Inhibitory Effects of C₂ to C₁₀ 1-Alkynes on Ammonia Oxidation in Two Nitrososphaera Species

Abstract

A previous study showed that ammonia oxidation by the Thaumarchaeota Nitrosopumilus maritimus (group 1.1a) was resistant to concentrations of the C₈ 1-alkyne, octyne, which completely inhibits activity by ammonia-oxidizing bacteria. In this study, the inhibitory effects of octyne and other C₂ to C₁₀ 1-alkynes were evaluated on the nitrite production activity of two pure culture isolates from Thaumarchaeota group 1.1b, Nitrososphaera viennensis strain EN76 and Nitrososphaera gargensis. Both N. viennensis and N. gargensis were insensitive to concentrations of octyne that cause complete and irreversible inactivation of nitrite production by ammonia-oxidizing bacteria. However, octyne concentrations (≥20 μM) that did not inhibit N. maritimus partially inhibited nitrite production in N. viennensis and N. gargensis in a manner that did not show the characteristics of irreversible inactivation. In contrast to previous studies with an ammonia-oxidizing bacterium, Nitrosomonas europaea, octyne inhibition of N. viennensis was: (i) fully and immediately reversible, (ii) not competitive with NH₄⁺, and (iii) without effect on the competitive interaction between NH₄⁺ and acetylene. Both N. viennensis and N. gargensis demonstrated the same overall trend in regard to 1-alkyne inhibition as previously observed for N. maritimus, being highly sensitive to ≤C₅ alkynes and more resistant to longer-chain length alkynes. Reproducible differences were observed among N. maritimus, N. viennensis, and N. gargensis in regard to the extent of their resistance/sensitivity to C₆ and C₇ 1-alkynes, which may indicate differences in the ammonia monooxygenase binding and catalytic site(s) among the Thaumarchaeota.

INTRODUCTION

With only a few isolates of ammonia-oxidizing archaea (AOA) available in pure/axenic culture, very little is known about the physiological and biochemical differences that might exist across the broad genotypic range of AOA (1–3) or how AOA physiology differs from that of ammonia-oxidizing bacteria (AOB). Although it is generally believed that both AOA and AOB initiate the oxidation of NH₃ with ammonia monooxygenase (AMO), there is evidence that suggests some aspects of NH₃ oxidation are probably different between them. For example, the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxide-3-oxyl (PTIO) inhibits NH₃ oxidation by AOA and has little effect on AOB activity (4–6), suggesting that the intermediary products of NH₃ oxidation might be fundamentally different in AOA and AOB. Furthermore, whereas NH₃-oxidizing activity in AOB is inhibited by low concentrations (0.4 to 10 μM) of the Cu chelator, allylthiourea (ATU) (4, 7–9), the ATU sensitivity of NH₃ oxidation by AOA is more variable. For example, NH₃-oxidizing activity of Nitrososphaera gargensis is only partially inhibited by 100 μM ATU (10), and 500 μM ATU is required to completely stop NH₃-oxidizing activity in Nitrososphaera viennensis strain EN76 (4). In contrast, NH₃ oxidation by an obligate acidophilic AOA, Nitrosotalea devanaterra, is completely inhibited by 50 to 100 μM ATU (11).

Recently, Taylor et al. (12) showed that NO₂⁻ production by the marine AOA, Nitrosopumilus maritimus, was unaffected by 20 μM aqueous concentration (C_aq) of C₈ and C₉ 1-alkynes, whereas NH₃-oxidizing activity of AOB is completely and quickly (≤30 min) inactivated by 1 to 5 μM C_aq of 1-alkynes with a chain length of C₂ to C₁₀ (13). A short-term assay, based upon the difference in sensitivity of AOA and AOB to 1-octyne, was subsequently developed to measure the contributions of AOA and AOB to soil nitrifying activity. With the availability of more AOA isolates in pure culture, we deemed it important to confirm if the octyne resistance properties of N. maritimus were similar among other AOA isolates, and particularly among the group 1.1b lineage of Thaumarchaeota, whose members are widely distributed in terrestrial environments (2, 14). We report here the results of studies carried out to assess the effects of 1-alkynes of various chain lengths on the NH₃-oxidizing activity of two axenic culture isolates of AOA from group 1.1b, namely, Nitrososphaera viennensis strain EN76 (15, 16) and Nitrososphaera gargensis (1, 10). Both strains showed similar, albeit not identical 1-alkyne chain length inhibition profiles to N. maritimus. However, both strains were partially inhibited by ≥20 μM octyne, which led to additional studies to compare the inhibitory properties of octyne with those of the more extensively studied alkyne, acetylene.

