Bacillus sphaericus 2362 is highly pathogenic to the larvae of mosquitos. The potential of this bacterium as a biological control agent could be improved by the availability of a stable cloning vector. Such a vector would allow the incorporation of traits into B. sphaericus which could benefit its large scale production or larvicidal characteristics. To evaluate the use of the Staphylococcus aureus plasmid pUB110 (4.5 kb) for this purpose, the stability (ability of the plasmid to remain intact and divide into daughter cells during bacterial cell division) of this plasmid and four plasmids derived from pUB110 was examined. The recombinant plasmids were developed in the laboratory of Dr. William Burke at Arizona State University.
Stability was examined under various conditions of cell growth. Throughout spore germination, vegetative growth, and sporulation (spore recycling) in host mosquito larvae cadavers, pUB110 and two of the derivatives, pLDT103 (7.6 kb) and pTST130 (6.5 kb), were found to be stable (100%, 99%, and 96% respectively). Plasmids pLDT117 (9.3 kb) and pTST112 (6.5 kb) were lost from B. sphaericus in 23% (pLDT117) and 35% (pTST112) of the colonies from resulting spores examined. During the construction of these unstable plasmids, a region of DNA which binds the host cell membrane in B. subtilis was interrupted.
Stability was also measured after 40 generations of vegetative growth in a chemostat. Each plasmid-bearing strain was grown at both 28°C and 38°C, with generations times of 42 min and 108 min. All plasmids were functionally stable under the conditions of extended vegetative growth that were successfully tested. The strain which carried pTST130 was unable maintain a steady growth rate in the chemostat with a generation time of 42 min. Therefore, reliable stability determinations could not be made at the higher growth rate for this strain.
As through spore recycling in mosquito larvae, strains harboring pUB110, pLDT103, and pTST130 were stable throughout the growth cycle in a shaken flask. Plasmid pLDT117 was found to be stable during exponential and early stationary phase. However, 10-15% of the colonies grown from resultant spores lost neomycin resistance (conferred by the plasmid). The strain carrying pTST112 was unstable throughout the growth curve. During exponential and early stationary phases, the plasmid was present in about 85% of the cells tested. The stability reported for spores of B. sphaericus 2362 [pTST112] was 78%. / Master of Science
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/41321 |
| Date | 02 March 2010 |
| Creators | Seyler, Richard W. |
| Contributors | Microbiology and Immunology |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | viii, 82 leaves, BTD, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | OCLC# 25119410, LD5655.V855_1991.S494.pdf |
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