Progress 06/01/03 to 05/31/08
Outputs
Progress Report Objectives (from AD-416) The objective of this cooperative research project is to generate sequence coverage to assemble the genome of the important honey bee pathogen Paenibacillus larvae subsp. larvae. The gram-positive bacterium, Paenibacillus larvae subsp. larvae, causes the most devastating larval honey bee disease, American Foulbrood (AFB). The second objective of this research project is to generate sequence coverage to assemble the genome of the important honey bee fungal pathogen Ascosphaera apis. A. apis causes another devastating honey bee disease, Chalkbrood. Genetic sequences for P. l. larve and A. apis would provide the first opportunity for interactive whole genome analysis of the insect host and its natural bacterial and fungal pathogens. This amendment involves the sequencing of three plasmids from P. larvae subsp. larvae, which are estimated collectively to have a length of roughly 200 kb. Approach (from AD-416) The strategy developed at BCM-HGSC will aim for 10X coverage of the ~5 MB P.l.larvae genome. A short-insert plasmid library (1-3 Kb, randomly sheared) will be constructed from a virulent isolate of the bacterial pathogen collected from a single honey bee colony with severe AFB symptoms (isolate #230010, Berkeley, CA). Other libraries will be made to more precise specifications for the P. larvae genome project. A total of 120,000 sequence reads are predicted. In addition to the construction of the draft DNA sequence, initial genome annotation and publication of the P.l.larvae genome are expected. The same strategy will be implemented to generate 5.5X coverage of the ~(22-24) MB A. apis genome. Virulent isolates of the fungal pathogen were collected from a single honey bee larvae infected with the Chalkbrood. A short-insert plasmid library (1-3 kb, randomly sheared) will be constructed from a single mating type of the fungus, isolated from the original culture. The second plasmid library will be constructed to generate 2X coverage of the genome isolated from the second fungal mating type. An additional plasmid library would be constructed to generate 1X coverage of the P. larvae native plasmid DNA. Significant Activities that Support Special Target Populations The DNA (bacterial and fungal) have been sent to the BCM-HGSC. Whole bacterial genome shotgun libraries were constructed in plasmid vectors, and paired-end sequences were generated by the Sanger dideoxy-terminator technique. Sequences of the fungal genome were produced in much the same way. Genomic sequences were generated for both pathogens (Paenibacillus larvae and fungal pathogen Ascosphaera apis), and genomes were assembled using the Atlas and Phrap assembly programs. The size of the P. larvae genome was estimated about 4 Mb. Fungal genome sequencing generated a total of over 156 Mb of high quality bases. They were assembled into contigs that totaled 21.6 Mb in length, which represents about 7x fungal genome coverage. The FTP files containing genome sequences were posted on the BCM-HGSC website. Sequencing data were also submitted to a public (NCBI) database. Genome sequencing and analysis will provide a unique opportunity to investigate host-pathogen interactions and to identify key molecules involved in host invasion, pathogenesis, and host escape mechanisms. Using this information, the potential exists to develop molecular tools to measure physiological stress in honey bees. This information would allow researchers to better define the impact of diseases on bee health and would provide beekeepers a method to manage colonies to prevent large-scale population crashes. ADODR organized two meetings and also communicated with collaborators using other means of communication, such as conference calls and E-mails.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) The objective of this cooperative research project is to generate sequence coverage to assemble the genome of the important honey bee pathogen Paenibacillus larvae subsp. larvae. The gram-positive bacterium, Paenibacillus larvae subsp. larvae, causes the most devastating larval honey bee disease, American Foulbrood (AFB). The second objective of this research project is to generate sequence coverage to assemble the genome of the important honey bee fungal pathogen Ascosphaera apis. A. apis causes another devastating honey bee disease, Chalkbrood. Genetic sequences for P. l. larve and A. apis would provide the first opportunity for interactive whole genome analysis of the insect host and its natural bacterial and fungal pathogens. This amendment involves the sequencing of three plasmids from P. larvae subsp. larvae, which are estimated collectively to have a length of roughly 200 kb. Approach (from AD-416) The strategy developed at BCM-HGSC will aim for 10X coverage of the ~5 MB P.l.larvae genome. A short-insert plasmid library (1-3 Kb, randomly sheared) will be constructed from a virulent isolate of the bacterial pathogen collected from a single