ABSTRACT
The hydrothermal vent chemolithoautotroph Thiomicrospira crunogena grows rapidly in the presence of low concentrations of dissolved inorganic carbon (DIC) (= CO2 + HCO3− + CO3−2). Its genome encodes α-carbonic anhydrase (α-CA), β-CA, carboxysomal β-like CA (CsoSCA), and a protein distantly related to γ-CA. The purposes of this work were to characterize the gene products, determine whether they were differentially expressed, and identify those that are necessary for DIC uptake and fixation. When expressed in Escherichia coli, CA activity was detectable for α-CA, β-CA, and CsoSCA but not for the γ-CA-like protein. α-CA and CsoSCA but not β-CA were inhibited by sulfonamide inhibitors. CsoSCA was also inhibited by dithiothreitol. When grown under DIC limitation in chemostats, T. crunogena transcribed csoSCA more frequently than when ammonia limited, while genes encoding α-CA and β-CA were not differentially transcribed under these conditions. Cell extracts from T. crunogena grown under both DIC- and ammonia-limited conditions had CA activity that was strongly inhibited by sulfonamides, though extracts from nitrogen-limited cells had some CA activity that was resistant, perhaps due to a higher level of β-CA activity. Based on predictions from the SignalP software program, subcellular location when expressed in E. coli, and carbonic anhydrase assays conducted on intact T. crunogena cells, α-CA is located in the periplasm. However, inhibition of α-CA by acetazolamide had only a minor impact on rates of DIC uptake or fixation. Conversely, inhibition of CsoSCA with ethoxyzolamide inhibited carbon fixation but not DIC uptake, consistent with this enzyme functioning to facilitate DIC interconversion and fixation within carboxysomes.
Thiomicrospira crunogena, a deep-sea hydrothermal vent sulfur-oxidizing chemolithoautotroph (12), lives in a spatially and temporally heterogeneous environment. In this organism's mesophilic habitat, dilute hydrothermal fluid (∼30°C) emitted from the crust interacts with cooler bottom water (2°C) (13) and creates turbulent eddies. As a result, the habitat chemistry oscillates between dominance by dilute hydrothermal fluid (dissolved inorganic carbon [DIC] = CO2 + HCO3− + CO3−2 = 2 to 7 mM; pH = 5 to 8) and bottom water (DIC = 2 mM; pH = 8) (10). Due to these changes in pH and DIC, the CO2 concentration sweeps between 20 and 1,000 μM, with a periodicity ranging from seconds to days (10, 13).
T. crunogena has a carbon concentrating mechanism (CCM), an adaptation likely to facilitate carbon fixation in the presence of these oscillations in DIC composition and concentration. In similarity to many species of cyanobacteria (3), T. crunogena can grow rapidly despite DIC concentrations of less than 20 μM; when grown in the presence of low concentrations of DIC, its whole-cell affinity for DIC (0.026 mM) is markedly smaller than that when it is cultivated at high DIC concentrations (0.66 mM) (8). T. crunogena can use both extracellular CO2 and HCO3− for carbon fixation and is able to accumulate intracellular DIC to concentrations 100 times higher than those of extracellular DIC (8). The inducible molecular mechanism responsible for generating high intracellular concentrations of DIC has yet to be characterized, since the genome does not encode any apparent orthologs to any of the HCO3− or CO2 transporters that have been characterized in cyanobacteria (29).
Carbonic anhydrase (CA) has also been implicated in facilitating DIC uptake and fixation and in pH homeostasis, both of which would be a benefit in the variable hydrothermal vent habitat. Five phylogenetically distinct classes of CA have been identified (α, β, γ, δ, and ζ), and enzymes belonging to three of these (α, β, and γ) have been detected among bacteria (16, 24, 31). Genes encoding homologs of all three of these classes of carbonic anhydrases are present in the T. crunogena genome (29), and it is of interest to determine whether they play a role in the T. crunogena CCM, since DIC uptake has not been well characterized for the Proteobacteria.
