Progress 12/01/06 to 11/30/11
Outputs
OUTPUTS: The first output was that draft genome sequences (at 7.5X coverage) were completed and assembled for the potato pathogen Streptomyces turgidiscabies strain Car8 and the sweet potato pathogen Streptomyces ipomoeae strain 91-03. These sequences were submitted to Genbank in the form of whole-genome shotgun sequences. The second output was that we closed and manually annotated the 674,223-bp transmissible pathogenicity island found in S. turgidiscabies strain Car8. The third output is that we have recently sequenced three additional S. ipomoeae isolates (strains 78-51, 88-35, and B12321) and closed the sequence for a smaller potential pathogenicity island present in S. ipomoeae. These additional genome sequences will soon be submitted to Genbank. PARTICIPANTS: PD Pettis was involved in providing overall direction of the project by working with the partner organization, other collaborators and graduate students involved in this project. Sequencing was performed by the J. Craig Ventor Institute under the immediate direction of Co-PD Badger. We also collaborated extensively with Dr. Rosemary Loria of Cornell University (now University of Florida), who had finished the S. scabies 87-22 genome sequence in a separate USDA-funded project. Jose Huguet-Tapia, a graduate student in Dr. Loria's lab, traveled to the PD's lab, where he and the PD manually annotated the S. turgidiscabies pathogenicity island (PAIst). Jose also performed all of the comparative bioinformatic analyses involving the various Streptomyces genome sequences. Dongli Guan, a graduate student in the PD's lab, fully characterized and then extracted high-quality genomic DNA for the S. turgidiscabies and S. ipomoeae strains used for genomic sequencing. TARGET AUDIENCES: The PD presented both poster and oral presentations of the results of this project to other researchers interested in microbial genomics and bioinformatics at the NSF/USDA CSREES Microbial Genome Sequencing Workshop held annually as part of the Plant and Animal Genome Conference. The PD has also incorporated a section on microbial genomics, including that involving Streptomyces plant pathogens, into both his undergraduate (BIOL 4215, course entitled Molecular Biology of Bacterial Disease) and graduate (BIOL 7800, course entitled Advance Microbial Genetics) courses. The project also provided a novel and extensive training ground for the graduate students involved, in particular for Jose Huguet-Tapia, whose Ph.D. dissertation is based largely on the results from this project (see fourth publication listed above). PROJECT MODIFICATIONS: Towards the end of 2010, we had completed most of our original specific objectives. The one remaining goal was to identify and close a putative pathogenicity island in S. ipomoeae strain 91-03, which would be analogous to the one found in S. turgidiscabies strain Car8 (PAIst). However, from extensive analysis of the draft sequence of 91-03 there did not appear to be an analogous pathogenicity island (or remants of an island due to possible intragenic recombination events). We therefore used some of our remaining funds to sequence three additional S. ipomoeae strains using a combination of 454 sequencing and Solexa sequencing. These three strains were isolated from separate geographical locations and would be used to confirm whether or not S. ipomoeae contains a pathogenicity island analogous to PAIst. From analysis of the resulting sequences we did not find evidence for a PAIst analog in S. ipomoeae. However, two of the three strains, along with the original sequenced S. ipomoeae strain 91-03, showed evidence of a smaller potential island located at a different chromosomal locus than PAIst. Interestingly, this alternative locus includes the biosynthetic gene cluster for the phytotoxin thaxtomin C. The sequence for this smaller putative pathogenicity island was then closed in the relevant strains and we are currently analyzing the results of this effort. We expect to eventually publish these additional findings.
