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Department of Bioengineering, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0412, USA
Correspondence
Bernhard Ø. Palsson
palsson{at}ucsd.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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arcA, fadR and arcA/fadR) indicated that (i) ArcA is a major transcription factor for the transcriptional regulation of fatty acid metabolism in the absence of oxygen, and (ii) ArcA and FadR cooperatively regulate the fad regulon under anaerobic conditions. To determine the direct interaction between ArcA and the promoters of the fad regulon genes, chromatin immunoprecipitation (ChIP) analysis was performed. ChIP analysis suggested that ArcA directly binds to the promoter regions of the fad regulon genes in vivo. An ArcA-binding motif was identified from known binding sequences and predicted putative binding sites in the promoter regions of the fad regulon genes. These results indicate that ArcA directly represses the expression of fad regulon genes during anaerobic growth.
The authors disclose a possible conflict of interest related to US Patent Application 20040072723.
These authors contributed equally to this study.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Thus, fatty acid metabolism is tightly regulated so as to allow the cells to adapt quickly to environmental changes (Henry & Cronan, 1992). Escherichia coli can utilize fatty acids as a sole carbon and energy source by means of the enzymes encoded by the fad regulon genes (Schulz, 1991; Magnuson et al., 1993; Clark & Cronan, 1996). The proteins encoded by fad regulon genes are essential for the transport, activation and 
-oxidation of fatty acids (DiRusso & Nystrom, 1998). In detail, exogenous fatty acids are transported into the cell by a specific transport process mediated by the outer-membrane-bound fatty acid transport protein FadL (Black, 1991), and activated by the inner-membrane-associated acyl-CoA synthetase FadD (Black et al., 1992), yielding acyl-CoA thioesters. The activated fatty acids are then catabolized via the -oxidation pathway mediated by the acyl-CoA dehydrogenase encoded by fadE (Campbell & Cronan, 2002) and the tetrameric enzyme complex encoded by fadB and fadA (Binstock et al., 1977; Pramanik et al., 1979), resulting in the production of acetyl-CoA. Acetyl-CoA is subsequently used to generate metabolic energy and precursors required for cell maintenance (Clark & Cronan, 1996). The genes of the fad regulon are repressed by FadR in the absence of long-chain fatty acids (LCFAs) (Campbell et al., 2003), which bind FadR, eliminating its activity (DiRusso et al., 1992; Xu et al., 2001). E. coli can also grow on fatty acids under anaerobic conditions provided that a terminal respiratory electron acceptor, such as nitrate, is available in the medium (Campbell et al., 2003). Furthermore, it has been reported that the anaerobic -oxidation pathway is mediated by fadJ, fadI and fadK genes, whose transcriptional regulation is independent of FadR control (Campbell et al., 2003; Morgan-Kiss & Cronan, 2004). Anaerobic fatty acid metabolism is distinct from the aerobic one in that (i) several of the fad enzymes are not required for anaerobic growth on fatty acids, (ii) anaerobic growth on fatty acids is not under FadR control, and (iii) fatty acids that have different chain lengths can be metabolized (Campbell et al., 2003).
As another transcription regulator that controls fatty acid metabolism, ArcA has been shown to strongly (>20-fold) repress the expression of 3-hydroxyacyl-CoA dehydrogenase encoded by the fadB gene, and to weakly repress acyl-CoA dehydrogenase encoded by the fadE gene, under anaerobic conditions. The ArcA protein of E. coli is a global transcription factor that mediates gene expression in response to changing redox conditions (Lynch & Lin, 1996b; Iuchi & Lin, 1988). ArcA is a component of the ArcBA two-component signal transduction system, with the membrane-associated ArcB tripartite sensor kinase comprising a transmitter domain, a receiver domain and a phosphotransfer domain (Ishige et al., 1994; Lin & Iuchi, 1991). A reduction in oxygen concentration induces the autophosphorylation of the transmitter and receiver domains of ArcB. The highly phosphorylated ArcB transfers the phosphate group to ArcA (Iuchi & Lin, 1992; Tsuzuki et al., 1995; Georgellis et al., 1997). Then ArcA-phosphate (ArcA-P) binds to promoter regions of particular genes, where it modulates expression. Once activated, ArcA-P acts primarily as a negative transcriptional regulator of target operons, which predominantly encode enzymes involved in aerobic respiration. In some cases, however, ArcA-P appears to function as a positive regulator of some genes, such as cydAB (encoding the cytochrome d oxidase complex) and pfl (encoding pyruvate formate-lyase) (Govantes et al., 2000; Drapal & Sawers, 1995).
