S2

THE Listeria monocytogenes GENOME

Professor Pascale COSSART Institut Pasteur Unité des Interactions Bactéries-Cellules 28 rue du Dr. Roux 75724 Paris cedex 15 France

Listeria monocytogenesis a foodborne pathogen responsible for severe human diseases resulting in meningitis, meningo-encephalitis, septicemia, and abortions with a high mortality rate : (30 %). It is also affecting sheep and cows and is of veterinary importance. This intracellular pathogen was discovered in 1936 and became in the sixties a model for immunologists interested in the induction of a cellular immune response. Since 1986, geneticists and cell biologists have been analyzing in great details the molecular and cellular basis of the infectious process. The main features are that this bacterium has the capacity to enter cells that are non phagocytic. This process results in the formation of a membrane vacuole which is rapidly lysed. When free in the cytosol, bacteria replicate and also move inside cells by a spectacular actin polymerization process. This actin driven motility also allows the bacterium to spread directly from cell to cell after formation of protrusions containing the bacterium and of two membrane vacuoles which in turn are lysed allowing release of the bacterium in a second infected cell. Interestingly most of the genes involved in these different steps are located on a 10Kb fragment and are coregulated by the same transcriptional activator. In contrast, the genes involved in entry are located in a different chromosomal region. In fact, the unique ? chromosome is circular and is 3,2 Mbase. In the frame work of a European consortium which received financial support from the European Commission (EC) and coordinated by the Pasteur Institute (P. Cossart as coordinator of the consortium and P. Glaser, as coordinator of data), the sequence of the genome has been determined by a shot gun approach. The work is at the stage of the last finishing/polishing step. The first results of the annotation will be discussed

S3

THE PHOTORHABDUS LUMINESCENS GENOME PROJECT

Institut Pasteur Laboratory of Genomics of Microbial Pathogens 25, rue du Dr Roux 75724 Paris Cedex 15 FRANCE

Enterobacteriaceae of the genus Xenorhabdus and Photorhabdus are considered as insect pathogens. These are highly pathogenic after inoculation but not after ingestion. Their natural vectors are nematodes belonging to the families of Steinernematidae and Heterorhabditidae in whose guts they establish symbiotic relationships. These nematodes cause small lesions to these insects which are then rapidly killed by the bacteria. These nematode-bacteria couples are possibly efficient auxiliaries in the fight against insect scavengers of cultures. They have broad targets, the most sensitive ones being Coleoptera and Lepidoptera while Diptera are more resistant. Little is known about their genetics, besides their kinship to E. coli, the model organism for Enterobacteriaceae. These bacteria also synthetize toxins, including fungicides and large-spectrum antibiotics which are especially active on Gram-positive bacteria but also on Gram-negative bacteria. Finally, these bacteria have the capacity to secrete numerous enzymes in the medium during the stationary phase. A progress report of the sequencing of the P. luminescens genome will be presented.

S6

USE OF GENOMIC SEQUENCING IN INVESTIGATIONS OF EPIDEMIOLOGY, VIRULENCE MARKERS AND PHYLOGENETIC RELATIONSHIPS OF AQUATIC BIRNAVIRUSES

Department of Biochemistry, Microbiology and Molecular Biology University of Maine Orono, Maine, USA, 04469

Aquatic birnaviruses cause serious diseases in a variety of fish species used worldwide in aquaculture. These viruses exhibit a high degree of antigenic heterogeneity and variation in biological properties such as pathogenicity, host range, and temperature of replication. The larger segment (A) of the bi-segmented dsRNA viral genome contains a large ORF encoding a polyprotein which is posttranslationally cleaved into the outer capsid protein, VP2, and an internal protein, VP3. To better understand genetic and biological diversity among these viruses, we determined the nucleotide and deduced amino acid sequences of cDNA of the ORF of genome segment A of the 9 type strains of Serogroup A, the ORF of a variety of representative isolates of the major serotypes, and the VP2 coding region of a variety of isolates representing all other serotypes. In a phylogenetic tree constructed from the sequences, these viruses clustered into 6 genogroups each comprised of several genotypes, which generally correlated with geographical origin and serological classification. Similar data have been obtained for a variety of monoclonal antibody neutralization escape variants of aquatic birnaviruses. Specific amino acid variations in VP2 of these variants were found to be associated with differences in ability to replicate in different fish cell cultures. These data provide the most comprehensive analysis to date of genetic variation among aquatic birnaviruses. Furthermore, we demonstrated that genomic sequencing of PCR amplification products of a relatively small sequence within the hypervariable region of the VP2 gene can be used as a rapid, specific and economical method for serotype and even strain identification of clinical isolates with the use of this genomic database and computer phylogenetic analysis programs.

S7

REVOLUTIONS IN MASS SPECTROMETRY-BASED STRATEGIES FOR BIOPOLYMER ANALYSIS

Mass Spectrometry Resource, Depts. of Biochemistry and Biophysics, Boston University School of Medicine, 715 Albany Street, R-806, Boston, MA 02118-2526

Within the last decade, following the near-simultaneous introduction of two new methods for the formation of stable ions from large molecules - electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), the utilization of mass spectrometry-based methods for biopolymer analysis has undergone explosive growth. This phenomenon is due to several factors: the mass range now accessible to the analyst, the precision of the measurements and their very high sensitivity, the ease of sample preparation and potential for high throughput (including robotic systems), and the growth of databases available over the Web. Although parallel methods exist for gene and protein sequencing, the role of MS continues to grow, especially in arenas where unusual DNA modifications may be encountered or post-translational modifications significantly affect protein structure, biodistribution and bioactivity. In response to the need to determine structural details as well as molecular weights (or molecular weight distributions), the development of instruments capable of multistage mass analysis (MSⁿ) has also accelerated. In tandem mass spectrometry experiments, selected ions are decomposed by collisions with gases, photons, electrons or surfaces, and the resulting product ions yield information on subunit sequence, branching (in the case of oligosaccharides or cross-linked proteins) and substituents. Fascinating new areas of MS research include conformational analysis of biopolymers and structural studies of non-covalent complexes, which may be preformed before introduction into the MS ion source, or may be formed between trapped ions. A survey of this rapid growth will be presented, illustrated by examples from current investigations.