α-CA enzymes are the best biochemically characterized carbonic anhydrases and are widespread among bacteria and animals (30). In the alphaproteobacteria Mesorhizobium loti and Rhodopseudomonas palustris, as well as the epsilonproteobacterium Helicobacter pylori, the α-CA enzymes are periplasmic (14, 18, 22). A role in DIC uptake is suggested for R. palustris, since autotrophic growth by this purple photosynthetic bacterium is inhibited when its periplasmic CA is inactivated either by acetazolamide or mutation, and growth rates are restored with elevated CO2 concentrations or lower pHs (22). For the α-CA gene present in the T. crunogena genome, an analysis with the SignalP software program (4) predicts the location of this CA to be periplasmic due to an amino-terminal hydrophobic region that is likely to be a signal peptide.
β-CA enzymes are found in bacteria, plants, and archaea (30) and include the distantly related β-like carboxysomal CA enzymes (CsoSCA) present in many autotrophic proteobacteria and some cyanobacteria (27, 32). In the sulfur-oxidizing betaproteobacterial autotroph Halothiobacillus neapolitanus, CsoSCA is present in small amounts in carboxysomes, which are protein-bound inclusions packed with RubisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) (32). CsoSCA converts HCO3− to CO2 within the carboxysome, where it is fixed by RubisCO (9, 11). A csoSCA gene in T. crunogena is present in a carboxysome operon (29), and carboxysomes are evident in electron micrographs of this microorganism (28); it is likely that CsoSCA plays a similar role in T. crunogena.
A gene encoding a second β-CA enzyme is present in the T. crunogena genome, in an apparent operon with cbbM, which encodes form II RubisCO, similar to what has been observed in the close relative Hydrogenovibrio marinus (35). Since form II RubisCO is most abundant in T. crunogena cells cultivated under high-DIC conditions (K. Scott, unpublished data), it is possible that this β-CA plays a role in T. crunogena growth under high-DIC conditions and is not part of an adaptation to low-DIC conditions.
γ-CA enzymes are present in the archaea and bacteria, and homologs have been found in eukaryotes (31). In methanogenic archaea, such as Methanosarcina thermophila, γ-CA is expressed preferentially when cells are grown with acetate as the carbon source and electron donor and may function to maintain low intracellular CO2 concentrations (1, 2). A γ-CA homolog is present in some cyanobacterial carboxysomes and has been demonstrated in Synechocystis sp. strain PCC6803 to be capable of binding either CO2 or HCO3−, and it may play a role in carboxysome function in these organisms (7). The γ-CA homolog in the T. crunogena genome is quite distantly related to the γ-CA genes in M. thermophila (27% identical) and Synechocystis sp. strain PCC6803 (32% identical), which suggests it may have an alternative (e.g., non-CA) activity in T. crunogena.
Consistent with their evolutionary independence, CA enzymes from the different classes respond differently to the presence of carbonic anhydrase inhibitors. Diverse members of the α class have been found to be sensitive to the sulfonamide inhibitors acetazolamide and ethoxyzolamide (20). Fewer members of the β and γ classes have been characterized with respect to inhibitor sensitivity. The few that have (archaeal β and γ classes; carboxysomal β-like) are also sensitive to these inhibitors, though their Ki values are often larger than those measured for α-class enzymes (11, 20, 36, 37).
This study was designed to address the remarkable diversity and function of carbonic anhydrase homologs in the T. crunogena genome. To verify that all four genes encoded functional CA enzymes and to characterize patterns of inhibition, the α-, β-, carboxysomal, and γ-CA genes were overexpressed in Escherichia coli. The response of transcription of CA genes to the concentration of DIC available during growth was monitored. CA activity was measured in T. crunogena cell extracts and in incubations with whole cells to infer the cellular locations of the enzymes, and carbon fixation and DIC uptake rates were measured in the presence of CA inhibitors to determine whether these processes were affected by CA inactivation.
MATERIALS AND METHODS
Analytical methods and reagents.DIC was quantified with a gas chromatograph (8). Protein concentrations were determined by using the RC DC protein assay (Bio-Rad, Hercules, CA).