MATERIALS AND METHODS

Chemicals.

NH₄Cl and linear aliphatic 1-alkynes (C₃ to C₁₀, ≥97%) were obtained from Sigma-Aldrich (St. Louis, MO), and acetylene (C₂H₂) was obtained from Airgas (Radnor, PA).

Cultures.

Nitrososphaera viennensis strain EN76, here referred to as N. viennensis, was cultivated at its optimal growth temperature of 42°C in a freshwater medium (FWM) (16, 17), supplemented with 2 mM NH₄⁺ and 1 mM pyruvate. Growth was routinely followed by measuring NO₂⁻ production as described previously (12). Nitrososphaera gargensis has now been obtained into pure culture (M. Palatinszky et al., unpublished data) and was cultivated at its optimal growth temperature of 46°C in a carbonate media (18) supplemented with 2 mM NH₄⁺. During routine growth of N. gargensis, further additions of 2 mM NH₄⁺ were made after NH₄⁺ was consumed. Cultures that had consumed a total of 8 to 10 mM NH₄⁺ were used for experimental purposes. The excess insoluble carbonate in the N. gargensis medium provides: (i) buffering against the acidification that occurs with 10 mM NO₂⁻ production and (ii) is thought to provide a growth-promoting surface for N. gargensis. The presence of the carbonate provides challenges during experimental manipulations. For example, harvesting and concentration of the culture leads to a dense suspension of carbonate that settles immediately upon onset of static conditions. This phenomenon makes it difficult to dispense uniform aliquots of cells to experimental replicates. Because of the challenges posed by the excess carbonate in the media, only a subset of the experiments were performed with N. gargensis. As expected, the N. gargensis cultures grown with 8 to 10 mM NH₄⁺ yielded higher protein concentrations (132.6 ± 21.0 μg of protein/ml of culture) than the N. viennensis cultures grown with 2 mM NH₄⁺ (9.0 ± 0.3 μg of protein/ml of culture). However, the N. gargensis cultures had much lower rates of NO₂⁻ production when expressed on a milligram protein basis. Protein concentrations were determined by using a Pierce BCA protein assay kit (Thermo Scientific, Waltham, MA) as described by the manufacturer.

Octyne sensitivity.

Octyne stocks were prepared as described previously (12). N. viennensis was cultivated at 42°C in 200-ml volumes of FWM. After accumulation of 1.8 to 2 mM NO₂⁻, the cultures were harvested by centrifugation (10,000 × g, 15 min) and concentrated 100-fold by resuspension in 2 ml of fresh FWM supplemented with 1 mM pyruvate but minus NH₄⁺. N. gargensis was cultivated at 46°C in 200 ml of carbonate medium supplemented with a total addition of 8 to 10 mM NH₄⁺, harvested by centrifugation (10,000 × g, 15 min), and concentrated 100-fold by resuspension in 2 ml of fresh carbonate medium minus NH₄⁺. Aliquots (100 μl) of N. viennensis or N. gargensis cell suspensions were added to 10-ml portions of their respective growth media supplemented with 1 mM NH₄⁺ that had been pre-equilibrated with octyne (0, 10, 20, or 40 μM) for 30 min at either 42 or 46°C for N. viennensis and N. gargensis, respectively. Bottles were incubated as described above and sampled periodically over 6 h to quantify the rate of accumulation of NO₂⁻. Additional incubations were carried out with N. viennensis at two different temperatures (35 and 46°C) to evaluate the sensitivity of NO₂⁻ production to 10 μM octyne. An experiment was performed at 42°C in the presence or absence of 1 mM pyruvate to determine whether the latter influenced the rate and sensitivity of NO₂⁻ production to 10 μM octyne.

Sensitivity of AOA isolates to C₂ to C₁₀ alkynes.