S10

MICROBIAL DEGRADATION OF PESTICIDES

Department of Soil, Water, and Climate, and Biological Process Technology Institute University of Minnesota 1991 Upper Buford Circle, 439 BorH St. Paul, MN 55108 (USA)

Due to their great metabolic diversity and versatility, microorganisms have the ability to transform a large number of disparate synthetic chemicals in the environment. In the last 50 years, human activity has added a large number of new organic chemicals to the environment. The use of halogenated pesticides and herbicides in the major agricultural regions of the world has resulted in the contamination of soil and water. One of the most cost effective approaches for cleaning-up contaminated soil and water involves the exploitation of microbial activity or a combination of microbial metabolism with chemical/physical treatment, in a process termed bioremediation. Despite numerous years of study, however, little information is available concerning the major biodegradative pathways by which microbes metabolize a large number of the currently used halogenated pesticides. This is due, in part, to the large number of pesticides used and the variety of ways in which microbes metabolize them. In addition, since some pesticides are new to the environment, microbes have not evolved the catabolic machinery necessary for their degradation. Recently, combined genetic and genomic approaches have been used to elucidate the genes and biochemical pathways by which microbes degrade pesticides. In addition, these approaches have been useful in elucidating means by which catabolic genes are recruited in microorganisms and assembled into biochemical pathways. Taken together, these approaches have proven useful in understanding the metabolic logic by which microbes degrade pesticides and have provided information crucial for the use of these microorganisms to remediate contaminated water and soil.

P1

SEQUENCE–TAGGED CONNECTORS FROM A RECOMBINANT COSMID LIBRARY

Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO 80523

Bacterial ring rot of potato is caused by the gram positive pathogen, Clavibacter michiganensis subsp. sepedonicus (Cms). Significant progress toward constructing a physical map of a pathogenic strain of Cms has been made using DNA fingerprinting of recombinant cosmids clones that are archived in 96 well plates as an ordered array of cosmids representative of a 13X fold coverage of the genome. Our progress in contig construction will be reported. End sequences of selected cosmid clones were determined and this yielded sequence–tagged connectors (STC) as well as sequence–tagged sites (STS). BLAST analysis of the sequence data revealed that the probability for finding similarity in the existing sequence databanks is high. This STC approach provides the framework upon which the complete genomic sequence may be constructed.

P2

GENOME ANALYSIS FOR Fibrobacter succinogenes S85 USING INVERSE PCR-DIRECT SEQUENCING METHOD

¹ Rumen Microbiology Research Team, STAFF-Institute, 446-1 Ippaizuka, Kamiyokoba, Tsukuba 305-0854, Japan

² Japan Collection of Microorganisms, RIKEN, Wako 351-0198, Japan

A genus Fibrobacter is obligate anaerobic bacteria and has been isolated only from the digestive tract of herbivorous mammals. This genus deeply branched from other genera on molecular phylogenetic tree. F. succinogenes is one of the major cellulolytic bacterium in the rumen. So far, over 10 genes for fiber digesting enzymes have been cloned and sequenced from F. succinogenes S85. This strain is presumed to have many more cellulolytic genes that are unstable in E. coli host-vector system. F. succinogenes deserves to be determined the complete genome sequence but whole genome shot-gun sequencing method may be unsuitable. We determined total 24-kb sequences of F. succinogenes S85 using inverse PCR-direct sequencing method. In the sequences, ORFs having ORF on another strand were found at high frequency, because of low usage of anti-stop-codons. ORFs confirmed function showed a certain pattern on codon frequency but ORFs of another strands showed different patterns. The unfunctional strands cloned into E. coli may be expressed some proteins which affect to existence of host. Running costs for inverse PCR-direct sequencing method is about 1.4 times as expensive as whole genome shot gun method (expect finishing) on our estimate. However, this method is quickly and non-cloning procedure, and require only a few implements. In addition, sharing the work among laboratories is easy on this method, because sequence determination and mapping operate together. Each laboratory can argue the determining region itself, and can analyze and appreciate the sequence immediately.

P3

CONSTRUCTION OF A BACTERIAL ARTIFICIAL CHROMOSOME LIBRARY OF THE LATE BLIGHT PATHOGEN, Phytophthora infestans

¹ Mycology, Bacteriology and Nemotology Unit, Scottish Crop Research Institute, Invergowrie, Dundee, UK

² Genomics Unit, Scottish Crop Research Institute, Invergowrie, Dundee, UK

³ 2Department of Phytopathology, Wageningen Agricultural University, The Netherlands

The Oomycete plant pathogen, Phytophthora infestans, causes late blight of potato and is a major disease of potato worldwide. The interaction between P. infestans and potato has been shown to follow a gene-for-gene relationship. Recent research has resulted in the construction of a genetic linkage map for P. infestans with markers closely linked to five avirulence genes. The genome of P. infestans is approximately 250 Mb and therefore a large insert size bacterial artificial chromosome (BAC) library is required to perform a chromosome walk to clone avirulence genes from this important pathogen. We have been building a BAC library of P. infestans T30-4, an F1 individual possessing several avirulence genes, in the vector pBeloBAC11 with the objective of ten-fold genome coverage and an average insert size of over 100 kb. With insert sizes over 100 kb, ten-fold coverage of the P. infestans genome could be expected with approximately 25 000 clones. Progress towards the construction of this library will be presented along with the rationale and plans for its exploitation.

P4

THE COMPLETE NUCLEOTIDE SEQUENCE OF PADP-1, A NOVEL CATABOLIC PLASMID ENCODING GENES FOR THE DEGRADATION OF ATRAZINE IN Pseudomonas sp. STRAIN ADP

¹ Department of Biochemistry, University of Minnesota, Saint Paul, MN 55108

² Biological Process and Technology Institute, University of Minnesota, Saint Paul, MN 55108

³ Department of Soil, Water and Climate, University of Minnesota, Saint Paul, MN 55108