Impacts
The 674,233-bp transmissible pathogenicity island from S. turgidiscabies strain Car8 (designated PAIst) is, to our knowledge, the largest pathogenicity island described to date. We found that it consists of two non-overlapping modules of 105,364 and 568,859 bp. These two modules are delimited by three copies of an 8-bp palindrome (TTCATGAA) that is also the integration site of the element. Based on this arrangement as well as previous experimental data, we strongly suspect that PAIst is not only transmissible as a single intact entity but also as individual modules. Through bioinformatic analysis we found that the 105,364-bp module is nearly identical to a genomic island found in the potato pathogen Streptomyces scabies strain 87-22, while the 568,859-bp module possesses only very limited synteny to that genome. Nevertheless, both modules contain numerous previously identified virulence genes or newly identified candidates. These results for PAIst were reported in the second publication listed below. We have also used our genome sequences in bioinformatic analyses aimed at defining the Streptomyces pathogenome. As an initial approach, we compared the predicted proteomes for the three sequenced plant pathogens (S. turgidiscabies Car8, S. ipomoeae 91-03 and S. scabies 87-22) and eight sequenced non-pathogenic Streptomyces species. From this analysis we identified 4,259 protein-encoding genes that are absent in the non-pathogens and are either shared among two or more of the pathogenic species or are unique to a specific pathogen. The majority of these genes encoded hypothetical proteins, some of which are conserved but for which the function still cannot be predicted. Nevertheless, a number of other genes were identified that are predicted to encode secreted proteins, transcriptional regulators, membrane transporters, and enzymes for secondary metabolite biosynthesis. Thus, many of these could represent important shared or unique virulence factors for plant pathogenic Streptomyces species. These initial bioinformatic results were presented in the first publication listed below. We are also in the process of performing a more detailed bioinformatic analysis involving these same plant pathogenic species and more non-pathogenic streptomycetes, and it is expected that a manuscript detailing this work will be submitted in the near future. Analysis of our genome sequences has also revealed important facets of known virulence factors. For example, while S. turgidiscabies, S. ipomoeae, and S. scabies all produce phytoxic compounds known as thaxtomins, the genome data confirmed that S. ipomoeae is missing one thaxtomin gene (txtC), which is present in the other two species. This result is consistent with the observation that S. ipomoeae produces a thaxtomin derivative which is less-modified than that produced by the other two species. The S. ipomoeae genome analysis for the txtC gene was reported in the third publication listed below.
Publications
- 1. Bignell, D.R.D., Huguet-Tapia, J.C., Joshi, M.V., Pettis, G.S., and Loria, R. 2010. What does it take to be a plant pathogen: genomic insights from Streptomyces species. Antonie van Leeuwenhoek 98: 179-194.
- 2. Huguet-Tapia, J.C., Badger J.H., Loria, R., and Pettis, G.S. 2011. Streptomyces turgidiscabies Car8 contains a modular pathogenicity island that shares virulence genes with other actinobacterial plant pathogens. Plasmid 65: 118-124.
- 3. Guan, D., Grau, B.L., Clark, C.A., Taylor, C.M., Loria, R., and Pettis, G.S. 2012. Evidence that thaxtomin C is a pathogenicity determinant of Streptomyces ipomoeae, the causative agent of Streptomyces soil rot disease of sweet potato. Mol. Plant-Microbe Interact. 25: 393-401.
- 4. Huguet-Tapia, J.C. 2010. Genome analysis in plant pathogenic Streptomyces. Ph.D. dissertation, Cornell University, Ithaca, N.Y.
