Progress 10/01/00 to 09/30/05
Outputs
Funds from this grant supported our efforts to sequence the genome of an uncultivated microorganism, Epulopiscium. These funds also supported the development protocols to label and track bacteria in complex environmental samples. (1) Epulopiscium spp. are the largest heterotrophic bacteria yet described. A distinguishing feature of the Epulopiscium group is their production of multiple, internal offspring as a means of cellular reproduction. Based on their phylogenetic position, among low G+C Gram positive endospore-forming bacteria, and the remarkable morphological similarity between developing endospores and Epulopiscium offspring, we hypothesized that intracellular offspring production in Epulopiscium evolved from endospore formation. We are taking an aggressive genomic approach to understanding the biology of this microorganism. In a collaborative effort with a research team at The Institute for Genomic Research (TIGR) we are determining the complete genome sequence
of one species of Epulopiscium. This information should shed light on the evolution of the unusual mode of reproduction seen in these bacteria and also help us determine the physiological potential of these intestinal symbionts. More specifically, what is the role of these bacteria in the intestinal tract of the host fish? Funds from this grant were used to collect cells to be used for genomic DNA library construction. Type B Epulopiscium cells were manually collected from surgeonfish intestinal content. The DNA extracted from these cells was used to generate a large insert clone library (a fosmid library) and small insert clone libraries to support the Epulopiscium genome project. Sequencing all Epulopiscium genomic DNA libraries are currently underway. (2) Large Epulopiscium cells contain an extraordinary amount of DNA and this DNA is located at the periphery of the cytoplasm of the cell. In an effort to understand the metabolism of this DNA we developed a method to label DNA of
anaerobic bacteria with the nucleotide analog bromodeoxyuridine, BrdU. The BrdU is then detected via immunofluorescence in whole cells to localize regions that have recently replicated. We have applied this protocol to natural populations of Metabacterium polyspora (a multiple endospore-forming bacterium and relative of Epulopiscium) and type B Epulopiscium. Using this method we have discovered that DNA replication occurs in M. polyspora as forespores develop. This result demonstrates a significant modification in the core endospore developmental program that may allow M. polyspora to respond to local nutrient availability as offspring development progresses. This change may allow M. polyspora to regulate the number of offspring it forms in concert with nutrient status of the cell. (3) We published a strain-specific assay to detect viable Lactobacillus on cattle feed, using enrichment culturing and PCR. The assay proved useful in tracking bacteria applied to complex feed mixtures that
contain a high background of other lactobacilli. The assay provides a means of tracking potential probiotics which will aid studies to determine their efficacy in controlling pathogen loads.
Impacts
One of the greatest challenges facing microbiologists is devising ways of studying and understanding the role of microorganisms in natural environments. During the course of these studies we developed methods to identify, track and study DNA replication in specific microbial populations in complex environmental samples. Specifically, we developed genomic libraries from an uncultured intestinal symbiont, Epulopiscium, and will use these libraries to determine its genome sequence. In preliminary studies of the genomic libraries we have identified novel polysaccharide degrading genes that could be used in industrial applications. We have developed a protocol that can be used to follow DNA replication in anaerobic bacteria. This will allow us to study the cell cycle and offspring development of intestinal microorganisms in their natural environment. Finally, we developed and published a strain-specific assay that allows us to enumerate a viable probiotic after it has been
applied to cattle feed. This protocol allows us to track the probiotic during the feed application process and could be used to track this population in the intestinal tract of cattle. By use of this assay we have identified feed processing steps that compromise the viability of the probiotic.
Publications
- Flint, J. F. and E. R. Angert. 2005. Development of a strain-specific assay for detection of viable Lactobacillus sp. HOFG1 after application to cattle feed. J Microbiol Meth 61: 235-243.