A preliminary experiment was conducted in bottles containing alkynes pre-equilibrated at 30, 42, and 46°C. Headspace alkyne concentrations were monitored by using gas chromatography. There was no significant difference in headspace alkyne concentrations between bottles incubated across this temperature range, indicating that temperature shifts over this magnitude did not change the gas/aqueous phase-partitioning in the assay bottles sufficient to modify the aqueous concentration (C_aq) of alkynes. N. viennensis and N. gargensis were grown and harvested as described above, and 100-fold-concentrated cell aliquots (100 μl) were added to 10-ml portions of their respective growth media supplemented with 1 mM NH₄⁺ that had been pre-equilibrated for 30 min with 10 μM C_aq of each alkyne (C₂ to C₁₀) in 125-ml bottles sealed with gray butyl stoppers (12). This specific alkyne concentration was chosen after it had been determined that 10 μM octyne had no effect on NH₃ oxidation by either N. viennensis or N. gargensis but was previously shown to be more than sufficient to inactivate AOB (12). Bottles were incubated at 42 or 46°C for N. viennensis and N. gargensis, respectively, and NO₂⁻ accumulation monitored over 8 h.

Characteristics of acetylene and octyne inhibition of NO₂⁻ production by N. viennensis. (i) Kinetic relationships between NH₄⁺ and alkyne inhibition of N. viennensis.

Aliquots of a N. viennensis cell suspension were exposed to different concentrations of either octyne (30 or 40 μM) or acetylene (1, 3, or 6 μM) in the presence of various concentrations of NH₄⁺ (0.01 to 2 mM) at 42°C. Initial rates of NO₂⁻ production were determined by sampling every 15 min for a total of 90 to 120 min, and the data were plotted in double-reciprocal Lineweaver-Burk plots.

(ii) Examining the influence of octyne on the relationship between NH₄⁺ concentration and acetylene on the inhibition of NO₂⁻ production by N. viennensis.

Aliquots of N. viennensis were exposed to 30 μM octyne with increasing concentrations of NH₄⁺ (0.25 to 2 mM) for 15 min, followed by the addition of 1 μM acetylene. NO₂⁻ accumulation was evaluated at 30-min intervals for 120 min at 42°C, and analysis of variance (ANOVA) of rates determined differences in response to acetylene, octyne, or the combination of acetylene and octyne. Controls included treatments without alkyne and a treatment containing 1 μM acetylene minus octyne.

(iii) Time course of recovery of NO₂⁻ production in N. viennensis following inhibition by acetylene or octyne.

Aliquots of a N. viennensis cell suspension were exposed to either 40 μM octyne or 6 μM acetylene with an initial NH₄⁺ concentration of 0.5 mM for 2 h, along with a minus-alkyne control also incubated for 2 h. This concentration of NH₄⁺ was chosen to minimize the potential for competition with acetylene. After the alkyne exposure period, bottles were degassed to remove the alkynes (12). Additional NH₄⁺ was added to increase the concentration to 2 mM for optimal growth conditions, and the recovery of NO₂⁻ production was monitored by sampling at 60-min intervals over 4 h.

(iv) Hydroxylamine-dependent NO₂⁻ production by acetylene- and octyne-treated N. viennensis.

It is well established that hydroxylamine-dependent NO₂⁻ production is unaffected by alkyne inactivation of AMO in AOB and has been used on several occasions to infer that alkynes specifically target AMO and have no effect on downstream enzymes in the NO₂⁻ production pathway (19, 20). Aliquots of N. viennensis cell suspensions were exposed to 18 μM acetylene or 40 μM octyne, with 2 mM NH₄⁺, and a range of hydroxylamine concentrations (20 to 400 μM), and evidence for hydroxylamine-dependent NO₂⁻ accumulation was sought by incubation at 42°C and sampling at 15-min intervals for 2 h. Abiotic controls containing only growth medium plus hydroxylamine were included. We were unable to measure significant hydroxylamine-dependent NO₂⁻ production in octyne-inhibited or acetylene-inactivated cultures (P > 0.3). Furthermore, hydroxylamine-dependent NO₂⁻ production could not be measured in N. viennensis cultures immediately after total depletion of NH₄⁺ and untreated with either acetylene or octyne.

Statistics.

SigmaPlot 12.5 (Systat Software, Inc., San Jose, CA) was used to perform statistical analyses. ANOVA was used to determine whether the differences in the mean values among the treatment groups were greater than would be expected by chance. If there was a statistically significant difference the Holm-Sidak method of all pairwise multiple-comparison procedure was used to isolate the group or groups that differed from the others. A nonlinear regression procedure was used to estimate N. viennensis K_m for NH₄⁺ in the presence of 1 μM acetylene. The best-fit slopes of the relationship between NO₂⁻ production and NH₄⁺ concentration in response to the presence of acetylene or octyne were estimated by linear regression of double-reciprocal plots.

