Isoprene is a volatile hydrocarbon of unknown function, produced by certain bacteria, plants and animals, sometimes in huge amounts—the Earth’s forests are estimated to emit >500 x 106 tons of isoprene per year. With funding from this program we explored the biochemistry and regulation of isoprene formation in the model bacterial system, Bacillus subtilis, with the goals of explaining the biological rationale for isoprene biogenesis and constructing an isoprene-overproducing microbial system. Although the role for isoprene formation in B. subtilis is still uncertain, our current model for regulation of this hydrocarbon’s synthesis is that isoprene production in B. subtilis is controlled by a combination of i) rapid regulation of isoprene synthase activity and ii) supply of the substrate for isoprene synthase, dimethyallyl diphosphate (DMAPP). This model parallels our current thinking about the control of isoprene formation in plant chloroplasts. In this reporting period we have been working to test part ii) of this model; this work has produced new results using genetic and analytical approaches. For examples, we have developed an analytical method to resolve DMAPP and its isomer, isopentenyl diphosphate, from each other in bacteria and plants. We have also shown that the IPP isomerase (type 2) of B. …