⁴ Department of Microbiology, University of Minnesota, Saint Paul, MN 55108

⁵ Clemson Genomics Institute, Clemson University, Clemson, South Carolina 29630

Pseudomonas sp. strain ADP degrades atrazine and uses this herbicide as a sole source of nitrogen for growth. The first three genes involved in the degradation of atrazine atzA, atzB and atzC have been localized to a 100 kb self-transmissible plasmid, pADP-1. The complete nucleotide sequence of pADP-1 is being determined in order to learn more about the organization and evolution of this plasmid. A shotgun-sequencing library of pADP-1 was made in pUC18. The library contained 4,608 clones, with an insert size of 1.5 to 3.0 kbp. Random automated DNA sequencing (forward and reverse) was done to get a 10x coverage of the pADP-1 genome. The sequence obtained was assembled into six large contigs. DNA sequence analysis indicated that regions containing genes necessary for replication and transfer functions had 99% identity to pR751, a classical IncP-beta plasmid originally isolated from Enterobacter aerogenes. The atzA, atzB and atzC genes are not organized in an operon-like structure. The atzA gene is located 8 kbp upstream of the atzB gene and the atzC gene is located at least 20 kbp downstream of the atzB gene. Three copies of the insertion element IS1071 were identified and flank the atzA and atzB genes. Sequences homologous to transposase genes present in Pseudomonas pseudoalcaligenes and Ralstonia sp. were identified upstream and downstream of the atzC gene respectively. The presence of insertion sequences flanking the atrazine catabolic genes suggests that pADP-1 evolved from pR751 after several transposition events.

P5

CONSTRUCTION AND DNA SEQUENCING OF A BAC CONTIG SPANNING THE PHYTOPHTHORA SOJAE GENOME

¹ Department of Plant Pathology University of California, Davis Davis, CA95616

² National Center for Genome Resources 1800 Old Pecos Trail, Santa Fe, NM 87505 USA

The more than 40 species of the oomycete Phytophthora cause serious diseases of a huge range of crop and ornamental plants. To facilitate isolation of such genes from the soybean pathogen Phytophthora sojae by map-based cloning, we are constructing a BAC contig of the entire genome of this organism, using a hybridization fingerprinting strategy. We are hybridizing a 13X BAC library with unique mixtures of random probes, most of them repetitive, and with ESTs. Computer software has been developed to collect, simulate and analyze the data. At present we have probed the library with 49 of the mixtures. Each mixture hybridized to around 300 - 500 BACs. 19% of the BACs so far have received the minimum number of hits needed to establish statistically significant overlaps (5 each). Of these BACs, 34% have been placed into contigs. We have confirmed the authenticity of three of the largest contigs by HindIII digestion. With the long term goal of sequencing the entire genome of P. sojae and selected sequences from other Phytophthora species, such as P. infestans we have established the Phytophthora genome initiative (PGI). We have begun preliminary sequencing of a 200 kb BAC contig spanning two avirulence genes from P. sojae. Sequencing of the first 60 kb BAC is nearly complete, and software has been developed for automatic processing, annotation and publishing of the sequence data via the web. In the region sequenced so far the gene density is extremely high. Both the sequencing data and the BAC hybridization data suggest that the P.sojae genome is composed of gene-rich regions separated by regions rich in repetitive sequences.

P6

BIOFILM AND PATHOGENICITY: THE Xylella fastidiosa GENOME MODEL

Centro de Biologia Molecular e Engenharia Genética (CBMEG), UNICAMP. CxP 6109, CEP 13083-970, Campinas – S.P., Brazil.

The gram-negative bacterium Xylella fastidiosa was the first plant pathogen targeted for complete genome sequencing. It is the causal agent of many economically important plant diseases including Pierce's disease of grapevine (PD), alfalfa dwarf, phony peach disease (PPD), periwinkle wilt (PW), leaf scorch of several plant species, and citrus variegated chlorosis (CVC). CVC is a major concern to the Brazilian citrus industry and is considered to be potentially the more devasting citrus disease. CVC has been found only in Brazil and Argentina. This work describes an operon containing the genes responsible for the synthesis and secretion of a novel exopolysaccharide (EPS), the fastidiosan gum. The similarity found with already described genes, coupled to preliminary chemical analysis, suggests that this EPS is comprised of a cellulosic (1->4)-beta-D-glucose backbone with a disaccharide side chain, possessing the sequence of Mannose-Galactose acid attached to alternate glucose residues. We postulate the implication of several X. fastidiosa genes and their products in biofilm formation and hypothesizes their role both in the disease symptoms development in the plant as in the spread of the bacteria by insect vectors.

P7

MOLECULAR ANALYSIS OF MICROBIAL DIVERSITY IN THE GASTROINTESTINAL TRACTS OF FOOD ANIMAL SPECIES.

¹ Department of Animal Sciences University of Illinois 1207 W. Gregory Drive Urbana, IL 61801

² Departments of Fisheries, Animal and Veterinary Science and Biochemistry, Microbiology and Molecular Genetics University of Rhode Island Building #14, East Farm Kington, RI 02881

Molecular tools based on small subunit (SSU) rDNA gene sequences offer a powerful and rapid tool for the analysis of complex microbial communities found in the gastrointestinal (GI) tracts of food animal species. Polymerase chain reaction (PCR) based techniques have been applied to the analysis of microbial communities in bovine, porcine, prawn, salmon and cod gastrointestinal tracts. Single cell PCR methodologies have addressed the diversity and phylogeny of unculturable ruminal ciliate protozoa and their archaeal and bacterial endosymbionts. Denaturing gradient gel electrophoresis (DGGE) has been used as a tool to profile microbial diversity of ruminal microbial communities from steers fed different diets; porcine GI ecology from pigs fed probiotics; prawns GI communities from individuals reared in commercial and controlled environments; and Atlantic salmon and cod which were fed aquaculture diets. Comparative sequence analysis of the excised DGGE amplicons can be used to presumptively identify predominant bacterial species. Direct cloning of SSU rDNA genes amplified from these complex communities can also be used to determine the extent of diversity in these GI communities. Subsequent PCR analysis using kingdom specific primer sets has successfully evaluate the diversity of Archaea and Eucarya from these gastrointestinal communities.

P8

GENOMIC DIVERSITY AMONG RUMINAL ISOLATES OF Fibrobacter succinogenes AND Prevotella ruminicola.