RESULTS

Sensitivity of N. viennensis and N. gargensis to octyne.

Initial work focused on the response of NO₂⁻ production by N. viennensis and N. gargensis to different concentrations of octyne (C₈). The rate of NO₂⁻ production by N. viennensis in the presence of 10 μM octyne was not significantly different (P > 0.05) than the rate in the no-alkyne control (175 ± 20 nmol/min/mg of protein, Fig. 1A) at the optimal growth temperature of 42°C. However, 20 and 40 μM octyne partially inhibited NO₂⁻ accumulation to 58 and 33%, respectively, of the activity of the no-alkyne control. Initial rates of NO₂⁻ production (0 to 6 h) by N. viennensis were also insensitive to 10 μM octyne at 35 or 46°C (P ≥ 0.1), and the presence or absence of 1 mM pyruvate had no effect on the inhibitory effects of octyne (P ≥ 0.1). The rate of NO₂⁻ production by N. gargensis was not affected by the presence of 10 μM octyne (P > 0.1) compared to the rate in the no-alkyne control (3.3 ± 1.1 nmol/min/mg of protein, Fig. 1B). As with N. viennensis, NO₂⁻ accumulation was partially inhibited by 20 and 40 μM octyne to 56 and 29%, respectively, of the activity of the no-alkyne control. In the case of both AOA isolates, NO₂⁻ production in the presence of 20 and 40 μM octyne continued at a constant rate for at least 24 h (data not shown), suggesting that octyne was not behaving as a turnover-dependent irreversible inactivator of AMO. Octyne concentrations in the headspace of the biological treatments at 24 h were no different than in abiotic controls (P > 0.17), providing no evidence of substantial consumption by the AOA. In addition, gas chromatographic analysis provided no evidence of trace contamination of octyne by other volatile compounds that might explain partial inhibition by ≥20 μM octyne.

FIG 1.

Response of NO₂⁻ production by N. viennensis (A) and N. gargensis (B) to different aqueous concentrations of octyne (Oct). Cell suspensions were added to their respective media pre-equilibrated with Oct in the presence of 1 mM NH₄⁺. Bottles were incubated at 42 or 46°C for N. viennensis and N. gargensis, respectively, and the accumulation of NO₂⁻ was monitored. Experiments were repeated three times (in triplicate) with similar results. Error bars represent the standard deviations of three replicate bottles of each treatment.

Sensitivity of AOA isolates to C₂ to C₁₀ alkynes.

To determine whether there was differential sensitivity to alkynes of various chain lengths, we compared the NO₂⁻ production response of N. viennensis and N. gargensis to C₂ to C₁₀ 1-alkynes in the presence of 1 mM NH₄⁺ (Fig. 2). There was a significant decrease in NO₂⁻ production compared to the no-alkyne control when N. viennensis was exposed to 10 μM C₂ to C₅ and C₇ 1-alkynes (P < 0.05), but there was no significant difference (P > 0.05) in NO₂⁻ accumulation when exposed to C₆, and C₈ to C₁₀ alkynes. The reduced rate of NO₂⁻ production by N. viennensis in the presence of 10 μM C₇ (69% ± 1% of control) continued at the same rate for at least 24 h, showing no sign of a time-dependent reduction of the rate as would be anticipated if C₇ was behaving as an inactivator of NO₂⁻ production (data not shown). Although 10 μM C₇ was inhibitory, 2.5 and 5.0 μM C₇ did not have a significant effect on the rates of NO₂⁻ accumulation (P > 0.07). Furthermore, as described above for octyne, there was no evidence for substantial consumption of C₇ in the N. viennensis treatments after 24 h inhibition (P > 0.15), and no evidence of contamination of C₇ by other alkynes that might explain partial inhibition. In the case of N. gargensis, there was a significant inhibitory effect of 10 μM C₂ to C₅ but no significant effect of C₆ to C₁₀ alkynes on NO₂⁻ accumulation (P > 0.05). In comparison, our previous work had also shown a significant inhibitory effect of C₂ to C₅ 1-alkynes on NO₂⁻ production by the marine AOA N. maritimus whereas 20 μM C₆ and C₇ 1-alkynes were partially inhibitory (56% ± 2% and 58% ± 4%, respectively), and 20 μM C₈ and C₉ 1-alkynes had no effect (12).

