Crosstalk of Escherichia coli FadR with global regulators in expression of fatty acid transport genes.

Escherichia coli FadR plays two regulatory roles in fatty acid metabolism. FadR represses the fatty acid degradation (fad) system and activates the unsaturated fatty acid synthetic pathway. Cross-talk between E. coli FadR and the ArcA-ArcB oxygen-responsive two-component system was observed that resulted in diverse regulation of certain fad regulon β-oxidation genes. We have extended such analyses to the fadL and fadD genes, the protein products of which are required for long chain fatty acid transport and have also studied the role of a third global regulator, the CRP-cAMP complex. The promoters of both the fadL and fadD genes contain two experimentally validated FadR-binding sites plus binding sites for ArcA and CRP-cAMP. Despite the presence of dual binding sites FadR only modestly regulates expression of these genes, indicating that the number of binding sites does not determine regulatory strength. We report complementary in vitro and in vivo studies indicating that the CRP-cAMP complex directly activates expression of fadL and fadD as well as the β-oxidation gene, fadH. The physiological relevance of the fadL and fadD transcription data was validated by direct assays of long chain fatty acid transport.

Introduction

Much of our current knowledge of bacterial fatty acid metabolism comes from studies with Escherichia coli [1]. The fatty acid degradation (fad) pathway is primarily responsible for the transport, activation and β-oxidation of fatty acids [2]. The known long-chain fatty acid (LCFA) transport system components are the outer-membrane transport protein, FadL [3], [4] and the inner-membrane associated acyl-CoA synthetase, FadD [5], [6]. In this system, the FadL transporter delivers exogenous long chain fatty acids across the cell membrane into the periplasmic space [3], [4]. From there by an unknown mechanism the LCFA enter the cytosol where they become activated to their acyl-CoA thioesters by FadD which allows entry of the acyl chains into the β-oxidation cycle [5], [6]. E. coli FadR coordinates the catabolic and anabolic fatty acid pathways (Fig. 1). In this dual role FadR acts as a repressor for the entire set of fad regulon genes and also functions as an activator for unsaturated fatty acid biosynthesis pathway by increasing transcription of both fabA [7], [8] and fabB [9] (Fig. 1). The physiological ligands that antagonize FadR binding to its cognate operators are LCFA CoA thioesters synthesized by the cytosolic acyl-CoA synthetase, FadD [7], [10]. In strains lacking either FadD or FadL addition of LCFA fails to induce fad regulon expression because the regulatory ligand cannot be synthesized. Therefore, extremely stringent regulation of fadD and fadL genes is precluded because this would block derepression.

Figure 1

Regulation of fatty acid metabolism by E. coli FadR.

. In the absence of a long chain acyl-CoA E. coli FadR represses the fad regulon genes [8], [23] whereas it activates transcription of fabA and fabB, the two genes of the unsaturated fatty acid synthetic pathway [7]–[9]. . Binding of long chain acyl-CoA species results in large changes in FadR structure resulting in dissociation of the protein from its operator sites. Dissociation increases fad regulon expression whereas expression of fabA and fabB is decreased. The ovals denote FadR whereas the small open circles represent the acyl-CoA pool.

Similar to most of the other fad regulon genes, fadL [4], [11], [12] and fadD [12], [13] are controlled at the transcriptional level by two different regulatory systems in addition to FadR, the oxygen-sensitive ArcA-ArcB two-component system and the cyclic AMP (cAMP) receptor protein-cyclic AMP (CRP-cAMP) complex. Our interest in fadL and fadD arose from the presence of two FadR-binding sites in the promoters of both genes whereas the other FadR regulon fad genes each have only a single site. This raised the question of regulatory interactions among the multiple regulators, FadR, ArcA and CRP-cAMP when bound to the respective promoter regions. Cho and coworkers [12] initially used quantitative RT-PCR to assay regulation of fadL and fadD by FadR and ArcA under anaerobic conditions and reported that deletion of either arcA, fadR alone, or both arcA and fadR resulted in increased fadL expression by 34-, 4- or 69-fold, respectively, whereas fadD transcription increased 69-, 4.5-, and 84-fold. These workers did not investigate the effects of CRP-cAMP. Classical catabolite repression of the fad pathway has long been known. Pauli and coworkers [14] reported that Fad enzyme levels were very low in wild type cells grown in glucose even in the presence of inducing levels of fatty acid (or in constitutive fadR mutant strains) and that glucose inhibition could be partially relieved by addition of cAMP. CRP mutant strains also had low fad enzyme activities. In this laboratory Clark [15] using early lacZ fusion technology showed that glucose acted at the transcriptional level in fadBA and fadE expression. Although as discussed below CRP-cAMP binding sites for several fad genes have been proposed, none had been directly tested for the ability to bind the complex. Therefore, it remained possible that CRP-cAMP regulation of the fad regulon was an indirect effect.

We report experiments defining the interactions of these regulatory proteins and their effects on fatty acid transport.

Materials and Methods

Bacterial strains and growth conditions

All the strains used here are E. coli K-12 derivatives (Table 1). The bacterial media were used for bacterial growth and analyses of β-galactosidase activity. These included LB medium (Luria-Bertani medium containing 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl per liter), Rich broth (RB medium containing 10 g of tryptone, 1 g of yeast extract, and 5 g of NaCl per liter) and minimal medium M9 [16] supplemented with 0.4% glucose or other carbon source, 0.1% Vitamin-Free Casamino Acids, 0.1 mM CaCl2, and 0.001% thiamine. When necessary, antibiotics were used as follows (in mg/liter): sodium ampicillin, 100; kanamycin sulfate, 50; chloramphenicol, 20 and tetracycline HCl, 15. To monitor transcriptional regulation by ArcA-ArcB, an anaerobic environment (5% H2, 75% N2, and 20% CO2) was generated in an anaerobic environmental system (Bio-Bag environmental chamber type A; Becton Dickinson). The E. coli strains were grown on modified LB media in which potassium nitrate (5 mM) was added as an electron acceptor [17]. Fatty acids such as oleic acid (Sigma) were neutralized with KOH, solubilized with Tergitol NP-40, and used as an inducer at 5 mM final concentration.