Bacterial strains and cultivation. Thiomicrospira crunogena XCL-2 was cultivated in artificial seawater medium supplemented with 40 mM thiosulfate and 10 mM Na HEPES, pH 8 (TASW) (8, 12). Cells were grown in chemostats (Bioflo 110; New Brunswick Scientific) under DIC limitation [0.1 mM DIC, 13 mM (NH4)2SO4] or ammonia limitation [50 mM DIC, 0.8 mM (NH4)2SO4]. The pH (= 8) and oxygen concentration (∼20 to 100 μΜ) were maintained in the growth chambers by using pH and O2 electrodes which directed 10 N KOH addition and O2 sparging (pure O2 was used for the DIC-limited cells, while 5% CO2, balance O2 was used for the ammonia-limited cells) (8).
One Shot Mach1 (T1 resistant) and BL21(DE3) One Shot E. coli (Invitrogen, Carlsbad, CA), used for transformation and expression studies, were cultivated in Luria broth supplemented with the appropriate antibiotic (see below) (26).
Expression of T. crunogena CA genes in E. coli.Genes encoding CA homologs, as well as cbbM, encoding form II RubisCO (negative control), were PCR amplified from T. crunogena genomic DNA (30 cycles: 95°C denaturation, 1 min; 50°C annealing, 30 s; and 72°C extension, 2 min, final extension, 8 min) and cloned into the pET SUMO plasmid (Table 1; Invitrogen, Carlsbad, CA). The constructs were then introduced into competent One Shot Mach1 (T1 resistant) E. coli. Transformants were selected from colonies growing on Luria plates with 50 μg/ml kanamycin and validated via PCR. Plasmid DNA was isolated with spin columns (Qiagen, Germantown, MD) and transformed into BL21(DE3) One Shot E. coli for expression. After verifying the gene presence with PCR, these E. coli cells were cultivated in Luria broth supplemented with 1% glucose and 50 μg/ml kanamycin. To induce gene expression in E. coli, once cultures reached an optical density at 600 nm (OD600) of ∼0.3, isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and the cultures were incubated overnight (room temperature, 100 rpm), harvested the next day (10,000 × g, 5 min, 4°C), and flash frozen with liquid nitrogen. To verify target gene expression, samples from each E. coli culture were analyzed via SDS-PAGE (26), followed by Western blotting using antisera directed against the polyhistidine tag added to the amino terminus of the proteins when they are expressed from the pET-SUMO vector (anti-His G-alkaline phosphatase-conjugated antibody; Invitrogen).
Primers and probes used to target T. crunogena carbonic anhydrase genes
Once transgenic CA gene expression was confirmed, CA was purified from these frozen E. coli cells. Frozen cell paste was resuspended in Ni column loading buffer (20 mM Na2HPO4, pH 7.4, 0.5 M NaCl, 20 mM imidazole) and sonicated on ice using acid-washed glass beads (≤106 μm; Sigma, St. Louis, MO) with 15-s blasts (sonic dismembrator; Fisher Scientific, Pittsburgh, PA) until cell extract viscosity was reduced. Samples were then centrifuged (10,000 × g, 15 min, 4°C) and injected into a nickel column (HisTrap FF, 25 ml; GE Healthcare, Waukesha, WI) equilibrated with Ni column loading buffer. After the column was washed with ∼100 ml loading buffer, His-tagged proteins were eluted with elution buffer (20 mM Na2HPO4, pH 7.4, 0.5 M NaCl, 0.5 M imidazole). Proteins were washed and desalted using Centricon Ultracel YM-10 membranes (Millipore, Billerica, MA), and their amino-terminal histidine tags were removed via SUMO protease according to the manufacturer's recommendations (Invitrogen, Carlsbad, CA).
