Cannon 9604450 Polyhydroxyalkanoates (PHA) are polyesters that accumulate within inclusion-bodies in many bacteria and archaea as carbon and energy reserves. Despite the fact that the gene for PHA synthase has been cloned and sequenced from more than twenty strains of bacteria, the genetic regulation and the links between PHA accumulation and other metabolic pathways are poorly understood. Most of our present knowledge in this area comes from studies of Pseudomonas and Alcaligenes. Apart from some inclusion-body structural studies, very little of our present knowledge comes from studies of the Bacillus genus. The decision to diversify and pursue this study of PHA accumulation in B. megaterium is likely to reveal important new insights into the regulation of PHA accumulation and its links with other cellular processes. An understanding of the regulation of PHA inclusion-body synthesis and its coordinated regulation with other aspects of cellular metabolism is the subject of this research. The following strategy is being undertaken: (1) It has been shown that PHA accumulates in a growth phase dependent manner, and the sequence and characterization of the phaB/phaC region of B. megaterium has been determined. Three novel genes and two divergent promoters associated with PHA accumulation have been identified. This research seeks to demonstrate the functions of the three novel gene products and to determine what regulates the two divergent promoters (pl and p2). The effects of over-expression (gene dosage) and deletion (knock-out by marker substitution), of each of these three genes, individually and in combinationq with each other, will be tested. Effects of these mutations will be measured in relation to transcription from the pl and p2 promoters, translation of the messages, PHA accumulation, and size and shape of inclusion-bodies throughout growth of the culture. (2) It is hypothesized that spore formation is not occurring during PHA accumulation, based on promoter studies of the genes involved. This suggests that PHA accumulation has a regulatory role in spore formation. This hypothesis will be tested by examining the relationship of transcription from the p2 promoter to spore formation. For example, replacement of p2 with a constitutive promoter would be expected to block sporulation, while a deletion mutation in the p2 promoter, may result in very high sporulation rates. (3) A PHA over-producing transposon mutant of B. megaterium has been isolated and the tagged DNA has been identified as a cluster of gas vesicle genes (gvp). The relevance of gas vesicles to PHA inclusion-bodies in B. megaterium is being examined. This is being done by measuring the effects of deletion or over-expression of gvp genes on PHA accumulation (as in 1, above). The accumulation in bacteria of the biodegradable polymer PHA is the result of the combined effects of pha gene products and prevailing environmental conditions, which are reflected in many cellular processes. The results of these experiments should increase our understanding of the integrated genetic regulation of these processes with PHA accumulation.
