pMOL30 taken alone (MIC: 1mM). Nevertheless the optimal resistance is still observed by the wild type strain carrying both pMOL28 and pMOL30 (1, 2 mM) while the plasmid free derivative (AE104) displays a MIC of 0.6 mM. We may wonder why 15 genes would be required to only double the resistance to copper. In order to see if all the genes are functional and to investigate their expression, transcriptomic analyses using quantitative PCR technique have been performed on these 15 genes with differents copper concentration and induction times. Transcriptomic data show that all the 15 genes are inductible by copper and may therefore participate to the resistance mechanism. Proteomic data showed that copper induction led to the expression of CopK, CopS, CopR, CopA and CopC. In-silico analysis, such as BLAST and phylogenic comparison from databases allowed to predict hypothetical functions to most of the corresponding proteins although some of them (copK, copT, copV, copL and copH ) look to be unique or to have an equivalent only in the genome of R.metallidurans. Insertional mutants were also made with the intent to hit every of the the 15 genes or putative ORFs. Two differents types of mutagenesis based on transposon insertion have been performed and resulted in the production of single and multiple mutants. Single mutant have been done using mutagenesis by stochiometric transposition of miniTn5-Tp (resistance to trimethoprim) in-vitro, and multiple mutants have been made using the transposition of TnphoA3 by conjugation. We obtain 48 mutants: 4 differents single mutants: copD, copR, copI and copT and several multiple mutants. We plate them on minimal medium containing several copper(II) concentrations: 0mM, 0.4mM, 0.6mM, 0.8mM, 1mM, 1.2mM and 1.5mM to determine their phenotype and compare them to the reference strains. Six phenotype have been observed: wild-type, copper sensitive (as the plasmid-free derivative of R.metallidurans), copper hyper-sensitive (two categories: those still able to grow with 0.4

mM of Cu and those unable to grow even in presence of 0.4 mM), regulation (mutants that display limited growth on the whole range of concentrations) and copper resistant (the clone are more resistant compare to the reference strain). This phenotypic diversity will help us to confirm the function of some of the genes and to understand the interactions between all the involved proteins. Further investigation will focus on the verification of multiple mutants to understand possible synergies, localisation of the proteins (cytoplasm, inner membrane, periplasm, outer membrane, ...) phoA as an indicator, over-expression / purification of genes products involved in copper resistance for in-vitro functional analysis and 3D-structure determination... Then the next step will be to investigate the possible role of others genes involved in copper resistance on the chromosome as well as on the two plasmids.

Virulence of recombinant bovine herpesvirus 1 deleted in the gene encoding glycoprotein E

Muylkens B. a, Meurens F. a, Pourchet A., Schynts F.1, Thiry E.

Departement of Infectious and Parasitic Diseases, laboratory of Virology, Faculty of Veterinary Medecine, University of Liège, Belgium

a Aspirant FNRS 1 Division of Animal Virology, CER, Marloie

Intramolecular recombination is a frequent event during the replication cycle of bovine

herpesvirus 1 (BoHV-1), the causative agent of infectious bovine rhinotracheitis. The

consequences of this process, related to Herpesviridae evolution, have to be assessed in the

context of large use of live marker vaccines for the control of this disease. The epidemiology

of BoHV-1 infection considers the existence of nasal co-infections between vaccine and

wildtype strains. This situation is able to generate recombinant viruses that could combine the

glycoprotein E negative phenotype of the vaccine strain and the virulent phenotype of the

wildtype strain. In order to evaluate the risk associated with the emergence of these new

viruses, gE negative recombinant viruses were generated from seven wildtype strains and one

mutant strain deleted in the genes encoding glycoproteins C and E. Seven co-infections were

performed in vitro. The progeny viruses were analysed by double immunofluorescence

staining in order to isolate gE negative recombinant viruses in each situation of co-infection.

Fourty three gE negative recombinant BoHV-1 were generated. Two PCR confirmed the

complete deletion in the gene encoding glycoprotein E. Virulence of these recombinant

viruses was tested in vitro and compared to profiles of virulence of parental strains. Three

measurements were performed on each virus to assess its virulence : titration, one-step growth

curve and plaque size determination. The results allowed the selection of the most highly

virulent gE negative recombinant virus in each situation of co-infection.

Ectoine release following osmotic downshock in a halotolerant Brevibacterium strain

Annelies Onraedt, Bart Walcarius, Wim Soetaert & Erick Vandamme Ghent University, Laboratory of Industrial Microbiology and Biocatalysis,

Department of Biochemical and Microbial Technology, Coupure links 653, B-9000 Gent, Belgium

e-mail to annelies.onraedt@UGent.be Ectoine is a hygroscopic low-molecular mass compound synthesized or accumulated by halophilic and halotolerant eubacteria for osmotic stress protection. This and other osmoprotective compounds are named compatible solutes, because of their compatibility with cellular metabolism, even at very high concentrations. The size of the intracellular ectoine pool in the bacterial cells depends on the osmotic pressure of their environment. This implicates that the cells need to be equipped with efficient uptake and efflux mechanisms to face the changes in osmolarity. We investigated the release of ectoine following an osmotic downshock using Brevibacterium sp. When Brevibacterium sp. cells, grown in the presence of 1 M NaCl (~2,4 osM), are subjected to an osmotic downshock, ectoine is released in the environment. This efflux is extremely fast; it is not significantly influenced by addition of external ectoine; no efflux is observed under isoosmolar conditions and the efflux remains unchanged after full uncoupling of membrane energetics by carbonylcyanide m-chlorophenylhydrazone (CCCP). These results indicate that an osmoregulated mechanosensitive channel, similar to those found in E. coli and Corynebacterium glutamicum, is responsible for the excretion of ectoine in Brevibacterium sp. The bigger the difference in medium osmolarity before and after the shock, the more ectoine is released. However, when the cells are suspended in deionized water, no more then 60% of ectoine synthesized intracellularly was released into the environment. The cells keep an important stock of ectoine intracellular, although there is no osmotic stress left. Following this release, ectoine was slowly taken up again. This uptake is inhibited by CCCP. We suggest that an energy requiring system is responsible for the uptake. Since ectoine is actively taken up, even in the absence of any osmotic stress, the activity of the uptake carrier is probably not osmotically regulated. Ectoine can serve as a sole source of carbon and nitrogen, so it is most likely that ectoine is consumed by the cells. This was confirmed by the finding that intracellular synthesized ectoine levels decrease after depletion of the carbon source in a standard fermentation medium. Our findings suggest that Brevibacterium species is equipped with an osmoregulated efflux channel and an efficient energy requiring uptake carrier, whose activity is not osmotically regulated.

