SummaryIn Escherichia coli, the main player in transcription regulation of fatty acid metabolism is the FadR protein, which is involved in negative regulation of fatty acid degradation and in positive and negative regulation of the cellular processes related to it, as well as in positive regulation of the biosynthesis of unsaturated fatty acids in a concerted manner with negative regulation of FabR. On the other hand, Bacillus subtilis possesses two global transcriptional regulators, FadR (YsiA) and FapR. B. subtilis FadR represses fatty acid degradation, whereas FapR represses almost all the processes in the biosynthesis of saturated fatty acids and phospholipids. Furthermore, Streptococcus pneumoniae FabT represses the genes of fatty acid biosynthesis that are clustered in its genome. Long-chain acyl-CoAs appear to be metabolic signals for fatty acid degradation by bacteria in general, and antagonize the FadR protein from either E. coli or B. subtilis. However, malonyl-CoA is a metabolic signal for fatty acid and phospholipid biosynthesis by Gram-positive low-GC bacteria, and it antagonizes FapR. These would be the primary aspects for understanding the elaborate and complex regulation of fatty acid metabolism in bacteria to maintain membrane lipid homeostasis.

The organization and function of the Bacillus subtilis YsiA regulon involved in fatty acid degradation were investigated. Northern and primer extension analyses indicated that this regulon comprises five operons, i.e. lcfA-ysiA-B-etfB-A, ykuF-G, yhfL, yusM-L-K-J, and ywjF-acdA-rpoE. YusJ and AcdA, YsiB and YusL, and YusK presumably encode acyl-CoA dehydrogenases, 3-hydroxyl-CoA dehydrogenase/enoyl-CoA hydratase complexes, and acetyl-CoA C-acyltransferase, respectively, which are directly involved in the fatty acid beta-oxidation cycle. In addition, LcfA and YhfL are likely to encode long chain acyl-CoA ligases. On gel retardation and footprinting analyses involving the purified YsiA protein, we identified cis-sequences for YsiA binding (YsiA boxes) in the promoter regions upstream of ysiA, ykuF, yusL, yhfL, and ywjF, the equilibrium dissociation constants (K(d)) for YsiA binding being 20, 21, 37, 43, and 65 nm, respectively. YsiA binding was specifically inhibited by long chain acyl-CoAs with 14-20 carbon atoms, acyl-CoAs with 18 carbon atoms being more effective; out of long chain acyl-CoAs tested, monounsaturated oleoyl-CoA, and branched chain 12-metyltetradecanoyl-CoA were most effective. These in vitro findings were supported by the in vivo observation that the knock-out of acyl-CoA dehydrogenation through yusJ, etfA, or etfB disruption resulted in YsiA inactivation, probably because of the accumulation of long chain acyl-CoAs in the cells. Furthermore, the disruption of yusL, yusK, yusJ, etfA, etfB, or ykuG affected the utilization of palmitic acid, a representative long chain fatty acid. Based on this work, ysiA, ysiB, ykuF, ykuG, yhfL, yusM, yusL, yusK, yusJ, and ywjF can be renamed fadR, fadB, fadH, fadG, lcfB, fadM, fadN, fadA, fadE, and fadF.

Both sevoflurane and isoflurane induced apoptosis in peripheral lymphocytes in dose-dependent and time-dependent manners in vitro.

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Bacillus subtilis LmrA is known to be a repressor that regulates the lmrAB and yxaGH operons; lmrB and yxaG encode a multidrug resistance pump and quercetin 2,3-dioxygenase, respectively. DNase I footprinting analysis revealed that LmrA and YxaF, which are paralogous to each other, bind specifically to almost the same cis sequences, LmrA/YxaF boxes, located in the promoter regions of the lmrAB operon, the yxaF gene, and the yxaGH operon for their repression and containing a consensus sequence of AWTATAtagaNYGgTCTA, where W, Y, and N stand for A or T, C or T, and any base, respectively (three-out-of-four match [in lowercase type]). Gel retardation analysis indicated that out of the eight flavonoids tested, quercetin, fisetin, and catechin are most inhibitory for LmrA to DNA binding, whereas quercetin, fisetin, tamarixetin, and galangin are most inhibitory for YxaF. Also, YxaF bound most tightly to the tandem LmrA/YxaF boxes in the yxaGH promoter region. The lacZ fusion experiments essentially supported the above-mentioned in vitro results, except that galangin did not activate the lmrAB and yxaGH promoters, probably due to its poor incorporation into cells. Thus, the LmrA/YxaF regulon presumably comprising the lmrAB operon, the yxaF gene, and the yxaGH operon is induced in response to certain flavonoids. The in vivo experiments to examine the regulation of the synthesis of the reporter β-galactosidase and quercetin 2,3-dioxgenase as well as that of multidrug resistance suggested that LmrA represses the lmrAB and yxaGH operons but that YxaF represses yxaGH more preferentially.