The regulatory details of fatty acid metabolism under anaerobic conditions have not been investigated under uniform culture conditions, and with isogenic strains that contain well-defined arcA deletions. In this study, we used arcA, fadR and arcA/fadR mutants to analyse the roles played by ArcA and FadR on gene expression of the fad regulon in response to oxygen. In addition, we used chromatin immunoprecipitation (ChIP) to provide in vivo evidence of ArcA binding to the promoter regions of fad regulon genes.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). E. coli DH5
was the host for propagating plasmids. All bacterial strains and plasmids used in this study are listed in Table 1. Deletion mutant strains were constructed using 
Red and the FLP-mediated site-specific recombination method (Datsenko & Wanner, 2000). In short, MG1655 cells containing pKD46 were made electrocompetent by growing them at 30 °C in Luria–Bertani (LB) broth containing 100 mg ampicillin l–1. At an OD600 of 0.6, the cells were washed three times with ice-cold 10 % (w/v) glycerol, and resuspended in the residual volume of the final wash. An FRT-flanked kanamycin gene was PCR amplified from pKD13 using primers listed in Table 2. The PCR products were purified with a Qiagen PCR purification kit, and then transformed into MG1655 competent cells by electroporation. After electroporation, the cells were transferred to 1 ml SOC (20 g tryptone l–1, 5 g yeast extract l–1, 0.5 g NaCl l–1, 3.6 g glucose l–1) and incubated in a rotator at 37 °C for 1 h, and then plated onto LB agar containing 50 mg kanamycin l–1. To confirm that the kanamycin gene was inserted into the correct region of the chromosome, PCR verification was carried out using primers within the inserted region and primers flanking the inserted region (Datsenko & Wanner, 2000). Mutants were cultured non-selectively at 37 °C, and then ampicillin sensitivity was used to test for loss of pKD46. To remove the kanamycin insert, the cells were transformed with the plasmid pCP20 which contains the FLP recombinase, which is inducible above 43 °C. Transformants were selected on ampicillin (100 mg ml–1) plates at 30 °C. Colonies were cultured non-selectively at 43 °C, and then tested for loss of both ampicillin and kanamycin resistance. All mutants were verified by PCR analyses using primers flanking the deleted region.
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). In short, the linear DNA fragments carrying extensions homologous to the last portion of the target gene, and to a downstream region from it, were amplified from the template plasmid (pBOP508) using PCR. The linear DNA fragment was transformed into the E. coli MG1655 carrying pKD46 by electroporation. After treatment of pCP20, the mutant was verified by PCR.
Media and growth conditions.
LB broth containing 10 g tryptone l–1, 5 g NaCl l–1 and 1 g yeast extract l–1 was used to generate the deletion mutants and the BOP308 strain harbouring 8myc-tagged ArcA. Antibiotics were used at the following concentrations (mg l–1): ampicillin, 100; kanamycin, 50; chloramphenicol, 25. Minimal medium was M9 medium supplemented with 0.2 % (w/v) glucose or 0.5 % (w/v) tryptone as sole carbon source (Pauli et al., 1974; Covert et al., 2004). In the case of anaerobic cultures, after the medium was flushed with nitrogen/carbon dioxide (9 : 1) mixture gas for 30 min to assure anaerobic conditions, the strains were grown at 37 °C with continuous sparging with the gas mixture, and agitation in the minimal medium.
Total RNA isolation and gene expression analysis.