Gluconobacter NAD-dependent polyol dehydrogenase catalyzed production of D-sorbitol and D-mannitol with coenzyme regeneration

S. Parmentier 1, F. Arnaut 2 & E. Vandamme 1 1. Department of Biochemical and Microbial Technology, Ghent University, Ghent,

BE 2. Puratos N.V., Groot-Bijgaarden, BE

Acetic acid bacteria are known for their unique sugar-metabolizing enzymes. The enzymes of Gluconobacter have been divided into two groups based on their location and function. One group consists of the particulate enzymes tightly bound to the bacterial membrane and linked to the respiratory chain. Those NAD(P)-independent enzymes have a practical importance to produce various oxidation products of carbohydrates. The second group consists of enzymes located in the cytoplasm catalyzing the intracellular metabolism of carbohydrates. The latter are NAD(P)-dependent and are known to occur in high levels. The cytosolic Gluconobacter oxydans LMG 1489 NAD-dependent D-mannitol (MDH) and D-sorbitol (SDH) dehydrogenase were evaluated for their ability to produce D-sorbitol and D-mannitol. D-Sorbitol and D-mannitol are six-carbon sugar alcohols, naturally occurring in small quantities in fruits and vegetables. They are used as a sweet builder in food and in various pharmaceutical preparations. A crude Gluconobacter cell extract was prepared and characterized. The pH-optimum for D-fructose reduction was situated around pH 6.0 and the enzyme was stable within the pH-range of 6.5 to 8.5. D-Fructose reduction was optimal at 50 °C and complete loss of enzymatic activity was observed after heating for 10 minutes at 70°C. The crude Gluconobacter enzyme extract was able to reduce NAD in the presence of D-sorbitol, D-mannitol, D-xylitol and D-arabitol and to oxidize NADH in the presence of D-fructose, L-sorbose and D-xylose. The enzymatic activity for reduction of D-fructose was 10 times lower with NADPH as compared to NAD. Since the enzymes are NADH-dependent, an efficient coenzyme regeneration system is needed for the production of D-mannitol and D-sorbitol. In this context, formate dehydrogenase (FDH) is an interesting enzyme to be used in a coupled enzyme reaction, since no byproducts remain in the reaction mixture.

D-D-mannitol/D-sorbitol

D-fructose

Na-formateCO2 + H2O

NADH NAD

FDH

MDH/SDH

The reaction was performed in a 0.1 M bis-tris buffer pH 6.5 at 25°C. D-Fructose was converted to D-mannitol and D-sorbitol and Na-formate was converted to CO2 and water at the same rate. The optimal substrate concentration was 100 mM for D-fructose and Na-formate. At a coenzyme concentration of 0.5 mM (>Km (KmMDH(NADH) = 0.01 mM, KmSDH(NADH) = 0.28 mM, KmFDH(NAD) = 0.029mM)) the enzymes were saturated with NAD(H), such that -from an economic point of view- a further increase in coenzyme concentration is not needed.

A new immune-escape mechanism : Rho GTPase family members are targets for Yersinia type III effectors to inhibit caspase-1

activation. Peter Schotte (1, 2), Geertrui Denecker (1,3), Peter Vandenabeele (3), Guy Cornelis

(4), Rudi Beyaert (2) (1) These authors contributed equally to this work as first authors.

(2)Molecular Signal Transduction in Inflammation, Department of Molecular Biomedical Research, VIB, Gent University, K.L. Technologiepark 927, B-9052

Zwijnaarde. Tel. 0032 9 3313772, Fax. 0032 9 3313609, E-mail: rudi@dmbr.Ugent.ac.be, Peter.Schotte@dmbr.Ugent.ac.be

(3)Unit of Molecular Signaling and Cell Death, Department of Molecular Biomedical Research, VIB, Gent University, Gent, Belgium.

(4)Division of Molecular Microbiology, Biozentrum, Universität Basel, Basel, Switzerland

A number of Gram-negative pathogens subvert the innate immune system of their host by a

virulence mechanism called type III secretion system (TTSS). In the archetypal Yersinia - Y.

pestis, agent of bubonic plague, Y. pseudotuberculosis and Y. enterocolitica - the TTSS is

encoded on a 70-kb virulence plasmid. By this mechanism, Yersinia bacteria adhering at the

surface of eukaryotic cells inject proteins - called Yops - across cellular membranes into the

cytosol of these cells. These Yops are powerful effectors that take control of the host cells by

hijacking the intracellular machinery. Pathogenic Yersinia disrupts signal transduction

pathways in macrophages, which blocks their response to infection. Here we show that

inhibition of Rho GTPases by the type III effectors YopE and YopT prevents the activation of

caspase-1 and maturation of IL-1beta by the macrophage. Accordingly, caspase-1 activation

was prevented by dominant negative Rac1, as well as by inhibition of Rho GTPases with C.

botulinum Toxin B or the prenylation inhibitor GGTI-2147. Constitutive active Rac1

stimulated caspase-1 protease activity. These results implicate a novel role for Rho GTPases

in the onset of the inflammatory and immune response and shed light on a new function of

different Yop effectors. Intriguingly, modulation of caspase-1 mediated inflammation might

also occur during infection with several other pathogens such as Clostridium spp., Salmonella

spp., Bacillus spp. and Staphylococcus spp., which encode proteins that are also known to

target specific Rho GTPases. Our findings may therefore give new insights in drug design for

treating infectious diseases.

Norovirus detected in bovine species in Belgium

A. Scipioni1, I. Bourgot1, D. Ziant2, G. Czaplicki3, P. Demol4, M. Lomba5, G. Daube2 and E. Thiry1

1 Virology, Department of Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, University of Liège, Belgium, 2 Food Microbiology, Department of Food Sciences, Faculty of Veterinary Medicine, University of Liège, Belgium, 3 Centre de

prévention et de guidance vétérinaire de Loncin, Liège, Belgium, 4 Medical Microbiology and Virology, Faculty of Medicine, University of Liège, Belgium, 5

