Progress 10/01/06 to 09/30/09
Outputs
OUTPUTS: Paenibacillus popilliae and Paenibacillus lentimorbus cause milky spore disease in the larval stage of various scarab beetles. Development of P. popilliae and P. lentimorbus into biological control agents would provide an alternate method for managing these pests in lieu of traditional pesticides. Polymerase chain reaction (PCR) amplification of P. popilliae genomic DNA and sequencing of the amplified DNA has isolated and characterized 25 different DNA segments of the P. popilliae genome. BLASTX analysis of these isolated P. popilliae DNA segments has helped determine identity numerous genes relative to reported protein sequences in the GenBank database. Transcription start codons have been tentatively identified and, where included, presumptive ribosome-binding sites have been located. DNA sequence and gene identification data have been disseminated via scientific publication in the DNA database, GenBank. Construction of an insertional mutant library has been discontinued. The insertional mutagen, Tn916, was not confirmed as inserting into the bacterial genome in a completely random manner despite switching the conjugative donor. Construction of a phenotypic marker and a gene insertion probe for P. popilliae was nearing completion. Fusion of the P. popilliae thymidine kinase (tdk) promoter region to green fluorescent protein (GFP) was completed and found to be functional in Escherichia coli. Insertion of the tdk-GFP fusion into Tn916 and placement this transposon construct into Enterococcus faecalis (for conjugative transfer into P. popilliae) was in progress. Partial assembly of a promoter-less GFP gene insertion marker, using frame-reading insertion, into Tn916 was underway. Dr. Dingman, in association with Dr. Richard Cowles (The Connecticut Agricultural Experiment Station) has had limited success at in vivo spore production. Production of large amounts of P. popilliae endospores was to provide a source of this pathogen for use in environmental control studies. Students and teachers were mentored through presentations of research findings. Additionally, conversations with Connecticut citizens were done to educate individuals on molecular biology principles and on current practices for pesticide treatment of scarab beetle infestations. PARTICIPANTS: Dr. Douglas W. Dingman (primary investigator) has performed all experiments and analyses associated with this project. Dr. Richard Cowles has provided scarab beetle larvae for endospore production via injection. Mrs. Cindy Musante provided technical support and assisted in setup of equipment/supplies associated with experimental design. TARGET AUDIENCES: Scientists studying bacterial genomics and scientists investigating molecular properties of bacterial entomopathogens are targets of this research. These data provide information to advance scientific investigations of genetic variability within pathogenic Paenibacilli. Via comparative analysis of gene sequences and genomic organization, scientists can use these data to better understand P. popilliae pathogenicity and disease characteristics of other insecticidal bacteria. Scientists can use the knowledge gained to produce molecular tools for altering and probing these bacteria to gain more information and to provide effective control agents against harmful insects. Future scientific research on P. popilliae, or related entomological bacteria, will have available these data for construction of a genomic profile and for use in making comparisons between bacterial genes. Target audiences include landscape managers, lawn care specialists, and homeowners, also. Through results from applied investigations, these target audiences will benefit from information that can be used to establish a biocontrol agent and protocol to combat scarab beetle larvae infestations. High school and grade school students and teachers gained exposure to scientific principles via mentoring and research presentations. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This investigation has presented 25 new DNA fragments of the P. popilliae genome, and representative gene identifications, to the international DNA database, GenBank. These data are freely available on the World-Wide-Web for individuals to view and use. This availability will impact researchers by providing more sequence data pertaining to P. popilliae for future investigations. Also, this information will provide protein sequences (deciphered from the DNA sequences) from a different bacterium for inclusion into families of classified proteins. The randomly isolated DNA segments obtained from P. popilliae can be used in future investigations to help produce a physical map of the genome prior to, or in conjunction with, complete genomic sequencing of P. popilliae. Availability of a phenotypically marked strain of P. popilliae will help investigations to tract disease progress within the insect. Construction of the tdk-GFP protein fusion was the outcome reached in providing a marked strain. Future scientific investigations can make use of this fusion to complete the phenotypic marking of P. popilliae. The ability to probe the genomic operation of P. popilliae would provide a powerful tool to investigate pathogenic coding and function of this bacterium. Development of a promoter-less GFP and Tn916 fusion for P. popilliae gene insertion would provide that tool and significantly impact scientists ability to study and understand the pathogenic function of this bacterium. The presented data are providing a better understanding of milky disease bacteria and will help in future scientific endeavors at producing an effective biocontrol for infestations of scarab beetle larvae.
Publications
- Dingman, D. W. 2009. Sixteen sequence submissions. Accession numbers; GU295420-GU295435. GenBank.
