Alkane-induced expression, substrate binding profile, and immunolocalization of a cytochrome P450 encoded on the nifD excision element of Anabaena 7120
© Torres et al; licensee BioMed Central Ltd. 2005
Received: 06 January 2005
Accepted: 24 March 2005
Published: 24 March 2005
Alkanes have been hypothesized to act as universal inducers of bacterial cytochrome P450 gene expression. We tested this hypothesis on an unusual P450 gene (cyp110) found on a conserved 11 kilobase episomal DNA element of unknown function found in filamentous cyanobacteria. We also monitored the binding of potential substrates to the P450 protein and explored the distribution of P450 protein in vegetative cells and nitrogen-fixing heterocysts using immuno-electron microscopy.
Hexadecane treatments resulted in a two-fold increase in mRNA, and a four-fold increase in P450 protein levels relative to control cultures. Hexane, octane and dodecane were toxic and induced substantial changes in membrane morphology. Long-chain saturated and unsaturated fatty acids were shown to bind the CYP110 protein using a spectroscopic spin-shift assay, but alkanes did not bind. CYP110 protein was detected in vegetative cells but not in differentiated heterocysts where nitrogen fixation occurs.
Hexadecane treatment was an effective inducer of CYP110 expression in cyanobacteria. Based on substrate binding profiles and amino acid sequence similarities it is hypothesized that CYP110 is a fatty acid ω-hydroxylase in photosynthetic cells. CYP110 was found associated with membrane fractions unlike other soluble microbial P450 proteins, and in this regard CYP110 more closely resembles eukarytotic P450s. Substrate stablization is an unlikely mechanism for alkane induction because alkanes did not bind to purified CYP110 protein.
Anabaena sp. strain PCC 7120 (Anabaena 7120) is an obligate photoautotrophic cyanobacterium that reduces atmospheric nitrogen in terminally differentiated cells known as heterocysts . Approximately every tenth cell along a filament differentiates into a heterocyst under nitrogen-fixing conditions. Three developmentally regulated DNA rearrangements occur within the heterocyst chromosome which excise DNA elements integrated within the nifD, fdxN and hupL genes [2, 3]. The element interrupting the nifD gene is 11,268 base pairs in length and carries its own site-specific recombinase, the xisA gene . The nifD element is found in most heterocyst forming cyanobacteria integrated within the nifD gene [5, 6]. It is absent from all non-heterocystous strains examined to date [7, 8]. While factors driving the evolution of the nifD element and its associated genes remain obscure, an essential role in diazotrophic growth can be ruled out since an Anabaena strain cured of the nifD element was shown to fix nitrogen under aerobic conditions in heterocysts of normal morphology .
Hexadecane-dependent induction of cyp110 transcripts in nitrogen-fixing and ammonia-supplemented cultures
We first tested the effects of hexane, octane, dodecane and hexadecane on the growth of Anabaena 7120 cultures. All alkane additions in this study were carried out at 0.2% (v/v), the recommended concentration for microbial P450 induction . In preliminary experiments, we determined that Anabaena 7120 exhibited rapid chlorosis with 0.2% hexane and octane. These treatments yielded only highly degraded RNA and were not further analyzed (data not shown). RNA samples from control, dodecane and hexadecane treated cultures were size fractionated and hybridized with cyp110. A psbA1 specific probe was used to assess RNA quality and for normalization.
The psbA1 gene, which encodes the D1 reaction center protein, was used as a load control and hybridization standard. As seen in Fig. 2(b) and 2(d), the psbA1 probe identifies a discrete 1.25 kb transcript in control RNA as previously shown . Relative amounts of mRNAs were estimated by densitometry of the scanned autoradiogram. Exposure times for detection of the cyp110 transcript were 12 times longer than for the highly expressed psbA1 mRNA.
The ratio of the cyp110/psbA1 signal intensities was 2.3 fold higher in hexadecane-treated cultures in the nitrogen-fixing culture (panels A&B) while a 2.2-fold increase was observed in the ammonium-supplemented culture (panels C&D). The dodecane treatment had little effect on cyp110 mRNA levels in either nitrogen-fixing or ammonium supplemented cultures. Similar results were obtained in 2 separate experiments with RNA isolated from independent cultures. The doubling of steady-state cyp110 mRNA levels in response to hexadecane treatment was independent of nitrogen status of the culture, consistent with previous data showing the entire nifD element is not required for nitrogen fixation in heterocysts .