Centre de prévention et de guidance vétérinaire de Ciney, Namur, Belgium

Noroviruses (NV), from the family Caliciviridae, are responsible of a highly contagious mild human gastroenteritis which is transmissible by the fecal-oral way. They are involved in the majority of foodborne diseases. Humans were considered until recently as the sole host for these viruses but viruses closely related to NV were recently found in stored stool samples from two calves, four pigs and in 44% of specimens from veal calf farms and 2% specimens from pig farm in The Netherlands. These results opened discussions about the potential for zoonotic transmission of these viruses. To investigate the situation in Belgium, 146 stool samples from diseased calves were assayed by reverse transcription-polymerase chain reaction amplification, using primers specific for the detection of NV from humans (Vinjé et al., 1996). In the same time, 469 human samples were also tested. Ten NV RNA were detected and confirmed by sequencing in human samples. NV RNA was also detected and confirmed in one sample of calf stool (B37). The sequences obtained were compared with Genbank database and alignments were generated. Preliminary molecular epidemiology study consisting of comparison of sequences obtained in this study and sequences of members of the Caliciviridae family was carried out. It shows 90% homology on 238 bp with the bovine Newbury virus (Genbank acces number AF097917). Until recently, humans were considered the only host of NV. Recent studies in Japan, United Kingdom and The Netherlands, however, demonstrated calicivirus sequences in the caecum of pigs and in stored pig and calf stool samples containing calicivirus-like particles by electron microscopy. Molecular characterization of calf enteric caliciviruses, named Newbury agent and Jena virus, revealed that they were genetically related and more closely associated with NV. In this study, we detected one Norwalk-like nucleotide sequence in calf stool sample that is close to Newbury agent. This study provides further evidence that NV is present in calves and not only restricted to humans. These findings suggest that there is a potential zoonotic transmission or that calves may be reservoir hosts of NV. VINJE, J. and M.P. KOOPMANS. Molecular detection and epidemiology of Small Round-Structured Viruses (SRSV) in outbreaks of gastroenteritis in the Netherlands. J.Infect.Dis. 174, 610-615, 1996

Occurrence and genetic diversity of Bacillus thuringiensis from national and landscape parks in the NE Poland

Izabela Swiecicka and Jacques Mahillon

MBLA, UCL, Croix Du Sud 2/12, B-1348 Louvain – la - Neuve Recent trends suggest that B. thuringiensis (B.t.) as the environmentally safe microbial pest control agents, will become increasingly important and the extensive use of it in agriculture, forestry, human health and food protection, will increase in the coming years. New isolates of this bacilli, especially from natural environments, can display previously unknown activity against a number of pests, which are not controlled with the available δ-endotoxins, and provide novel alternatives for coping with the problem of insect resistance. B.t. was originally isolated from intenstinal content of free-living rodents and insectivores, settled in Biebrza National Park and Lomza Landscape Park of the Narew River Volley (the north - estern Poland). This region called "The Green Lungs of Poland" contains a rich unique biodiversity, therefore, a high diversity of B.t. can be expected. The purpose of the present study was to assess the occurrence of B.t. in small free-living animals (rodents and insectivorous) and determine their genotypic diversity on the bases of cry genes content and DNA profiling. To identify cry-type genes, PCR with pairs of universal primers for cry homology group, has been exploited. Genotypic relatedness of bacilli under study have been indicated on the basis of DNA patterns determined by pulsed - field gel electrophoresis (PFGE). In total, almost one hundred strains classified as B.t. were isolated from the intestines of small mammals. B.t.-positive samples were found in 10 % animals. Bt index, corresponding to the procentage of B.t. strains on the total number of bacilli, was estimated to be 10%. DNA of one third of isolates did not amplify the cry primers used in the study. None of the strains was positive to cry3 and cry8,7 primers. The rest isolates were grouped in eight cry - profiles. The most frequent cry gene were cry1 and cry9. Among the isolates nine different DNA profiles (A-I) were found for strains from animals caught in both parks. The number of DNA fragments observed in PFGE patterns varied from 5 to 8 within a range of 10 to 1100 kb. In conclusion: 1. The presence of B.t. in the alimentary tract of small free living animals is transient,

indicating that the food of these animals varied in content of this bacteria. 2. B.t. isolated in NE Poland revealed a high genetic diversity in cry genes profile. 3. Although a high diversity among strains has been observed, several independent isolates

turned out to be clonal.

Secretion of biologically active porcine interleukin-6 by Lactococcus lactis

Termont, Sofie1 and Remaut, Erik1

1 Applied Molecular Bacteriology Unit, Department for Molecular Biomedical Research, Flanders Interuniversity Institute for Biotechnology (VIB) and Ghent

University, Technologiepark 927, B-9052 Zwijnaarde (Ghent), Belgium. Sofie.Termont@dmbr.ugent.be

Lactococcus lactis is a non-pathogenic, non-colonizing gram-positive bacterium that is widely used in dairy industries. Because of their presence in the human diet, they are harmless and designated GRAS (“generally recognized as safe”). For this reason, lactococci can be developed as a live delivery system for production of important heterologous biological proteins in a safe way [1]. We successfully cloned porcine interleukin-6 in Lactococcus lactis. The expression of this porcine interleukin is plasmid driven and his codon usage is optimized for L. lactis. Biologically active pIL-6 could be identified on Western blot and expression levels were quantified with sandwich ELISA. Moreover, we were able to chromosomally integrate the expression cassette of pIL-6 by replacement of the thymidylate synthase gene thyA in the genome of Lactococcus lactis [2, 3]. The resulting strain contains the porcine IL-6 gene fused to the usp45 secretion leader and is therefore able to secrete pIL-6 [4]. As the thyA gene is essential for growth of L. lactis, the new biologically contained L. lactis strain is strictly dependent on thymidine for growth and survival [5]. REFERENCES: 1. Schotte, L., et al., Secretion of biologically active murine interleukin-10 by

Lactococcus lactis. Enzyme Microb Technol, 2000. 27(10): p. 761-765. 2. Law, J., et al., A system to generate chromosomal mutations in Lactococcus lactis

which allows fast analysis of targeted genes. J Bacteriol, 1995. 177(24): p. 7011-8. 3. Ross, P., F. O'Gara, and S. Condon, Cloning and characterization of the thymidylate

synthase gene from Lactococcus lactis subsp. lactis. Appl Environ Microbiol, 1990. 56(7): p. 2156-63.

4. van Asseldonk, M., et al., Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subsp. lactis MG1363. Gene, 1990. 95(1): p. 155-60.

5. Steidler, L., Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10. Nature biotechnology, 2003. 21(7): p. 785-789.

Nongrowth energy dissipation as a consequence of substrate release in continuous culture

Jacques Thierie and Michel J. Penninckx

Université Libre de Bruxelles, Faculté des Sciences –Interfacultaire des Bioingénieurs, Laboratoire de Physiologie et Ecologie microbiennes (UPEM), B-