¹ Rumen Microbiology Research Team, STAFF-Institute, Tsukuba, Ibaraki 305-0854, Japan

² Department of Animal Sciences, University of Illinois, Urbana, IL 61801, USA

³ Collection of Microorganisms, RIKEN, Wako, Saitama 351-0198, Japan

Fibrobacter succinogenes and Prevotella ruminicola represent two major groups of ruminal bacteria involved into the plant cell wall breakdown process in the rumen. Representatives of the former such as fibrobacteria possess truly cellulolytic activities and are considered to be involved into the first steps of polysaccharide degradation. Representatives of the second group such as prevotellas are unable to degrade the native forms of substrate and are involved into further degradation of oligosaccharides as well as in a number of other metabolic functions of the rumen. Earlier studies demonstrated the high degree of diversity among the ruminal fibrobacteria and prevotellas including genetic diversity at the level of SSU rDNA, different G+C content of chromosomal DNA, and different sets of cellulase genes in isolates. In this study, genomic diversity among four strains of F. succinogenes and seven strains of P. ruminicola was studied using rare-cutting restriction endonucleases and pulsed-field gel electrophoresis (PFGE). Despite the close phylogenetic relatedness of strains within the two genera, they found to be highly polymorphic at the level of genomic DNA restriction fragment length (RFLP). To identify the possible sources of genomic diversity, we performed comparative analysis of genetic polymorphism within the SSU rDNA and cellulase genes as well as the gene order in the vicinity of a cellulase gene using PCR and Southern hybridization. Probably both factors, the high degree of polymorphism in coding sequences as well as the gene order rearrangements, contribute to genomic diversity found in these ruminal bacteria.

GENOMIC DIFFERENTIATION OF HOST SPECIFIC TOXINS-PRODUCING ALTERNARIA SPECIES BY URP-PCR FINGERPRINTS AND PROBES DERIVED FROM THE POLYMORPHIC PCR BANDS

¹ Division of Molecular Genetics, National Institute of Agricultural: Science & Technology, RDA, Suwon 447-707, Korea.

² Division of Applied Biology and chemistry of , Chungnam National University, Taejon 305-764, Korea

³ Seoulin Research Institute of Life Science, Seoulin Scientific Co., Ltd, Seoul 134-030, Korea

Several Alternaria fungi that have been known to produce host-specific toxins (HSTs) that are active only in specific plant species or varieties. However, classification of the Alternaria spp. is still remained to be confused, although morphological methods and molecular tools have been employed to distinguish among HSTs-producing Alternaria fungi or between them and nonpathogenic A. alternata. Thus, this study was carried out to clarify genetic relationship among the HSTs-producing Alternaria spp.by PCR fingerprinting method. Twelve URP (universal rice primer) pimers of 20 mer derived from repetitive sequence of rice were used to obtain PCR polymorphism among 32 isolates of four HSTs producing Alternaria, i.e., A. kikuchiana (AK-toxin), A. mali (AM-toxin), A. longipes (AT-toxin), A. alternata f. sp. Lycopersici (AAL-toxin), nonpathogenic A. alternata. Four URP primers (URP1F, URP2R, URP8R and URP9F) amplified polymorphic PCR bands depending on each Alternaria spp.. Particularly, URP9F primer produced unique PCR profiles in isolates of A. kikuchiana and A. longipes that were clearly differentiated from other Alternaria spp. Phylogenic analysis based on PCR polymorphic bands amplified by URP primers revealed that isolates of AM-toxin producing A. mali and nonpathogenic A. alternata were closely related, but isolates of A. kikuchiana and A. longipes were distantly related to them. Furthermore, the polymorphic PCR band detected by URP9F were selected to use as DNA probes. The probes isolated from PCR bands of A. mali and A. kikuchiana produced specific RFLP patterns on isolates of A. kikuchiana and A. longipes that are distinquisable from other Alternaria spp.. In conclusion, DNA markers used in this study could effectively applied to differentiate genomes of HSTs-producing Alternaria species.

P10

GENETIC DIVERSITY OF BLAST (Magneporthe grisea) POPULATIONS IN THAILAND

¹ Lampang Agricultural Research and Training Center, PO. Box 89, Muang, Lampang, 52000, Thailand.

² DNA Fingerprinting Unit , National Center for Genetic Engineering and Biotechnology at Kasetsart University, Kampangsaen, Nakornpathom, 73140, Thailand.

³ National Center for Genetic Engineering and Biotecnology (BIOTEC), National Science and Technology Development Agency, Bangkok, 10400 ,Thailand

Magneporthe grisea ( anamorph : Pyricularia grisea) is the causal agent of blast disease, one of the most devastating disease throughout the world. Understanding genetic structure and variation is the key for controlling this disease in rice. We are particularly interested in characterizing and diversifying blast isolates collected from Central, North and Northeast of Thailand to study their genetic structure and variation. One hundred and twenty-three isolates selected from 635 monospore were characterized using RAPD and AFLP. Blast population was highly diverse and dynamic. Geographical isolates could not be adequate for representing virulence group, suggesting that few provincial isolates could not be enough for identifying broad-spectrum resistance in rice germplasm. The relationship between the clustering of isolates and pathotype will be discussed.

P11

EVOLUTION OF TOMATO SPECIALIZATION IN Phytophthora infestans

Department of Plant Pathology 334 Plant Science Bldg. Cornell University Ithaca, NY 14853

The oomycete Phytophthora infestans is a devastating pathogen of potato and tomato worldwide. Several clonal lineages of P. infestans are particularly devastating on tomato, and have been described as tomato specialized. Members of these lineages do not produce a necrotic lesion, but rather grow more biotrophically on the tomato leaflet. Isolates of the P. infestans clonal lineage US-8 (not tomato specialized) produce a necrotic lesion that is typically visible within 48-72 hr after inoculation of the tomato plant, and sporulation typically occurs at the margin of the lesion. In contrast, tomatoes infected with a tomato specialized strain (US-17) often produce no necrosis at all. The entire leaflet may support sporulation for several days without necrosis. We are investigating this phenotype by comparing expression profiles of both host and pathogen genes from tomato plants infected with a tomato specialized isolate of P. infestans (US-17) vs. a non-specialized isolate (US-8). Initial EST sequencing results from a cDNA library generated 12 hours post-inoculation indicated that the majority of the clones were tomato genes that were not specific to the host-pathogen interaction. Of 100 clones sequenced, 70 were of tomato origin (based on BLASTN searches) and 30 had no significant homology to any other sequence in the database.

