Progress 09/15/01 to 05/31/05
Outputs
Clavibacter michiganensis subspecies are major phytopathogens impacting the health of several economically important crops. Most species in the group grow slowly in vitro and are difficult to dissect genetically. The objective of this research is to obtain the complete genome sequence of C. michiganensis subsp. sepedonicus (Cms). Cms causes bacterial ring rot of potato, which is considered to be the most important disease of seed potatoes worldwide. The ATCC 33113 strain was chosen for sequencing because it is virulent and accessible to the scientific community. The project was conducted through a collaboration of scientists at Colorado State University, the Sanger Centre, U.K., and Ohio State University, with expertise in Clavibacter biology and high-throughput DNA sequencing and annotation of high-GC gram-positive bacteria. A whole-genome shotgun strategy, generating 8-10 fold genome coverage from several libraries in pUC and m13 was used. The sequencing project
website is www.sanger.ac.uk/projects/C_michiganensis. Several libraries were constructed for the project, including small (2-2.8 kb) insert pUC19 libraries and several large insert libraries with 6, 9-10, and 38-42 kb inserts. At present there are 55,801 reads totaling 29.324 Mb and giving a theoretical coverage of 99.98% of the genome. The assembly phase is completed. The finished genome is 3.40 Mb and consists of a circular chromosome (3.25 Mb), the circular plasmid pCS1 (50.35 kb) and the linear plasmid pCSL1 (94.6 kb). The genome is 0.8 Mb (30%) larger than originally predicted. The predicted gene set was obtained, and manual and automated annotation of the genome sequence has been completed. On publication of the analysis and annotation, a fully annotated sequence will be released to the EMBL/GenBank/DDBJ public databases. Bioinformatic studies revealed the presence in Cms of eleven homologues of pat-1, a gene associated with pathogenicity in the related tomato pathogen, C.
michiganensis subsp. michiganensis (Cmm). Chromosomal pat-1 homologues in Cms are not clustered within a single region as in Cmm. Instead, pat-1 homologues are distributed around the chromosome of Cms, though there is some clustering in two regions. Whole genome comparisons between the potato and tomato pathogen were performed. Our analyses so far have shown the presence of extremely high sequence similarities between common genes in Cmm and Cms, often translating into greater than 85.2% identity at the nucleotide level. A somewhat surprising find from these studies is the extensive number of rearrangements in Cms as compared to Cmm. From the available annotation, we have learned that Cms contains about 102 copies of insertion elements (IS) which are transposases belonging to at least 3 different families. In contrast, Cmm does not contain these insertion elements. This is consistent with previous studies showing the insertion element IS1121 in Cms is not present in Cmm. We are in the
process of examining the expansion of IS elements in Cms relative to the chromosomal rearrangements, and whether this expansion is associated with loss of gene function.
Impacts
There is a zero tolerance for bacterial ring rot in the seed potato industry. Ensuring that seed is free of the pathogen (Cms) currently depends on positive reactions in a limited number of antigen or DNA-based assays, none of which target DNA sequences important in pathogen virulence. Having the complete genome sequence of Cms can lead to identification of genes required for disease development and provide new targets for pathogen detection.
Publications
- Laurila. J., M. C. Metzler, C. A. Ishimaru, and V.-M. Rokka. 2003. Infection of plant material derived from Solanum acaule with Clavibacter michiganensis ssp. sepedonicus: temperature as a determining factor in immunity of S. acaule to bacterial ring rot. Plant Pathol. 52:496-504.
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Progress 01/01/04 to 12/31/04
Outputs
Clavibacter michiganensis subspecies are major phytopathogens impacting the health of several economically important crops. Most species in the group grow slowly in vitro and are difficult to dissect genetically. The objective of this research is to obtain the complete genome sequence of C. michiganensis subsp. sepedonicus (Cms). Cms causes bacterial ring rot of potato, which is considered to be the most important disease of seed potatoes worldwide. The ATCC 33113 strain was chosen for sequencing because it is virulent and accessible to the scientific community. The project is being conducted through a collaboration of scientists at Colorado State University, the Sanger Centre, U.K., and Ohio State University, having expertise in Clavibacter biology and high-throughput DNA sequencing and annotation of high-GC gram-positive bacteria. A whole-genome shotgun strategy, generating 8-10 fold genome coverage from several libraries in pUC and m13 was used. The shotgun phase
is complete, and a database of reads is available for searching at Sanger's Blast Server, or for downloading from their FTP site. The sequencing project website is www.sanger.ac.uk/projects/C_michiganensis. Several libraries were constructed for the project, including small (2-2.8 kb) insert pUC19 libraries and several large insert libraries with 6, 9-10, and 38-42 kb inserts. At present there are 55,801 reads totaling 29.324 Mb and giving a theoretical coverage of 99.98% of the genome. The assembly phase is completed. The finished genome is 3.40 Mb and consists of a circular chromosome (3.25 Mb), the circular plasmid pCS1 (50.35 kb) and the linear plasmid pCSL1 (94.6 kb). The genome is 0.8 Mb (30%) larger than originally predicted. The predicted gene set has been obtained. Manual and automated annotation of the genome sequence is underway. About 50% of the predicted CDS have been annotated. On completion and publication of the analysis and annotation, a fully annotated sequence will
be released to the EMBL/GenBank/DDBJ public databases. Bioinformatic studies revealed the presence in Cms of eleven homologues of pat-1, a gene associated with pathogenicity in the related tomato pathogen, C. michiganensis subsp. michiganensis. Chromosomal pat-1 homologues in Cms are not clustered within a single region as in Cmm. Instead, pat-1 homologues are distributed around the chromosome of Cms, though there is some clustering in two regions.