Samples for RNA transcript analyses were taken from exponentially growing cells. Total RNA and cDNA preparation was performed as described previously (Covert et al., 2004). Each quantitative PCR (qPCR) reaction contained 0.5 µM of each forward and reverse primer (qPCR primer set in Table 2
), 150 ng cDNA, and 25 µl 2x SYBR Master Mix (Qiagen). All qPCR reactions were done in triplicate on a Bio-Rad Lightcycler, with the following profile: 1 cycle at 95 °C for 15 min, 45 cycles at 94 °C for 20 s, 54 °C for 20 s, and 72 °C for 30 s. A standard curve was calculated for each primer, and a measure of PCR efficiency was obtained from it. Using the standard curve, the relative cDNA quantity was obtained for each gene by normalizing it to the quantity of acpP cDNA in the same sample.
ChIP analysis.
Cells carrying ArcA-8myc fusion (BOP308) were grown in M9 minimal medium containing 2 g glucose l–1 at 37 °C (Cho et al., 2006; Covert et al., 2004). When the aerobic and anaerobic cultures reached an OD600 of 
0.6 and 0.25, respectively, the cells were cross-linked by 1 % formaldehyde at room temperature for 30 min. Following quenching of the unused formaldehyde with 125 mM glycine at room temperature for 5 min, the cross-linked cells were harvested by centrifugation, and washed three times with 50 ml ice-cold Tris-buffered saline. The washed cells were resuspended in 0.5 ml lysis buffer composed of 50 mM Tris/HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 µg RNaseA ml–1, protease inhibitor cocktail (Sigma), and 1 kU Ready-Lyse lysozyme (Epicentre). The cells were incubated at room temperature for 30 min, and then treated with 0.5 ml 2x immunoprecipitation buffer composed of 100 mM Tris/HCl (pH 7.5), 200 mM NaCl, 1 mM EDTA, 2 % (v/v) Triton X-100, and protease inhibitor cocktail. The lysate was then sonicated four times (20 s each time) in an ice bath to fragment the chromatin complexes. Cell debris was removed by centrifugation at 15 800 g at 4 °C for 10 min, and the resulting supernatant was used as the cell extract for the immunoprecipitation. To remove non-specific DNA binding to the Dynabeads M-280 Streptavidin magnetic beads (Dynal Biotech), the washed magnetic beads were added into the sonicated cell extract. After 5 h incubation at 4 °C, the supernatant was recovered, and 10 µg biotin-conjugated anti-c-myc antibody (Santa Cruz Biotechnology) was added. The supernatant was then incubated at 4 °C overnight, and the washed magnetic beads were added. After overnight incubation at 4 °C, the beads were washed twice with the lysis buffer [50 mM Tris/HCl (pH 7.5), 140 mM NaCl, 1 mM EDTA, 1 % (v/v) Triton X-100, and protease inhibitor cocktail], once with the wash buffer I [50 mM Tris/HCl (pH 7.5), 500 mM NaCl, 1 % (v/v) Triton X-100, and 1 mM EDTA], once with wash buffer II [10 mM Tris/HCl buffer (pH 8.0), 250 mM LiCl, 1 % (v/v) Triton X-100, and 1 mM EDTA], and once with TE buffer [10 mM Tris/HCl (pH 8.0), 1 mM EDTA], in order. After removing the TE buffer, the immunoprecipitated (IP) DNA–protein complexes were eluted from the beads by 50 µl elution buffer [50 mM Tris/HCl (pH 8.0), 10 mM EDTA, and 1 % SDS], and incubated at 60 °C for 20 min. The enriched DNA–protein complexes were mixed with 120 µl TE buffer containing 1 % SDS, and then reverse cross-linked at 65 °C overnight. After reversal of the cross-links, proteins in the DNA sample were removed by incubation with 120 µl proteinase K solution [10 mM Tris/HCl (pH 8.0), 1 mM EDTA, 2 % (w/v) glycogen, and 100 µg proteinase K ml–1) for 2 h at 37 °C. The sample was then purified with a PCR purification kit (Qiagen). To monitor the enrichment of promoter regions, 1 µl IP DNA and a ChIP-qPCR primer set (see Table 2) were used to carry out gene-specific qPCR. qPCR was performed in triplicate on a Bio-Rad Lightcycler with the following profile: 1 cycle at 95 °C for 15 min, 45 cycles at 94 °C for 20 s, 50 °C for 20 s, and 72 °C for 40 s.