P12

CHARACTERIZATION OF Paracoccidioides brasiliensis ISOLATES USING RAPD MOLECULAR MARKERS AND rDNA SEQUENCING.

Departamento de Genética-UNESP (Campus de Botucatu-SP) Distrito de Rubião Júnior s/n Botucatu São Paulo-Brazil 18600-000

CHARACTERIZATION OF Paracoccidioides brasiliensis ISOLATES USING RAPD MOLECULAR MARKERS AND rDNA SEQUENCING. Monteiro¹, J.P.; Palmieri¹, D.A.; Nóbile¹, P.M.; Kurokawa², C.S.; Lopes¹, C.R. 1-Depto. Genética, IBB-UNESP (Botucatu-SP); 2-Depto. Microbiologia e Imunologia, IBB-UNESP (Botucatu-SP). The paracoccidioidomycosis is a deep mycosis of great incidence in Latin America caused by the dimorphic fungus Paracoccidioides brasiliensis. The disease affects mainly male rural workers. At room temperature the fungus presents itself as a conidium producing mycelium (the infectious form), while at body temperature or while in culture at 37^oC, it changes its form into a yeast-like microorganism. So far, little is known about the biology of this fungus and the use of molecular techniques in its characterization is still in its early stages. In the present work the characterization of ten isolates was promoted using the RAPD technique and automated sequencing of both internal transcribed spacers and 5.8s rDNA. RAPD amplifications were endured using 46 arbitrary primers. Grouping analysis was done using Link’s similarity coefficient and the Neighbor Joining method. Amplifications of the ITS containing rDNA region were carried out using previously described primers. The PCR products were sequenced using the same primers as template and the Neighbor Joining method was used for grouping analysis. ITS sequences showed little differences among the isolates placing them as one group inside the ascomycete order Onygenales. The RAPD data obtained divides the species into two well distinguished groups as previously reported. It also shows that Paracoccidioides brasiliensis is a microorganism specially susceptible to genetic rearrangements in order to gain or loose its virulence. Financial support: CAPES

P13

FAST IDENTIFICATION OF BACTERIAL STRAINS

Fidelity Systems, Inc. 7961 Cessna Ave. Gaithersburg, MD 20879

Currently, the most advanced method of establishing the phylogenetic position of a microorganism consists in the PCR amplification of rRNA genes with universal primers and sequencing the purified amplicon directly or after cloning into a plasmid. rRNA sequencing has a potential to replace earlier less reliable methods based on probe hybridization in wide biological and medical applications. Unfortunately, the PCR based sequencing has a number of intrinsic limitations: amplification of any contamination with universal primers, generation and amplification of chimeras, non-proportional amplification of mixed samples, to name a few. To circumvent these problems we have developed a new method for the identification of bacterial strains. The method consists in the isolation of total bacterial DNA and using it as a template in sequencing reaction. We will describe a set of specially designed universal primers that allow reading sequences from any source. The protocols and examples of fast strain identification will be presented. In addition to the identification of isolated strains, the new method allows to detect single nucleotide polymorphisms and gross genome rearrangements in mixed bacterial samples.

P14

ASSEMBLY AND PRELIMINARY SEQUENCE ANALYSIS OF SEQUENCED BAC SUBCLONES FROM PHYTOPHTHORA SOJAE

¹ National Center for Genome Resources, 1800A Old Pecos Trail, Santa Fe, NM 87505

² Department of Plant Pathology, University of California, Davis, CA, 95616 USA

Phytophthora sojae is the major pathogen of Soybeans in the Midwestern United States, causing tens of millions of dollars in annual crop losses. In conjunction with a large scale sequencing effort of a BAC contig spanning the P. sojae genome (detailed in the Arredondo et al. poster at this meeting), we have begun assembly and preliminary analysis of this data. From the perspective of analysis, Phytophthora presents a unique challenge in that taxonomically it appears to be equally distant from both plants and higher fungi. Most of the current gene prediction algorithms have been developed and optimized for use with mammalian, higher plant or yeast genomes. In addition, there is relatively little Phytophthora sequence data available in the public databases which can be used to build training sets for such algorithms. Through the Phytophthora Genome Intiative there is EST data publicly available that can be used to validate the occurance of predicted genes. A comparison based on a number of different prediction tools will be shown. Using the new sequence information we have also tested the taxonomic placement of Phytophthora with respect to several recently completed microbial genomes and against Arabidopsis.

P15

THE PHYTOPHTHORA GENOME INITIATIVE: INFORMATICS FOR DISTRIBUTED SEQUENCING PROJECTS

National Center for Genome Resources, 1800A old Pecos Trail, Santa Fe, NM 87505.

Collaborative study of microbial genomes requires coordination and access to information from widely distributed laboratories. Information technology can facilitate such collaborative ventures with tools for curation, analysis, and annotation that provide networked access to information repositories and analysis services. The Phytophthora Genome Initiative is a distributed collaboration to study the genome and evolution of a destructive class of plant pathogenic oomycete. NCGR helped intiate a sequencing project/shared informatics source to promote understanding and control of this pathogen. In the pilot phase of the project, two laboratories prepared P. infestans EST and P. sojae BAC libraries and sent them to a third laboratory for sequencing. Results of sequencing reactions were transferred to NCGR for analysis and curation. The system automatically performs simple analyses (i.e., vector removal and similarity searching) that can then be retrieved by the investigator using a web browser. Here we describe the analysis and retrieval system, summarize results from comparing the species, and outline future plans, including the incorporation of new tools into the analysis pipeline. The enabling force of a distributed information system that allows scientists to share analysis results has not yet been widely realized. The system described here is intended for reuse, and could provide support for distributed research 'collaboratories' to help understand microbial genomes. More information is available at www.ncgr.org/pgi.