CYP110 protein is only detected in hexadecane-treated cultures and localized in the membrane fraction
Heterologous expression of the CYP110 protein in E. coli produced a substantial amount of membrane associated P450 protein . This is a clear distinction between CYP110 and its closest sequence homolog, P450BM3, which has been extensively studied in part because it is a soluble P450 model protein . To determine if CYP110 is membrane associated in Anabaena 7120, protein from membrane and soluble fractions separated by centrifugation were subjected to Western blot analysis. As shown in Fig. 3D, the membrane fraction appeared to contain all of the immuno-reactive P450 protein in the Anabaena 7120. This stands in contrast to P450BM3 and most other microbial P450 proteins, and in this regard, CYP110 more closely resembles the eukaryotic P450 proteins.
Spectral characterization of CYP110 protein and substrate binding assay
Spectral Shift and Binding Constants of CYP110 with SeveralCompounds.
Cetyltrimethyl Ammonium Bromide
Sodium Dodecyl Sulfate
Saturated Fatty Alcohols C6-C18
N-methyl Lauryl Amide
N,N-Dimethyl Lauryl Amide
Fatty amines and fatty amides are known to interact with P450 ω-hydroxylases via Type II spectral shifts [19, 20], and this was also true for CYP110. The minimal chain length for this interaction was 5 since n-butylamine did not cause a spectral shift while n-pentylamine did bind. There was a clear trend for tighter binding with longer hydrocarbon chain length, as the CYP110-dodecylamine interaction had the lowest tested Ks value (0.012 mM). CYP110 bound fatty amines 10 fold better than P450non, which has a binding constant of 1.3 mM towards octylamine . Twelve-aminododecanoic acid causes a Type I spectral shift instead of a Type II suggesting an altered mode of binding. Lu et al.  showed that terminal amino fatty acid derivatives interact weakly with the heme iron of cytochromes P450.
The binding of CYP110 towards other fatty acid derivatives was also examined. Table 1 displays a list of compounds that displayed no spectral shift, including fatty alcohols or saturated n-alkanes. No spectral shift was observed with 11- or 12-hydroxydodecanoic acid or with methyl laurate. The unsaturated fatty acid 11-dodecenoic acid was also tested with no shift observed. Two other amines that did not display spectral shifts were the polyamine, putrescine, and cetyltrimethylammonium bromide (CTAB). Sodium dodecyl sulfate (SDS) does bind CYP110 and causes a Type I spectral shift. Although no binding constant was determined, it was observed that the maximal shift to 388 nm occurred below 0.5 mM and reverted back to 416 nm above this concentration, a phenomenon likely to be linked to the critical micelle concentration for SDS (data not shown).
Alkane effects on morphology and localization of CYP110
Detection of anti-CYP110 polyclonal gold conjugates.
(Avg. no. of gold particles)
Anabaena 7120 untreated
Anabaena 7120 dodecane treatment
Anabaena 7120 hexadecane treatment
Nonspecific immuno-staining was estimated by incubating sections with preimmune rabbit IgG or with polyclonal anti-CYP110 antibodies that had been pre-adsorbed with purified CYP110 protein produced in E. coli. Very low background was seen in both the preimmune rabbit IgG primary incubation (Fig. 5h), and sections treated with preabsorbed anti-CYP110 antibodies (Fig. 5i). No clusters of immunogold conjugates were seen in any of the control sections. These results clearly show the majority of CYP110 protein is localized in vegetative cells and corroborate Northern and Western data showing elevated CYP110 expression in hexadecane-treated cultures.