1180 Brussels, Belgium (Fax + 32 (0)2 373 33 09; E-mail: upemulb@resulb.ulb.ac.be)

Except in particular situations [3], bacterial metabolism is devoted to growth; one part of the substrates used to provide the precursor metabolites and the other part being degraded to supply the energy necessary for biosynthesis. In a typical basic situation, the bacterial metabolic flux is only composed by two main flux: an anabolic flux (producing the biomass) and a catabolic flux (or fueling, producing the energy).In the past, it appeared as “logical” to suppose that these two main flux were perfectly coupled in order to achieve maximum efficiency and economy. Yet, this assumption was contradicted by the discovery of resting cells, which are able to utilize energy sources in the complete absence of growth [3,2]. The advent of continuous-culture techniques in the 1950s (and especially the chemostat) permitted to cultivate bacteria under defined growth rates and to keep the cultures in steady states of well defined composition. It rapidly appeared that nongrowth energy dissipation was a very common phenomenon. Moreover, it was shown that the energy dissipation generally depended on growth rate. The nongrowth energy dissipation question is now more than fifty years old and is still the subject of many debates. So far as we know, we have encountered two manners to tackle the problem in literature. The first could be presented as a physiological approach and terms as “energy spilling”, “wastage”, “slip reactions”, futile cycles” and “overflow metabolism” are used together with “maintenance energy” and “endogenous metabolism”. The physiological approach has provided some good illustration of energy dissipation but seem unable to offer an unique and coherent picture of the phenomenon. The second consider the problem from a thermodynamical point of view and especially in the framework of non-equilibrium thermodynamics. Conceptually, resting cells, for example, may be considered as dissipative structures [1] for which the “spilled” energy is used to maintain the spatial structure of the cell. From a more specific perspective, the mosaic non-equilibrium thermodynamics of bacterial growth [5] discuss clearly the correlation between energy dissipation and efficiency of a system (the optimal state does not necessarily corresponds to the maximal efficiency state). In this viewpoint, energy dissipation represents optimization costs of some process. The present communication is based on previous results [4] and show how the release of a previously substrate taken up by the cell (that is to say, a substrate cycling) may induce energy dissipation and can take all the “spilled” energy into account. Our main result showed how a substrate release leads to uncoupling of anabolic and catabolic flux. The new mechanism we proposed may also be considered as a possible process devoted to the regulation of energy dissipation. Bibliography. [1] Glansdorf P and Prigogine I (1971). Structure, stabilité et fluctuations. Masson et Cie. Eds, Paris [2] Russell JB & Cook GM (1995). Energetics of bacterial growth: Balance of anabolic and catabolic reactions. Microbiol. Rev. 59:48-62 [3] Russell JB & Strobel HJ (1990). ATP-ase dependent energy spilling by the ruminal bacterium Streptoccocus bovis. Arch. Microbiol. 153:378-383 [4] Thierie J (2000). Cellular cycling of substrate as a possible cryptic way for energy spilling in suspended cellular continuous cultures. Biotechnol. Letters 22:1143-1149 [5] Westerhoff HV, Lolkema JS, Otto R, & Hellingwerf KJ (1982). Thermodynamics of growth. Non-equilibrium thermodynamics of bacterial growth. The phenomenological and the mosaic approach. Biochim. Biophys. Acta 683:181-220.

Infection with PRRS virus increases components of the receptor complex for bacterial lipopolysaccharides in the lungs of pigs

Steven Van Gucht, Kristien Van Reeth, Geoffrey Labarque, Hans Nauwynck, Maurice

Pensaert

Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium

Introduction Porcine reproductive and respiratory syndrome virus (PRRSV) is a respiratory virus of swine that plays an important role in multifactorial respiratory disease. European strains of PRRSV cause mild or no respiratory signs on their own, but can sensitize the lungs for the production of proinflammatory cytokines and respiratory signs upon exposure to bacterial lipopolysaccharides (LPS) (1,2). LPS are endotoxins of Gram-negative bacteria. They are present in high concentrations in organic dust in swine confinement units and released locally in the lungs during bacterial infection. The inflammatory effect of LPS depends on the binding to the LPS receptor complex. Both CD14 and LPS-binding protein (LBP) are components of this receptor complex. CD14, also called the primary LPS receptor, is expressed on the membrane of monocytes, macrophages and neutrophils. Binding of LPS to CD14 can be enhanced by LPS-binding protein (LBP), a soluble acute phase protein produced by liver and lung epithelial cells. LBP increases the effect of LPS a 100-fold. In this study, we quantified the levels of CD14 expression and LBP in the lungs of pigs throughout a PRRSV infection. Materials and methods Twenty-four gnotobiotic pigs were inoculated intranasally with PRRSV (106 50% tissue culture infectious doses per pig, Lelystad strain) or phosphate-buffered saline (PBS) and euthanatized 1 to 52 days later. Lungs were examined for CD14 expression (immunofluorescence and image analysis), LBP (ELISA) and virus replication. CD14-positive cells were characterized using different cell markers. Results In the lungs of PBS-inoculated pigs, cells with clear CD14 expression were scarce (15 11 cells/mm2) and confined to single cells in the interstitium. Most resident (sialoadhesin-positive) macrophages expressed little CD14 on their membranes. Throughout the PRRSV infection, there was a massive infiltration of highly CD14-positive cells in the lungs. These cells were clustered in the interstitium and positive for the monocyte-macrophage marker SWC3a, but negative for the macrophage marker sialoadhesin. This process resulted in an increase of CD14 expression in the lung tissue starting from 3 days post inoculation (DPI), peaking at 9 DPI (40 times higher than the PBS control pigs) and back to normal at 40 DPI. All pigs had detectable levels of LBP in their BAL fluids. PBS-inoculated pigs had 105 144 ng LBP/ml. Pigs euthanatized between 7 and 14 DPI had 3 to 12 times higher levels of LBP compared to the PBS-inoculated pigs. Both CD14 and LBP peaked at 9-10 DPI and were correlated tightly with the amount of virus replication in the lungs. Conclusion PRRSV causes a clear increase of CD14 and LBP, two components of the LPS receptor complex, in the lungs during infection. The increase of CD14 expression is mainly due to an infiltration of highly CD14-positive monocytes. As both CD14 and LBP potentiate the inflammatory effects of LPS, their increase in the lungs could explain why PRRSV sensitizes the lungs for the production of proinflammatory cytokines and respiratory signs upon exposure to LPS.

References 1. Labarque G., Van Reeth K., Van Gucht S., Nauwynck H. and Pensaert M., 2002. Porcine reproductive-respiratory syndrome virus infection predisposes pigs for respiratory signs upon exposure to bacterial lipopolysaccharide. Vet. Microbiol. 88 (1): 1-12. 2. Van Gucht S., Van Reeth K. and Pensaert M., 2003. Interaction between porcine reproductive-respiratory syndrome virus and bacterial endotoxin in lungs of pigs: potentiation of cytokine production and respiratory disease. J. Clin. Microbiol. 41 (3): 960-966.