P16

INTERPRO, AN INTEGRATED RESOURCE OF PROTEIN DOMAINS AND FUNCTIONAL SITES

EMBL Outstation - The European Bioinformatics Institute, Hinxton, Cambridge CB10 1SD, UK

The databases PROSITE, Pfam, PRINTS, ProDom, SWISS-PROT and TrEMBL joined forces to launch a new Integrated Resource of Protein Domains and Functional Sites, abbreviated InterPro. The growing amount of sequence data without any functional characterization leads to less and less meaningful hits with the conventional similarity searches like BLAST and fastA. The usual approach to gain a good coverage is to analyze unknown sequences by as many motif recognition tools as possible. This would also increase the statistical significance, provided we know which prediction of tool A is equivalent to which prediction of tool B. The goal of InterPro is to solve this problem. For each set of equivalent patterns, profiles, fingerprints, and hidden Markov models, we created a dedicated InterPro entry. Each entry contains links to the parent databases, descriptions of the specific methods, functional annotation, literature references, and a list of all matched SWISS-PROT and TrEMBL protein sequences. The entries are classified as protein family, domain, repeat, or post-transcriptional modification site. We also maintain links between InterPro entries where appropriate. For instance, the phosphotransferase system domain (IPR000032) points to the PTS-EI acceptor site (IPR001020) and the PTS-phosphorylation site (IPR002114). Initially, we computed a list of corresponding signatures. However, this list contained too many errors, mainly caused by the different biological concepts of the parent databases. Therefore, we decided to check this list manually by a group of by now sixteen scientists, who also merged and rewrote the functional annotation. InterPro is accessible on the web at http://www.ebi.ac.uk/interpro and http://srs.ebi.ac.uk. The XML flat file can be downloaded via anonymous ftp at ftp.ebi.ac.uk/pub/databases/interpro. If you have questions or comments, please feel free to contact interpro@ebi.ac.uk.

P17

PhytoPaths: A DATABASE FOR PLANT-MICROBE INTERACTIONS AND GENOMES

USDA ARS Western Regional Research Center, Albany, CA 94710-1105 USA

The ACEDB (Durbin and Thierry-Mieg, 1991-) program has been historically useful in establishing a number of public genome databases. Program enhancements allow the database to operate under a wide variety of computer environments (Unix, Win9x/NT, MacOS, HTTPd, Java). A database, named PhytoPaths, has been created under the ACEDB environment which displays plant-microbe interactions. Several individual genome databases displayed at the USDA-ARS Center for Bioinformatics and Comparative Genomics (CBCG) do include information about plant-microbe interactions, and a few have specialized in focusing on specific plant-microbe interactions (PathoGenes, Giese et al., RiceBlastDB, Yap et al.). The PhytoPaths database contains information on plant-microbe interactions covering several host and pathogen species. Current descriptions include over 2,000 specific interactions. PhytoPaths attempts to utilize recent findings about the nature of plant disease resistance and pathogen defense responses to establish a system for modeling and examining host-pathogen interactions. The database may be used to identify interactions, define genes involved in the interactions, and establish genetic maps for the pathogens and hosts. Additional features include literature databases, disease images, host range evaluations, germplasm interactions, nucleic acid sequences, and comparative mapping tools. The database may be viewed at the PhytoPaths WWW home page.

P18

DEVELOPMENT OF A PHYSICAL MAP AND SEQUENCE TAG CONNECTOR FRAMEWORK FOR THE BRADYRHIZOBIUM JAPONICUM GENOME

¹ Clemson University Genomics Institute Room 100 Jordan Hall Clemson, SC 29634

² Center for Legume Research Univ Tennessee M409 Walters Lifew Sci Bldg Knoxville, TN 37996

³ Biological Process Technology Institute University of Minnesota 1991 Upper Buford Circle, 439 Borlaug Hall St. Paul, MN 55108

We have constructed a physical framework for the Bradyrhizobium japonicum (Bj) genome. A bacterial artificial chromosome (BAC) library was constructed from megabase Bj DNA partially restricted with HindIII. The BAC library contains 4,608 clones with an average insert size of 148 kbp providing 78 genome equivalents based on an estimated genome size of 8.7 Mbp. A high-resolution physical map of the genome has been constructed with BAC contigs using a BAC fingerprinting approach. Over 40 genetic markers have been placed on the physical map by hybridizing known gene probes to high-density BAC colony filter arrays. In order to discover new genes in the Bj genome and to construct a sequence tag connector (STC) framework, the ends of 1,536 BAC inserts have been sequenced (forward and reverse) to generate a total of 3,072 sequences. This provides an STC on average every 2.8 kbp across the Bj genome. The use of this approach allows the low-cost development of information rich molecular scaffolds for microbial genomes. These detailed scaffolds may then be used as tools to direct and finish full-scale shotgun sequencing efforts.

P19

AFLP: A VERSATILE TOOL FOR GENOMIC RESEARCH IN MICROORGANISMS

¹ Keygene N.V., Agro Business Park 90, P.O. Box 216, 6700 AE Wageningen, the Netherlands

² Beagle b.v.b.a. Stierstraat 23 2018 Antwerp Belgium

AFLP® is a powerful fingerprinting technique, which can be applied to DNA as well as to cDNA. Because of its versatility, AFLP allows addressing various questions in genome research in microorganisms using only a single technology. Here we present two different applications of the AFLP technique in genome research. These applications are: - Strain identification using AFLP. - Identification of differentially expressed genes using cDNA-AFLP. AFLP generates very reproducible high-density fingerprints, which can be used to discriminate between various strains at the subspecies level. Moreover, AFLP enables identification of unknown strains that originate from different habitats. cDNA-AFLP enables genome-wide monitoring of gene expression in micro-organisms. It is very well suited to investigate gene expression in microorganisms even if no sequence information is available. Specifically designed bio-informatic tools ensure a high-throughput differential expression analysis. Sequence information generated through the cDNA-AFLP screening can be used to construct specific micro-arrays. AFLP® is a registered trademark of Keygene N.V.

P20

HEAT SHOCK, OSMOTIC, AND OXIDATIVE STRESS; THE RELATIONSHIP BETWEEN STRESS RESPONSES USING MICROARRAY ANALYSIS IN Saccharomyces cerevisiae

UNIVERSITY OF ARIZONA, DEPATRMENT OF BIOCHEMISTRY, TUCSON, ARIZONA 85721

Microarrays representing 6,144 open reading frames (ORFs) were used to analyze and compare changes in global gene expression associated with heat shock, osmotic, and oxidative stress. In order to determine the amount of similarity between the three stress responses, the top 300 upregulated ORFs for each stress were classified into 23 functional categories as outlined by the MIPS Database. For all three stresses, the upregulated ORFs most often fell into the functional categories of cell rescue and defense (total 60 ORFs), carbohydrate metabolism (total 60 ORFs), and energy (total 58 ORFs) with the overlap, or degree of similarity, equal to 20%. Categories least common in the upregulated ORFs were translation (1 ORF) and cell growth (7 ORFs). Variation between the stress responses was also observed. Approximately 68% (11/16) of the upregulated ORFs in the Major Facilitator Superfamily category were unique to osmotic stress, 81% (9/11) of the upregulated ORFs involved in cellular biogenesis were unique to oxidative stress, and 100% (8/8) of the upregulated ORFs associated with the TCA Cycle were found only from the heat shock data. Overall, we found 260 functionally unknown, upregulated ORFs with a subset of 16 unknown ORFs common to all three stresses. This subset is the target of our ongoing work to characterize the genes and biochemical processes involved in stress responses in yeast.