Table 1

Strains and plasmids used in this study.

Bacteria or plasmids	Relevant characteristics	References/sources
E. coli strains
Topo10	F−, ΔlacX74, a cloning host for recombinant plasmids	Invitrogen
BL21(DE3)	Protein expression host	Lab collection
MG1655	Wild type of E. coli K-12	CGSCa, Lab collection
MC4100	F−, araD139, Δ(argF-lac)169	[25]
DH5α (λ-pir)	Δlac host for pAH125 and its derivatives	[25], [37]
MC1061	Wild type of E. coli K-12, Δlac	[23]
MFH8	UB1005, fadR::Tn10	[7]
BW25113	A wild type strain of E. coli K-12	CGSCa, [38]
JW2341-1	F− Δ(araD-araB)567 ΔlacZ4787(::rrnB-3) LAM- ΔfadL752:: kan rph-1 Δ (rhaD-rhaB) 568 hsdR514	CGSCa, [38]
JW1794-1	F− Δ(araD-araB)567 ΔlacZ4787(::rrnB-3) LAM- ΔfadD730::kan rph-1 Δ (rhaD-rhaB) 568 hsdR514	CGSCa, [38]
FYJ55	fadH-lacZ transcriptional fusion	[17]
FYJ56	fadH-lacZ transcriptional fusion, ΔfadR::Tn10	[17]
FYJ57	JW1176-1, ΔfadR	[17]
FYJ59	JW4361-1, ΔarcA	[17]
FYJ60-2	JW5702-4, Δcrp	[17]
FYJ65	fadH-lacZ transcription fusion, Δcrp	[17]
FYJ76	FYJ60-2, Δcrp ΔarcA726::kan	[17]
FYJ77	FYJ76, ΔarcA Δcrp	[17]
FYJ78	FYJ77, ΔarcA Δcrp ΔfadR::Tn10	[17]
FYJ82	fadBA-lacZ transcriptional fusion, ΔarcA	[17]
FYJ83	fadBA-lacZ transcriptional fusion, Δcrp	[17]
SI203	fadBA-lacZ transcriptional fusion	[17], [39]
SI207	fadBA-lacZ transcriptional fusion, ΔfadR::Tn10	[17], [39]
FYJ103	JW2341-1 carrying pCP20ts, ΔfadL	This work
FYJ104	FYJ103, fadL-lacZ transcriptional fusion	This work
FYJ105	FYJ104, ΔfadR::Tn10, fadL-lacZ transcription fusion	P1vir(MFH8)×FYJ104b
FYJ118	MC1061, ΔfadL::km	This work
FYJ119	FYJ118 carrying pCP20, ΔfadL	This work
FYJ132	JW1116-1 carrying pCP20ts	This work
FYJ133	FYJ59, ΔarcA, fadL-lacZ transcriptional fusion	P1vir(FYJ104)×FYJ59c
FYJ134	FYJ60-2, Δcrp, fadL-lacZ transcriptional fusion	P1vir(FYJ104)×FYJ60-2c
FYJ139	FYJ59, ΔarcA ΔfadR::Tn10	P1vir(MFH8)×FYJ59b
FYJ140	FYJ60-2, Δcrp ΔfadR::Tn10	P1vir(MFH8)×FYJ60-2b
FYJ141	FYJ139, ΔarcA ΔfadR::Tn10, fadL-lacZ transcriptional fusion	P1vir(FYJ104)×FYJ139c
FYJ142	FYJ140, Δcrp ΔfadR::Tn10, fadL-lacZ transcriptional fusion	P1vir(FYJ104)×FYJ140c
FYJ158	DH5α (λ-pir) carrying pAH-PfadD	[22]
FYJ159	MC4100 with fadD-lacZ transcriptional fusion at the chromosomal attB λ site.	[22]
FYJ161	FYJ57, ΔfadR, fadD-lacZ transcriptional fusion	P1vir(FYJ159)×FYJ57c
FYJ162	FYJ59, ΔarcA, fadD-lacZ transcriptional fusion	P1vir(FYJ159)×FYJ59c
FYJ163	FYJ60-2, Δcrp, fadD-lacZ transcription fusion	P1vir(FYJ159)×FYJ60-2c
FYJ164	FYJ139, ΔarcA ΔfadR::Tn10, fadD-lacZ transcriptional fusion	P1vir(FYJ159)×FYJ139c
FYJ165	FYJ140, Δcrp ΔfadR::Tn10, fadD-lacZ transcriptional fusion	P1vir(FYJ159)×FYJ140c
FYJ166	FYJ77, Δcrp ΔarcA, fadD-lacZ transcription fusion	P1vir(FYJ159)×FYJ77c
FYJ167	FYJ78, Δcrp ΔarcA ΔfadR::Tn10, fadD-lacZ transcription fusion	P1vir(FYJ159)×FYJ78c
FYJ169	FYJ77, Δcrp ΔarcA, fadL-lacZ transcriptional fusion	P1vir(FYJ104)×FYJ77c
FYJ170	FYJ78, Δcrp ΔarcA ΔfadR::Tn10, fadL-lacZ transcriptional fusion	P1vir(FYJ104)×FYJ78c
FYJ187	MC4100 carrying pINT-ts	[22]
FYJ238	Topo 10 carrying pET28-crp	This work
FYJ239	BL21(DE3) carrying pET28-crp	This work
FYJ294	DH5α (λ-pir) carrying pAH-PfadL	This work
FYJ295	MC4100 with fadL-lacZ transcriptional fusion at the chromosomal attB λ site.	This work
Plasmids
pET28(a)	T7 promoter expression KmR vector	Novagen
pKD46	bla PBAD gam pSC101, oriTS, AmpR	[40]
pKD13	bla FRT ahp FRT PS1 PS4 oriR6K, KmR	[40]
pCP20	bla cat cI857 λP R flp oriTS, AmpR, Cm	[19], [41]
pKG137	Transcription fusion plasmid, ahp FRT lacZY + this oriR6K, KmR	[19], [40]
pINT-ts	Temperature sensitive helper plasmid expressing Intλ, AmpR	[37]
pAH125	A promoter-less lacZ reporter plasmid in E. coli, KanR	[25], [37]
pAH-PfadD	pAH125 carrying fadD promoter region, KanR	[22]
pAH-PfadL	pAH125 carrying fadL promoter region, KanR	This work
pET28-fadRec	pET28 carrying wild type fadR	[23], [25]
pET28-crp	pET28 encoding CRP	This work