FIG 2.

Response of NO₂⁻ production by N. viennensis and N. gargensis when exposed to C₂ to C₁₀ 1-alkynes. Cultures were harvested and aliquots of cell suspensions were added to treatments containing their respective media with 10 μM C_aq of C₂ to C₁₀ alkyne and 1 mM NH₄⁺. N. viennensis and N. gargensis were incubated at 42 and 46°C, respectively, and NO₂⁻ accumulation was measured. Experiments were repeated at least three times with similar results. Bars represent the average activity in three replicate treatments compared to the no-alkyne control. Error bars represent the standard deviations of the average.

Characteristics of acetylene and octyne inhibition of NO₂⁻ production by N. viennensis.

Because NO₂⁻ production rates in both strains were partially inhibited by ≥20 μM octyne but showed no sign of time-dependent rate reduction, several experiments were conducted to compare in more detail the characteristics of acetylene and octyne inhibition in N. viennensis.

(i) Kinetic relationships between NH₄⁺ and alkyne inhibition of N. viennensis.

The first experiment compared the concentration-dependent inhibition of NO₂⁻ production by acetylene and octyne. In the absence of acetylene or octyne the rate of NO₂⁻ production by N. viennensis was saturated with NH₄⁺ concentrations from 0.01 to 2.0 mM (Fig. 3A). In the presence of 1 μM acetylene and 0.25 mM NH₄⁺, the initial rate of NO₂⁻ production (V_i) was inhibited to 47% of the control, but the inhibition could be completely overcome by increasing the NH₄⁺ concentration to 1 mM (Fig. 3A). When acetylene concentrations were increased to 3 and 6 μM, inhibition could not be completely overcome by increasing the NH₄⁺ concentration to 2 mM. These results suggest that acetylene and NH₄⁺ are competitive substrates for binding at the active site. Although V_max was achieved in the presence of 1 μM acetylene by raising the NH₄⁺ concentration, the apparent K_m increased from ≤0.01 mM to 0.55 ± 0.08 mM NH₄⁺. Although there are no published values of the K_s for NH₄⁺/NH₃ in these particular AOA isolates, a maximum velocity of NO₂⁻ production was achieved with 10 μM NH₄⁺/NH₃ in the absence of acetylene (Fig. 3A). When the responses of the rates of NO₂⁻ production to a range of acetylene and NH₄⁺ concentrations were plotted in a double-reciprocal plot (Fig. 3B), the lines representing each inhibitor concentration had a different slope, also diagnostic of competitive inhibition. In contrast, there was no significant effect of NH₄⁺ concentration (P > 0.07) on V_i at any inhibitory octyne concentration, indicating that octyne inhibition could not be overcome with the addition of higher concentrations of NH₄⁺ (Fig. 3C). When the responses of NO₂⁻ production to a range of octyne and NH₄⁺ concentrations were plotted in a double-reciprocal plot the intercepts on the y axis are distinctively different (Fig. 3D). This result would be expected if inhibition by octyne was noncompetitive with respect to NH₄⁺.

FIG 3.

Response of initial velocities of NO₂⁻ production by N. viennensis to acetylene (A and B) and octyne (C and D) at increasing concentrations of NH₄⁺. (A) Inhibition by 1 μM acetylene is overcome by increasing the NH₄⁺ concentration. (B) Lineweaver-Burk double-reciprocal plot shows that the slopes for each acetylene concentration differ, indicating competitive inhibition. (C) V_i decreases with higher octyne concentrations, but octyne inhibition cannot be overcome by increasing NH₄⁺. (D) Lineweaver-Burk double-reciprocal plots intercept the 1/V₀ axis at different values indicating noncompetitive inhibition. Error bars represent the standard deviations of three replicates.

(ii) Examining the influence of octyne on the relationship between NH₄⁺ concentration and acetylene on the inhibition of NO₂⁻ production by N. viennensis.