Purification and autophosphorylation of 6x His-tagged ArcA.
Purification of 6x His-tagged ArcA was performed as described by Yamamoto et al. (2005). E. coli BL21(DE3) harbouring pKH55-21 was grown in LB broth supplemented with 100 mg ampicillin l–1, at 30 °C. After induction with 1 mM IPTG at OD600 0.8, the cells were harvested after 4 h, washed, and resuspended in lysis buffer [10 mM Tris/HCl (pH 7.5), 1 mM EDTA, 100 mM NaCl and 1 mM PMSF]. The cells were then lysed using lysozyme and sonication, and 6x His-tagged ArcA was purified by the Ni-NTA method. The recovery and purity of the proteins were checked by SDS-PAGE. The purified protein fractions were pooled, and dialysed against storage buffer [50 mM Tris/HCl (pH 7.5), 100 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, 2 mM DTT and 20 % (w/v) glycerol] at 4 °C. His-tagged ArcA was then phosphorylated by a standard phosphorylation reaction in which 6x His-tagged ArcA (60 µg ml–1) was incubated for 1 h at 30 °C in a buffer containing 100 mM Tris/HCl (pH 7.5), 10 mM MgCl2, 125 mM KCl and 50 mM disodium carbamoyl phosphate (Sigma).
Electrophoretic mobility shift assay (EMSA).
For the binding studies with ArcA-P, each promoter region of the fad regulon genes was amplified by PCR, using the genomic DNA as a template, and a biotin-labelled ChIP-qPCR primer set (see Table 2). The PCR products (1 ng) were then purified, and incubated with the ArcA-P for 30 min at 30 °C in binding buffer containing 100 mM Tris/HCl (pH 7.5), 100 mM KCl, 10 mM MgCl2, 2 mM DTT and 10 % (w/v) glycerol. The DNA–protein complexes were analysed by electrophoresis on a 6 % DNA retardation gel (Invitrogen). The DNA was transferred to nylon Hybond-N+ membrane (Amersham Biosciences), and visualized by using a Lightshift chemiluminescent EMSA kit (Pierce).
Western blot analysis.
Each sample was subjected to electrophoresis in a 10 % polyacrylamide gel (Invitrogen), and the resolved proteins were electrotransferred to a Hybond-ECL membrane (Amersham Biosciences). The ECL Western detection kit, mouse monoclonal 9E10 antibody (Santa Cruz Biotechnology) and horseradish-peroxidase-conjugated sheep anti-mouse immunoglobulin G (Amersham Biosciences) were used to detect the tandem-myc-tagged proteins. The BCA protein assay kit (Pierce) was used to measure the amount of protein in the isolated protein solution.
Computational methods.
The ArcA-P position weight matrix (PWM), developed from 39 sequences of 13 ArcA-P controlled operons (see Table 3), was used to score the promoter regions of the fad regulon genes (Liu & De Wulf, 2004). The matrix-screening method (www.BindGene.org) predicts the affinity of ArcA-P for any 15 bp DNA sequence in 300 bp upstream region of each fad regulon gene.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Busch & Saier, 2002; Clark & Cronan, 1996; DiRusso et al., 1992; Cronan & Subrahmanyam, 1998; Higashitani et al., 1993). The transcription of fadL is induced by Crp in response to cAMP levels, and repressed by FadR in the absence of exogenous fatty acids. Furthermore, the transport of LCFAs is abolished during high external osmotic pressure, due to repression by OmpR (Higashitani et al., 1993). In addition to FadL, the fatty acid transport system requires the inner-membrane-associated FadD, whose transcription is repressed by FadR in the absence of fatty acid (Cronan & Subrahmanyam, 1998). Under anaerobic conditions, repression by an oxygen-sensing transcription factor(s) would be plausible, given that E. coli is unable to utilize fatty acid as a carbon source without a final electron acceptor (Campbell et al., 2003). None of the transcription factors involved in fatty acid regulation (Crp, OmpR and FadR) is oxygen sensing, though, recently, the ArcBA two-component signal transduction system, which senses oxygen indirectly through a change in redox potential, has been implicated in the negative regulation of aerobic fatty acid metabolism (Georgellis et al., 2001; Clark & Cronan, 1996; Malpica et al., 2004). This hypothesis is also supported by gene expression levels of fadL and fadD found in previous microarray studies (Covert et al., 2004).