CGSC denotes Coli Genetic Stock Center, Yale University.

Selection for tetracycline resistance.

Selection for kanamycin resistance.

Plasmids and genetic manipulations

The pCR2.1-TOPO vector (Invitrogen) was used for PCR cloning and sequencing whereas expression vector pET28a (Novagen) was used for protein preparation (Table 2). All plasmids constructed were validated by PCR analyses plus direct DNA sequencing.

Table 2

Primers used in this study.

Primers	Primer sequences (5′–3′)
fadL-PF	CGT TGA TTT CCT CTG TAT GTG C
lacZ-R	GAC CAT GAT TAC GGA TTC ACT G
fadL-promoter-F (SalI)	CCG GTCGAC CGT TGA TTT CCT CTG TAT GTG
fadL-promoter-R (EcoRI)	AACC GAATTC GCG AGA GCA GAC TTT GTA AAC
fadD-promoter-F (SalI)	CCG GTCGAC GTT GCG GTA CAA AAC CAG CA
fadD-promoter-R (EcoRI)	AACC GAATTC CTC TAA AAT GCG TGT TCG TCG
crp-F1 (BamHI)	CG GGATCC ATG GTG CTT GGC AAA CCG CA
crp-R1 (XhoI)	CCG CTCGAG TTA ACG AGT GCC GTA AAC GAC
fadL-FadR-site1-F	GCA ACA TTC CAG CTG GTC CGA CCT ATA CTC TCG CC
fadL-FadR-site1-R	GGC GAG AGT ATA GGT CGG ACC AGC TGG AAT GTT GC
fadL-FadR-site2-F	CTC TCG CCA CTG GTC TGA TTT CTA AGA TGT ACC TC
fadL-FadR-site2-R	GAG GTA CAT CTT AGA AAT CAG ACC AGT GGC GAG AG
fadD-FadR-site1-F	GTA ATT ATC AAG CTG GTA TGA TGA GTT AAT ATT ATG
fadD-FadR-site1-R	CAT AAT ATT AAC TCA TCA TAC CAG CTT GAT AAT TAC
fadD-FadR-site2-F	GAA ACA GCG GCT GGT CCG CTG TTT CTG CAT TCT
fadD-FadR-site2-R	AGA ATG CAG AAA CAG CGG ACC AGC CGC TGT TTC
fadD-CRP-site-F	GTA AAG ATA AAA ATA AAT AGT GAC GCG CTT CGC AAC CTT TTC GTT GGG
fadD-CRP-site-R	CCC AAC GAA AAG GTT GCG AAG CGC GTC ACT ATT TAT TTT TAT CTT TAC
fadL-CRP-site1-F	CTG CAA AAT CGG ATA AGT GAC CGA AAT CAC ACT TAA AAA TGA TCT
fadL-CRP-site1-R	AGA TCA TTT TTA AGT GTG ATT TCG GTC ACT TAT CCG ATT TTG CAG
fadL-CRP-site2-F	CCC TAC ACT TCG CGC TCC TGT TAC AGC ACG TAA CAT AGT TTG TAT AAA AAT AAA TC
fadL-CRP-site2-R	GAT TTA TTT TTA TAC AAA CTA TGT TAC GTG CTG TAA CAG GAG CGC GAA GTG TAG GG
fadH-CRP-site1-F	GTA ACC TGG ATA AAA CGC GAC AAG CGT CGC ATC CGG CGT TAT CAC CGG
fadH-CRP-site1-R	CCG GTG ATA ACG CCG GAT GCG ACG CTT GTC GCG TTT TAT CCA GGT TAC
fadH-CRP-site2-F	CAC CGG GCG TAT TCT TTT TGA ATC CCA TCA CAA ACC CCG CAC TCC
fadH-CRP-site2-R	GGA GTG CGG GGT TTG TGA TGG GAT TCA AAA AGA ATA CGC CCG GTG
T7-F	TAA TAC GAC TCA CTA TAG GG
T7-R	GCT AGT TAT TGC TCA GCG G

The sequences underlined are restriction sites, and the bold letters are predicted FadR or the CRP-cAMP sites.