P21

COMPUTIONAL APPROACHES FOR IDENTIFICATION OF THE ACTIVE SITE OF AlbA FOR INACTIVATION OF ALBICIDIN PHYTOTOXIN

Institute of Molecular Agrobiology, 1 Research Link, The National University of Singapore, Singapore 117604

Leaf scald caused by a bacterial pathogen, Xanthomonas albilineans, is a major sugarcane disease. Albicidins, a family of phytotoxin produced by the pathogen, has played an important role in the disease development. The albA from Klebsiella oxytoca is an albicidin resistance gene which encodes an albicidin binding protein. Its strong affinity and selectivity towards albicidin render it a potential as a useful candidate to confer a novel form of phytotoxin and disease resistance in sugarcane. Kenetics and stoichiometric analysis of binding reaction of AlbA indicates the presence of a single high affinity binding site. Binding of albicidin to AlbA is pH dependent. There was a significant increase in albicidin binding at pH 5-6 which is in the pKa range of a typical histidine residue, suggesting that histidine residues might be important in the high affinity binding reaction. To investigate the molecular mechanism of the binding reaction, we have purified the AlbA protein. Several amino acids specific reagents have been used to probe the amino acid residues involved in albicidin binding reaction. Evidence indicate that only one out of the total 6 histidine residues in AlbA is likely directly involved in the binding reaction with albicidins. For efficient identification of the histidine residue involved, we attempt to use a computer-assisted approach, based on the characteristics of substrate as well as that of histidine residues in the secondary structure of AlbA. The accuracy of computer prediction will be determined by site-directed mutagenesis.

P22

RECENT ADVANCES IN THE MOLECULAR ANALYSIS OF PATHOGENICITY MECHANISMS OF Clavibacter xyli SUBSP. xyli, CAUSAL ORGANISM OF RATOON STUNTING DISEASE OF SUGARCANE, DEMOSTRATE THAT THIS PATHOGEN IS A PRIME CANDIDATE FOR GENOME SEQUENCING.

¹ Bureau of Sugar Experiment Stations 50 Meiers Road Indooroopilly, Queensland 4068 AUSTRALIA

² Cooperative Research Centre for Tropical Plant Protection John Hines Building University of Queensland St. Lucia, Queensland 4072 AUSTRALIA

³ Botany Department University of Queensland Brisbane, Queensland 4072 AUSTRALIA

⁴ Joint Center for Crop Improvement University of Melbourne Dept. of Agric. Forestry & Horticulture Parksville, Victoria 3052 AUSTRALIA

From a simple modification of the culture medium, which allowed growth Clavibacter xyli subsp. xyli (Cxx) as single colonies six years ago, through to being able to complement Cxx transposon mutants and cloning and sequencing "pathogenicity" genes, the work on Cxx has steadily progressed. Being able to transform a bacterium may seem trivial to some, but in the case of Cxx, a nutritionally fastidious, slow growing (it takes 3 to 4 weeks to get visible single colony growth on plate cultures), xylem limited bacterium that produces no reliable symptoms on its host plant sugarcane, transformation was a major milestone. Since that early success, we have been able to get transposon mutagenesis working, developed an assay to screen mutants for loss of "pathogenicity", identified a mutant that can no longer colonise sugarcane (therefore is nonpathogenic), cloned the wild type copy of the mutated gene and used this gene to complement that mutant. We now have the complete nucleotide sequence of that gene and we are finishing the sequencing of the regions both upstream and downstream from the gene (˜40 Kb) to identify other genes that may play a role in pathogenicity. Initial analysis of the sequence data from the flanking region indicates that we have tagged transporter system including: ATP-binding protein msmk; ABC transporter ATP-binding protein HI1252; Peptide transport system permease protein sapC; Transport ATP-binding protein CydD; Hypothetical outer membrane usher protein in agai-mtr. Whether this transport system plays a role in pathogenesis is unknown at this time. The transposon insertion in this region may simply make Cxx more auxotrophic than it already is. Sequencing the genome of Cxx would be a much quicker and more economic way to unravel how this bacterium recognises and causes disease on sugarcane.

P23

A NOVEL ENZYME INACTIVATES QUORUM-SENSING SIGNAL AND ATTENUATES VIRULENCE OF Erwinia carstovora

Institute of Molecular Agrobilogy, 1 Research Link, The University of Singapore, Singapore 117604

N-acyl-homoserine lactones, known as autoinducers (AIs), are widely conserved signal molecules that present in quorum-sensing system of many Gram-negative bacteria. AIs are involved in the regulation of a diverse range of biological functions, including expression of pathogenic genes in plant pathogens Xanthomona campestris, Pseudomonas solanacearum, several Erwinia species and human pathogen Pseudomonas aeruginosa. A bacterial isolate, 240B1, is found capable of enzymatic inactivation of AIs. The gene (aiiA) for AI inactivation from 240B1 has been cloned and shows to encode a novel protein of 250 amino acids. Expression of aiiA gene in transformed Erw. carolovora strain SCG1 significantly inhibits AIs release, suppresses extracellular pectolytic enzyme activities, and attenuates pathogenicity of Erw. carolovora in potato, eggplant, Chinese cabbage, carrot, celery, cauliflower and tobacco. Site-directed mutagenesis shows that the conserved aspartate and histidine residues are required for AiiA activity. Our results indicate that the signal-inactivation approach represents a promising novel strategy for prevention of the diseases in which virulences are regulated by AIs.