- Dingman, D. W. 2009. Nine sequence submissions. Accession numbers; GU299523-GU299531. GenBank.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Paenibacillus popilliae, and the closely related Paenibacillus lentimorbus, cause milky disease in larvae of certain scarab beetles. Use of P. popilliae and P. lentimorbus as biological control agents would help lower use of traditional pesticides and provide an alternate method for managing these pests. Scarab beetle larvae and adults cause extensive horticultural and agricultural damage and traditional pesticides are the only effective means for management. Investigations are being conducted to provide a broad-based understanding of the molecular biological, physiological, and environmental adaptation properties of milky disease bacteria. Information gained will help future research by providing a foundational understanding of these bacteria. Polymerase chain reaction (PCR) amplification of P. popilliae genomic DNA and sequencing of the amplified DNA has identified five new genes (a serine/threonine protein phosphatase, a spore-germination protein, a transposase, a collagenase, and a erythromycin esterase). The collagenase is being explored further as a potential virulence factor. The finding of an erythromycin esterase is unusual in that P. popilliae is not known to be resistant to erythromycin. The transposase has been grouped into the IS21 family of transposons and an insertion site and sequence has been found. The start codon for the transposase was identified. Chromosome walking via PCR amplification of flanking sequences has extended the sequences of two previously identified genes. The DNA sequence containing the heat-shock protein has been extended 487 bp and has resulted in identification of the protein start codon. The DNA sequence containing the virB11-like gene (a type-IV secretory protein) has been extended 529 bp. No new genes or gene features have been identified in this extension. These five newly isolated genes will be mapped onto the crude physical map of P. popilliae in conjunction with other mapped genes. Gene sequence information will be disseminated via scientific publication in the DNA database, GenBank. Dr. Dingman, in association with Dr. Richard Cowles (The Connecticut Agricultural Experiment Station) and Dr. Jerry Silbert (The Watershed Partnership, Inc.), is initializing experiments to use regionalized isolates of milky disease bacteria for testing a correlation between environmental adaptation and virulence. In vivo spore production experiments are in progress. High school students and teachers were mentored through presentations of research findings in informal discussions. In addition, telephone conversations with Connecticut citizens were conducted to explain molecular biology principles and current practices for pesticide treatment of scarab beetle infestations. PARTICIPANTS: Dr. Douglas W. Dingman (primary investigator) has performed all experiments and analyses associated with this project. Dr. Richard Cowles has collaborated on endospore production efforts for efficacy tests and has provided scarab beetle larvae for injection. Mrs. Cindy Musante provided technical support and assisted in setup of equipment/supplies associated with experimental design. TARGET AUDIENCES: Scientists studying bacterial genomics will find these results useful. These data provide information to advance scientific investigations of bacterial genetic organization and pathogenicity via comparative analysis of genomic content. This scientific advancement is on fundamental and applied frontiers. Scientists, to alter these bacteria and provide a product that is effective for use as a control agent, can use the knowledge gained. Secondary target audiences include landscape managers, lawn care specialists, and homeowners who, through results from applied investigations, will possibly gain a biocontrol agent effective against scarab beetle larvae. This can lower the use of traditional pesticides on these insect pests and lower environmental accumulation. The Watershed Partnership (involved with lowering pollutant runoff into Long Island Sound) have received regular updates on milky disease efficacy testing experiments. High school and grade school students and teachers gained exposure to scientific principles via mentoring and research presentations on several occasions. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This investigation has identified new genes in P. popilliae and has provided a better understanding on the molecular properties of the genome. The randomly isolated genes from P. popilliae are being used to produce a physical map of the genome. This map will significantly help in the assembly of a complete genomic sequence of this bacterium. Complete genomic sequences are the foundation for understanding molecular properties and characteristics in a bacterium. This amplification has added a third gene (i.e., collagenase) to the list of genes potentially involved in the pathogenic properties of milky disease organisms. Work to extend DNA sequences and possibly isolate other pathogenicity genes via linkage to pathogenicity islands using the lysM domain-containing gene and virB11-like gene can now continue with the collagenase gene. The presence of a naturally occurring transposon that is part of a large family of transposons adds insight into the genomic commonality and evolutionary properties of this bacterium. This transposase may present another tool for molecular manipulations of P. popilliae. The collected data are providing a better understanding of how milky disease cause disease and will help in future molecular manipulations to produce an effective biocontrol. The DNA sequence data obtained for milky disease bacteria will be submitted to GenBank.