Both FYJ159 and FYJ295 are, respectively, derivatives of E. coli strain MC4100, in which either a fadD–lacZ transcriptional fusion [18] or a fadL-lacZ transcriptional fusion, was integrated into the chromosomal attλ site (Table 1). Strain FYJ104 which carries a chromosomal fadL-lacZ transcription fusion (Table 1) was constructed using the FLP-mediated site-specific recombination method [19]. The kanamycin resistance cassette was removed from the fadL::km strain JW2341-1 (Table 1) by expression of the FLP recombinase encoded by plasmid pCP20 (Table 1) to give strain FYJ103, which retained a single FLP recombinase target (FRT) site. The FRT site was used for site-specific integration of the lacZ fusion plasmid, pKG37 (an improved version of pCE71) containing a FRT site upstream of a promoterless lacZY genes, a kanamycin resistance gene, and the R6K origin of replication (Table 1) [19]. The transformants were screened on LB agar plates containing kanamycin and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) at 37°C to obtain the chromosomal fadL-lacZ fusion strain FYJ104. The fusion plasmid was stably integrated due to its R6K origin and loss of the temperature-sensitive pCP20 plasmid [19]. The fadL promoter-lacZ junction was validated by a PCR using primers, fadL-P plus lacZ-R (Table 2).

P1vir phage transduction

P1 transductions were carried out as described by Miller [20] with minor modifications. Strains FYJ133, FYJ134, FYJ141, FYJ142, FYJ169 and FYJ170 were generated by transduction of strains FYJ59 (ΔarcA), FYJ60-2 (Δcrp), FYJ139 (ΔarcA ΔfadR::Tn10), FYJ140 (Δcrp ΔfadR::Tn10), FYJ77 (Δcrp ΔarcA), and FYJ78 (Δcrp ΔarcA ΔfadR::Tn10), respectively (Table 1) with a P1 lysate grown on FYJ104 (fadL-lacZ) with selection for kanamycin resistance. Similarly, a P1 lysate grown on FYJ159 (fadD-lacZ) was used for transduction of the following strains FYJ57, FYJ59, FYJ60-2, FYJ139, FYJ140, FYJ77 and FYJ80 with selection for kanamycin resistance to give strains FYJ161, FYJ162, FYJ163, FYJ164, FYJ165, FYJ166 and FYJ167, respectively (Table 1). Transduction of strains FYJ104, FYJ59 and FYJ60-2 with a P1 lysate grown on MFH8 (fadR::Tn10) with selection for tetracycline resistance gave strains FYJ105 (ΔfadR::Tn10, fadL-lacZ), FYJ139 (ΔarcA ΔfadR::Tn10) and FYJ140 (Δcrp ΔfadR::Tn10), respectively.

β-Galactosidase assays

Mid-log phase cultures in LB, RB or minimal media (with or without supplementation with various carbon sources), were collected by centrifugation, washed twice with Z Buffer [21] and assayed for β-galactosidase activity after lysis with sodium dodecyl sulfate-chloroform [21]. The data were recorded in triplicate with no less than three independent experiments.

Protein expression and purification

Hexahistidine-tagged E. coli FadR (and/or CRP) proteins were produced in E. coli BL21 (DE3) carrying the expression plasmid pET28-fadRec (and/or pET28-crp) (Table 1) by induction of bacterial cultures at an OD600 nm of 0.8–1.0 with 0.3 mM IPTG at 30°C for 3 h [17], [22]. The cells were pelleted washed twice with ice cold PBS buffer (101.4 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 8% glycerol, pH 7.4), dissolved in the same buffer and lysed using a French pressure cell. The extracts were centrifuged to remove bacterial debris and the supernatants loaded onto a nickel chelate column (Qiagen). Following washing with ten column volumes of with PBS buffer containing 50 mM imidazole, the FadR proteins were eluted with 150 mM imidazole. Appropriate eluted protein fractions were pooled and dialyzed against PBS buffer then concentrated by ultrafiltration (30 kDa cut-off, Amicon Ultra) [17]. The protein purity was judged by 12% SDS-PAGE, followed by staining with Coomassie brilliant blue R250 (Sigma, St. Louis, MO). Both FadR and CRP proteins were characterized by liquid chromatography quadrupole time-of-flight mass spectrometry of tryptic peptides and chemical cross-linking as described previously [18].

Electrophoretic mobility shift assays

These assays of the interaction between the fadD and fadL promoters, FadR and the cAMP-CRP complex were done essentially as previously reported [18], [23]. All of the FadR (and/or CRP) probes were prepared by annealing two complementary primers (Table 2) by incubation in TEN buffer (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 8.0) at 95°C for 5 min followed by slow cooling to 25°C and then DIG labeling by terminal transferase with DIG-ddUTP (Roche). DNA probes (Table 2) for assay of DNA binding by the CRP-cAMP complex were similarly synthesized. The digoxigenin-labeled DNA probes (either 0.1 or 0.2 pmol) were incubated with either DNA binding protein in binding buffer (Roche) for 15 min at room temperature and then analyzed by native PAGE (6.5% PAGE for the CRP probes and 7% PAGE for all other probes). The separations were then visualized as previously described [18], [23].