To confirm the predicted periplasmic location of α-CA, two constructs with this enzyme were fabricated. In the first construct (alpha-sig), the 5′ PCR primer included the signal peptide, while for the second construct (alpha-nosig), the 5′ primer annealed immediately downstream of the predicted cleavage site (Table 1). α-CA gene expression and enzyme purification using the alpha-nosig construct were conducted as described above. Periplasmic, membrane, and whole-cell fractions were generated from E. coli cells carrying the alpha-sig construct. Since periplasm preparations are optimal on early exponential phase cells (21), the growth and induction parameters were modified from those used for the other constructs. Alpha-sig cells were grown to an OD600 of ∼0.1, induced with 1 mM IPTG, and incubated at room temperature while agitated (100 rpm) until an OD600 of 0.4 was reached. Cells were harvested by centrifugation (10,000 × g, 5 min, 4°C), and cells were resuspended to an OD600 of 10 in an ice-cold hyperosmotic solution (0.75 M sucrose, 10 mM HEPES [pH 7.5], 1 mM dithiothreitol [DTT]). Lysozyme was added to a final concentration of 0.2 mg/ml, and cells were incubated on ice for 2 min. Two volumes of ice-cold hypotonic solution (1.5 mM EDTA [pH 7.5], 1 mM DTT) was added slowly to rupture the outer membrane and release the periplasmic proteins (21). Protoplasts were removed by centrifugation (12,000 × g, 2 min, 4°C). To verify that periplasm preparations were not contaminated by cytoplasmic proteins released by lysed cells, malate dehydrogenase assays (15) were conducted with the samples. A membrane fraction and high-speed supernatant were also prepared from this construct by suspending intact cells in CA assay buffer (5 mM HEPES, pH 8.0, 0.1 mM ZnSO4) and sonicating them on ice as described above. Crude extract was centrifuged to remove intact cells (6,000 × g, 30 min, 4°C). The resulting low-speed supernatant was decanted and centrifuged at high-speed (75,000 × g, 90 min, 4°C) to pellet membranes. High-speed supernatant was decanted, and the membrane pellet was washed three times with CA assay buffer. Periplasm preparations were unsuccessful with freshly harvested T. crunogena cells.
CA assays and inhibition.Carbonic anhydrase activity was measured in samples from E. coli cells expressing T. crunogena CA genes, as well as T. crunogena cells. T. crunogena cells were resuspended in assay buffer (5 mM HEPES, pH 8.0, 0.1 mM ZnSO4) and sonicated on ice using acid-washed glass beads (≤106 μm; Sigma, St. Louis, MO) with three 15-s blasts (sonic dismembrator; Fisher Scientific, Pittsburgh, PA). Portions of 2 ml crude extract (T. crunogena) or appropriately diluted samples from expression constructs in E. coli were placed in a 4°C reaction vial with a stir bar and a pH electrode. Once the temperature was stable at 4°C, 1 ml ice-cold CO2-saturated distilled water was injected, and the pH was monitored (19). For reactions where inhibitors were used, 2-ml samples were stirred for 1 min after inhibitor addition, and then CO2-saturated distilled water was added and the pH was monitored as it fell. Bovine CA (1 μg/ml, final concentration), used as a positive control, was dissolved in 5 mM HEPES, pH 8.0, 0.1 mM ZnSO4. For a negative control, CA activity was measured in samples that had been autoclaved for 1 h and diluted to their original volume with distilled water. Units of activity [U = (tac − t)/t, where t and tac are the time (in seconds) required for the pH to decrease from 8.0 to 7.0 in the sample and autoclaved control, respectively] (5) were calculated and normalized for the protein concentration in the assay (U mg−1). For CA assays conducted with whole cells, T. crunogena was harvested via centrifugation (5,000 × g, 15 min, 4°C) and washed three times in assay buffer that was rendered isosmotic with growth medium by adding NaCl (5 mM HEPES, pH 8.0, 0.1 mM ZnSO4, 65 mM NaCl).
Transcription of CA genes in T. crunogena. Thiomicrospira crunogena cells were harvested by centrifugation (10,000 × g, 5 min, 4°C), flash frozen in liquid nitrogen, and stored at −80°C. RNA was isolated from low- and high-DIC cells by using the Ribopure system (Ambion, Austin, TX), and cDNA was reverse transcribed using the Improm II RT system (Promega, Madison, WI), with primers targeting the gene of interest (Table 1). TaqMan primers and probe for quantitative reverse transcriptase PCR (qRT-PCR) were designed using the Primer Express software program (Table 1) (ABI, Carlsbad, CA). qRT-PCR was carried out with custom TaqMan primers and probe, TaqMan universal master mix containing AmpliTaq Gold DNA polymerase, and the Step One real-time PCR system (ABI, Carlsbad, CA), using the following parameters as recommended by the manufacturer: a two-step holding stage (50°C for 2 min and 95°C for 10 min) and a two-step cycling stage (95°C for 15 s and 60°C for 1 min, 40 cycles).