Publications
- Dingman, D. W. 2008. DNA fingerprinting of Paenibacillus popilliae and Paenibacillus lentimorbus using PCR-amplified 16S-23S rDNA intergenic transcribed spacer (ITS) regions. J. Invertebr. Pathol. doi:10.1016/j.jip.2008.09.006
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Paenibacillus popilliae and Paenibacillus lentimorbus are very closely related bacteria that naturally occur in the soil and cause disease (i.e., milky disease) in larvae of certain scarab beetles. Scarab beetle larvae and adult beetles cause extensive horticultural and agricultural damage costing hundreds of millions of dollars annually. Use of traditional pesticides is currently the only effective means for managing these insect pests. However, public concerns regarding health and environmental impacts associated with pesticide use have necessitated exploration of alternate methods for managing these important pests. Use of P. popilliae and P. lentimorbus, as biological controls, fit this niche. In order to establish these bacteria as control agents, a broad-based understanding (e.g., characterization of molecular biological, physiological, and environmental adaptation properties) is needed. Therefore, investigations were conducted to provide information that will be
helpful for future molecular investigations or to gain an understanding of bacterial/environmental interactions. Construction of an insertional mutant library of P. popilliae using random insertion of the transposon Tn916 was started. Although initial isolates failed to demonstrate randomness of insertion via Southern hybridization testing, I am continuing insertional mutagenesis using a different conjugative donor. The new donor presents a better possibility for randomization of insertions. An unfinished library of approximately 2000 isolates is being tested via Southern hybridization for randomness of Tn916 insertion. Polymerase chain reaction (PCR) amplification of genomic DNA has identified 2 new genes (a lysM domain-containing protein and a heat-shock ATPase) from P. popilliae. Inverse PCR amplification of isolates obtained from the unfinished Tn916 insertional mutant library has identified 4 new genes (a ribosome associated GTPase, a hypothetical cytosolic protein, a haloacid
dehalogenase-superfamily hydrolase, and dimethyladenosine transferase). These 6 newly isolated genes are currently being mapped onto the crude physical map of P. popilliae in conjunction with 13 currently mapped genes. When physical mapping of the genome is completed, this information will be disseminated via scientific publication in a peer-reviewed scientific journal. Dr. Dingman, in association with Dr. Steven Alm (University of Rhode Island), mentored Ms. Andrea Bixby (graduate student, University of Rhode Island) on research involving molecular identification and the use of natural milky disease isolates to control scarab beetle infestations. High school students were mentored through participation on the Science Research Review Panel for the CT High Scholl Innovation Expo and grade school students were instructed via participation in the CPTV science exposition.
PARTICIPANTS: The primary investigator on this project, Dr. Douglas W. Dingman, performed all experiments associated with this project. Dr. Neil Schultz assisted with Southern hybridization experiments to test for randomness of Tn916 insertion into the P. popilliae genome. Mrs. Cindy Musante provided technical support and assisted in setup of equipment/supplies associated with experimental design.
TARGET AUDIENCES: These findings target other scientists working on milky disease bacteria for fundamental or applied purposes. These data have provided information that scientists can use to advance investigations of milky disease bacteria on fundamental and applied frontiers. This knowledge will be used to direct changes on how these bacteria are manipulated or utilized by scientists to provide a product that is effective for use as a control agent. The effectiveness of commercially available milky spore powder using the current stock strain was discussed with Richard Vento (Director Laboratory Operations) and Dr. F. Obenchain (consultant), representatives of Reuter-St. Gabriel Laboratories. Dr. T. Michaildes (Univ. California) was contacted involving a growing problem with misidentification of P. lentimorbus in environmental samples. Secondary targets include landscape managers, lawn care specialists, and homeowners who, through results from applied investigations, will benefit
from the use of these biocontrols against scarab beetle larvae. The long-term benefits of improved biocontrol applications will minimize the use of traditional pesticides against these insect pests. High school and grade school students were mentored on several occasions.
Impacts
This investigation about the P. popilliae genome has provided necessary information with which to advance research into disease-causing properties of this microorganism. It has resulted in the random isolation of genes from P. popilliae, using non-specific PCR, for production of a genomic map. This random amplification precedes use of degenerate PCR to amplify and isolate genes associated with pathogenicity that may be present in milky disease organisms. The lysM domain-containing gene is potentially associated with pathogenicity via receptor recognition properties. Isolation of this gene, and use of the previously identified virB11-like gene, is directing work to extend the DNA sequences and possibly isolate other pathogenicity genes via linkage to pathogenicity islands. All collected data have built upon current knowledge of these microorganisms. This is helping us better understand how to use and manipulate milky disease for biocontrol. The molecular biological data
obtained from this research have increased the understanding of environmental distribution and microbial evolution of milky disease organisms. This will help prevent, or bypass, problems associated with development and use of milky disease bacteria for biocontrol. The finding that milky disease bacteria are geographically distributed based on phylogeny is being used to test a new approach for increasing the efficiency of regionalized isolates to cause disease. DNA sequence data for 20 16S rDNA accessions of milky disease bacterial were submitted to GenBank.
Publications
- Dingman, D. W. 2007. Geographical distribution of milky disease bacteria in the eastern United States based on phylogeny. J. Invertebr. Pathol. doi:10.1016/j.jip.207.09.002
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