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Progress 12/01/08 to 11/30/09
Outputs
OUTPUTS: As we reported last year, we closed the large transmissible pathogenicity island present in the chromosome of S. turgidiscabies strain Car8 (PAIst) and completed assemblies of our draft genome sequences for S. turgidiscabies Car8 and Streptomyces ipomoeae strain 91-03. A critical component of this grant is performing comparative analyses using our draft genome sequences and closed pathogenicity island sequence. Our first output for this year is that we completed several comparative analyses for the S. turgidiscabies pathogenicity island. Our second output is that we also completed a preliminary comparative analysis using our 2 draft sequences as well as the genome sequences for the pathogen Streptomyces scabies and for 3 non-pathogenic Streptomyces spp. (Streptomyces coelicolor, Streptomyces avermitilis, and Streptomyces griseus). This latter analysis involved creating ortholog families using OrthoMCL (Li et al., 2003, Genome Res. 13: 2178-2189) with the idea that conserved protein families specific for pathogenic Streptomyces species could be identified. This work was performed in collaboration with Dr. Rosemary Loria at Cornell University, who recently spearheaded sequencing of the S. scabies genome. PARTICIPANTS: PD Pettis continued to provide overall direction for the project. For comparative genomic and PAI analyses, the PD collaborated extensively with Dr. Rosemary Loria of Cornell University and, in particular, one of her graduate students (Jose Huguet), who has a strong interest in microbial genome bioinformatics. Jose spent a portion of the previous year working in the PD's lab on manual annotation of the PAIst, and he has been performing the comparative analyses described here. This grant has thus provided a unique training opportunity for Jose, who is set to graduate with a Ph.D. from Cornell in 2010. Several of his dissertation chapters will be based on his bioinformatic work concerning the genomes of S. turgidiscabies, S. ipomoeae, as well as S. scabies. Manuscripts concerning characterization of the PAIst as well as comparative analyses involving the S. turgidiscabies and S. ipomoeae genome sequences are currently in preparation and should be submitted in 2010. TARGET AUDIENCES: Results for this project were reported as an oral presentation to other researchers interested in microbial genomics and bioinformatics at the NSF/USDA CSREES Microbial Genome Sequencing Workshop (Jan. 10-11, 2009), which is held annually in San Diego, CA as part of the annual Plant and Animal Genome Conference. The presentation was entitled "Genomics of Streptomycete Plant Pathogens." PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The comparative analysis of the PAIst revealed several previously unknown features. First, we found that a region of the PAIst is syntenic with an integrated plasmid from Corynebacterium glutamicum. This syntenic region includes putative genes for both replication and conjugation. The implication of this result is that PAIst now appears to be a kind of integrative and conjugative element (ICE), which may also have replicative ability. Secondly, we found the 3' portion of PAIst is syntenic with portions of both the S. coelicolor and S. avermitilis chromosomes. Finally, as reported last year, we found that the 674-kb PAIst has a modular structure consisting of two sub-modules of 105 kb and 569 kb in size. Comparative analysis has now shown that the 106-kb module shows 99% similarity to the genomic island located at 1,566576 bp in the chromosome of S. scabies. The syntenic regions in both species include the virulence genes nec1 and tomA. Meanwhile, a portion of the 569-kb module of PAIst shows strong similarity to the genomic island located at position 6,531,990 bp in S. scabies. The regions of similarity in both species include the biosynthetic genes for the phytotoxin thaxtomin A. All of these comparative results taken together provide important insights regarding the mechanism and evolution of transmissible pathogenicity in the Streptomyces genus. The comparative analysis involving the draft sequences for S. turgidiscabies and S. ipomoeae along with the genome sequences for S. scabies and three non-pathogenic Streptomyces spp. allowed us to identify 270 ortholog gene families that were specific to the three pathogenic species. At least some of these gene families may represent functions that are important for pathogenicity in these bacteria. Interestingly, we found that four of these pathogen-specific families involved pectate lysase genes. Phylogenetic analysis for each of these four families revealed that two of the pathogen-specific pectate lyase families are more closely related to fungal pathogen pectate lyase orthologs, while the other two are more closely related to orthologs from other bacterial species. These results may thus provide additionial insights as to how pathogenicity has evolved in the streptomycetes. As many more genome sequences for more Streptomyces spp. are now becoming available, we are currently peforming additional comparative analyses, which include the newly available sequences.