To verify that amplification efficiencies were similar for primer/probe sets directed against the 16S rRNA gene (= calibrator) and target genes (α-CA, β-CA, and csoSCA genes), qPCR using these primer/probe sets was conducted on a serial dilution of template cDNA. In all cases, a plot of ΔCT (= CT target − CT calibrator, where CT is the qPCR cycle where fluorescence of the reaction has crossed the value considered to be baseline) versus log [template cDNA] had a slope close to zero, indicating that primer/probe amplification efficiencies were similar for all primer/probe sets (17).
To determine the concentration of primer/probe and template that resulted in CT values falling within 10 to 20 cycles, a dilution series of 16S primer/probe and cDNA concentrations was run. Based on the results of these experiments, 50 ng of cDNA template, 900 nM primers, and 250 nM probe were used. To verify that the 16S gene was expressed at the same level for both DIC- and ammonia-limited cells and was therefore suitable for use as a calibrator, the CT value for qPCR directed against 16S in cDNA from DIC- and ammonia-limited cells at a range of template concentrations was captured. No difference in CT values, and therefore 16S gene expression, was detected. The amount of 16S RNA in cDNAs from DIC- and ammonia-limited cells was found to be ∼40% of total cDNA used in each qPCR, as determined by quantifying 16S rRNA by creating a calibration curve with 16S rRNA generated with a MEGAscript T7 kit (Ambion, Austin, TX).
Fold differences in transcription between DIC- and ammonia-limited cells were calculated as 2− ΔΔCT, where ΔΔCT = [(CT target − CT calibrator) − (CT targetRef − CT calibratorRef)]; CT target and CT calibrator are the CT values for target and 16S amplification in ammonia-limited cells, and CT targetRef − CT calibratorRef are the corresponding values from DIC-limited cells (17).
Carbon fixation assays and inhibition.To measure the effects of CA inhibitors on DIC uptake and fixation, DIC- and ammonia-limited T. crunogena bacteria were cultivated in chemostats as described above, and a 350-ml portion of culture (OD600 of ∼0.1) was harvested by centrifugation (5,000 × g, 15 min, 4°C). Cells were washed 3× with ice-cold wash buffer (artificial seawater medium, buffered with 10 mM HEPES, pH 8) unsupplemented with thiosulfate or DIC, and resuspended to a final volume of 3 ml in wash buffer. This cell suspension was sparged with soda lime-scrubbed (CO2-free) air for 30 min to minimize the DIC concentration. A 20-μl portion of cell suspension was added to 1.98 ml TASW supplemented with radiolabeled DIC at a range of concentrations (0.07 to 11 mM for low-DIC cells; 0.47 to 43 mM for high-DIC cells; specific activity = 2 to 30 Ci/mol). For incubations with inhibitors, 20 μl of cell suspension was either added to TASW supplemented with acetazolamide (AZA) (250 μM) or ethoxyzolamide (EZA) (250 μM) to measure any immediate effects from these compounds or incubated on ice for 1 h in the presence of 250 μM inhibitor and added to TASW brought to this concentration of inhibitor as well. A 4-min time course was taken by injecting 0.4-ml portions of the incubation into 0.5 ml glacial acetic acid in scintillation vials at 1-min intervals. After allowing the 14C-labeled DIC to dissipate overnight, scintillation cocktail was added for quantification of the acid-stable 14C via scintillation counting.
To measure the effects of acetazolamide and ethoxyzolamide on DIC uptake, cell suspensions were prepared and sparged with CO2-free air as described above. Ten μl cell suspension was added to 200 μl TASW supplemented with 0.3 mM radiolabeled DIC (10 Ci/mol), layered on top of 65 μl silicone oil (Dow Chemical SF 1156) overlaying 20 μl killing solution (2:1 [vol:vol] 1 M glycine, pH 10, Triton) in a 0.6-ml microcentrifuge tube (8). After 30 s, these tubes were centrifuged for 40 s and flash frozen with liquid nitrogen. Cell pellets were clipped into scintillation vials primed with 50 μl β-phenethylamine and 3 ml scintillation cocktail (to measure fixed plus intracellular inorganic carbon) or 0.5 ml glacial acetic acid (to measure fixed, acid-stable carbon). After the pellets were clipped into glacial acetic acid, they were vigorously vortexed to disperse them, and 14CO2 was allowed to dissipate overnight before addition of scintillation cocktail. To estimate the intracellular volume, which is necessary for calculating the intracellular concentration of DIC, cells were also incubated with [14C]sorbitol (to measure periplasmic volume) and 3H2O (to measure periplasmic plus cytoplasmic volume) and centrifuged through silicone oil as described above. The cytoplasmic volume was calculated by subtracting the sorbitol-permeable space from the 3H2O-permeable space (8).