To confirm and quantify more accurately the transcript levels of fadL and fadD, qPCR was used to determine the threshold cycle (CT) for each gene. The CT was normalized to expression of the acyl carrier protein (acpP), which is unaffected by the diauxic shift in the wild type and arcA mutants (Covert et al., 2004). Expression of the fad regulon genes is strongly repressed by glucose via Crp, in both the presence and absence of oleate. However, this catalytic repression can be reduced by using growth medium containing tryptone, allowing the analysis of a fadR deletion on gene expression of the fad regulon (Pauli et al., 1974). When wild-type cells were cultured under anaerobic conditions, and in medium containing tryptone as carbon source, expression of fadL and fadD was reduced to 1 and 7 % of the expression under aerobic conditions, respectively (Fig. 1a, b). However, under anaerobic conditions, the level of expression significantly increased in the arcA, fadR and arcA/fadR mutants: the expression levels of fadL and fadD were increased 34- and 69-fold in the arcA mutant, and 69- and 84-fold in the arcA/fadR double mutant, respectively. In contrast, under aerobic conditions, no significant change in gene expression was observed in the arcA mutant, but expression levels of fadL and fadD were elevated four- and threefold in the fadR mutant, respectively, and both genes had a fivefold increase in the arcA/fadR double mutant. Pauli et al. (1974) and Sallus et al. (1983) reported that fadD and fadL expression increased three- and threefold in the fadR mutant, respectively, under aerobic conditions. These results suggest that ArcA, either directly or indirectly, reduces fatty acid transport in the absence of oxygen through the repression of fadL and fadD expression.
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; Robison et al., 1998; Shen & Gunsalus, 1997), along with the BindGene algorithm (McGuire et al., 1999; Lockwood & Frayling, 2003). Within the promoter region of fadL, a putative ArcA-binding site was located at the –31 position (GTTGCAAGTATGTTT), along with two FadR-binding sites located at the –9 (AGCTGGTCCGACCTATA) and +16 (CACTGGTCTGATTTCTA) positions (Fig. 2a). Within the fadD promoter region, two putative ArcA-binding sites were identified at the –16 (GTTAACATAATATTA) and –30 positions (GTTAATATTATGTTA), along with two FadR-binding sites at the –28 (AGCTGGTATGATGAGTT) and –114 (GGCTGGTCCGCTGTTTC) positions (Fig. 2b).
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). After reversal of the formaldehyde-mediated cross-links between the ArcA-8myc fusion and DNA, the IP DNA was used for qPCR. qPCR was employed to measure the differential amounts of DNA fragments of the fadL and fadD promoter regions present in IP DNA of the ArcA-8myc and the wild-type strain. Primers specific to the promoter regions of acpP and rrsA were used as negative controls, since expression of these genes has been shown to be unaffected by oxygen depletion in the wild type and arcA mutants (Covert et al., 2004). In addition, primers specific to promoter regions of mdh and sdhCDAB, both known to be ArcA-binding regions, were used as positive controls (Park et al., 1995; Shen & Gunsalus, 1997). As expected, the enrichment ratio between the IP DNA of the ArcA-8myc and the wild-type strain, of the acpP and rrsA promoter regions, exhibited no significant difference between aerobic and anaerobic conditions (Fig. 2c). In the case of mdh and sdhCDAB promoter regions, the enrichment ratio of anaerobic conditions was 12- and 13-fold higher, respectively, than that of the aerobic conditions (Fig. 2c). Using primers specific to the fadL and fadD promoter regions, qPCR showed enrichment ratios of 11- and 12-fold, respectively, in anaerobic conditions (Fig. 2c). When PCR was performed on the IP DNA of the wild type and the ArcA-8myc strain, a PCR product was seen only in the ArcA-8myc sample (Fig. 2d). Furthermore, we used an EMSA to determine interaction with the fadL and fadD promoter, and this confirmed binding of ArcA to the fadL and fadD promoter region (Fig. 2e). Taken together with the expression data discussed above, the results indicate that ArcA directly represses the gene expression of fadL and fadD by binding to the promoter regions under anaerobic conditions.