New endogenous interferon alpha subtypes expressed irrespective of viral infection

Vincent van Pesch, Hanane Lanaya and Thomas Michiels

Christian de Duve Institute of Cellular Pathology, Université Catholique de Louvain, MIPA-VIRO 74-49, 74, avenue Hippocrate, B-1200, Brussels, Belgium

Type-I interferons or IFNs-, are a family of cytokines induced by viral infection and primarily involved in antiviral defense of the cells. IFNs- were also reported to be produced constitutively at low levels in mouse and human cells. These so-called endogenous IFNs are thought to exert important homeostatic functions in the uninfected host as well. We previously showed 1 that the leader (L) protein of Theiler's virus inhibits the production of type-I interferons by infected cells. The L protein specifically inhibits transcription of the IFN -4 and IFN- genes which are known to be activated early in response to viral infection and to be required for transcriptional activation of the other IFN- subtype genes. By searching IFN genes that were not repressed during Theiler's virus infection, we identified three uncharacterized IFN- genes that are constitutively expressed in uninfected mouse cells, in vitro and in vivo 2. Two of these genes correspond to pseudogenes that were tentatively called IFN-(2)and IFN-(3). IFN-�(2) transcripts are the most abundant IFN- transcripts detected in several mouse organs in the absence of viral infection. The third gene, expressed constitutively at low levels codes for a new IFN- subtype (tentatively called IFN-13) with antiviral activity. IFN-13 displays unusual characteristics: first, it is transcribed constitutively at very low levels and transcription of this gene is not responsive to viral infection; second, this IFN carries two predicted N-glycosylation sites in contrast to other IFN- subtypes which contain either a single or no N-glycosylation sites. The peculiarities of IFN-13 suggest an unusual role for this new constitutive interferon. We further took advantage of the mouse genome sequencing project to characterize the various IFN- subtypes expressed in murine cells. A total of 14 IFN- genes and 3 IFN- We started to characterize those IFN- subtypes to address the question of the reason for the multiplicity of IFN genes present in the mouse and human genomes. 1. van Pesch, V., O. van Eyll, and T. Michiels, The leader protein of Theiler's virus inhibits immediate-early alpha/beta interferon production. J Virol 75:7811-7817 2. van Pesch, V., and T. Michiels, Characterization of IFN-13, a novel constitutive murine interferon alpha subtype. J. Biol. Chem. 2003 in press (manuscript freely available at www.jbc.org)

Gelatinase B (MMP-9) and neutrophil collagenase (MMP-8) regulate the biological activities of CXC-chemokines

Van den Steen, P.E., Proost, P., Wuyts, A., Van Damme, J., Opdenakker, G.Rega Institute, University of Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium

During bacterial and viral infections, chemokines attract leukocytes to fight the invading microorganisms. Chemokines also activate the leukocytes, resulting in the production and secretion of proteolytic enzymes as part of the innate immune defence mechanisms. However, overproduction of these proteases may contribute to the pathology of e.g. septic shock. We have demonstrated that bacterial (LPS) and viral products (double stranded RNA) induce gelatinase B in leukocytes, and that this enzyme appears in vivo within minutes after experimental bacteremia. Here, we describe that two proteases, gelatinase B (matrix metalloproteinase-9, MMP-9) and neutrophil collagenase (MMP-8), modify the biological activity of chemokines. Neutrophils secrete gelatinase B and neutrophil collagenase after stimulation with CXC-chemokines, e.g. interleukin-8/CXCL8 (IL-8). Gelatinase B cleaves and potentiates IL-8 generating a positive feedback. These findings are further extended by comparison of the processing of the CXC-chemokines human and mouse granulocyte chemotactic protein-2/CXCL6 (GCP-2) and the closely related human epithelial-cell derived neutrophil activating peptide-78/CXCL5 (ENA-78) with that of human IL-8. Human GCP-2 and ENA-78 are cleaved by gelatinase B at similar rates as IL-8. In addition, GCP-2 is cleaved by neutrophil collagenase, though at a lower rate. The cleavage of GCP-2 is exclusively aminoterminal and does not result in any change of biological activity. In contrast, ENA-78 is cleaved by gelatinase B at 8 positions with varying rates. Sequential cleavage of ENA-78 may physiologically result in an early potentiation and at later time intervals into inactivation of the chemokine. Remarkably, in the mouse species, which lacks IL-8 and where murine GCP-2/LIX replaces IL-8 from the human species as the most potent neutrophil activating chemokine, also aminoterminal clipping and 2-fold potentiation by gelatinase B was observed. In addition to the similarities in the potentiation of IL-8 in humans and GCP-2 in mice, the conversion of mouse GCP-2/LIX by mouse gelatinase B is so far the fastest for any reported combination of chemokines and MMPs. This rapid conversion was also performed by crude neutrophil granule secretion under physiologic conditions, extending the relevance of this proteolytic cleavage to the in vivo situation. An alternative aminoterminal cleavage of mGCP-2/LIX by neutrophil degranulate was observed, which was inhibitable by pefabloc. The protease responsible for this cleavage was identified as neutrophil elastase. References: Van den Steen et al., 2000, Neutrophil gelatinase B potentiates interleukin-8 tenfold by aminoterminal processing, whereas it degrades CTAP-III, PF-4 and GRO- and leaves RANTES and MCP-2 intact. Blood 96:2673-2681. Van den Steen et al., 2003, Gelatinase B/MMP-9 and neutrophil collagenase/MMP-8 process the chemokines human GCP-2/CXCL6, ENA-78/CXCL5 and mouse GCP-2/LIX and modulate their physiological activities. Eur. J. Biochem. 270:3739-3749. Paemen et al., 1997, Induction of gelatinase B and MCP-2 in baboons during sublethal and lethal bacteraemia. Cytokine 9:412-415.

Absence of viral antigens on the surface of EHV-1-infected Blood mononuclear cells during viremia: a potential strategy of immune

escape?

K.M. van der Meulen, H.J. Nauwynck, M.B. Pensaert

Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium

Equine herpesvirus type 1 (EHV-1) is a major pathogen of horses. Upon infection, the virus

replicates in the respiratory tract. Primary replication is followed by a leukocyte-associated viremia. Carried by infected peripheral blood mononuclear cells (PBMCs), EHV-1 spreads to internal organs where it may induce abortion or nervous system disorders. Infection with EHV-1 is the major cause of infectious abortion in horses and causes serious economic losses to horse breeders worldwide. Vaccination with the currently available vaccines is able to limit replication of EHV-1 in the respiratory tract, however, it cannot prevent EHV-1-induced viremia. Therefore, abortion regularly occurs in vaccination-immune horses.

Previous in vitro studies demonstrated that 70 % of EHV-1-infected PBMCs lack viral antigen expression on their cell surface. Absence of surface expression protects infected PBMCs from complement-mediated lysis in vitro and represents a potential strategy of immune evasion. It was examined if infected PBMCs lack surface expression of viral antigens during viremia upon experimental inoculation of ponies. Moreover, surface expression of major histocompatibility complex class I (MHC-I) was examined.