honey bee colony with severe AFB symptoms (isolate #230010, Berkeley, CA). Other libraries will be made to more precise specifications for the P. larvae genome project. A total of 120,000 sequence reads are predicted. In addition to the construction of the draft DNA sequence, initial genome annotation and publication of the P.l.larvae genome are expected. The same strategy will be implemented to generate 5.5X coverage of the ~(22-24) MB A. apis genome. Virulent isolates of the fungal pathogen were collected from a single honey bee larvae infected with the Chalkbrood. A short-insert plasmid library (1-3 kb, randomly sheared) will be constructed from a single mating type of the fungus, isolated from the original culture. The second plasmid library will be constructed to generate 2X coverage of the genome isolated from the second fungal mating type. An additional plasmid library would be constructed to generate 1X coverage of the P. larvae native plasmid DNA. Significant Activities that Support Special Target Populations This report serves to document research conducted under a Specific Cooperative Agreement between USDA-ARS and Baylor College of Medicine Sequencing Center. Additional details of research can be found in the report for the current project, 6204-21000-009-00D, Pests, Parasites, Diseases, and Stress of Honey Bees used in Honey Production and Pollination. The objective of this project is to generate sequence coverage to assemble genomes of the important honey bee bacterial pathogen, Paenibacillus larvae, and fungal pathogen, Ascosphaera apis. The gram-positive bacterium, Paenibacillus larvae, causes the most devastating larval honey bee disease, American Foulbrood (AFB). Chalkbrood of honeybees is caused by the fungus, Ascosphaera apis. Various chemicals have been tested for control or inhibition of chalkbrood with little success; no chemicals for the treatment of chalkbrood have been registered for use in the USA. Genomic sequences for these two honey bee pathogens will provide the first opportunity for whole genome analysis of the insect host and its natural pathogens. This research is expected to add significantly to the basic understanding of host/pathogen interactions and lead to the identification of the microbial toxins and other virulence factors. Genome Sequencing Projects (both bacterial and fungal) involved Whole Genome Shotgun (WGS) by sequencing both ends of the inserts from a plasmid library, prepared by Baylor Sequencing Center. Sequences were assembled using the Atlas and Phrap assembly programs. The initial analysis of a large number of random sequences using genome assembly and oligonucleotide frequency analysis gave estimation of the P. larvae genome size about ~5 MB. A. apis genome size was determined about ~24 MB. The FTP files containing P. larvae and A. apis sequences were posted on the BCM-HGSC website. Draft sequences of P. larvae and A. apis were also deposited in the NCBI, GenBank database. The ADODR organized meetings to develop strategies for genome sequencing and to discuss the approach for analysis and annotation of pathogen genomes. We have also regularly discussed progress of the project using telephone and E-mail communications.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 4d Progress report. This report serves to document research conducted under a Specific Cooperative Agreement between USDA/ARS and Baylor College of Medicine Human Genome Sequencing Center. Additional details of research can be found in the report for the parent project 6204-21000-009-00D, Pests, Parasites, Diseases, and Stress of Honey Bees Used in Honey Production and Pollination. Draft sequences of P. larvae and A. apis were produced by standard procedures and have been deposited in GenBank. Genomic DNA was isolated from the virulent strain of P. larvae at the USDA, ARS, Beltsville, MD; purified and bacterial species were confirmed using PCR methods at the USDA, ARS laboratories in Beltsville, MD, and Weslaco, TX. The DNA has been sent to BCM-HGSC collaborator. Whole genome shotgun libraries were constructed in plasmid vectors, and paired-end sequences were generated by the Sanger dideoxy-terminator technique and assembled using the Atlas and Phrap
assembly programs. P. larvae genome is 4.0 Mb and is likely nearly completely represented in the assembly. The FTP files containing P. larvae sequences were posted on the BCM-HGSC web site, which gave us the opportunity to conduct preliminary search for P. larvae genes that are involved in bee pathogenesis and encode for various virulence factors including metallopeptidases, Vegetative Insecticidal Toxin Proteins (VIP's), and Ricin-type toxins. The draft sequence of A. apis was produced in much the same way. A total of over 156 Mb of high quality bases were produced and assembled into contigs that totaled 21.6 Mb in length, which represents about 7x fungal genome coverage. We have made amendments to this agreement with the objective to sequence 200 Kb of bacterial plasmid DNA.
