Starving bacteria are important ecologically, in biotechnology, and as models of biological differentiation. Upon starvation, the non- sporeforming bacterium Escherichia coli differentiates into markedly resistant cells by expressing some 50 genes in early starvation. The KatF protein, which is believed to be a novel sigma factor, is required for the synthesis of several of these proteins, since defect in the katF gene substantially prevents starvation protein induction and resistance development. This finding suggests but does not prove the involvement of KatF- regulated starvation proteins in the development of cellular resistant state. Direct evidence will be sought for this role. A katF deletion strain will be constructed. A drug resistance marker will be inserted into the cloned katF gene which will be crossed into the E. coli chromosome, using a recB recC sbcB strain, followed by transfer of the mutated gene into the wild type background. The katF structural gene under the control of a titratable promoter will be introduced into the deletion strain. Different amounts of KatF will be produced during starvation, and correlation to the degree of resistance to starvation, oxidative, heat and osmotic stresses will be determined. Two-dimensional polyacrylamide gel electrophoresis patterns will be determined in selected instances. This may shed light on which protein spots are especially important in the development of starvation and other resistances. Similiar experiments will be done during exponential phase of growth to see if overproduction of KatF can confer resistance during growth as well. There is evidence that KatF synthesis may be regulated at the postranscriptional level. To explore this, transcriptional and tranlational fusions of katF to lacZ will be constructed using the polymerase chain reaction technology. %%% Bacteria in nature exist primarily in a starving state. What biochemical characters are expressed under starvation conditions and how this expression is regulated are therefore important in the understanding of bacterial activities in nature which include besides disease causation, recycling of elements and environmental detoxification. Starving bacteria are also important in microbial based industrial fermentations because their use permits rapid and cheap biotransformations using special bioreactors. Some fifty new genes are expressed by Escherichia coli when this bacterium is starved. A subset of these may be concerned with synthesis of proteins that make the cells more resistant to many different kinds of stresses. How might these proteins make cells more resistant to stresses is an important biological question which can be conveniently explored in this model system. In this proposal, the aim is to clearly determine if a specific subset of starvation proteins is involved in stress resistance. This subset is controlled by a novel genetic element called KatF. By using techniques of molecular biology, the amount of KatF in the cells will be precisely regulated, the concentration of the proteins it regulates will be qunatified, and a determination will be made of whether their concentration correlates with the degree of resistance acquired by the cells. Identification of proteins that determine the cellular resistant state can make it possible to manipulate cellular resistance; and an insight into their biochemical role can provide information about fundamental cellular functions.