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Progress 01/01/04 to 12/31/04
Outputs
Over the past year, funds from this grant supported our efforts to determine the complete nucleotide sequence of the genome of an uncultivated microorganism, Epulopiscium. These funds also supported the development of a protocol for in situ labeling of DNA in live anaerobic bacteria. We hope to use this protocol to follow DNA replication in uncultured bacteria found in the intestinal tract of herbivores. (1) Epulopiscium spp. are the largest heterotrophic bacteria yet described. A distinguishing feature of the Epulopiscium group is their production of multiple, internal offspring as a means of cellular reproduction. Based on their phylogenetic position, among low G+C Gram positive endospore-forming bacteria, and the remarkable morphological similarity between developing endospores and Epulopiscium offspring, we hypothesized that intracellular offspring production in Epulopiscium evolved from endospore formation. We are taking an aggressive approach to understanding
the biology of this microorganism. In a collaborative effort with a research team at The Institute for Genomic Research (TIGR) we are determining the complete genome sequence of one species of Epulopiscium. This information should shed light into the evolution of the unusual mode of reproduction seen in these bacteria and also help us determine the physiological potential of these intestinal symbionts. More specifically, what is the role of these bacteria in the intestinal tract of the host fish? Using samples collected in October 2003, we manually collected 20,000 Epulopiscium cells. DNA extracted from these cells was used to generate a large insert clone library (a fosmid library) to support the Epulopiscium genome project. Sequence data from this large insert library will provide a scaffold to assist assemble of the shotgun sequence data from small insert libraries. Some of the Epulopiscium samples were also provided to collaborators at TIGR. This supported the development of
several new small insert genomic libraries. TIGR is currently sequencing all of these libraries. Epulopiscium spp. are giants in the bacterial world. (2) We are interested in understanding more about the biology of these cells. Large Epulopiscium cells contain an extraordinary amount of DNA and this DNA is located at the periphery of the cytoplasm of the cell. We believe that this is a significant adaptation that supports the high metabolic activity of these large cells. In an effort to understand the metabolism of this DNA we are developing a method to label DNA of anaerobic bacteria with the nucleotide analog bromodeoxyuridine, BrdU. Antibodies to detect BrdU in situ are commercially available. This type of protocol has been successfully used in mammalian cells to follow DNA replication and degradation but has seen limited application to bacteria. Preliminary experiments with Clostridium lentocellum, a relative of Epulopiscium, have demonstrated the utility of this approach. In
future experiments we will apply this protocol to Epulopiscium cells in their natural environment.
Impacts
Using Epulopiscium as a model system we are developing genome-based approaches to study the biology of a microorganism that is not yet available in laboratory culture. With its large cell size and the presence of large amounts of cellular DNA and RNA we can study Epulopiscium at the single-cell level. This puts us in a unique position to develop more sensitive techniques for studying bacteria taken from natural environments. This genome-based approach provides access to the vast and untapped genetic resources of the uncultured microbial world.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
Over the past year, funds from this grant supported our efforts to determine the complete nucleotide sequence the genome of an uncultivated microorganism. We have manually collected over 50,000 individual Epulopiscium cells from ethanol-fixed intestinal contents of the surgeonfish Naso tuberosus. A portion of the DNA extracted from these cells was amplified (replicated) and used to generate a small-insert clone library. Draft sequence from three small insert libraries were generated in collaboration with The Institute for Genomic Research (TIGR). Assembly of the sequence reads into large contigs proved problematic. This may be due to modification of the genomic DNA during the amplification process. In October of 2003, with additional support from the NSF and a Cornell College of Agriculture and Life Sciences Travel Grant Faculty Travel Grant, I was able to collect additional surgeonfish intestinal contents (with Epulopiscium cells). These cells will be used to
generate more genomic DNA for library construction. Progress has been made toward developing a rapid sorting technique for Epulopiscium cells. So far we have relied on micromanipulation of individual cells from fixed gut contents samples to generate enough material for genomic analyses. Initial efforts to raise polyclonal antiserum against Epulopiscium cells were unsuccessful. As an alternative we have cloned three flagellin gene homologues from Epulopiscium. These genes were cloned into expression vectors and used to produce the Epulopiscium flagellin polypeptides in E. coli. The purified polypeptides were used to generate antisera in rabbits. The antisera have been tested and are specific for each polypeptide and are of high titer. This serum has been used to coat magnetic beads for developing an immunomagnetic separation technique. In addition we have been working with a company to develop an automated cell sorting protocol for separating Epulopiscium cells based on in situ
hybridization-conferred fluorescence and DNA staining.