RESULTS
Expression of T. crunogena CA genes in E. coli.When the α-CA, β-CA, and csoSCA genes were expressed in E. coli, the proteins they encode were apparent via SDS-PAGE and Western blot analysis (Fig. 1), and CA activity was measurable (Table 2). No activity was detected when either the γ-CA-like gene or cbbM was expressed in E. coli.
SDS-PAGE (A) or Western blot (B) analysis of T. crunogena carbonic anhydrase enzymes heterologously expressed in E. coli. All were expressed with an amino-terminal polyhistidine tag (+ tag), which added 13 kDa to the molecular mass of each protein, and were subsequently cleaved with a protease to remove the tag. Primary antibody for the Western blot was directed against the polyhistidine tag.
Activity and inhibition of T. crunogena carbonic anhydrase enzymes heterologously expressed in E. coli
α-CA activity (from the “alpha-nosig” construct [Table 1; see also Materials and Methods]) was stable after purification and removal of the polyhistidine tag, while β-CA was active while polyhistidine tagged but precipitated and lost activity during SUMO protease treatment. CsoSCA activity was present in crude extract but did not survive purification (Table 2). Accordingly, enzyme inhibition was measured in crude cell extracts (CsoSCA), polyhistidine-tagged purified protein (β-CA), or purified, cleaved protein (α-CA).
α-CA activity was found to be sensitive to both ethoxyzolamide and acetazolamide (Table 2), with activity completely inhibited at ethoxyzolamide concentrations as low as 2.5 μM (data not shown). β-CA activity, however, was not inhibited either by ethoxyzolamide or acetazolamide; surprisingly, the presence of these compounds stimulated activity (Table 2). CsoSCA activity was low and was inhibited by both ethoxyzolamide and dithiothreitol (Table 2), similar to carboxysomal carbonic anhydrase activity from Halothiobacillus neapolitanus (11).
In E. coli, the cellular location of α-CA expressed from the alpha-sig construct, in which α-CA was cloned with its predicted signal peptide, was primarily periplasmic (119 Wilbur-Anderson units [W-A U]/mg protein), with a small amount associated with the membrane fraction (11 W-A U/mg protein). These activities are consistent with translocation to the periplasm via the Sec complex, since the Sec complex would translocate the bulk of the protein to the periplasm but leave it tethered to the membrane via the signal peptide; subsequent cleavage via signal peptidase would liberate the protein from the cell membrane (25, 34). Indeed, α-CA from the alpha-sig construct did not bind to the Ni column, which supports this model of N-terminal cleavage after translocation (which would also remove the poly-His tag).
CA activity was not detectable in the periplasm of E. coli cells expressing the γ-CA-like gene, precluding the possibility that the activity measured from the alpha-sig construct was due to native activity from E. coli. Malate dehydrogenase activity was undetectable in periplasm preparations from alpha-sig cells but high (5 μmol/min mg protein) in high-speed supernatant from lysed alpha-sig cells, suggesting that the periplasm preparations were free from cytoplasmic contamination.
Transcription of CA genes in T. crunogena.The transcription of csoSCA was particularly sensitive to the DIC concentration during growth; DIC-limited cells had substantially higher csoSCA RNA levels than ammonia-limited cells (173-fold increase; ΔΔCT = −7.4 ± 1.7 standard deviation [SD]; n = 3). Transcription levels of α-CA were similar for both (1.2-fold increase under DIC-limited conditions; ΔΔCT = −0.3 ± 1.8 SD; n = 3). DIC-limited cells may transcribe β-CA somewhat less than ammonia-limited cells do, but the differences were not statistically significant (0.4-fold under DIC-limited conditions; ΔΔCT = 1.4 ± 1.2 SD; n = 3).