Impacts
There is a zero tolerance world-wide for bacterial ring rot in the seed potato industry. Ensuring that seed is free of the pathogen (Cms) currently depends on positive reactions in a limited number of antigen or DNA-based assays, none of which target DNA sequences important in pathogen virulence. Having the complete genome sequence of Cms can lead to identification of genes required for virulence and provide new targets for pathogen detection.
Publications
- Ishimaru, C. A., Knudson,D. L., Brown,S. E., Francis,D.M., and Parkhill, J. 2004. Genome sequencing of Clavibacter michiganensis subsp. sepedonicus. Phytopathology (94:S123).
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Progress 01/01/03 to 12/31/03
Outputs
Clavibacter michiganensis subspecies are major phytopathogens, impacting the health of several economically important monocots and dicots. Most species in the group grow slowly in vitro and are difficult to dissect genetically. The objective of this research is to obtain the complete genome sequence of C. michiganensis subsp. sepedonicus (Cms). Cms causes bacterial ring rot of potato, which is considered to be the most important disease of seed potatoes world wide. The ATCC 33113 strain was chosen for sequencing because it is virulent and accessible to the scientific community. The project is being conducted through a collaboration of scientists at Colorado State University, the Sanger Centre, U.K., and The Ohio State University, having expertise in Clavibacter biology and high-throughput DNA sequencing and annotation of high-GC gram-positive bacteria. The complete genomic sequence is being generated at the Sanger Centre by a whole-genome shotgun strategy, generating
8-10 fold genome coverage from several libraries in pUC and m13. End-sequences from large insert BAC and lambda libraries have been generated, providing a scaffold to assemble the sequence contigs produced by the assembly of shotgun data. Gap closure is being achieved through a combination of primer walking, PCR, and shotgun strategies. The completed sequence will be analyzed and annotated using in-house tools available at the Sanger Institute. Library preparation and validation is completed. Shotgun phase is completed. A database of reads is available for searching on the Sanger Blast Server, or for download from the Sanger FTP site. At present there are 55,801 reads, totaling 29.324 Mb giving a theoretical coverage of 99.89% of the genome. Test assemblies of the present coverage yields 46 contigs > 2kb with a total size of 3.546 Mb. Closure is anticipated 28 months from start date. Annotation of the sequence is anticipated 28-36 months from start date. Current assemblies give a
genome size 0.8 Mb (30%) larger than originally predicted. The overall %GC content is 72.2%. The sequence of the linear plasmid in Cms has been finished and annotated. Preliminary automated annotation of the genome sequence has begun. On completion and publication of the analysis and annotation, a fully annotated sequence will be released to the EMBL/GenBank/DDBJ public databases. An enhanced set of web pages allowing full access to the sequence and annotation will be available at the Sanger Centre website.
Impacts
There is a zero tolerance world-wide for bacterial ring rot in the seed potato industry. Ensuring that seed is free of the pathogen (Cms) currently depends on positive reactions in a limited number of antigen or DNA-based assays, none of which target DNA sequences important in pathogen virulence. Having the complete genome sequence of Cms can lead to identification of genes required for virulence and provide new targets for pathogen detection.
Publications
- Laurila. J., M. C. Metzler, C. A. Ishimaru, and V.-M. Rokka. 2003. Infection of plant material derived from Solanum acaule with Clavibacter michiganensis ssp. sepedonicus: temperature as a determining factor in immunity of S. acaule to bacterial ring rot. Plant Pathol. 52:496-504.
- Ishimaru, C. A., Parkhill, J., Francis, D. M., Knudson, D. L. 2003. Complete genome sequence of Clavibacter michiganensis subsp. sepedonicus 3rd ASM and TIGR Conference on Microbial Genomes. USDA-CSREES Microbial Sequencing Awardee Workshop. New Orleans, Louisiana. January 2003.