Impacts
Using Epulopiscium as a model system we are developing genome-based approaches to study the biology of a microorganism that is not yet available in laboratory culture. With its large cell size and the presence of large amounts of cellular DNA and RNA we can study Epulopiscium at the single-cell level. This puts us in a unique position to develop more sensitive techniques for studying bacteria taken from natural environments. This genome-based approach provides access to the vast and untapped genetic resources of the uncultured microbial world.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
Over the past year, funds from this grant supported our efforts to determine the complete nucleotide sequence the genome of an uncultivated microorganism. We have manually collected over 50,000 individual Epulopiscium cells from ethanol-fixed intestinal contents of the surgeonfish Naso tuberosus. We have also devised a protocol for purification of DNA from these organisms. A portion of the DNA extracted from these cells has been amplified (replicated) and will be used to generate a small-insert clone library. Another portion of this DNA will be used to build a large-insert (fosmid) library. These libraries will be used in a project to determine sequence the entire genome of Epulopiscium. Progress has been made toward developing a rapid sorting technique for Epulopiscium cells. So far we have relied on micromanipulation of individual cells from fixed gut contents samples to generate enough material for genomic analyses. We have begun to inject purified Epulopiscium
cells into a rabbit to raise polyclonal antiserum against Epulopiscium. This serum will be used to coat magnetic beads for developing an immunomagnetic separation technique. We have also cloned and sequenced three flagellin homologues from Epulopiscium cellular DNA. These genes have been cloned into an expression vector system which would allow us to generate polypeptides for further antiserum production. These surface proteins are an alternative target for immunoselective isolation of cells. Pulsed-field gel electrophoretic analyses of the Epulopiscium genome have not been fruitful so we are exploring other ways to determine the size of the Epulopiscium genome. We are using a combination of quantifying DNA extracted from isolated cells and real-time quantitative PCR to determine the unit genome size of Epulopiscium. From these studies we have estimated that an individual cell contains up to 80,000 copies of an approximately 4 Mb genome. Further mapping studies are in the planning
stages.
Impacts
Using Epulopiscium as a model system we are developing genome-based approaches to study the biology of a microorganism that is not yet available in laboratory culture. With its large cell size and the presence of large amounts of cellular DNA and RNA we can study Epulopiscium at the single-cell level. This puts us in a unique position to develop more sensitive techniques for studying bacteria taken from natural environments. This genome-based approach provides access to the vast and untapped genetic resources of the uncultured microbial world.
Publications
- Mendell, J. and Angert, E. R. 2002. Real-time PCR Quantification of the ftsZ Gene in Single Cells of Epulopiscium. Poster Abstract Boston Bacterial Meeting.
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Progress 01/01/01 to 12/31/01
Outputs
During the past year we have focused on determining the genome size of Epulopiscium and other closely related bacteria by pulsed-field gel electrophoresis (PFGE) techniques. We have been able to determine the genome size of Clostridium lentocellum and C. propionicum (both are approximately 2.8 Mb). However this approach has not produced distinct bands for Epulopiscium samples. Even uncut Epulopiscium DNA appears degraded. The activation of endogenous nucleases often interferes with PFGE mapping of Clostridium genomes. We believe a similar process is happening in the Epulopiscium samples as the cells are harvested from their host. We are trying other approaches to gain information about the complexity of this sheared DNA. We are working toward building a fosmid library of Epulopiscium genomic DNA. Fosmids are a modified type of bacterial artificial chromosome. The vector contains the origin of replication from the E. coli F episome. For this library we will use DNA
extracted from a surgeonfish gut contents sample. This sample will be enriched for Epulopiscium cells. The genomic DNA will be sheared, end repaired and size selected. DNA fragments approximately 40 kb in length will be cloned into the fosmid vector. The ligation products will be packaged into phage heads and used in transfections of E. coli cells. This is a very efficient means of transferring large plasmids into E. coli. Once inside E. coli the DNA is maintained as a low copy number plasmid. We have used a commercially available kit to generate fosmid libraries of C. lentocellum and C. propionicum DNA. I visited the Los Alamos National Laboratory December 2001 to establish a link with the National Flow Cytometry Labs. We will be sending them Epulopiscium samples to test the feasibility of using flow cytometric methods to sort Epulopiscium cells from surgeonfish gut contents.
Impacts
(N/A)
Publications
- No publications reported this period
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