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Progress 10/01/04 to 09/30/05
Outputs
4d Progress report. This report serves to document research conducted under a Specific Cooperative Agreement between ARS and Baylor College of Medicine Sequencing Center. Additional details of research can be found in the report for the parent project 6204-21000-009-00D, "Pests, Parasites, Diseases, and Stress of Honey Bees Used in Honey Production and Pollination." The objective of this project is to generate sequence coverage to assemble the genome of the important honey bee bacterial pathogen Paenibacillus larvae. The size of the P. larvae genome was estimated about 5 MB. Genomic DNA was isolated from the virulent strain of P. larvae by Dr. Jay Evans (USDA/ARS, Beltsville), purified, and bacterial species was confirmed using PCR methods in two USDA/ARS laboratories (Beltsville and Weslaco). The DNA has been sent to BCM-HGSC collaborators, where two short-insert libraries (1-3 Kb) were generated. The initial analysis of a large number of random sequences (23000)
using genome assembly and oligonucleotide frequency analysis gave a somewhat contradicting estimation of the genome size. This and other issues currently are being investigated. Some of the discrepancies in the initial results from the first two libraries will be clarified by the attempts to make a new library with the larger inserts (3-5 Kb) followed by the P. larvae genome assembly and manual gene annotation. The FTP files containing P. larvae sequences (contig sizes in the range of 400 bp up to 57 Kb) were posted on the BCM-HGSC Web site, which gave us the opportunity to conduct preliminary search for P. larvae genes that are involved in bee pathogenesis and encode for various virulence factors including metallopeptidases, Vegetative Insecticidal Toxins (VIP's), and Ricin-type toxins. We have made amendments to this agreement with the objective to sequence genome of the important fungal honey bee pathogen Ascosphaera apis and extended the term date to 5/2006. The size of the
A. apis genome was estimated about (22-24) MB. We have isolated both mating type strains of Ascosphaera apis from locally infected honey bee colonies (Dr. Aronstein, USDA/ARS, Weslaco), and fungal species was confirmed using PCR methods (Dr. D. Murray, USDA/ARS, Weslaco). Genomic DNA from these strains was isolated and sent to BCM-HGSC collaborators. They have created short-insert plasmid library (3-5 Kb) for one of the mating types of the fungus. There was some trouble with the shearing of the DNA, which has prevented completion of the second mating type library. Therefore, we are currently preparing more genomic DNA of this second mating type. Sequencing of the first plasmid library is in progress.
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Progress 10/01/03 to 09/30/04
Outputs
4. What were the most significant accomplishments this past year? D. Progress Report This report serves to document research conducted under a Specific Agreement between ARS and Baylor College of Medicine Sequencing Center. Additional details of research can be found in the report for the parent project 6204-21000-009-00D, "Pests, Parasites, Diseases, and Stress of Honey Bees Used in Honey Production and Pollination." The objective of this project is to generate sequence coverage to assemble the genome of the important honey bee bacterial pathogen Paenibacillus larvae. The size of the P. larvae genome was estimated about 5 MB. Genomic DNA was isolated from the virulent strain of P. larvae by Dr. Jay Evans (USDA/ARS, Beltsville), purified, and bacterial species was confirmed using PCR methods in two USDA/ARS laboratories (Beltsville and Weslaco). The DNA has been sent to BCM-HGSC collaborators, where two short-insert libraries (1-3 Kb) were generated. The initial
analysis of a large number of random sequences (23000) using genome assembly and oligonucleotide frequency analysis gave a somewhat contradicting estimation of the genome size. This and other issues currently are being investigated. Some of the discrepancies in the initial results from the first two libraries will be clarified by the attempts to make a new library with the larger inserts (3-5 Kb) followed by the P. larvae genome assembly and manual gene annotation. The FTP files containing P. larvae sequences (contig sizes in the range of 400 bp. up to 57 Kb) were posted on the BCM-HGSC Web site, which gave us the opportunity to conduct preliminary search for P. larvae genes that involved in bee pathogenesis and encode for various virulence factors including metallopeptidases, Vegetative Insecticidal Toxins (VIP's), and Ricin type toxins. We have made amendments to this agreement with the objective to sequence genome of the important fungal honey bee pathogen Ascosphaera apis and
extended the term date to 5/2006.
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