Four ponies that had been previously exposed to EHV and one naive control pony were inoculated with the Belgian EHV-1 strain 97P70. PBMCs were collected at different time points between 0 and 28 days post inoculation and surface expression of viral antigens and MHC-I was determined on infected PBMCs by immunofluorescence labeling.

Expression of viral antigens on the surface of EHV-1-infected PBMCs was observed in one of the four ponies previously exposed to EHV (1/8 infected PBMCs). In the other three ponies, all examined EHV-1-infected PBMCs lacked visually detectable levels of plasma membrane-anchored viral antigens (0/7, 0/8 and 0/8 infected PBMCs, respectively). Also in the naive control pony, none of the EHV-1-infected PBMCs showed surface expression of viral antigens (0/18 infected PBMCs). MHC-I was found on 80-100 % of EHV-1-infected PBMCs in all ponies. This resembled the percentage of MHC-I-positive cells in non-infected PBMCs (83-100 %).

In conclusion, absent surface expression of viral antigens may allow EHV-1-infected PBMCs to escape from antibody-dependent immune responses and, therefore, may explain how EHV-1 can reach the pregnant uterus or the central nervous system despite the presence of EHV-1-specific antibodies.

Analysis of The complete genome sequence of bacteriophage vwb

W. Van Dessel1; L. Van Mellaert1; W. Streit2 and J. Anné1 1Laboratorium voor Bacteriologie, Katholieke Universiteit Leuven, Rega Instituut,

Minderbroedersstraat 10, B-3000 Leuven, Belgium. 2Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen,

Grisebachstrasse 8, 37077 Göttingen, Germany. The actinophage VWB, isolated from a soil sample enriched with Streptomyces venezuelae is a narrow host bacteriophage only infecting Streptomyces venezuelae ETH14630 and Streptomyces exfoliatis ATCC12672. It is a temperate phage belonging to the Siphoviridae family (Anné et al., 1984). Using attP and the phage encoded integrase a very efficient site-specific integration vector was developed earlier, allowing the integration of genes of interest into the attB site consisting of a tRNAArg (Van Mellaert et al., 1998). To eventually identify other genes of interest, the complete nucleotide sequence of VWB (GenBank nr. AY320035) was determined, resulting in a 49220 bp DNA sequence with a 71.2% GC-content. This is, next to C31 (Smith et al., 1999), the second Streptomyces phage sequenced so far. Sequence analysis was performed mainly using heuristic models. Characteristically, these models are not ‘trained’ by means of a set of already annotated genomes. Instead they use a theoretical algorithm with parameters derived from the sequence to be annotated. This would allow predictions in small anonymous genomes, such as those of bacteriophages (Besemer and Borodovsky, 1999; Besemer et al., 2001). These analyses suggest the presence of 61 open reading frames (ORFs) covering 84.3% of the phage VWB genome. Furthermore, the obtained ORFs were subjected to several annotation efforts. By using a combination of software packages, the known issue of low similarity to homologous genes, even to those of other phages, was somewhat tempered. As a consequence, a homologous gene to 35 or the 61 ORFs could be detected in the GenBank, and in several cases a potential function could be assigned. It could be noticed that ORFs predicted to encode similar functions, were grouped in blocks. This allowed a preliminary comparison of VWB to other phages, suggesting a similar block arrangement as in the genomes of low GC content Siphoviridae. These findings also support the modular theory of phage evolution (Lucchini et al., 1999, Botstein 1980). References Anné J., Wohlleben W., Burkardt H.J., Springer R., Puhler A. (1984). Morphological and molecular characterization of several actinophages isolated from soil which lyse Streptomyces cattleya or S. venezuelae. J. Gen. Microbiol., 130, 2639-2649. Besemer, J., Borodovsky, M. (1999). Heuristic approach to deriving models for gene finding. Nucl. Acids Res., 27, 3911-3920. Besemer J., Lomsadze A., Borodovsky M. (2001). GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucl. Acids Res., 29, 2607-2618. Botstein D. (1980). A theory of modular evolution for bacteriophages. Ann. N.Y. Acad. Sci., 354, 484-490. Lucchini, S., Desiere, F., Brüssow, H. (1999). Similarly organized lysogeny modules in temperate Siphoviridae from low GC content Gram-positive bacteria. Virology, 263, 427-435. Smith M.C., Burns R.N., Wilson S.E., Gregory M.A. (1999). The complete genome sequence of the Streptomyces temperate phage straight phiC31: evolutionary relationships to other viruses. Nucl. Acids Res., 27, 2145-2155.

Van Mellaert L., Mei L., Lammertyn E., Schacht S., Anné J. (1998). Site-specific integration of bacteriophage VWB genome into Streptomyces venezuelae and construction of a VWB-based integrative vector. Microbiology, 144, 3351-3358.

Further evidence for a role of cytokines in the pathogenesis of swine influenza: results from vaccination-challenge studies

Kristien Van Reeth and Maurice Pensaert

Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium

Introduction Vaccination against swine influenza virus (SIV) generally reduces virus replication upon challenge. In experimental infection studies of influenza virus naive pigs, we found a strong correlation between peak virus titres in the lungs, proinflammatory cytokine levels in lung lavage fluids and disease (1). Also in humans, interferon-alpha (IFN-), tumour necrosis factor-alpha (TNF-), interleukin-1 and –6 (IL-1 and –6) are considered important mediators of influenza symptoms (2). Here we have studied the effect of vaccination on cytokine production after an influenza virus challenge, to further confirm the relationship between virus replication, cytokines and disease. Materials and methods Forty-five pigs were vaccinated twice with a commercial inactivated SIV vaccine or with experimental vaccines, and 31 control pigs were not vaccinated. Three weeks after the last vaccination, all pigs were challenged intratracheally with SIV. Twenty-four hours after the challenge, we determined clinical scores, lung virus titres, neutrophil counts and bioactive levels of IFN-, TNF-, IL-1 and IL-6 in lung lavage fluids. Results Virus was isolated from the lungs of all 31 challenge controls (mean titre 6.9 log10 TCID50) and from 24/45 vaccinated pigs (mean titre 3.4 log10 TCID50). Fever, respiratory disease and neutrophil infiltration of the lungs developed in 29/31 controls, but only in 10/45 vaccinates. Compared with the unvaccinated controls, vaccination reduced mean IFN- and IL-6 levels at least 100 times and TNF- levels 10 times. Virus titres were directly correlated with disease severity and with IFN-, TNF- and IL-6. IL-1 levels, in contrast, were similar in unvaccinated and vaccinated pigs and they were not correlated with other parameters of disease. Conclusion These studies provide further evidence for a causal relationship between virus replication, cytokine production and symptom formation. References 1. Van Reeth K., Nauwynck H.J., Pensaert M.B., 1998. Bronchoalveolar interferon-, tumor necrosis factor-, interleukin-1 and inflammation during acute influenza in pigs: a possible model for humans? J. Inf. Dis. 177: 1076-1079. 2. Hayden F.G., Fritz R., Lobo M.C., et al. 1998. Local and systemic cytokine responses during experimental human influenza A virus infection. Relation to symptom formation and host defense. J. Clin. Invest. 101: 643-649.

