Ornston 9603980 We shall expand knowledge of an Acinetobacter chromosomal segment that allows growth of the bacteria with many different chemicals from the environment. Presently sequenced and open to genetic investigation is the pca-qui-pob cluster containing genes associated with catabolism of quinate (qui), shikimate (qui) and 4-hydroxybenzoate (pob) through protocatechuate (pca). Downstream from pobA are genes provisionally designated caf and required for catabolism of chemically analogous plant products (caffeate, coumarate and ferulate) through metabolic reactions that give rise to protocatechuate. After this DNA has been cloned and sequenced, it will be subjected to genetic analysis by a simple procedure that allows selection of strains in which caf genes are defective. The results will expand knowledge of both the evolutionary source and the biochemical mechanisms of enzymes that catabolize caffeate, coumarate and ferulate. The enzymes are likely to share ancestry with enzymes associated with the (-oxidation of fatty acids. Thus the combination of evolutionary and genetic investigations is designed to elucidate how genes for a classical metabolic sequence have been adapted to novel metabolic challenges. Variations upon the metabolic theme of ( oxidation are represented in catIJF and pcaIJF, nearly identical chromosomal segments that exchange sequence information by gene conversion. The potential contribution of this intracellular horizontal sequence transfer to concerted divergence of catIJF and pcaIJF in divergent Acinetobacter cell lines will be compared with the rates of divergence of caf genes with analogous biochemical functions. Knowledge emerging from the foregoing studies will define further a chromosomal island of catabolic diversity, the pca-qui-pob-caf gene cluster containing genetic information allowing growth of Acinetobacter with a wide range of compounds in the environment. The nutrients support the growth of many but not all representatives of the genus Acinetobacte r. We shall characterize genetic variants of the pca-qui-pob-caf gene cluster from different Acinetobacter cell lines. Particular attention will be directed to strains exhibiting phenotypes indicating that portions of the cluster are absent. We shall determine the extent to which the missing phenotypic properties reflect the absence of DNA required for their maintenance. This evidence will indicate if the known phenotypic variations are the consequence of abrupt genetic discontinuities that may help to distinguish members of different genomospecies within the complex genus Acinetobacter. If genetic variations in the pca-qui-pob-caf cluster are concordant with biological separation of Acinetobacter genomospecies by other criteria, it may be possible to infer how evolution of different cell lines within the genus was influenced by different sets of nutritional opportunities within the environment. This project will expand knowledge of evolutionary mechanisms that led to the extraordinary ability of bacteria to remove chemicals from the environment. Application of such knowledge may improve the biological cleanup of environmental chemicals and also may assist in development of processes for bacterial conversion of natural resources to chemicals with improved value for society.