CA activity and inhibition in DIC- and ammonia-limited T. crunogena.CA activity was measurable in crude extract from T. crunogena cells (Fig. 2). CA activity in crude extracts from DIC-limited T. crunogena (0.5 W-A U/mg protein) was completely inhibited by 250 μM ethoxyzolamide but not by dithiothreitol, consistent with this activity being due primarily to α-CA. CA activity was also detectable in cell extracts from ammonia-limited cells (total CA activity = 1.2 W-A U/mg protein). Similar to the case with DIC-limited cells, substantial inhibition by ethoxyzolamide and not DTT suggests that this activity is dominated by α-CA. For crude extract from ammonia-limited cells, some CA activity was resistant to both ethoxyolamide and dithiothreitol, consistent with the presence of β-CA activity (Fig. 2). CA activity in intact DIC- and ammonia-limited cells was completely inhibited by 250 μM acetazolamide (Fig. 2).
Carbonic anhydrase activity and inhibition in T. crunogena crude extract (A) or whole cells (B). Enzyme activity was measured in cell extracts and intact cells grown under DIC limitation (solid bars) or ammonia limitation (open bars). CA activity was measured in the presence and absence of dithiothreitol (DTT) (10 mM), ethoxyzolamide (EZA) (250 μM), and acetazolamide (AZA) (250 μM). Error bars indicate standard errors.
Effect of CA inhibitors on DIC uptake and fixation.For intact T. crunogena cells, ethoxyzolamide had a pronounced and immediate inhibitory effect on carbon fixation rates in both DIC- and ammonia-limited cells, while acetazolamide did not (Fig. 3). These lower carbon fixation rates do not appear to result from inhibition of DIC uptake, since the concentration of intracellular DIC was not measurably affected by ethoxyzolamide, though acetazolamide did exert a small effect (Fig. 4).
Effect of acetazolamide (AZA) and ethoxyzolamide (EZA) on carbon fixation by intact T. crunogena cells. Inhibitors (250 μM) were added to intact cell suspensions, and carbon fixation was measured immediately or after incubation on ice for 1 h. (A) Cells grown under DIC limitation (DIC during growth, ∼0.1 mM). (B) Cells grown under ammonia limitation (DIC during growth, ∼60 mM). The x axes in the figure differ, and the y axes are identical. The standard deviations of the carbon fixation rates are too small to be visible as error bars.
Effects of thiosulfate (TS), 250 μM acetazolamide (AZA), and ethoxyzolamide (EZA) on the size of the intracellular DIC pool and carbon fixation in 20-s incubations of intact DIC-limited (A) or ammonia-limited (B) T. crunogena cells. Thiosulfate was present for cells incubated with AZA and EZA. Extracellular [DIC] = 0.3 mM. Error bars indicate standard errors, and “*” and “#” indicate nonoverlapping 95% confidence intervals for the intracellular DIC concentrations and carbon fixed, respectively, compared to results for “40 mM TS.”
DISCUSSION
Of the four CA homologs present in the T. crunogena genome, three are demonstrated here to encode functioning CA enzymes. DIC-limited cells transcribe csoSCA more actively than ammonia-limited cells do, while α- and β-CA transcription is insensitive to the conditions tested here. All lines of evidence strongly suggest a periplasmic location for α-CA. Thus far, it appears that only the CsoSCA enzyme is implicated in DIC uptake or fixation. Τranscription levels for csoSCA are greatly enhanced under DIC-limited conditions, consistent with its role in a carbon-concentrating mechanism. It appears that CsoSCA, which is sensitive to EZA (Table 2), plays a role in carbon fixation but not DIC uptake, since EZA did not inhibit DIC uptake in intact cells, though it did affect the rate of carbon fixation (Fig. 4). The greater degree of inhibition of carbon fixation by EZA in DIC-limited T. crunogena cells than in ammonia-limited cells (40% inhibition and 23% inhibition, respectively) may be due to CsoSCA playing more of a role in DIC-limited cells, which is consistent with enhanced transcription of csoSCA and other carboxysomal genes (data not shown) under DIC-limited conditions.
