Transcriptional regulation of the fad regulon genes of Escherichia coli by ArcA

1. Transcriptional regulation of the fad regulon genes of Escherichia coli by ArcA Chao WANG Sept. 13, 2006

2. Background Fatty acids and their derivatives play essential roles in anumber of cellular processes, including cell signalling,transcriptionalcontrol, cell membrane synthesis and protein Modification. Thus, fatty acid metabolism istightly regulated so as to allow the cells to adapt quickly toenvironmental changes. Escherichiacoli can utilize fatty acids as a sole carbon and energysource by means of the enzymes encoded by the fad regulongenes.

3. Exogenousfatty acids are transported into the cell by a specific transportprocess mediated by the outer-membrane-bound fattyacid transport protein FadLand activatedby the inner-membrane-associated acyl-CoA synthetaseFadD, yielding acyl-CoA thioesters. Theactivated fatty acids are then catabolized via the β-oxidationpathwaymediated by the acyl-CoA dehydrogenase encodedby fadEand the tetramericenzyme complex encoded by fadB and fadA, resulting in the production ofacetyl-CoA. Acetyl-CoA is subsequently used to generatemetabolic energy andprecursors required for cell maintenance. The genes of the fadregulon are repressed by FadR in the absence of long-chainfatty acids (LCFAs), which bindFadR, eliminating its activity

4. E. coli can also grow on fatty acids under anaerobicconditions provided that a terminal respiratory electronacceptor, such as nitrate, is available in the medium. Furthermore, it has been reportedthat the anaerobic β-oxidation pathway is mediated byfadJ, fadI and fadK genes,whose transcriptional regulationis independent of FadR control. • Anaerobic fatty acid metabolismis distinct from the aerobic one in: • several ofthe fad enzymes are not required for anaerobic growth onfatty acids • anaerobic growth on fattyacids is not underFadR control • fatty acids that have different chainlengths can be metabolized

5. As another transcription regulator that controls fatty acidmetabolism,ArcA has been shown to strongly (>20-fold)repress the expression of 3-hydroxyacyl-CoA dehydrogenaseencoded by the fadB gene, and to weakly repress acyl-CoAdehydrogenase encoded by the fadE gene, under anaerobicconditions. The regulatory details of fatty acid metabolism underanaerobicconditions have not been investigated underuniform culture conditions, and with isogenic strains thatcontain well-defined arcA deletions. In this study, arcA, fadR and arcA/fadR mutants areused to analyse theroles played by ArcA and FadR on geneexpression of thefad regulon in response to oxygen. In addition,chromatin immunoprecipitation (ChIP) is used to provide in vivoevidence of ArcA binding to the promoter regions of fadregulon genes.

6. Methods Bacterial strains and plasmids. Construction of an E. coli strain harbouring 8myc-taggedArcA. Media and growth conditions. Total RNA isolation and gene expression analysis. ChIP analysis. Purification and autophosphorylation of 66 His-tagged ArcA. Electrophoretic mobility shift assay (EMSA). Western blot analysis. Computational methods. The ArcA-P position weight matrix(PWM), developed from 39 sequences of 13 ArcA-P controlledoperons, was used to score the promoter regions of thefad regulon genes. The matrix-screeningmethodpredicts the affinity of ArcA-P for any15 bp DNA sequence in 300 bp upstream region of each fad regulongene.

7. Results ArcA downregulates the transcription of fattyacid transport genes underanaerobicconditions. Direct in vivo binding of ArcA to the upstreamregions of fadL and fadD inthe absence ofoxygen. Downregulation of the β-oxidation pathway byArcA. Direct in vivo binding of ArcA to the upstreamregions of the genesinvolved in the β-oxidationpathway in the absence of oxygen. Downregulation of anaerobic β-oxidativeenzymes by ArcA in the absenceof finalelectron acceptors. Direct in vivo binding of ArcA to the upstreamregions of anaerobic β-oxidative genes in theabsence of oxygen.

8. Discussion Evidence • FadRrepression coincides with ArcA repression for the fatty acidmetabolism under anaerobic conditions; • ArcA-P is aDNA-binding protein specific to the upstream of fadregulon genes involved in fatty acid metabolism of E. coli; • under anaerobic conditions, transcription of fad regulongenes is increased upon the inactivation of ArcA, whereas,under aerobic conditions, transcription of those genes is notaffected by the inactivation of ArcA; • the fadR deletionaffects both aerobic and anaerobic transcription of fadregulon genes; • under anaerobic conditions, thetranscript levels of fad regulon genes are synergisticallyincreased upon the deletion of both arcA and fadR.

9. A transcriptional regulation model of fattyacidmetabolism in E. coli under aerobic and anaerobic

10. Under aerobic conditions, the absenceof LCFAs induces FadR to repress transcription of the fadregulon genes by direct binding to their promoterWhen LCFAs are present in growth medium,theFadR protein is released from the promoter regions,resulting in transcriptional induction of the fad genes.Under aerobic conditions, the ArcBA twocomponent signaltransduction system is inactivated by a high redox potential. Whenever an exogenouselectron acceptor is unavailable, the cell curtails its respirationprocess in favour of fermentation, thereby diverting thecarbon source for biosynthesis. During fermentation, ArcA and Fnrregulate its metabolism. Once active, ArcAdirectly binds to the promoter regions of fad regulon genes,resulting in transcriptional repression. When no fatty acidsare present, FadR remains active under anaerobicconditions,and co-regulates the fad regulon, along with ArcA-P.In thepresence of fatty acids, FadR becomes inactive, and isreleased from promoter regions, whereas ArcA-P continuesto repress the transcription of these genes.

11. In addition to the regulation by FadR and ArcA under aerobicandanaerobic conditions, many fad genes are positivelyregulated by the Crp regulatory protein in response to changingcyclic AMP levels. Growth on glucose strongly represses the synthesis offad enzymes, which indicates that the Crp regulatory systemexerts its normal positive control of carbon utilization. LCFA transport (fadL) is repressed byOmpRin response to the high external osmotic pressure.

12. Summary Investigated the regulation of fatty acidmetabolismby oxygen, and found that fatty acid transportand degradation is repressed by ArcA in the absence ofoxygen. It is knownthat compounds that must be firstactivated to acetyl-CoArequire a suitable electron acceptorto be utilized as a solecarbon source. Consequently, it wouldbe natural tohypothesize that enzymes involved in themetabolism of othercarbon sources requiring activation toacetyl-CoA are also members of the ArcA regulon.

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