Pseudorabies virus (PRV)-specific antibodies suppress intracellular viral protein EXPRESSION in PRV-infected porcine blood monocytes

G.R. Van de Walle, H.W. Favoreel, H.J. Nauwynck, M.B. Pensaert

Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Belgium

Blood monocytes infected with pseudorabies virus (PRV), a swine alphaherpesvirus, may

carry PRV via the blood to internal organs, even in vaccinated animals with a strong humoral immune response. This implies that PRV-infected monocytes are not efficiently eliminated by the antibody-dependent immune system. Infected monocytes express viral proteins on their cell surface, rendering these cells recognizable to antibody-dependent lysis. However, we showed earlier that upon antibody binding, viral proteins gB and gD mediate rapid internalization of the majortity of antibody/viral protein-complexes from the cell surface. This process inhibits efficient antibody-dependent cell lysis (=immune-masked monocytes).

Here, we examined in vitro the long term fate of such immune masked monocytes. Therefore, PRV-infected monocytes were incubated with porcine PRV-specific or PRV-negative antibodies. All monocytes were productively infected, as assessed by the expression of late genes. However, 30% of the infected monocytes, cultivated in the continuous presence of virus-specific antibodie,s survived up to 194 hpi, end of experiment (compared to 0% for cells cultivated with PRV-negative antibodies). Of these surviving cells, ±75% did no longer express microscopically detectable viral late proteins from 144 hpi onward.

These data suggest that PRV-specific antibodies suppress viral protein expression in infected monocytes, perhaps helping the virus to persist and reach internal organs in vaccinated animals.

Pseudorabies virus US3-mediated actin stress fiber breakdown may facilitate spread of the virus in vitro

G. Van Minnebruggen1, H.W. Favoreel1, L. Jacobs2, H.J. Nauwynck1, M.B. Pensaert1 1Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Belgium

2Institute for Animal Science and Health (ID-DLO), Lelystad, The Netherlands.

Pseudorabies virus (PRV) is an alphaherpesvirus of swine, closely related to the human

herpes simplex and varicella-zoster viruses. We have demonstrated that, during a PRV infection of different cell types, the PRV-encoded US3 serine/threonine protein kinase induces breakdown of a specific component of the cellular cytoskeleton: the actin stress fibers.

From literature, it is known that during infection, PRV US3 is expressed as a long (< 5% of US3 protein) and a short isoform (> 95% of US3 protein). We have PCR-amplified both US3 isoforms and cloned them in a mammalian expression vector. Transfection of these constructs in swine kidney cells showed that both US3 isoforms are able to induce actin stress fiber breakdown in the absence of other PRV proteins. In addition, we found that abundant localization of US3 in the nucleus is a prerequisite in order to induce stress fiber breakdown. Next, we investigated whether PRV US3-mediated actin stress fiber breakdown is important for direct cell-to-cell spread of the virus, an efficient yet poorly understood process that allows direct spread of alphaherpesviruses to neighbouring cells without contact with the immune surveilled extracellular environment (e.g. virus-neutralizing antibodies). Therefore, we performed plaque assays in the presence of virus-neutralizing antibodies using wild type and US3 null viruses, either in the absence or presence of Y27632 (a Rho-specific inhibitor that induces stress fiber breakdown). US3 null plaques were much smaller compared to wild type plaques, and Y27632 significantly increased US3 null plaque size whereas it did not affect wild type plaque size.

In conclusion, the PRV US3 protein kinase can induce actin stress fiber breakdown in PRV-infected and US3-transfected cells, and PRV US3-mediated actin stress fiber breakdown may be involved in direct cell-to-cell spread of the virus.

Genetic differentiation of Arthrospira strains

M. Waleron ac, K. Waleron ac, G. Duysens a, L. Hendrickx b, M. Mergeay b, A. Wilmotte a

a CIP, Institute of Chemistry B6, University of Ličge, B-4000 Ličge, Belgium b Laboratory for Microbiology, Division Waste & Clean-up, & Section of

Radiobiology, Division Radioprotection, Belgian Nuclear Research Centre, SCK/CEN, Boeretang 200, B-2400-MOL, Belgium

c Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk & Medical School of Gdansk, Kladki 24, 80-822 Gdansk Poland

The genus Arthrospira consists of several species: A. funiformis, A. fusiformis, A. geitleri, A. gomontiana, A. indica, A. jenneri, A. khanne, A. massartii, A. maxima, A. miniata, A. platensis, A. tenuis (Jeeji-Bai, 1999) which are difficult to distinguish. A molecular characterisation based on the ITS (Internally Transcribed spacer in rRNA gene) from 50 Arthrospira strains revealed that there were 4 genotypes grouped into 2 clusters (I and II). Inside each of them, 2 subclusters were observed (I.A, I.B and II.A, II.B). The 5th genotype was a consequence of the presence of two ITS forms II.A and II.B in the cells of the same strain (Scheldeman et al. 1999; Baurain et al. 2002). To study the genetic variability of several Arthrospira strains belonging to different ITS clusters, we have used several different methods. We have studied the polymorphism of 3 DNA loci: gyrA, recA and rpoC. Also 2 fingerprinting methods have been used: ERIC-PCR and HIP-PCR. New pairs of PCR primers were designed for the amplification of gyrA, recA and rpoC. They allowed us to amplify and sequence a fragment of each gene. A comparison of the resolution power of the different DNA loci with the data of the ITS sequences was carried out. The ERIC-PCR has been used for fingerprinting and diversity studies of different cyanobacterial species (Rasmussen & Svenning, 1998) and other bacterial genera (Versalovic et al. 1991). The use of ERIC-PCR to study the diversity of 18 Arthrospira strains shows that strains belonging to the 4 ITS subclusters may have different ERIC patterns. Indeed, the ERIC patterns are very similar inside the ITS clusters I.A and I.B, but quite heterogenous within clusters II.A and II.B. It is interesting to note that for 2 strains belonging to cluster II.A/B, one (PCC 7345) has a pattern similar to the one of cluster II.A and the second (SAG 257.80) is similar to Cluster II.B. Thus, ERIC-PCR might allow to differentiate Arthrospira strains with higher resolution than ITS. Another fingerprinting method, HIP-PCR, has been successfully used to distinguish cyanobacterial species and strains (Smith et al. 1998). Bhaya et al. (2000), described several modified primers based on HIP sequences, which we have used for differentiation of Arthrospira strains. Our preliminary results suggest that this method could be useful for this purpose and will have similar discriminatory power as ERIC-PCR. Bhaya D. Vaulot D, Amin P, Watanabe T., A. & Grossman A.R. (2000) Isolation of regulated genes of the cyanobacterium Synechocystis sp. strain PCC 6803 by differential display. J. Bacteriol. 182: 5692-5699. Baurain, D., Renquin, L., Scheldeman, P., Belay A. & Wilmotte A. (2002) Remarkable conservation of internally Transcribed Spacer sequences of Arthrospira (“Spirulina”) (Cyanophyceae, cyanobacteria) strains from four continents and of recent and 30 years-old dried samples from Africa. J. Phycology 38: 384-393 Jeeji-Bai, N. (1999). A taxonomic revision of the genus Arthrospira based on certain new criteria. In Charpy, L. & Larkum, A. W. D. [Eds.] Marine Cyanobacteria. Bull. Instit. Océanogr. Monaco, special issue n°19:47-52. Rasmussen, U. & Svenning M.M. (1998). Fingerprinting of cyanobacteria based on PCR with primers derived from short and long tandemly repeated repetitive sequences. Appl. Environ. Microbiol. 64: 265-272. Scheldeman, P., Baurain, D, Bouhy, R., Scott, M., Mühling, M.,Whitton, B.A., Belay, A. & A. Wilmotte (1999) Arthrospira ('Spirulina') strains from four continents are resolved into only two clusters, based on amplified ribosomal DNA restriction analysis of the internally transcribed spacer. FEMS Microbiol. Lett. 172: 213-222. Smith J.K., Parry J.D., Day, J.G. & Smith R.J. (1998). A PCR technique based on the Hip1 interspersed repetitive sequence distinguishes cyanobacterial species and strains. Microbiol. 144: 2791-2801. Versalovic J., Koeuth, T., Lupski, J.R. (1991). Distribution of repetitive DNA sequences in Eubacteria and application to fingerprinting of bacterial genomes. Nucl Acid Res 19, 6823-6831.