Most well characterized microbial P450s enzymes were isolated from heterotrophic organisms and are involved in the oxidative metabolism of xenobiotic compounds for energy and growth. Examples include camphor-degrading P450CAM  and two herbicide inducible P450 genes in Streptomyces griseolus . There is no evidence to date for cytochrome P450-dependent oxidative metabolism in the cyanobacteria. A P450-dependent N- demethylation activity is known in unicellular green algae that activates the pro-herbicide metflurazon into norflurazon . Two genera of marine cyanobacteria Microcoleus and Phormidium were isolated from oil spills along the Arabian sea and reported to contribute to oil degradation . While the first step in alkane degradation in many microbial systems is a P450-dependent hydroxylation [11, 26, 27], there are no data on the mode of alkane metabolism nor confirmation of hydrocarbon oxidation by axenic isolates of these cyanobacterial strains. Recent evidence does show that mixtures of these cyanobacteria and organotrophic bacteria of the genera Rhodococcus, Arthrobacter, Pseudomonas, and Bacillus are more effective than either group alone towards alkane oxidation in inorganic growth media .
The available evidence suggests that CYP110 does not participate in the degradation of alkanes. CYP110 has low overall sequence similarity to other known alkane hydroxylase enzymes like the CYP52 family . Alkanes are quite toxic to Anabaena 7120, and purified CYP110 protein expressed in E. coli binds fatty acids but not alkanes using a spin-shift assay. The membrane localization and spin-shift binding data suggest that long-chain polyunsaturated fatty acids are the most likely endogenous substrates for CYP110. The endogenous function of CYP110 in Anabaena 7120 remains a mystery. Sequence comparisons indicate the protein is related to the fatty acid omega hydroxylase proteins. One possibility is that ω-hydroxylated fatty acids are further oxidized to dicarboxylic acids which then undergo β-oxidation from both ends yielding acetate, as demonstrated in eukaryotic peroxisomes .
Cytochrome P450 proteins in diazotrophic bacteria have been suggested to play an ancillary role in nitrogen fixation. Many years ago, Appleby proposed that a cytochrome P450 enzyme functions as an alternative high-affinity oxidase in an efficient, leghemoglobin-facilitated pathway of oxidative phosphorylation in bacteroids of Bradyrhizobium japonicum . The idea was based on spectroscopic evidence for the presence of a P450 protein in B. japonicum bacteroids but not in aerobically grown cells and the blocking of the high-affinity oxidase system by the P450 inhibitor, N-phenylimidazole. More recently both bacteroids and anaerobically grown B. japonicum cells were shown to contain two immunologically distinct cytochromes P450, which were cloned and designated cyp112 and cyp114 . Neither protein is expressed in aerobically grown B. japonicum cells, consistent with the Appleby hypothesis. Transposon mutagenesis of the cyp112 locus was found to block expression of both Cyp112 and Cyp114. Nevertheless, the mutant strain produced effective nodules on soybeans. These results show neither protein is essential for symbiotic function under the conditions of plant growth used in these experiments. Subsequent analysis of the B. japonicum P450 cluster identified a third P450 gene and other open reading frames suggesting an operon involved in terpenoid biosynthesis . Like the Anabaena 7120 strain missing the nifD element, a quantitative phenotype related to nitrogen fixation under specific culture conditions cannot be excluded from the available evidence. B. japonicum and Anabaena 7120 are not the only diazotrophic bacteria harboring cytochrome P450 genes near clusters of nitrogen fixation-related genes. The large Sym plasmid from Rhizobium sp. NGR234 confers the ability to associate symbiotically with plants. It contains two cytochrome P450 genes whose function(s) are not known .
Alkanes have been hypothesized to act as universal inducers of microbial P450 genes . Consistent with this hypothesis, we observed a small but reproducible 2-fold enhancement of cyp110 transcription induced by 0.2% hexadecane. We did not test lower concentrations of hexane or dodecane to determine if non-toxic concentrations would have P450-inducing activity in Anabaena 7120. We cannot rule out the possibility that the lower solubility of longer chain alkanes in aqueous solutions may have been more important for the activity of hexadecane as an inducer than chain-length per se. The CYP110 protein displayed the highest affinity towards long-chain unsaturated fatty acids and was localized primarily in photosynthetic vegetative cells of Anabaena 7120. Unlike most prokaryotic P450 proteins, CYP110 was found to be associated with membrane fractions consistent with the solubility characteristics of its preferred substrates.