Downregulation of the -oxidation pathway by ArcA
The -oxidation pathway of E. coli consists of the enzymes acyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase and 2,4-dienoyl-CoA reductase, encoded by the genes fadE, fadBA and fadH, respectively (Clark & Cronan, 1996). All genes in this pathway are repressed by FadR in the absence of fatty acids (Henry & Cronan, 1992). Under anaerobic conditions, it has been suggested that ArcA represses the -oxidation pathway, but there is little direct evidence regarding this regulation (Lin & Iuchi, 1991; Covert et al., 2004).
To investigate ArcA regulation of the -oxidation pathway, qPCR was used to measure transcript levels of fadE, fadB, fadA and fadH in the wild type, arcA, fadR and arcA/fadR. A CT was calculated for each gene, and normalized to that of acpP, as was done for fadL and fadD. Relative to aerobic conditions, transcript levels of fadE, fadB, fadA and fadH, under anaerobic conditions, were reduced in the wild type to 7, 1, 1 and 10 %, respectively (Fig. 3). Anaerobically, the level of expression significantly increased in arcA, fadR and arcA/fadR strains, indicating their dependence on the availability of oxygen. Aerobically, none of the genes showed a significant difference in transcript levels between the wild type and arcA, which was not surprising given that ArcA has very little activity in the presence of oxygen (Iuchi & Lin, 1991). However, all genes showed increased transcript levels in the fadR and arcA/fadR mutants relative to the wild type (Fig. 3), and this is consistent with the current understanding of FadR regulation of these genes (Henry & Cronan, 1992). These results suggest that ArcA, either directly or indirectly, represses the -oxidation pathway in the absence of oxygen, through the negative regulation of fadE, fadB, fadA and fadH.
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-oxidation pathway in the absence of oxygen; Robison et al., 1998; Shen & Gunsalus, 1997). Within the fadE promoter region (300 bp upstream), a putative ArcA-binding site was located at the –80 position (GTTTACAATATAATT), upstream of a FadR binding site located at the –25 position (AAGTGGTCAGACCTCCT) (Fig. 4a). The fadBA promoter region contained a putative ArcA-binding site located at the –23 position (GTTAAAAATATGCAA), also located upstream of a FadR-binding site (ATCTGGTACGACCAGAT), located at the +19 position (Fig. 4b). The upstream region of fadH contains a putative ArcA-binding site at the –17 position (GTAAAATAATTGTTA), which, unlike the other two sites found, is located downstream of a FadR-binding site (AACTCATCCGACCACAT) at the –31 position (Fig. 4c).
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). When PCR was done on the IP DNA of both the ArcA-8myc and the wild-type strain, a PCR product was seen only in the ArcA-8myc sample (Fig. 4e). Furthermore, we also used an EMSA to determine interaction with the fadE, fadH and fadBA promoters and this confirmed binding of ArcA to the fadE, fadH and fadBA promoter region (Fig. 4f). Taken together with the expression data discussed above, we concluded that ArcA directly downregulates the gene expression of fadE, fadB, fadA and fadH by binding to the promoter regions.