An additional experiment was conducted to confirm that the mechanisms of octyne and acetylene inhibition differed. We reasoned that if octyne does not interact at the same site as acetylene it should not affect the competitive behavior between acetylene and NH₄⁺. N. viennensis was treated with octyne in some treatments before adding acetylene (Table 1). In the no-alkyne control V_max was saturated by all NH₄⁺ concentrations (0.25 to 2 mM NH₄⁺, P > 0.3). When N. viennensis was treated with 1 μM acetylene, there was a statistically significant effect of NH₄⁺ concentration on V_max (P ≤ 0.005), as shown earlier (Fig. 3). In contrast, the addition of 30 μM octyne decreased V_max (P < 0.01), but there was no significant protective effect of increasing NH₄⁺ concentration on the rate of NO₂⁻ production (0.25 to 2 mM NH₄⁺, P > 0.3). When N. viennensis was treated with octyne prior to acetylene, there was a statistically significant protective effect of increasing NH₄⁺ concentrations on the rate of octyne-insensitive, acetylene-sensitive NO₂⁻ production (P ≤ 0.005), indicating that the competitive behavior between NH₄⁺ and acetylene was not modified by the presence of octyne. This result gives additional support, suggesting that octyne is not functioning akin to acetylene as a competitive inhibitor of NH₄⁺/NH₃ oxidation.

TABLE 1.

Comparison of the response of rates of NO₂⁻ production by N. viennensis in a minus-alkyne control to treatments exposed to 1 μM acetylene, 30 μM octyne, or a combination of acetylene and octyne at different concentrations of NH₄^+a

NH4+ concn (mM)	NO2− production (nmol/ml/h) after exposure to various alkyne treatments
	Control	Acetylene	Octyne	Acetylene + octyne
2.0	13.1 (1.9)A	11.5 (2.5)A	6.5 (2.6)A	6.7 (1.1)A
1.0	15.5 (3.8)A	11.7 (2.4)A	4.9 (1.3)A	4.9 (0.6)A
0.5	14.0 (2.3)A	7.2 (1.7)AB	5.1 (1.2)A	1.6 (0.7)B
0.25	16.9 (0.8)A	4.1 (1.3)B	4.9 (1.5)A	1.0 (0.3)B

^aThe acetylene-octyne samples were exposed to 30 μM octyne for 15 min before the addition of 1 μM acetylene. Values in parentheses represent the standard deviations of three replicates. Different superscript letters indicate significant differences (P ≤ 0.03) between different NH₄⁺ concentrations within a specific alkyne treatment. Values with superscript letters in common are not significantly different.

(iii) Time course of recovery of NO₂⁻ production in N. viennensis following inhibition by acetylene or octyne.

It is known that acetylene is an irreversible inactivator of AMO of AOA (12, 21) and AOB (22). Recovery from inactivation requires new AMO protein synthesis resulting in a lag of several hours for resumption of maximum NO₂⁻ production. Previously, Vajrala et al. (21) showed that the eukaryotic protein synthesis inhibitor, cycloheximide, prevented recovery of NO₂⁻ production in N. maritimus after acetylene inhibition. In the case of N. viennensis, the same concentration range of cycloheximide slowed but did not prevent recovery of NO₂⁻ production after inactivation with acetylene (data not shown). The lack of an unambiguous protein synthesis inhibitor for N. viennensis made us reliant upon the comparative time course of recovery of NO₂⁻ production after exposure to acetylene or octyne. After exposure of N. viennensis to either 6 μM acetylene or 40 μM octyne, NO₂⁻ production was inhibited to 0 or 21%, respectively, of the control (Fig. 4). After the alkynes were removed by degassing, the initial rates of NO₂⁻ production during the first hour of the recovery period averaged 32% (acetylene) and 96% (octyne) of the no-alkyne control, respectively, indicating that the majority of octyne inhibition was quickly reversible. In the case of acetylene, 3 h were required for the NO₂⁻ production rate to reach the rate of the control treatment. A “starved” control incubated without NH₄⁺ for 2 h immediately accumulated NO₂⁻ at the same rate as the minus alkyne control upon the addition of NH₄⁺ (data not shown).

FIG 4.

Response of NO₂⁻ production by N. viennensis after acetylene or octyne exposure. Cells were exposed for 2 h to either 6 μM acetylene or 40 μM octyne in media with 0.5 mM NH₄⁺. Alkyne and no-alkyne treatments were then degassed and NH₄⁺ increased to 2 mM. NO₂⁻ accumulation was monitored after alkyne exposure for 4 h. Bars represent the average activity in three replicate treatments. Error bars represent the standard deviations of the average. Lowercase letters indicate the significance (P ≤ 0.02) of sampling time within a specific treatment. Values that have lowercase letters in common are not significantly different.