For RNA and protein isolation, cultures of Anabaena 7120 (4 L) were grown in Chu #10 media with continuous light, stirred at 30°C and bubbled with a 1% CO2/air mixture illuminated at approximately 100 μE s-1 m-1. Nitrogen supplemented cultures were grown with 2.5 mM NH4(SO4)2 to suppress heterocyst differentiation. Treatments were carried out after cultures reached A700 = 0.7. Control and test cultures (0.2 % dodecane or hexadecane [v/v] for 2 hours) were maintained in the same growth conditions during the treatment interval.
An amended RNA isolation procedure  was used for total RNA isolation. Briefly, cells were cooled by adding an equal volume of ice followed by transferring the cell paste into a mortar and freezing with liquid nitrogen. Cells were ground to a fine powder and placed in 10 mL RNA lysis buffer-pH 5.2 (0.1 M Tris, 0.1 M LiCl, 5 mM EDTA, 0.1 M NaCl, 0.1 M sodium acetate, 1 % SDS, 0.2% β-mercaptoethanol). After vortexing (2 min), 5 mL hot (60 C) NTE saturated-phenol, pH 6.0, was added, followed by 3 min of vortexing. Samples were placed at 60°C for 10 min, vortexed and 7 mL chloroform:isoamyl alcohol (24:1) added and the aqueous phase was subjected to repeated phenol/chloroform extractions until no visible material remained at the interface. Total RNA was precipitated overnight in 2 M LiCl at 4°C. RNA pellets were washed with 2 M LiCl and ethanol precipitated. After a 70% ethanol wash, the RNA was brought up in diethyl pyrocarbonate treated water.
Samples of total RNA (15 μg) were separated by agarose formaldehyde denaturing gel electrophoresis. The gel was equilibrated twice in 10× SSC for 15 min and RNA was transferred onto a nylon membrane (Boehringer Mannheim) by capillary transfer. Probes were labeled using the DIG-High Prime Mix (Boehringer Mannheim) following the manufactures' recommendations. The cyp110 probe was derived from the 2 kbp HindIII pAn 207.4 DNA fragment (Fig. 1) containing most of the cyp110 gene sequence . The psbA probe used is a 2 kbp pAn 625 Eco R1 fragment  containing the coding region for the D1 reaction center protein in photosystem II. RNA blots were first probed with cyp110, stripped, and reprobed with the psbA probe. The hybridized probes were detected using the CDP-Star (Boehringer Mannheim) chemiluminescent substrate, followed by autoradiography. Prehybridizations, hybridizations, and washes were carried out as described by the manufacturer. Signal intensities from northern blots were quantified using Intelligent Quantification software (Genomic Solutions).
P450 protein analysis
Proteins for Western blotting were isolated from N2-fixing Anabaena 7120 cultures treated with 0.2% hexadecane. Cultures (2 L) were grown to an A 700 = 1.0 – 1.2 prior to hexadecane addition. Fractions of 250 mL were collected at 0, 2, 12, 24, 36, 48 and 60 h after treatment. Cells were harvested by centrifugation and resuspended in 5 mL of protein buffer: 20 mM MOPS (pH 7.4), 10% glycerol, 1 mM DTT, 20 mM KCl, 1 mM PMSF, 1 mM apoprotin and 1 mM pepstatin. Samples were lysed by three passages through a French pressure cell. Soluble and membrane fractions were separated by centrifugation at 100,000 × g for 1.5 h. Pelleted membranes were resuspended in protein buffer supplemented with 0.2% deoxycholate by gentle shaking overnight at 4°C. The CYP110 protein was expressed from a pUC8 expression vector and purified as described elsewhere . Antiserum to this protein was raised in a New Zealand white rabbit using subcutaneous injections of 100 μg purified CYP110 protein in Freund's complete adjuvant. Two identical booster injections were made at 3 and 6 weeks after the first. Immune serum was collect at 6 and 12 weeks after the initial injection. Protein samples (50 μg) were separated on 12% SDS-PAGE  and blotted using standard methods . Antiserum raised against P450 protein was used at a 1:100 dilution and detected with a peroxidase-linked anti-rabbit secondary antibody (Amersham) and the ECL Western Blot system (Amersham) using X-ray film.
