Performing Department
BIOCHEMISTRY & GENETICS
Non Technical Summary
The need for "natural alternatives" to replace synthetic pesticides requires research investigations on biological controls. For maximum exploitation, molecular biological characterizations of these biological control agents are needed. This project seeks to provide a foundational map of the P. popilliae chromsome and to produce a library of chromsomal fragments from which to readily obtain genes of interest. This library will be aligned with the chromsomal map and a system for identifying and isolating genes will be tested.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The larval and adult stages of scarab beetles (order Coleoptera, family Scarabaeidae) cause considerable economic losses to agricultural and horticultural industries. Two decades ago, the annual damage to turfgrass in the U. S. was estimated to be one-quarter of a billion dollars. No estimate has been made on the economic loss these insects have also caused to ornamental plantings, fruit production, and other segments of agriculture. At present, the only effective control for scarab beetles is the use of synthetic insecticides. However, public worries about the health and environmental hazards associated with the use of these chemicals have increased the demand for the development of safer biological alternatives. Many synthetic pesticides are being, or have been, banded or greatly restricted from use without a good biocontrol replacement. Considerable attention is being focused on the development and use of biological control agents as "natural alternatives" to
replace synthetic pesticides. Paenibacillus popilliae, originally isolated and characterized as causing milky disease in larvae of Japanese beetles (Popillia japonica Newman), has been used in the past as a biocontrol and has potential for development into an efficient biocontrol agent. Using light and electron microscopy to physically define the mechanism involved in the initial stages of milky disease, Splittstoesser and associates showed cellular invasion of larval intestinal epithelial cells by P. popilliae (i.e., host cell attachment, phagocytic engulfment by an epithelial cell, and detainment of the bacterium within the phagocytic vesicle) to be the key mechanism whereby this bacterium crosses the intestinal epithelial lining and causes disease. Unfortunately, the molecular properties surrounding this mechanism in P. popilliae are not known. Knowledge about these pathogenic properties will advance our ability to manipulate this bacterium and help with the development of an
effective biological control agent. This knowledge will also have long-term fundamental benefits on the scientific understanding of molecular evolution of enteropathogenic disease mechanisms by extending disease comparison studies into insect pathogenicity. However, before this knowledge can be fully obtained a detailed characterization of the P. popilliae genome is needed. The objective of this investigation is to provide the necessary framework from which to conduct investigations on the mechanism of pathogenicity encoded in P. popilliae. The specific objectives aimed at performing this task are as follows: A) To construct a physical map of the bacterial genome and identify the location of several gene markers on this map. B) To create a Bacterial Artificial Chromosome (BAC) library of the bacterial genome that is coordinated to the physical map of the genome. Several known genes will be isolated from this library as initial studies for isolation of genes associated with
pathogenicity. C) To test the insertional mutagenesis capability of Tn916 prior to development of this transposon into an integrative vector for gene identification and isolation.
Project Methods
Construction of a physical map of the P. popilliae genome will be performed using the restriction enzymes I-CeuI, Pme I, and Pac I. Complete and partial digestion of P. popilliae genomic DNA with I-CeuI followed by PFGE separation and measurement and comparison of fragment sizes between complete and partial digests will be used to construct the physical map of I-CeuI restriction sites on the genome of P. popilliae. Complete and partial digestion of genomic DNA with Pme I and Pac I will also be done to determine fragment order and restriction enzyme site locations on the genome. Coordination of these restriction sites with the I-CeuI physical map of restriction sites will involve using purified I-CeuI fragments and digestion with Pme I and Pac I. The physical map of the P. popilliae genome will show the positions of these three enzymes. The P. popilliae genomic map will be further characterization by placement of the genetic markers vanF, cry18Aa, tdk, and the rrn
operon ITS regions. Hybridization probes for these markers will be constructed by PCR amplification and after digestion of P. popilliae genomic DNA with Pme I plus Pac I and PFGE, the separated DNA bands will be tested by Southern hybridization analysis to identify restriction fragment(s) on which the gene markers are located. Production of a BAC library for P. popilliae will be performed using the BAC vector, pIndigoBAC536. P. popilliae genomic DNA will be partially digested using HindIII and DNA restriction fragments of approximately 100 Kbp will be obtained using PFGE and extraction of DNA from agarose. Following insertion of these genomic fragments into the BAC vector and electroporation into E. coli, approximately 350 clones will be isolated. The clones will be spot inoculated into 96-well microtiter plates, grown for 3 hrs, and then be frozen at -80C in 25% glycerol. To test the library, Southern hybridization analysis will be performed using colony blots of the 350 clones and
hybridization probes for the vanF, cry18Aa, tdk, 16S, 23S, and 5S rRNA genes and for the 3 unique ITS regions. Prior to development of Tn916 into a tool for exploring P. popilliae genomic DNA, the transposon will be tested for suitability as an insertional mutagen. P. popilliae is naturally resistant to vancomycin and disruption of any of the genes responsible for this resistance will render the microbe sensitive to this antibiotic. Tn916-containing P. popilliae strains Tc1001 (resistant to tetracycline; tetr) and Em1001 (resistant to erythromycin; ermr) will be filter-mated and isolates resistant to both tetracycline and erythromycin (i.e., tetr-ermr) will be selected. Both strains are resistant to vancomycin. All tetr-ermr P. popilliae isolates will be tested for sensitivity to vancomycin and sensitive isolates will be screened by PCR amplification of the vanF gene cluster and Southern hybridization analysis for the presence of Tn916 in the vanF gene cluster. Identification of Tn916
in the vanF gene cluster will demonstrate that this transposon is suitable for further development into a molecular tool.