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 12. 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 cyp110 probe identified a range of transcript sizes with a maximum of 9 kb seen in mRNA from both nitrogen-fixing and ammonia supplemented Anabaena 7120 cultures Fig. 2(a) and 2(c). The size of the transcript identified by the cyp110 probe and the genetic organization of the nifD-element suggest that all of the open reading frames to the right of xisA on the nifD element are coordinately transcribed on a polycistronic mRNA. In support of this idea, a probe internal to the ORF6 coding region (Fig. 1) identified the same range of transcript sizes (data not shown). Since the xisA mRNA has not been detected with Northern blots 9, it is likely that the 9 kb transcript begins downstream of the xisA gene and terminates 3' to the orf designated adx in Fig. 1.

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 15. 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 9.

The hexadecane effect on cyp110 gene expression was further examined using CYP110-specific antibodies to probe for the presence of P450 protein in nitrogen-fixing Anabaena 7120 cultures. As shown in Fig. 3A, the 0.2% hexadecane treatment resulted in the appearance of an immuno-reactive protein band of 50 kDa that co-migrated with a purified CYP110 protein sample. The P450 protein band intensity increased at least 2 fold within 2 hours of treatment, and at least 4 fold 24 hours after treatment with hexadecane when compared to the background, zero-time expression shown in lane 3 (Fig. 3C). A western blot with protein samples from an untreated control culture failed to detect any immuno-reactive P450 protein (Fig. 3B). These results were replicated with 2 independent cultures.

Heterologous expression of the CYP110 protein in E. coli produced a substantial amount of membrane associated P450 protein 16. This is a clear distinction between CYP110 and its closest sequence homolog, P450_BM3, which has been extensively studied in part because it is a soluble P450 model protein 11. 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 P450_BM3 and most other microbial P450 proteins, and in this regard, CYP110 more closely resembles the eukaryotic P450 proteins.

Absorption spectra studies were recorded for CYP110 protein expressed and purified from E. coli carrying a pUC-based expression vector 16. As shown in Fig. 4A, the CYP110 protein has a typical cytochrome P450 absorbance profile in the visible region for the oxidized, reduced and carbon monoxide bound states. CYP110 also shows the characteristic peak at 450 nm in the CO difference spectrum (Fig. 4B). We monitored the visible spectrum while titrating a CYP110 solution with tetradecanoic acid. As shown in Fig. 4C, this resulted in a blue shift of the peak absorbance in the oxidized spectrum to 388 nm, similar to previously described P450 substrate-dependent Type I spectral shifts. This spectral shift was the same in either buffer A or buffer A + 0.2% deoxycholate. When buffer A was supplemented with 0.2% Triton X-100, no spectral shift was observed (data not shown). The titration yields the binding constant (Ks) for each substrate producing a spectral shift. A typical difference spectrum generated from the titration and the resulting double reciprocal plot are shown in Fig. 4D.

The calculated binding constants for the interaction of CYP110 with potential substrate molecules of different hydrocarbon chain length are listed in Table 1. For the saturated fatty acids tested, CYP110 displays the highest affinity for tetradecanoic acid with a K_s of 0.091 mM. The unsaturated fatty acids also bound well to CYP110. Overall, CYP110 had the highest affinity for alpha-linolenic acid (Ks 0.062 mM) and arachidonic acid (Ks = 0.051 mM), long chain fatty acids with 3 and 4 double bonds, respectively. For comparison, P450_BM3 has a K_s of 0.002 mM for arachidonic acid 17. The Ks values towards arachidonic acid for the mammalian cytochrome P450s from family 4 are in the same range 18.

Fatty amines and fatty amides are known to interact with P450 ω-hydroxylases via Type II spectral shifts 1920, 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 P450_non, which has a binding constant of 1.3 mM towards octylamine 21. Twelve-aminododecanoic acid causes a Type I spectral shift instead of a Type II suggesting an altered mode of binding. Lu et al. 20 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).

Given the toxicity of hexane to Anabaena 7120, transmission electron microscopy was used to document changes in cell structure associated with the alkane treatments shown to induce the CYP110 expression. Vegetative cells from control, dodecane and hexadecane treated cultures are shown in Fig. 5a,5b and 5c respectively. Many dark spherical inclusions are seen in the dodecane-treated cells (Fig. 5b). The dodecane treatment also distorts and thickens the cellular and photosynthetic membranes, creating a ruffled effect in both outer and inner membranes. Hexadecane treatment did not have the same dramatic effect on membrane morphology, however the close stacking of photosynthetic membranes seen in the control micrograph (Fig. 5a) is lost with hexadecane treatment (Fig. 4c).

Gold-conjugated anti-CYP110 antibodies were used to determine the relative protein expression levels in vegetative cells with and without alkane treatment and to assess vegetative cell versus heterocysts expression patterns Anabaena 7120. Immunoelectron micrographs showed a greater number of anti-CYP110 polyclonal gold conjugates in dodecane and hexadecane vegetative cells (Fig. 5e and 5f) than in control samples (Fig. 5d). Expression levels were quantified by counting gold particles in multiple micrographs as shown in Table 2. Immunogold particles most often clustered around dark-staining material in Anabaena 7120 vegetative cells grown in the presence of 0.2% (v/v) dodecane or hexadecane (Fig. 5e,f). We found fewer immunogold particles/cluster in dodecane-treated samples (avg. = 22) than hexadecane treated samples (avg. = 50). Heterocysts showed significantly fewer gold particles than vegetative cells, and the number of gold particles in heterocysts did not increase with hexadecane treatment (Fig. 5g, Table 2).

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.

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 37 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 10. The psbA probe used is a 2 kbp pAn 625 EcoR1 fragment 15 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).

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 16. 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 38 and blotted using standard methods 39. 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.

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 40.

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. 29 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.

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 16. 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.

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.