Oxidized, reduced and carbon monoxide reduced spectra
The purified soluble fraction of CYP110 was diluted 1:10 in buffer A (20 mM MOPS pH 7.4, 20% Glycerol, 2 mM DTT, 20 mM KCl, and 2 mM PMSF). The oxidized spectra was measured with a Hewlett Packard Diode Array Spectrophotometer. Five milligrams of sodium dithionite was added, and the reduced spectra measured. CO was then bubbled into the reduced sample at a rate of 1 bubble per second for 30 seconds. The oxidized spectra was subtracted from the CO reduced spectra to yield the peak at 450 nm. The concentration of cytochrome P450 was determined by using the extinction coefficient, 91 mM-1cm-1, as determined for rat liver P450 .
Substrate binding studies
Spectrophotometric titration of CYP110 with fatty acids, fatty amines, fatty amides, fatty alcohols, and n-alkanes with varying chain lengths (C5-C20) was preformed in buffer A with and without 0.2 % deoxycholate. Purified soluble CYP110 was diluted to a concentration of 3 μM, and the baseline spectrum was measured. Substrates were then titrated into the P450 from a 100 mM stock solution in ethanol. The n-alkanes were solubilized via the method of Scheller et al.  by sonication of the stock solution for 10 minutes on ice to enhance solubility. The substrates were titrated to concentrations varying from 5 μM to 2 mM depending upon the compound being tested. Fatty acids, amides, alcohols, esters and n-alkanes were titrated from 50 μM to 2 mM while fatty amines were titrated from 1 μM to 700 μM.
Immunocytochemical electron microscopy
Nitrogen-fixing Anabaena 7120 batch cultures were grown at ambient CO2 levels the presence or absence of 0.2% dodecane or hexadecane (v/v) under continuous light with stirring at 30 C for 30 days. Immediately upon harvesting, cells were fixed in 4% paraformaldehyde, 0.6% glutaraldehyde, 0.033 M phosphate, 0.1 M sucrose for 2 hours at 4°C. Samples were then washed three times in 7% sucrose, 0.033 M phosphate buffer. Following an ascending ethanol dehydration series (50%, 70%, 95%), the samples were infiltrated in LR white acrylic plastic (Electron Microscopy Sciences). Cells were then embedded in gelatin capsules for 24 hours at 50°C. Solidified LR-white samples were sectioned with a diamond knife and placed on carbon coated, nitrocellulose-covered, nickel grids. Sections were blocked with 1% BSA/10% goat serum in TBST (0.1% Tween, 16.5 mM Tris (pH 7.5), 137 mM NaCl, 2.7 mM KCl). The sections were incubated (2 h at room temperature) with 35 μL of the following antibodies: A. Anti-CYP110 polyclonal sera used in western blot analyses (1:400 dilution in BSA-TBST), B. preimmune rabbit IgG (1:400 dilution in BSA-TBST), or C. Immune serum preabsorbed with 2.5 mg purified CYP110 protein . Following the primary antibody treatment sections were washed at least four times in BSA-TBST and incubated for one hour (room temperature) in a drop of secondary antibody goat anti-rabbit IgG (Sigma). The sections were then viewed on a Hitachi H 7000 transmission electron microscope at 80 kV.
Conventional electron microscopy
Following growth conditions described for immunocytochemical studies, approximately 107 Anabaena 7120 cells were pelleted and fixed by placing in 1 mL 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at room temperatures for 1 hour. Cells were washed twice with cacodylate buffer (15 min/wash) and embedded with 1% agar. Agar embedded samples were cut into small cubes (~1 mm) and postfixed in 1% osmium tetroxide 0.1 M cacodylate (pH 7.4) for two hours at room temperature. Samples were rinsed with deionized water and dehydrated in an ascending ethanol series. Cells were infiltrated with 50% Spurr's media (Electron Microscopy Sciences): 50% absolute ethanol, followed by at least four more infiltrations with fresh 100% Spurr's media. To allow polymerization of the Spurr's media, samples were incubated overnight at 70 C. The samples were sectioned using a diamond knife and picked up on copper grids for viewing as above.
The authors wish to acknowledge the skillful assistance of Hank Adams and the NMSU Electron Microscope Facility. This work was supported by NIH Grant GM08136–23.
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