Progress 10/01/06 to 09/30/07
Outputs
The goal of this project was to produce a high sequence depth genome sequencing survey of Taylorella equigenitalis, aetiological agent of Contagious Equine Metritis. It was anticipated that at least a portion of each of the estimated 1600 genes would be generated by the 454 sequencing approach. Prior to this project, only two genes had been identified for this pathogen. The sequencing phase of the project was carried out by the DNA sequencing Center at Oklahoma University, under the directorship of Bruce Roe and yielded results that exceeded anticipations, due in part to the implementation of novel methodology and the absence of complex repeats in the genome (that could not be predicted prior to project initiation). Overall, approximately 1.6 Mb of DNA Sequence was generated, which is greater than 99 percent of the genome and was contained in approximately 50 segments. Overall, there was greater than 10x coverage of each sequence. The approx. 1600 Open Reading Frames
within the sequence have been delineated and compared to the current databases, allowing an initial prediction of function. Since the success of this phase exceeded expectations, attempts to close the genome are now underway in the PI's laboratory, using PCR and comparative genomic (bioinformatics) approaches to link segments. As a direct consequence of this project, the PI was invited to attend the 1st International Conference on Contagious Equine Metritis in Lelystad, Netherlands in July 2007. Discussions with the delegates there confirmed the potential utilization of the DNA sequence for further diagnostic or molecular epidemiological applications. A collaboration with Dr. John Moore (Belfast, N. Ireland) and Dr. Motoo Matsuda (Japan) is testing the utilization of novel CRISPR region for strain discrimination among Taylorella isolates, as is widely used for molecular epidemiological analyses of Mycobacterium tuberculosis (spoligotyping). A meeting with the Taylorella group of the
Technology Development Unit of the Veterinary Laboratory Agency (the main government research unit in this area in the United Kingdom), is also planned for mid-March 2008. Once the genome sequence is completed (which is beyond the original scope of the project), the data set will be deposited in the public database GenBank. If it is not possible to 'close' the genome, then the draft sequence set (in contigs) will be deposited in GenBank.
Impacts
The major result of the project period was the generation of an almost complete genome sequence of the Taylorella equigenitalis Type strain. Advances in technology and the fortuitous absence of extended repetitive regions enabled the genome sequence information to be assembled into fewer than 50 major segments. Analysis of the genes within this dataset revealed several novel findings: (i) local synteny (conserved gene arrangement) with the pathogen Bordetella pertussis, (ii) presence of multiple specialized secretion systems for the potential export of factors involved in the pathobiology of the organism, including a Tat export system, Type IV, Type V and the newly recognized Type VI secretion systems, (iii) presence of adhesions for adherence to host tissues, (iv) systems for Iron acquisition (transferring binding and uptake systems) and (v) a CRISPR region that in other pathogens is proving to be a sensitive marker for molecular epidemiological applications. Prior to
this work, the only known sequence for this organism was that encoding the ribosomal RNA genes. With greater than 99 percent of the genome sequence in hand, it was possible to perform an initial comparison to gene sequences of Taylorella asinigenitalis. The latter organism has only been identified in the last 5 yrs, and although initially though to be restricted to donkeys, has recently been recovered from a horse in Sweden. Only one sequence from T. asinigenitalis had been deposited in GenBank and so it was not possible to compare the two known Taylorella species at the molecular level. A limited (approx. 20 gene) survey was performed by sequencing clones from a T. asinigenitalis genomic library. For each gene identified, the corresponding gene from T. equigenitalis was in hand, allowing the similarity to be compared on a gene by gene basis. The initial analysis revealed that protein coding sequences are 85-95 percent identical at the amino acid level, reflecting enough sequence
divergence to generate diagnostic PCR assays to discriminate between the two species. Although not included as part of the original proposal, in addition to generating the genome sequence data, our laboratory sought to develop the first genetic transformation system for T. equigenitalis. To date, the broad-range plasmid pBBR1MCS (Chloramphenicol resistance, medium copy number approx. 30, approx. 4.7 kb) has been used to transform electrocompetent Taylorella equigenitalis strain ATCC 35865. Transformants were demonstrated to possess extrachromosomal DNA and had the same overall restriction fragment profile as the wild type cells and similar Southern hybridization signal with a specific probe from the tatC gene. Initial studies indicate that the low copy number plasmid pRK415 is able to transform T. equigenitalis to tetracycline resistance. Studies are underway to confirm the initial transformation results. In summary, our laboratory has developed the first method for the genetic
manipulation of any Taylorella species which is fully anticipated to be a critical adjunct to the genome sequence in further investigation of the pathobiology of these bacteria.
Publications
- No publications reported this period
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