Fatty acid transport assays

Fatty acid transport was assayed as described by Klein et al. [24] with minor modifications. To avoid complications by β-oxidation strains that carried a fadBA disruption (strains FYJ82, FYJ83 and SI203) were used (Table 1). To test ArcA-P regulation of fatty acid transport, FYJ82 strain (ΔarcA) was compared with the wild type strain SI203 (Table 1). Overnight cultures were inoculated into 10 ml of RB liquid media supplemented with potassium nitrate (5 mM) as electron acceptor and kept in a fully anaerobic chamber at 37°C for ∼10 hrs [17]. The anaerobic environment (5% H2, 75% N2, and 20% CO2) was generated by an anaerobic environmental chamber (Bio-Bag type A, Becton Dickinson) [17]. Cultures in exponential phase were treated with 100 mM chloramphenicol for 10 min prior to assay. 1-14C-Oleic acid (American Radio-labeled Chemicals) was injected into the anaerobic bacterial cultures using a syringe fitted with a fine needle (30G1 PrecisionGlide) to a final concentration of 45.5 µM. The cultures were mixed well by vigorous vortex mixing and incubated at room temperature for about 15 min with anaerobiosis monitored by an anaerobic indicator (0.001% resazurin). Finally, the bacteria were collected by centrifugation (4.200× g, 16 min), and washed for five times with iced RB medium. One ml of the cultures were subjected to membrane phospholipid extraction [18], [25] after measuring culture absorbance (A600) and adjusted to an absorbance of 1.5. The phospholipids were then separated from any residual fatty acid by thin layer chromatography [18], [25]. Assay of the effects of the CRP-cAMP system on logarithmic phase cultures aerobically grown in LB liquid proceeded in a similar manner.

Bioinformatic analyses

The known or predicted DNA binding sites recognized by either FadR or ArcA (or CRP) were all from the E. coli literature. Multiple alignments were done using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html), and the resultant output was processed by program ESPript 2.2 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi), generating the final BLAST version.

Results and Discussion

Regulatory complexity in the LCFA transport promoters, fadL and fadD

The fadL and fadD genes encode the proteins known to be required for LCFA transport (Fig. 2A). The fadL and fadD transcriptional start sites are located 95 bp and 60 bp upstream of the translation start sites, respectively [6], [26] (Fig. 2B). Despite a seemingly straightforward role in metabolism, both promoters contain demonstrated or annotated binding sites for three different transcription factors, FadR, ArcA and CRP (http://www.ecocyc.org). Moreover, fadL and fadD promoters are the only fad regulon promoters that contain two distinct FadR binding sites [6], [26]. In the fadL promoter the FadR sites are separated by only 8 bp whereas in the fadD promoter the two FadR sites are separated by 68 bp (Fig. 2B and C). The locations of the ArcA binding sites determined by Cho and coworkers [12] also differ. The fadL ArcA site is 5 bp from FadR site 1 whereas the fadD ArcA site overlaps FadR site 2 by one bp, raising the possibility of crosstalk between the two repressor proteins (Fig. 2). The fadL CRP site was reported to lie downstream of the transcription start site [4] (Fig. 2B), a position incompatible with the usual activator function of CRP. Searches using the CRP consensus sequence of Zheng et al. [27] produced another fadL candidate binding site upstream of the transcription start site. Although this seemed a much more plausible position for activation of transcription, experimental verification of the CRP binding site was required. Indeed, although CRP-cAMP regulation of the fad regulon genes at the physiological level has been known for many years [14], none of the proposed sites have been experimentally validated.

Figure 2

The fadL and fadD promoters of E. coli.

. Genomic contexts of fadL and fadD on the E. coli chromosome (the two genes are separated by 570 kb). The transcription start sites (S) are indicated by arrows. Dotted lines denote spanning with long distance. and . Sequences of the fadL promoter and the fadD promoter, respectively. All the demonstrated sites bound by either FadR or ArcA are labeled with black rectangles, whereas the annotated CRP binding sites that were validated in this study are labeled with blue rectangles. A dotted rectangle labels a fadL CRP binding site incorrectly predicted by EcoCyc (http://www.ecocyc.org). White letters shaded in red denote the identical residues, red letters shaded in white mean similar bases, whereas white-shaded black letters denote unrelated. Dots denote gaps. “M” denotes the translation initiation site. The numbers are given relative to the transcription start site (S). The fadL and fadD FadR binding sites and their relative spacing are largely conserved in the genomes of Citrobacter, Kelbsiella and Enterobacter.

The two FadR-binding sites of fadL and fadD result in only modest repression

To obtain a parallel comparison of the FadR binding sites of the two promoters we used electrophoretic mobiltity shift analyses (EMSA) to assay the binding abilities of the individual sites over a range of FadR concentrations (Fig. 3). The two fadL FadR sites bound FadR with equivalent affinities (essentially complete binding of the probes by 50 nM FadR, Fig. 3B and D). In contrast, although fadD site 1 showed a binding affinity comparable to that of the fadL sites (Fig. 3F), FadR binding by fadD site 2 (Fig. 3H) was >10-fold weaker than the other three sites (Fig. 3B, D and F). Addition of 25–50 µM oleoyl-CoA resulted in loss of FadR binding by all four sites (Fig. 3C, E, G and I). Therefore, our data are in good agreement with the reported DNase I foot-printing results [6], [11] and provide data on the effects of the acyl-CoA regulatory ligand that is lacking in the foot-printing experiments. To monitor expression of fadL and fadD, each promoter was fused to a LacZ reporter gene to allow expression to be assayed by β-galactosidase activity (Fig. 4). Deletion of FadR resulted in only modest derepression of the two LCFA transport system genes (2 to 2.5 fold for fadL and 2 to 3-fold for fadD) in medium with acetate as sole carbon source (Fig. 4A and B) whereas expression of fadBA and fadH increased by 5 to10-fold (Fig. 4C and D). Increased expression of genes fadL and fadD in the ΔfadR strain was also seen when the carbon source was either glucose or glycerol (Fig. 4A and B), These observations are generally consistent with those obtained upon oleate induction of strains carrying a functional FadR (Fig. 4E).

