Functions of the Clostridium acetobutylicium FabF and FabZ proteins in unsaturated fatty acid biosynthesis

<p>Abstract</p> <p>Background</p> <p>The original anaerobic unsaturated fatty acid biosynthesis pathway proposed by Goldfine and Bloch was based on in <it>vivo </it>labeling studies in <it>Clostridium butyricum </it>ATCC 6015 (now <it>C. beijerinckii</it>) but to date no dedicated unsaturated fatty acid biosynthetic enzyme has been identified in Clostridia. <it>C. acetobutylicium </it>synthesizes the same species of unsaturated fatty acids as <it>E. coli</it>, but lacks all of the known unsaturated fatty acid synthetic genes identified in <it>E. coli </it>and other bacteria. A possible explanation was that two enzymes of saturated fatty acid synthesis of <it>C. acetobutylicium</it>, FabZ and FabF might also function in the unsaturated arm of the pathway (a FabZ homologue is known to be an unsaturated fatty acid synthetic enzyme in enterococci).</p> <p>Results</p> <p>We report that the FabF homologue located within the fatty acid biosynthetic gene cluster of <it>C. acetobutylicium </it>functions in synthesis of both unsaturated fatty acids and saturated fatty acids. Expression of this protein in <it>E. coli </it>functionally replaced both the FabB and FabF proteins of the host in <it>vivo </it>and replaced <it>E. coli </it>FabB in a defined in <it>vitro </it>fatty acid synthesis system. In contrast the single <it>C. acetobutylicium </it>FabZ homologue, although able to functionally replace <it>E. coli </it>FabZ in <it>vivo </it>and in <it>vitro</it>, was unable to replace FabA, the key dehydratase-isomerase of <it>E. coli </it>unsaturated fatty acid biosynthesis in <it>vivo </it>and lacked isomerase activity in <it>vitro</it>.</p> <p>Conclusion</p> <p>Thus, <it>C. acetobutylicium </it>introduces the double of unsaturated fatty acids by use of a novel and unknown enzyme.</p

Cosmid-Based System for Transient Expression and Absolute Off-to-On Transcriptional Control of Escherichia coli Genes

Cosmids are plasmids that contain the phage λ sequences (cos) required for packaging of the phage DNA into the virion. Induction of a λ prophage in an Escherichia coli strain carrying a cosmid results in lysates containing phage particles that are filled with cosmid DNA. However, the lysates also contain a large excess of infectious phage particles which complicate use of the packaged cosmids. I report that cosmids packaged by induction of a strain carrying a prophage with an altered cos region results in lysates containing very high levels (>10(10)/ml) of particles that contain cosmid DNA together with very few infectious phage particles. These lysates can be used to transduce cosmid DNA into all of the cells of a growing culture with minimal physiological disturbance. When the cosmid carries a conditionally active origin of replication, transductional introduction of the cosmid under nonreplicative conditions provides a system of transient expression. Transient expression has been used to make a recA strain temporarily recombination proficient and to temporarily introduce a site-specific recombinase. Transductional introduction of a cosmid also allows absolute off-to-on transcriptional control of nonessential genes. Two examples are given showing that when a strain carrying a null mutation in the gene of interest is transduced with a packaged cosmid carrying a functional copy of that gene, the expression of the gene rapidly goes from absolutely off to high-level expression. Additional possible uses of in vivo-packaged cosmids are proposed

The Structure of Mammalian Fatty Acid Synthase Turned Back to Front

On page 1667 of this issue, Stuart Smith and colleagues [1] demonstrate that the animal fatty acid synthase is a head-to-head dimer rather than the head-to-tail dimer depicted in textbooks. This has important ramifications for the mechanisms of other multifunctional enzymes such as polyketide synthases [2]