P24

POSITIVE SIGNAL TRANSDUCTION IN Agrobacterium tumefaciens AUTOINDUCER BIOSYNTHESIS AND TI PLASMID CONJUGAL TRANSFER

Institute of Molecular Agrobiology 1 Research Link The National University of Singapore Singapore 117604

Autoinducers, also known as quorum-sensing signals, have been found in more and more Gram-negative bacteria and regulate many biological functions, including bioluminescence, Ti plasmid conjugal transfer, bacterial pathogenicity, biofilm maturation, and swarming. The Agrobacterium tumefaciens autoinducer (AAI) synthase gene, traI, has been identified and localized at a Tra region of Ti plasmid, which is positively regulated by the transcription factor, TraR, in the presence of AAI. In an effort to find out other components involved in the AAI signal biosynthesis, Tn5 mutagenesis approach has been used for identification of AAI biosynthesis mutants, either over-production or production deficiency. It is found that a single Tn5 insertional mutation in mutant M180 results in about 90% decrease in AAI biosynthesis. The efficiency of Ti plasmid conjugal transfer of the mutant is also significantly lower than that of the parent strain K588. The expressions of traR and traI gene in strain M180 are not affected, based on RT-PCR analysis. External addition of AAI to strain M180 fails to enhance AAI biosynthesis and Ti plasmid conjugal transfer, suggesting that mutated gene is required for effective external AAI signal transduction. The update results on gene cloning and further characterization will be presented.

P25

SYSTEMATIC IDENTIFICATION OF EXTRACELLULAR PROTEINS FROM EXPRESSED SEQUENCES OF THE OOMYCETE PLANT PATHOGEN Phytophthora infestans

Department of Plant Pathology Ohio State University-OARDC 1680 Madison Ave. Wooster, OH 44691

Interactions between plants and microbial pathogens involve complex signal exchanges at the plant surface and intercellular space interface. Surface components of a pathogen must play important roles in development of a complete infection cycle and recognition by resistant plants. We aim at identifying extracellular proteins from Phytophthora infestans, an economically important oomycete pathogen. Targeting extracellular proteins will increase the probability of identifying proteins essential for virulence and survival of the pathogen. In collaboration with other colleagues in the Phytophthora field, we initiated the Phytophthora Genome Initiative (PGI) which aims at launching a Phytophthora Genome Project. Recently, a pilot cDNA sequencing project has been completed for P. infestans (Kamoun et al. 1999 Fungal Genetics and Biology, in press). Unlike many eukaryotes, the 5' untranslated regions in Phytophthora transcripts are typically short, ranging from about 50 to 70 nucleotides. We developed an algorithm to identify secreted and membrane proteins based on identifying potential start codons and presence of signal peptides using the SignalP V1.1 software program software program (Nielsen et al. 1997 Protein Engineering 10:1-6). Analysis of 1000 ESTs from the PGI database revealed that 37% of the ESTs show no similarity to known sequences in public databases. We applied the algorithm to this set of sequences and found more than 50 novel genes encoding extracellular proteins. Functional genetic assays will be used to determine the role of the novel extracellular proteins in virulence/avirulence.

P26

THE BIOSYNTHETIC GENE CLUSTER FOR THE ANTIFUNGAL AND ANTITUMORAL POLYKETIDE GELDANAMYCIN IN Streptomyces hygroscopicus VAR. geldanus EF-76

Groupe de recherche en biologie des actinomycètes, Département de biologie, Université de Sherbrooke, Sherbrooke, Québec, Canada, J1K 2R1

The geldanamycin producer strain Streptomyces hygroscopicus var. geldanus EF76 isolated from potato rhizosphere in Québec (Canada) strongly inhibits the growth of Phytophthora fragariae var. rubi and protects raspberry against infections caused by the pathogen. Degenerated primers designed from conserved regions of ketoacylsynthase and acyltransferase genes from type I polyketide synthases (PKS) yielded a 1.3 kb PCR product when genomic DNA from EF-76 was used as template. This fragment was used to select potential clones for the biosynthesis of geldanamycin. An EF-76 genomic clone carrying a 45 kb insert was recovered and partially sequenced. Several regions of this insert showed high levels of homology with catalytic domains of PKS genes responsible for the biosynthesis of antibiotics such as oleandomycin, rapamycin, rifamycin and erythromycin. The insert also carries a gene coding for an enzyme exhibiting an amino acid sequence homology with Bacillus 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase. This enzyme could be involved in the biosynthesis of the shikimate-derived starter unit necessary for geldanamycin biosynthesis. A putative geldanamycin-resistance determinant was also positioned within the 45kb DNA fragment. Sequencing is still in progress and sequence analysis will allow us to delimit the DNA regions that govern the biosynthesis of geldanamycin in EF-76

P27

INVESTIGATING THE PATHOGENICITY OF Erwinia carotovora SUBSPECIES BY GENOME MAPPING USING BAC LIBRARIES

¹ Unit of Mycology, Bacteriology and Nematology; Scottish Crop Research Institute; Invergowrie; Dundee; Scotland; UK. DD2 5DA

² Genomics Unit; Scottish Crop Research Institute; Invergowrie; Dundee; Scotland; UK. DD2 5DA

The bacterial plant pathogen Erwinia carotovora subspecies atroseptica (Eca) has a host range limited to potato, on which it causes blackleg in the field and soft rot in storage. In contrast, Erwinia carotovora subspecies carotovora (Ecc) has a wider host range, can also cause soft rot on potato but does not cause blackleg. The molecular basis for these phenotypic differences is unknown. We have created BAC (Bacterial Artificial Chromosome) libraries for Eca and Ecc and are using them to generate complete physical maps of both genomes. Each clone in the Eca library has been fingerprinted by amplified fragment length polymorphism, and contigs have been constructed from the fingerprint data. Methods for closing gaps between contigs are being investigated. Known or putative pathogenicity genes or sub-species specific genes (obtained from international databases and from other research in our group) are being positioned onto the Eca physical map following hybridization to the BAC clones. Regions of interest, including a hrp operon and putative avirulence genes, have been identified and are currently being sequenced by random shearing and shotgun cloning of BAC insert DNA. The Eca and Ecc physical maps are being compared with the fully sequenced Escherichia coli genome to examine similarities in the genomic organisation of these closely related plant and human pathogens, and to identify novel E. carotovora-specific sequences.