Figure 3

E. coli fadL and fadD both carry two functional FadR-binding sites.

. sequence alignments of several known FadR binding sites from E. coli fad regulon. White letters with red background denote strictly conserved bases whereas yellow background letters denote highly conserved residues. The dual FadR sites of fadL and fadD are highlighted in bold italics. and . Gel shift assays of FadR binding to both fadL promoter sites. and . Gel shift assays of FadR binding to both fadD promoter sites. Long chain fatty acyl-CoA species block binding of FadR to the two fadL sites ( and ), as well as to the two fadD sites ( and ). FadR was used at concentrations of 0 (denoted by a minus sign), 1, 2, 5, or 10 pmol. FadR was incubated with 0.1 pmol of DIG-labelled probe in a total volume of 10 µl. For the acyl-CoA experiments the components were: probe, 0.1 pmol; FadR, 1 pmol and acyl-CoA, 50 pmol. Designations: C9:0; nonanoyl–CoA; C10:0, decanoyl-CoA; C16:0, palmitoyl-CoA; C16:1, palmitoleoyl-CoA; C18:0, stearoyl-CoA; C18:1, oleoyl-CoA.

Figure 4

FadR repression and oleate induction of fatty acid transport system.

and . Repression of fadL and fadD by FadR in cultures grown on various carbon sources. and . Parallel experiments with two β-oxidation genes fadBA and fadH are given for comparison. Strains FYJ104 (wild type) FYJ105 (ΔfadR::Tn10) were used for fadL whereas strains FYJ159 (wild type) and FYJ161 (ΔfadR) were used for fadD. Strains SI203 (wild type) and SI207 (ΔfadR) were used for fadBA whereas strains FYJ55 (wild type) and FYJ56 (ΔfadR) were used for fadH. . Induction of fadL and fadD expression by oleate. Oleate induction of the fatty acid transport system genes was compared with those of both fadBA and fadH expression. Oleic acid (5 mM) was added as the sole carbon source and compared to 5 mM acetate. Four strains expressing wild type FadR (FYJ295 (fadL-lacZ), FYJ159 (fadD-lacZ), SI203 (fadBA-lacZ), and FYJ55 (fadH-lacZ), were used. All strains were grown under aerobic condition (10 ml of culture in a 50 ml flask shaken at 200 rpm at 37°C). ß -Galactosidase (ß-gal) assays were conducted in triplicate and the error bars indicate standard deviations.

Expression of fadL and fadD is directly activated by the CRP-cAMP complex

The global regulator cAMP-CRP complex [28] can act as either an activator [29] or a repressor [30] in expression of genes involved in many E. coli metabolic pathways. Although putative class I cAMP-CRP binding sites had been proposed upstream of some fad regulon genes and activation of fadH transcription was observed in vivo [17], direct physical evidence for DNA binding of these promoters by the cAMP-CRP complex was lacking. We therefore used EMSAs with purified CRP in the presence or absence of cAMP to test the proposed sites. SDS-PAGE analyses indicated that the apo-form of CRP of molecular weight ∼23 kDa was homogenous (Fig. 5A). Chemical cross-linking showed the protein was dimeric as previously reported [28] (Fig. 5B). Liquid chromatography mass spectrometry analysis of tryptic peptides demonstrated that our recombinant protein matched E. coli CRP with 66% coverage (Fig. 5C).

Figure 5

Expression and characterization of E. coli CRP.

. SDS-PAGE profile of purified CRP. . Chemical cross-linking assays of purified E. coli CRP. The minus sign decodes no addition of the chemical cross-linker EGS, whereas the EGS concentrations were 1, 2, 5, 10, 15, and 20 µM. The cross-linking reaction mixtures were loaded on 12% SDS-PAGE. M denotes the Pre-stained broad range protein standards (BioRad). . MS identification of E. coli CRP protein. The tryptic peptides matching the CRP sequence are given in bold underlined type.

Higashitani and coworkers [4] predicted a fadL CRP binding site (Fig. 2B) centered at 57 bp downstream of the fadL transcription start site (termed fadL2) that is covered by our fadL probe 2, Fig. 6C). In contrast, we favored a site centered 157 bp upstream (Fig. 2C and 6A) covered by fadL probe 1 (Fig. 6B). In the fadH promoter, we previously proposed a CRP binding site (called fadH site 1 covered by fadH probe 1, Fig. 6A and E) and subsequently have predicted a second site (called fadH2 and covered by fadH probe 2, Fig. 6A and F). Given that sequence alignments of these sites showed only six conserved bp (Fig. 6A), the function of these sites required direct testing by EMSAs. As expected from numerous prior investigations (e.g., Lawson et al. [31]), CRP lacked DNA binding activity in the absence of its cAMP ligand (Fig. 6). Gel shift assays confirmed that the CRP-cAMP complex efficiently bound fadL probe 1 (Fig. 6B), but failed to bind fadL probe 2 (Fig. 6C). Therefore, fadL site 1 is a functional CRP-cAMP complex binding site whereas the site predicted by Higashitani and coworkers [4] is nonfunctional. Unlike Class II sites which overlap the −35 hexamer and Class III sites which have tandem CRP binding sites, the position of fadL site 1 identifies it as an atypical site that is located too far upstream to be reached the C-terminal domain (CTD) of the α–subunit of RNA polymerase which seems to have maximum stretch of 90–120 bp upstream of the promoter. In contrast the demonstrated fadD CRP-cAMP complex binding site [27] can be clearly identified as a Class I site (Fig. 2C and 6D). Both proposed fadH promoter sites bound the CRP-cAMP complex and therefore comprise a bona fide Class III site of the type that is composed of two Class I sites (some Class III sites have a Class II site coupled to a Class I site) (Fig. 6E and F).

Figure 6

The cAMP-CRP complex binds the fadL, fadD and fadH promoters.