Publications
- No publications reported this period
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Progress 12/01/07 to 11/30/08
Outputs
OUTPUTS: While assemblies were created for our draft sequences of the potato pathogen Streptomyces turgidiscabies strain Car8 and the sweet potato pathogen Streptomyces ipomoeae strain 91-03 during the last reporting period, we soon realized that they did not show the expected synteny with other sequenced streptomycete genomes. As detailed in the next section, a different approach was used during this reporting period which allowed us to create assemblies with the expected synteny. This was our first significant output during this current reporting period. The second significant output was that we closed the sequence of the large transmissible pathogenicity island present in the chromosome of S. turgidiscabies Car8. We also manually annotated all of the detectable open reading frames in the island (see next section). S. turgidiscabies strain Car8 represents the model for transmissible pathogenicity in this genus, and so the completed sequence and associated characterization of its pathogenicity island is a significant advancement in our understanding of the virulence determinants involved and the mechanism by which they are laterally transferred. PARTICIPANTS: The PD (Pettis) provided overall direction of the project while the co-PD (Badger) at the J. Craig Venter Institute directed the day-to-day operations there with respect to determining the assemblies for the draft genomes as well as closing the sequence for the S. turgidiscabies strain Car8 pathogenicity island. In particular, the co-PD was instrumental in determining the correct method for creating assemblies of the draft genomes that showed the expected synteny with other streptomycetes genome sequences. For manual annotation of the S. turgidiscabies Car8 pathogenicity island, the PD collaborated extensively with Dr. Rosemary Loria of Cornell University and members of her lab. In particular, a Ph.D. graduate student in Dr. Loria's lab (Jose Huguet) who has interest in microbial genome bioinformatics, spent one month in the PD's lab working with the PD on manual annotation of this island. The graduate student thus received important training and experience regarding bioinformatics of streptomycete plant pathogens. The PD is also collaborating with the Loria lab as we begin to do comparative genomic analyses of streptomycete pathogenic and non-pathogenic species (i.e., the next objective of the proposal). TARGET AUDIENCES: Results from this project were reported in the form of a poster to other researchers interested in microbial genomics at the annual NSF/USDA CSREES Microbial Genome Sequencing Workshop, which is held annually in San Diego, CA as part of the annual Plant and Animal Genome Conference. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The new assemblies for the two draft genomes showed the expected synteny with the genome sequences of other Streptomyces species such as Streptomyces scabies. In particular, our draft sequence of S. ipomoeae strain 91-03 showed synteny throughout its entire length with S. scabies, while our draft sequence of S. turgidiscabies Car8 also showed synteny throughout with the caviat that a large inversion has apparently occurred in the middle of the S. turgidiscabies genome. The pathogenicity island of S. turgidiscabies Car8 measures 674,218 bp and contains 737 identifiable ORFs. The island is organized as a modular type of integrative conjugative element (ICE), including a terminally positioned integrase gene; specifically, the island comprises 105- and 569-kb modules, which based on previous evidence from the Loria lab at Cornell University, can be laterally transmitted either together or independently from one another. Based on manual annotation (including BLAST search analysis of individual ORFs), the majority (i.e., 60%) of ORFs within the island are of unknown function. Another 12% are involved in various metabolic processes (known virulence loci such as tomA, fas, and txt are included in this category), while 7% of the ORFs are predicted to be involved in regulation (two-component systems, other transcription factors). A number of other functional categories were also identified including transposition (6%), transport (5%),DNA modification and repair (3%), site-specific recombination (the island integrase gene is included here)(2%), protein modification (2%), lantibiotic biosynthesis (1%), electron transport (1%) and peptidoglycan biosynthesis (1%). The impact of determining the entire sequence of the S. turgidiscabies Car8 pathogenicity island and characterizing its ORFs is that this knowledge will facilitate the eventual elucidation of the pathogenic mechanisms by which streptomycete plant pathogens cause disease. The sequence has also provided critical information (e.g., modular structure of the pathogenicity island, identification of an integrase gene) that will facilitate the unraveling of the mechanism used for lateral transmission of the island.