This work is currently supported by ESA/ESTEC (contract number n°15680/01/NL/ND),KBN 3 P04B 022 24 and by the CGRI II.18/KBN 018/2003-2004. Thanks to Dr. Paul Janssen for critical reading.

Changes associated with temperature or H2O2 stress on the physiology of A. platensis

Ruddy Wattiez, (1, *), Sarah Baatout (2,*), Larissa Heindrickx (3),

Florence Marty (3), Max Mergeay (2, 3). (1) Laboratory of biological chemistry, University of Mons-Hainaut, (2) Laboratory

of radiobiology and (3) Laboratory for microbiology, Belgian Nuclear Research Center, SCK-CEN, Mol, Belgium.

Cell intact mass spectrometry and flow cytometry provide powerful means to measure a wide range of cell characteristics in microbiological research. In order to estimate fine physiological changes associated with temperature and H2O2 stresses, cell intact mass spectrometry and flow cytometry were employed to estimate the extent of damage on the maintenance of membrane integrity and potential, esterase activity, intracellular pH and production of superoxides in Arthrospira platensis, chosen for its potential use in life support systems in space. Suspensions of A. platensis were submitted to a 1-hour temperature stress (from -170°C to 70°C) or H202 stress (at various concentrations from 0 to 880 mM). The laser desorption technologies could be used for the characterization of intact micro organisms by generation of different ion spectra of molecules (especially proteins) desorbed from bacteria. Specific cell fingerprint of A. platensis were analysed in different stress conditions in linear MALDI configuration. For flow cytometry, fluorochromes, including propidium iodide, rhodamine-123, 3,3'-dihexyloxacarbocyanine iodide, fluorescein diacetate, carboxy-fluorescein diacetate or hydroethidine were chosen as analytical parameters for identifying the physiological state and the overall fitness of individual cells. The individual cell's physiological state was assessed with a Coulter EPICS XL analyser based on the differential uptakes of these fluorescent stains. A. platensis exhibited varying staining intensities following temperature or oxidative stress. Membrane permeability and potential, esterase activity, intracellular pH and production of superoxide anions were increased to high levels after freezing (-170°C, -80°C and sometimes -20°C) or heat (45°C, 50°C, 60°C and 70°C) treatments. Following oxidative stress, membrane permeability, membrane potential (measured following DioC(6)3 staining, esterase activity and superoxide anion production were increased in function of the H202 concentration. In conclusion, we showed that there is a range of significant physiological alterations that occur after temperature or H2O2 stress. Mass spectrometry as well as flow cytometry are two complementary technologies for monitoring physiological changes induced by stress conditions. (*) These authors equally contributed to the work. This work is currently supported by ESA/ESTEC (contract number n°15680/01/NL/ND).

The history of agricultural management reflected in the endophytic community of Zea mays L.

Wittebolle Lieven*, Seghers Dave* and Verstraete Willy*

* Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, 9000 Ghent, Belgium

(Lieven.Wittebolle@UGent.be) The aim of this study was to examine the prevalence of endophytes in Zea mays L. and to evaluate the influence of agricultural managements on the endophytic community of the plant. The endophytic communities of the roots and kernels of Zea mays L. have been studied with molecular techniques (PCR-DGGE). First a surface disinfestation procedure was developed, optimized and then applied to different maize samples. This surface disinfestation has been validated both in a classical and molecular way. For that purpose, the washing water of the final step of the disinfestation protocol was plated and also used as template in a PCR with general bacterial primers. Afterwards the sterile plant tissues were homogenized with the use of a Stomacher® Lab-Blender. In a first phase, both structure and diversity of the endophytes were examined in relation to the soil and rhizosphere. PCR-DGGE-analysis showed that the majority of the root endophytes could also be found in the rhizosphere and the bulk soil. Still the maize root seemed to represent a distinct community. These results confirmed the hypothesis that the plant has a controlling function on its own endophytic influx. Sequence analysis revealed that

the -Proteobacteria dominate the maize roots. Diversity analysis based on DGGE gels

elucidated that starting in the bulk soil, via the rhizosphere to the endorhiza, the diversity of

the bacteria declined whereas that of the fungi increased. In a second phase, the influence of the agricultural management on the endophytic community was investigated. A difference in application of herbicides hardly had an effect on the endophytic community. On the other hand, a difference in fertilizer application (mineral versus organic) was reflected in the community of the type I methanotrophs. By application of mineral in stead of organic fertilization, the community structure changed and its diversity decreased. With the cluster analysis of the DGGE profiles a major effect was determined of the field of origin and the type of maize cultivar of which the endophytes had been analysed. This was respectively called the field and the cultivar effect. These results show the possibility of using molecular fingerprinting on endophytes as a tool to examine the agricultural treatment applied on soils. Future work will elucidate if this methodology can be used as a quality control for biologically grown crops, such as tomatoes.