. Sequence analyses of the CRP binding sites of fadL, fadD and fadH. White letters shaded in black denote strictly conserved bases, bold letters in grey represent highly similar residues, and dots mean gaps. The newly deduced CRP-binding site (fadL1 of A) binds the cAMP-CRP complex (panel ) whereas the CRP-binding site predicted earlier [17] (abbreviated fadL2) does not (panel ). D Binding of the fadD promoter region by the cAMP-CRP complex. and . The fadH promoter region contains two functional CRP-binding sites, fadH1 and fadH2, respectively. The DIG-labeled probe shifted by the cAMP-CRP complex is indicated by an arrow. All EMSA experiments were carried out using 6.5% native PAGE and representative results are shown. The protein samples were incubated with 0.6 pmol of DIG-labeled probe in a total volume of 20 µl that contained 200 pmol cAMP (when added). The right hand four lanes of each contained (left to right) 10, 5, 10 and 20 pmol of CRP, respectively.

To evaluate the effects of CRP on expression of the fatty acid transport genes, in vivo assays of β-galactosidase activities of the fadD and fadL lacZ fusions in Δcrp mutant strains were compared to strains expressing wild type CRP (Fig. 7). As a control we also assayed fadH expression and found that expression decreased in the absence of CRP (Fig. 6E and F) (Fig. 7D). As expected, the activities of both transport promoters were modestly decreased upon the loss of crp (Fig. 7A and B) verifying that CRP activates fadL and fadD transcription. Although in agreement with others (http://www.ecocyc.org), we failed to identify any CRP-binding site candidates in the fadBA promoter, expression of fadBA in the crp deletion strain is decreased relative to the wild type strain (Fig. 7C). This may indicate an indirect activation of the fadBA promoter by the CRP-cAMP complex. It seems possible that the indirect activation is due to action of the FIS protein, expression of which is activated by CRP binding to three discontinuous sites [27]. Indeed, a putative FIS binding site (TTGCATATTTTTAACACAA, −36 to 18) lies within the fadBA promoter and FIS is a known activator of fadBA transcription [32].

Figure 7

Activation of fatty acid transport system gene expression by the CRP-cAMP complex.

and . Decreased expression levels of fadL and fadD, respectively, were seen in the absence of CRP-cAMP. Strains FYJ104 (wild type) and FYJ134 (Δcrp) carry the fadL-lacZ transcriptional fusion whereas FYJ159 (wild type) and FYJ65 (Δcrp) contain the fadD-lacZ transcriptional fusion. and . Expression of fadBA and fadH are positively regulated by the functional CRP-cAMP complex. Strains SI203 (wild type) and FYJ83 (Δcrp) carry the fadBA-lacZ transcriptional fusion whereas FYJ55 (wild type) and FYJ65 (Δcrp) contain the fadH-lacZ transcriptional fusion. The cultures were grown in either LB or RB media. beta-Galactosidase activities were from at least three independent experiments, and the error bars indicate standard deviations.

Expression of fadL and fadD are additively repressed by FadR and ArcA under anaerobic conditions

The weak effects of FadR inactivation on expression of fadL and fadD suggested that other regulatory proteins may be involved and thus we tested the effects of deletion of the genes encoding the other proteins proposed to bind these promoters. The oxygen-sensitive two-component system ArcA-ArcB was reported to negatively regulate transcription of several fad regulon genes, including fadL, fadD, fadBA and fadH [12]. That report [12] also contained in vitro and in vivo evidence that the phosphorylated ArcA regulator (ArcA-P) bound the promoters of the target genes (Fig. 8A). We recently reported cross-talk between FadR and ArcA in expression of other fad regulon genes and found that the interaction could be either additive (fadH) (Fig. 8E) or synergistic (fadBA) (Fig. 8D) [17]. The fadD promoter FadR site 2 and the ArcA site overlap by a single bp (Fig. 2C) as also seen in fadH [17] whereas in the fadL promoter the ArcA and FadR site 1 sequences are separated by 5 bp (Fig. 2B). Thus, cross-talk between FadR and ArcA in fadL and/or fadD expression seemed likely.

Figure 8

Negative regulation of fadL and fadD by ArcA-P under anaerobic conditions.

Alignments of the ArcA-P binding sites of with those of other fad regulon genes. Red background indicates strictly conserved bases whereas yellow background denotes less conserved bases. Modulation of LacZ activity of the fadBA-lacZ transcriptional fusion by different regulatory proteins under anaerobic conditions. The fadBA-lacZ strains were SI203 (WT), SI207 (ΔfadR), FYJ82 (ΔarcA), FYJ83 (Δcrp), FYJ84 (ΔarcA ΔfadR), FYJ85 (Δcrp ΔfadR), FYJ86 (Δcrp ΔarcA) and FYJ87 (Δcrp ΔarcA ΔfadR), respectively. Transcriptional control of the fadH-lacZ fusion by different regulatory proteins under anaerobic conditions. The fadH-lacZ fusion strains were FYJ55 (WT), FYJ56 (ΔfadR), FYJ64 (ΔarcA), FYJ65 (Δcrp), FYJ68 (ΔarcA ΔfadR), FYJ81 (Δcrp ΔfadR), FYJ79 (Δcrp ΔarcA) and FYJ80 (Δcrp ΔarcA ΔfadR), respectively. Effect on fadL transcriptional levels by different regulatory proteins under anaerobic conditions. The fadL-lacZ were FYJ104 (WT), FYJ105 (ΔfadR), FYJ133 (ΔarcA), FYJ134 (Δcrp), FYJ141 (ΔarcA ΔfadR), FYJ142 (Δcrp ΔfadR), FYJ169 (Δcrp ΔarcA), and FYJ170 (Δcrp ΔarcA ΔfadR), respectively. Regulation of fadD transcription by different regulatory proteins under anaerobic conditions. The fadD-lacZ fusion strains were FYJ159 (WT), FYJ161 (ΔfadR), FYJ162 (ΔarcA), FYJ163 (Δcrp), FYJ164 (ΔarcA ΔfadR), FYJ165 (Δcrp ΔfadR), FYJ166 (Δcrp ΔarcA), and FYJ167 (Δcrp ΔarcA ΔfadR), respectively. The strains were grown on LB agar plates supplemented with 5 mM nitrate (KNO3) as the electron receptor. Anaerobic environments were generated using Bio-Bag environmental chamber type A as described [17]. beta-Galactosidase activities were recorded from at least six independent assays and are expressed as means ± standard deviations. *, P<0.005; **, P<0.001.

