Heme serves Salmonella typhimurium as the cofactor both for cytochromes (required for respiration) and for catalase and superoxide dismutase (involved in defense against oxygen's toxic effects). In Salmonella, the heme pathway also leads to siroheme and to vitamin B12, which is an essential nutrient for humans. Surprisingly, very little is known about the regulation of this branched pathway in enteric bacteria. Since Salmonella is well- suited to genetic analysis, we propose a primarily genetic approach to understanding the control of heme synthesis. This study is also relevant to the larger problem of Salmonella's life in the colon. Most of what we know about enteric bacteria has been observed during growth in air. As a facultative anaerobe, Salmonella respires to electron acceptors when they are present, but can also grow by fermentation in their absence. In nature, electron acceptors may be present only intermittently (in the diet, or as oxygen diffusing from the epithelial surface). The ability for rapid development of respiratory capacity may be very important. We'll attempt to exploit heme regulation as an approach to more general questions about control of gene function by oxygen Our initial studies have focused on the first step of the pathway, synthesis of ALA. We've learned that Salmonella has two routes of ALA synthesis, and we suspect that this duality is important in regulating flow through the pathway to different end products. The major route of ALA synthesis (both aerobically and anaerobically) depends on the hemeA and hemL genes. We'll characterize these genes and their regulation, and study the enzyme, ALA synthase. The second route of ALA synthesis is expressed only anaerobically. It may be related to the C5 route of ALA synthesis used in photosynthetic organisms. We've isolated Mud-lac fusions to most of the genes of the heme pathway, and we'll use these select and characterize mutations regulating expression of heme genes. Having a set of well characterized hem mutants and simple methods for working with them also allow rapid molecular biological characterization of these genes and the heme pathway enzymes.
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