In contrast to csoSCA, the α-CA gene is equally transcribed in DIC- and ammonia-limited cells, and the α-CA enzyme has the dominant carbonic anhydrase activity measureable in crude extracts from these cells, since CA activity in crude extracts was strongly inhibited by EZA, which targets α-CA and CsoSCA, but not by DTT, which inhibits CsoSCA. Whole-cell potentiometric assays, as well as assays from cell fractions from the alpha-sig construct, confirm the location of α-CA to be periplasmic, due to CA inhibition by AZA (Fig. 2), which is relatively membrane nonpermeating (23). Unlike R. palustris, whose periplasmic α-CA functions to convert bicarbonate to CO2, which facilitates CO2 diffusion into the cytoplasm (22), in T. crunogena, α-CA does not appear to play a major role in DIC uptake, since incubating cells in the presence of AZA, which inhibits α-CA, has only a minor effect on the size of the intracellular DIC pool and carbon fixation (Fig. 3 and 4).
The possibility that α-CA does not play a major role in DIC uptake and fixation is also supported by the genomic context of the α-CA gene; in T. crunogena, the α-CA gene is not located in the genome near any other genes whose products are involved in carbon fixation (29). A possible role in pH homeostasis, as seen for α-CA in Helicobacter pylori (6), is unlikely in T. crunogena, since the α-CA gene is transcribed at similar levels in cells grown at pHs 6.5 and 8 (data not shown). Another role might be to “trap” CO2 that is diffusing out of T. crunogena cells. Perhaps the α-CA may be converting periplasmic CO2 into bicarbonate, which is then transported into the cell. However, such a role is not supported by the high rates of DIC uptake and fixation measured for T. crungena incubated in the presence of AZA. The role α-CA plays in T. crunogena is elusive and merits further investigation.
In similarity to α-CA, it does not appear that β-CA plays a role in DIC uptake in T. crunogena cells. β-CA is transcribed similarly in DIC- and ammonia-limited cells; if it were a necessary part of the T. crunogena CCM, it would be expected to be transcribed more frequently in DIC-limited cells. Instead, β-CA activity may be higher in ammonia-limited cells, since 15% of CA activity is resistant to EZA in crude extract from ammonia-limited cells while no CA activity is apparent in crude extract from DIC-limited cells treated with this inhibitor (Fig. 2). A role for β-CA in ammonia-limited (high-DIC) cells is suggested by its genome context, since the β-CA gene is directly downstream from the gene encoding a form II RubisCO (29). The β-CA gene may be transcribed slightly more frequently under ammonia-limited (high-DIC) conditions, consistent with what has been noted for Hydrogenovibrio marinus (35). Perhaps cytoplasmic β-CA maintains intracellular DIC at near-chemical equilibrium, which would ensure that the form II RubisCO present under these conditions, which is likely to have a low affinity for CO2 (33), would still be able to meet the organic carbon demands of the cell.
T. crunogena has three distinct CA enzymes, and it is clear that CsoSCA is associated with carboxysomes and plays a major role in carbon fixation, particularly in DIC-limited cells. However, carbon fixation is affected in both DIC- and ammonia-limited cells by EZA, which is consistent with CsoSCA being associated with carbon fixation in both (Fig. 3). The csoSCA gene has been shown to be transcribed in both DIC- and ammonia-limited cells independently of other genes contained in the carboxysome operon (data not shown). Indeed, a core promoter is present immediately upstream from this gene, indicating that the csoSCA gene may be independently regulated from the other carboxysome genes.
Both the α-CA and β-CA genes appear to be constitutively expressed; the pH does not affect the level of transcription of these genes, and the DIC concentration has at most a minor (β) or no (α) apparent effect. Neither appears to play a role in DIC uptake and fixation. Knockout mutations of all three CAs are currently underway for a better understanding of the role α-CA and β-CA fulfill in T. crunogena. The functioning of α- and β-CA in this organism warrants further study to uncover their roles in facilitating the survival of this deep-sea microorganism.
ACKNOWLEDGMENTS
We are extremely grateful to the National Science Foundation for their support of this project (NSF-MCB-0643713 to K.M.S.).
We thank the anonymous reviewers for their suggestions.
FOOTNOTES
- Received 9 January 2010.
- Accepted 5 April 2010.
- Copyright © 2010 American Society for Microbiology