To test for cross-talk strains carrying arcA and/or fadR null mutations plus the chromosomal fadL-lacZ (or fadD-lacZ) transcriptional fusions were constructed. In general agreement with the report of Cho et al. [12], we found that the absence of arcA under anaerobic conditions resulted in increased transcription of fadL and fadD by 2 to 3-fold and >20-fold, respectively, (Fig. 8B and C). Our levels of derepression are somewhat less than those reported by Cho and coworkers [12] which we attribute to the gene used by these workers as their internal reference [17], [33]. However, our data differed from those of Cho and coworkers in the effects reported on FadR regulation of fadL transcription under anaerobic conditions when ArcA is functional (we see no effect versus their reported 4-fold increase). Although our statistical analysis argued that these two regulatory proteins control transcription of fadL and fadD in an additive manner, the wide variations in the data and the overlapping error bars provide caveats to this interpretation.

In the absence of the cAMP receptor protein (CRP) complex (cAMP-CRP), FadR and ArcA-ArcB repression of fadL and fadD expression was relatively weak (Fig. 8) and similar to that seen with fadBA and fadH [17]. Thus it seems that regulation by both FadR and ArcA-ArcB rely on the cAMP-CRP complex to activate transcription such that it can be further modulated.

Physiological relevance of ArcA and the CRP-cAMP complex to fatty acid transport

Uptake of [1-14C]oleic acid was assayed to evaluate the physiological consequences of ArcA-ArcB and the CRP-cAMP complex on fatty acid transport (Fig. 9). To rule out potential interference by β-oxidation, the strains studied lacked fadBA (Table 1). Incorporation of [1-14C]oleic acid into the membrane phospholipids of the ΔarcA strain (FYJ82) was consistently >2-fold greater that that of its parental strain under anaerobic conditions (Fig. 9A). Further analyses by thin layer chromatography showed that incorporation into the three major membrane phospholipids phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL) (Fig. 9B), consistent with anaerobic repression of fadL (and/or fadD) by ArcA-P (Fig. 8B and C).

Figure 9

Effects of ArcA and CRP on incorporation of [1-14C]oleic acid into membrane phospholipid.

Incorporation in the ΔarcA strain, FYJ82, and the wild type strain, SI203. At least three independent experiments were carried out and the data are expressed as mean ± standard deviation. A representative autoradiogram of a TLC separation of the 14C-labeled phospholipids of a panel experiment. The phospholipid species are phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL). A representative autoradiogram is given. Incorporation in the Δcrp strain, FYJ83 and the wild type strain, SI203. At least five independent experiments were performed and the data are expressed in means ± SD. A representative autoradiogram of a TLC separation of the 14C-labeled phospholipids of a panel experiment. Bacterial strains used in panel and were kept under anaerobic condition (details seen in Materials and Methods), whereas experiments in panel and were routinely conducted under aerobic condition.

In agreement with the observed CRP-cAMP activation of fadL and fadD transcription (Fig. 7A and B), quantitative determination of total 14C-labeled bacterial membrane phospholipids showed that the level of oleic acid incorporation in the strain lacking crp is only about one third that of the parental strain (Fig. 9C and D). To our knowledge these are the first direct physiological data that directly show that these two global regulators modulate LCFA transport pathway in E. coli.

Conclusions

Transcriptional regulation of fatty acid transport in E. coli involves three distinct regulatory systems, the specialized FadR system and the two global systems, ArcA and CRP-cAMP. FadR action is straightforward, it acts as a classical LacI-type repressor and only weakly represses fadD and fadL expression. CRP-cAMP regulation is also straightforward, fatty acids are a low status carbon source and E. coli prefers to use the highest status carbon source, glucose. In the presence of glucose (or in the absence of CRP), the other two regulators have little transcription to regulate. The apparent surprise is the stringent repression by ArcA seen under anaerobic conditions even in the absence of FadR because E. coli has a pathway to degrade fatty acids under anaerobic conditions [34] and low expression of FadD and FadL would seem likely to compromise function of the anaerobic pathway. However, under anaerobic conditions a new acyl-CoA synthetase, FadK, is induced that replaces FadD [35]. Unlike FadD which is inactive with short chain fatty acids [24], [35], FadK strongly prefers short chain length acids and such acids are the preferred growth substrates of the anaerobic β-oxidation pathway [34] although LCFA can also be utilized. Short chain acids readily enter fadL strains [36] and thus decreased expression of FadL is irrelevant for these growth substrates. However, degradation of LCFA such as oleate via the anaerobic pathway should require FadL. These apparent contradictions can be reconciled by our finding that ArcA represses fadL expression less than fadD expression (only about 4-fold) (Fig. 8B and C) and that FadK activates oleate poorly [35]. Hence, the significant level of FadL expressed under ArcA repression probably provides sufficient oleate transport to satisfy the poor catalytic activity of FadK with this substrate and allow the observed slow anaerobic growth on oleate [35].