Downregulation of anaerobic -oxidative enzymes by ArcA in the absence of final electron acceptors
The anaerobic -oxidation pathway was recently identified in E. coli (Campbell et al., 2003). This pathway is composed of at least three genes: fadJ, fadI and fadK. These genes are homologues of the fadB, fadA and fadD genes which are required for aerobic fatty acid -oxidation and transport. It is known that the transcription of fadJ and fadI is controlled by FadR in the presence of oxygen, whereas that of fadK is independent of FadR control (Campbell et al., 2003; Morgan-Kiss & Cronan, 2004). The fadJ, fadI and fadK genes are induced by fatty acids under anaerobic conditions, provided that a terminal respiratory electron acceptor, such as nitrate, is available (Campbell et al., 2003). Under fermentation conditions, it would be plausible that additional transcription factor(s) repress the genes for anaerobic fatty acid degradation because (i) E. coli is unable to utilize fatty acids as a carbon source without a final electron acceptor, such as nitrate (Campbell et al., 2003), and (ii) previous microarray studies have suggested that transcription of fadJ and fadI is repressed in the absence of oxygen, and that ArcA may be involved (Covert et al., 2004; Iuchi & Lin, 1988). We therefore investigated the role that ArcA may play in the negative regulation of fadJ, fadI and fadK under anaerobic conditions.
To determine the transcript levels of fadJ, fadI and fadK, qPCR was used to determine a CT number for each gene and normalize it to that of acpP. The transcript levels of fadJ and fadI in the wild type, under anaerobic conditions, were 6 and 15 %, respectively, compared with expression under aerobic conditions (Fig. 5a, b). The level of expression significantly increased in the fadR, arcA and arcA/fadR mutants, under anaerobic conditions. Under aerobic conditions, no significant change in gene expression was observed in the arcA mutant relative to the wild type. However, transcript levels of fadJ and fadI were elevated five- and eightfold in the fadR mutant, and seven- and tenfold in the arcA/fadR mutant, respectively. These results are consistent with previous studies showing that both fadJ and fadI are negatively regulated by FadR (Campbell et al., 2003). Under anaerobic conditions, transcript levels of fadJ and fadI were significantly increased 39- and 67-fold in the arcA mutant, and 39- and 67-fold in the arcA/fadR mutant, respectively. However, the transcript levels of fadK showed no significant change in any of the mutants (data not shown). Thus, it appears that ArcA, either directly or indirectly, negatively regulates the transcription of the anaerobic -oxidation pathway genes fadJ and fadI in the absence of oxygen. Interestingly, under anaerobic conditions, the expression of fadK is independent of FadR and ArcA.
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-oxidative genes in the absence of oxygen; Robison et al., 1998; Shen & Gunsalus, 1997). Within the promoter region (300 bp upstream) of fadJI, a putative ArcA-binding site was located at the –52 position (GTAAAAAAATGGTTA), upstream of a FadR-binding site located at the –27 position (ATCAGGTCAGACCACTT) (Fig. 6a). The qPCR of BOP308 IP DNA, using primers specific to the promoter region of fadJI, indicated that the level of enrichment ratio under anaerobic conditions was approximately 14-fold higher than under aerobic conditions (Fig. 6b). When PCR was performed on the DNA samples, PCR products were obtained from both the ArcA-8myc and the input DNA (i.e. total DNA). When PCR was done on IP DNA of the ArcA-8myc and the wild-type strain, a PCR product was seen only in the ArcA-8myc sample (Fig. 6c). Unlike fadJI, the fadK promoter, under anaerobic conditions, showed no significant enrichment ratio relative to the aerobic conditions (data not shown). As discussed above, the promoter region of the fadJI is close to that of fadL, thus we used an EMSA to determine interaction with the fadJI and fadL promoter, and this confirmed binding of ArcA to the fadJI and fadL promoter region (Fig. 6d). Taken together with the expression data discussed above, it is clear that ArcA directly represses gene expression of fadJ and fadI by binding to their promoter.