Publications
- No publications reported this period
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Progress 12/01/06 to 11/30/07
Outputs
OUTPUTS: Isolates of Streptomyces turgidiscabies strain Car8 and Streptomyces ipomoeae strain 91-03 were first verified for relevant biochemical and genetic markers. These markers included production of the phytotoxin thaxtomin and the presence of known virulence loci. Genomic DNA from each strain verified in this manner was then isolated to a high degree of purity and sent to our collaborator at the J. Craig Venter Institute (JCVI; formerly the Institute for Genomic Research), where it was used to construct small-insert and medium-insert plasmid libraries as well as fosmid libraries. For each genome, 40,000 random Sanger sequencing reactions per library or 120,000 total reactions per genome were performed and analyzed. From the individual reads, the S. turgidiscabies sequence has been assembled into a total of 4 scaffolds with the total span of scaffolds measuring 10,817,450 bp. The S. ipomoeae sequence has been assembled into one scaffold with a size of 10,425,661 bp. Both of these
assemblies were recently auto-annotated using relevant software, and we are now studying the annotated genome sequences. We are also beginning the next phase of the project, which will involve finishing the sequence for the pathogenicity island (PAI) present in each genome. If remaining funds are available, we will also perform one run of 454 pyrosequencing on each genome sequence in order to close the remaining gaps outside of the PAIs. For interested researchers, sequence data is being put on a JCVI ftp server. Once completed, the genome sequences will be available through Genbank and a JCVI ftp server.
PARTICIPANTS: The PD (G.S. Pettis) directed the project, including working with the Ph.D. student (D. Guan), who is supported by this project and who performed the biochemical and genetic verifications of the strains and purified the genomic DNA for sequencing. The co-PD (J. Badger) at our partner organization, The J. Craig Venter Institute, directed the day-to-day operations for library construction, random sequencing and annotation of the genomes. The PD received periodic updates and quality control summaries particularly with regard to the random sequencing, and then following consultation with the co-PD made any necessary strategy decisions. Training in annotation and analysis of genome sequences will be provided for the graduate student, who will participate in the manual curation of the PAI sequences for the two genomes. As mentioned earlier, we are also collaborating with Dr. Rosemary Loria of Cornell University by providing the S. turgidiscabies PAI sequence for comparative
analyses aimed at characterizing the PAI rearrangements in S. scabies.
TARGET AUDIENCES: Results from this project were reported in the form of a poster to other researchers interested in genomics at the annual NSF/USDA CSREES Microbial Genome Sequencing Workshop, which is held every year in San Diego, CA as part of the annual Plant and Animal Genome Conference. In the coming year, we expect to begin our k-8 outreach component.
Impacts
Our project has already had one unanticiptated outcome, which involves a newly formed collaboration. Dr. Rosemary Loria of Cornell University had recently sequenced the genome of the world-wide potato scab pathogen Streptomyces scabies (strain 87-22) in collaboration with the Sanger Institute. However, from the genome sequence it was apparent that the S. scabies PAI sequence had rearranged and was no longer intact. Dr. Loria and colleagues were unable to characterize the PAI rearrangements further since they had no intact PAI sequence with which to compare. She is now using our S. turgidiscabies PAI sequence of some 670 kb, which we are in the process of finishing, for comparative studies in order to determine the specific PAI rearrangements present in S. scabies 87-22. This approach will allow Dr. Loria and colleagues to complete the description of the genome sequence for this important plant pathogen and thereby get this work submitted for publication. We anticipate
that our project team will also contribute to the description of the S. scabies genome and comparison of its rearranged PAI to the intact one of S. turgidiscabies, and therefore we will be sharing authorship on the S. scabies manuscript with Dr. Loria and her colleagues. Such publication will be in addition to the manuscript(s) that we will prepare upon completion of our project, which will involve comparative genomic analysis involving the S. turgidiscabies and S. ipomoeae genomes as well as the other streptomycete and relevant non-streptomycete genomes already available.
Publications
- No publications reported this period
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