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Progress 01/01/02 to 12/31/02
Outputs
Clavibacter michiganensis subspecies are major phytopathogens, impacting the health of several economically important monocots and dicots. Most species in the group grow slowly in vitro and are difficult to dissect genetically. The objective of this research is to obtain the complete genome sequence of C. michiganensis subsp. sepedonicus (Cms). Cms causes bacterial ring rot of potato, which is considered to be the most important disease of seed potatoes world wide. The ATCC 33113 strain was chosen for sequencing because it is virulent and accessible to the scientific community. The project is being conducted through a collaboration of scientists at Colorado State University, the Sanger Centre, U.K., and the Ohio State University, having expertise in Clavibacter biology and high-throughput DNA sequencing and annotation of high-GC gram-positive bacteria. The complete genomic sequence of Clavibacter michiganensis subsp. sepedonicus is being generated by a whole-genome
shotgun strategy, generating 8-10 fold genome coverage from several libraries in pUC and m13. Sequencing reads have been produced using Big-dye terminators on ABI3700 capillary machines. End-sequences from large insert BAC and lambda libraries will also be generated, providing a scaffold to assemble the sequence contigs produced by the assembly of shotgun data. Gap closure will be achieved through a combination of primer walking, PCR, and shotgun strategies. The completed sequence will be analyzed and annotated using in house tools available at the Sanger Centre. Library preparation and validation is completed. The shotgun phase is in progress, and a database of reads is available for searching on the Sanger Blast Server, or for download from the Sanger FTP site. At present there are 45,927 reads, totaling 23.299 Mb giving 6.8x coverage, a theoretical coverage of 99.89% of the genome. The overall %GC content is 72.2%, a finding that is typical of this group of organisms. BLASTX using
the current Cms sequences against the complete Mycobacterium tuberculosis proteome of known and predicted proteins yielded a 35% hit rate.
Impacts
There is a zero tolerance world-wide for bacterial ring rot in the seed potato industry. Ensuring that seed is free of the pathogen (Cms) currently depends on positive reactions in a limited number of antigen or DNA-based assays, none of which target DNA sequences important in pathogen virulence. Having the complete genome sequence of Cms can lead to identification of genes required for virulence and provide new targets for pathogen detection.
Publications
- Ishimaru, C. A., Brown, S. E., and Knudson, D. L. 2002. Linear plasmid in Clavibacter michiganensis subsp. sepedonicus. The International Conference on the Status of Plant, Animal, and Microbe Genome Research, Plant, Animal, and Microbe Genomes X. San Diego, CA. P5.
- Ishimaru, C. A., Parkhill, J., Francis, D. M., Brown, S. E., and Knudson, D. L. 2002. Complete genome sequence of Clavibacter michiganensis subsp. sepedonicus. The International Conference on the Status of Plant, Animal, and Microbe Genome Research, Plant, Animal, and Microbe Genomes X. San Diego, CA. W211.
- Brown, S. E., Knudson, D. L., and Ishimaru, C. A. 2002. Linear plasmid in the genome of Clavibacter michiganensis subspecies sepedonicus. 2002. J. Bacteriol. 184:2841-2844.
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Progress 01/01/01 to 12/31/01
Outputs
Plant pathogenic members of the high-GC gram-positive bacteria are vastly unexplored. Despite their agricultural importance, the molecular biology of these bacteria has lagged behind that of the gram-negative bacteria. Most species in the group grow slowly in vitro and are recalcitrant to classical genetic manipulation methods. Clavibacter michiganensis subspecies are major phytopathogens, impacting the health of several economically important monocots and dicots. The objective of this research is to obtain the complete genome sequence of C. michiganensis subsp. sepedonicus (Cms). Cms causes bacterial ring rot of potato, which is considered to be the most important disease of seed potatoes. The ATCC 33113 strain was chosen for sequencing because it is virulent and accessible to the scientific community. The project is being conducted by scientists at Colorado State University, the Sanger Centre, U.K., and Ohio State University, having expertise in Clavibacter biology
and high-throughput DNA sequencing and annotation of high-GC gram-positives. In this first stage of the project, a culture of ATCC33113 was obtained from ATCC. Purified total genomic DNA from ATCC 33113 was produced at Colorado State University and, after ensuring that it is free of live bacteria, was shipped to the Sanger Centre. The next phase of the project will entail DNA library construction, whole genome shotgun sequencing, and gap closing. When the sequence is complete it will be analyzed and annotated using tools available at the Sanger Centre. Once the sequence of Cms is completed and annotated it will allow the first comprehensive genomic comparisons between animal and plant pathogens in the Actinomycetales.
Impacts
Having the genome sequence of Cms will enable identification of those genetic sequences that are required for pathogenicity. It will also help identify sequences that enable the pathogen to persist for long periods in a latent state. The expectation is that identifying regulatory sequences and genes that are conserved among Cms and other related plant and animal pathogens will provide new avenues for disease control.
Publications
- No publications reported this period
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