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-oxidation of LCFAs (C12–C18), with a final electron acceptor, such as oxygen or nitrate. When consecutive cycles of -oxidation have shortened an LCFA to the four-carbon stage, acetoacetyl-CoA induces the ato operon, whose products are required for the final step of converting the four-carbon intermediate to two molecules of acetyl-CoA (Clark & Cronan, 1996; Campbell et al., 2003). It has been shown that FadR acts both as a repressor of the genes of the fatty acid degradation (fad) pathway, and as a transcriptional activator of fatty acid biosynthetic (fab) pathway (Henry & Cronan, 1992). In the absence of oxygen, ArcA is bound to its cognate sites acting to repress transcription of the fad regulon genes, even under non-inducing conditions (without fatty acids). Based on the results presented here, we suggest a transcriptional regulation model of fatty acid metabolism in E. coli under aerobic and anaerobic conditions (Fig. 7). Under aerobic conditions, the absence of LCFAs induces FadR to repress transcription of the fad regulon genes by direct binding to their promoter (Henry & Cronan, 1992). When LCFAs are present in growth medium, the FadR protein is released from the promoter regions, resulting in transcriptional induction of the fad genes. Under aerobic conditions, the ArcBA two-component signal transduction system is inactivated by a high redox potential (
) (Georgellis et al., 2001; Iuchi & Lin, 1991; Malpica et al., 2004), and is unable to interact with promoter regions to regulate the fad genes. Whenever an exogenous electron acceptor is unavailable, the cell curtails its respiration process in favour of fermentation, thereby diverting the carbon source for biosynthesis. During fermentation, E. coli uses two global transcription factors, ArcA and Fnr, to regulate its metabolism. The ArcBA two-component signal transduction system is activated in the absence of oxygen due to a decrease in redox potential. Once active, ArcA directly binds to the promoter regions of fad regulon genes, resulting in transcriptional repression. When no fatty acids are present, FadR remains active under anaerobic conditions, and co-regulates the fad regulon, along with ArcA-P. In the presence of fatty acids, FadR becomes inactive, and is released from promoter regions, whereas ArcA-P continues to repress the transcription of these genes. The model presented here is supported by the following evidence: (i) FadR repression coincides with ArcA repression for the fatty acid metabolism under anaerobic conditions; (ii) ArcA-P is a DNA-binding protein specific to the upstream of fad regulon genes involved in fatty acid metabolism of E. coli; (iii) under anaerobic conditions, transcription of fad regulon genes is increased upon the inactivation of ArcA, whereas, under aerobic conditions, transcription of those genes is not affected by the inactivation of ArcA; (iv) the fadR deletion affects both aerobic and anaerobic transcription of fad regulon genes; and (v) under anaerobic conditions, the transcript levels of fad regulon genes are synergistically increased upon the deletion of both arcA and fadR.
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; Morgan-Kiss & Cronan, 2004). In those studies, it was suggested that ArcA represses fadK transcription under anaerobic conditions. However, the ChIP results, and the transcript level of fadK in the wild type and arcA mutant, indicate that its transcription is independent of ArcA regulation.
In addition to the regulation by FadR and ArcA under aerobic and anaerobic conditions, many fad genes are positively regulated by the Crp regulatory protein in response to changing cyclic AMP levels (Clark & Cronan, 1996; Pauli et al., 1974). Growth on glucose strongly represses the synthesis of fad enzymes, which indicates that the Crp regulatory system exerts its normal positive control of carbon utilization. It is also known that LCFA transport (fadL) is repressed by OmpR in response to the high external osmotic pressure (Clark & Cronan, 1996; Higashitani et al., 1993). Thus, taken together with published literature, the results presented here enhance further our understanding of the regulatory subnetwork controlling fatty acid metabolism (Fig. 7).
In summary, we have investigated the regulation of fatty acid metabolism by oxygen, and found that fatty acid transport and degradation is repressed by ArcA in the absence of oxygen. It is known that compounds that must be first activated to acetyl-CoA require a suitable electron acceptor to be utilized as a sole carbon source. Consequently, it would be natural to hypothesize that enzymes involved in the metabolism of other carbon sources requiring activation to acetyl-CoA are also members of the ArcA regulon.
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REFERENCES |
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) and anaerobic (
) conditions. Error bars indicate SD.