DISCUSSION

In an earlier study, we showed that NO₂⁻ production by the marine AOA, N. maritimus (Thaumarchaeota group 1.1a) was very resistant to inhibition by ≥C₇ 1-alkynes in comparison to the AOB, N. europaea and Nitrosospira multiformis (12). In the present study, we confirm that NO₂⁻ production by pure culture isolates of N. viennensis strain EN76 (15, 16) and N. gargensis (10), both members of Thaumarchaeota group 1.1b, show the same overall trend, being highly sensitive to ≤C₅ and much more resistant to longer-chain-length alkynes. They are also insensitive to concentrations of the C₈ alkyne, octyne, that cause complete and irreversible inactivation of NH₃ oxidation in AOB. These observations provide direct evidence that octyne-resistant NO₂⁻ production is a property of members of the phylogenetically distinct Thaumarchaeota group 1.1b and strengthens the idea that octyne might be exploited to differentiate between the relative activities of AOA and AOB in different natural environments where groups 1.1a and 1.1b are found.

Our observations that higher concentrations of octyne partially inhibit AOA activity in a manner that is (i) time independent, (ii) fully reversible, (iii) not competitive with NH₄⁺, and (iv) without effect on the competitive interaction between NH₄⁺ and acetylene raises the possibility that kinetic and catalytic properties of AMO from AOA and AOB might differ. For example, the observation that octyne inhibition of NO₂⁻ production in N. viennensis is noncompetitive with respect to NH₄⁺ concentration (23), suggests that octyne binds to an alternate (non-NH₃) binding site on AMO. This observation relates to earlier kinetic work of Keener et al. (24), who showed that, whereas NH₄⁺ and acetylene competed for the same binding site on AMO of N. europaea, NH₄⁺ concentration did not affect the rate of inactivation of N. europaea AMO by either 1- or 3-hexyne. These authors suggested that N. europaea AMO contained different binding sites or different access channels to the active site for small substrates (e.g., NH₃, CH₄, and acetylene) versus larger substrates (e.g., 1- and 3-hexyne). Indeed, Keener and Arp (25) also showed that most alternate substrates for N. europaea AMO are noncompetitive inhibitors of NH₃ oxidation. The specific reason(s) why octyne inhibits rather than inactivates AOA AMO are unknown and require further study.

Our data on the inhibitory properties of alkynes of different chain length on AOA AMO are worthy of discussion in context with the topic of substrate range of the membrane-bound, Cu-containing methane monooxygenase (pMMO). Whereas it makes intuitive sense to compare characteristics of AMO from AOA and AOB, a suggestion has been made recently that AOA amo sequences are more closely related to the particulate methane monooxygenase (pmo) of the Verrucomicrobia phylum than to AOB amo sequences (26). It has been known for many years that pMMOs of the well-studied methanotrophs, Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), exhibit a hydrocarbon substrate range that extends beyond CH₄ and yet, are restricted to short-chain alkanes (C₂ to C₅) and alkenes (C₂ to C₄) (27–29). It has been suggested that the chain-length-limited substrate range of pMMO is due to the active site of the enzyme being located in a hydrophobic pocket that cannot accommodate alkanes of >C₅, and there is speculation that the pocket might have separate binding sites for CH₄ versus the larger substrates (28–31). Perhaps, it is not unreasonable to link our own data with the above-mentioned properties of pMMO to suggest that AOA AMO might also contain a binding pocket resembling pMMO that is either too small to freely accommodate ≥C₆ 1-alkynes or constrained sufficiently to allow binding but does not allow turnover of the longer 1-alkynes. Clearly, since AMO of N. europaea oxidizes C₂ to C₈ alkanes and is also inactivated by C₂ to C₈ 1-alkynes, it is reasonable to conclude that the substrate range and catalytic properties of the two AMOs are different.

Furthermore, the reproducible differences in alkyne-chain-length sensitivity among the three isolates of Thaumarchaeota may also indicate differences in the properties of the AMO among the AOA. Whether such differences have ecological significance awaits further study.

Acetylene specifically and irreversibly inactivates AMO of N. europaea and under that inactivated condition N. europaea demonstrates hydroxylamine-dependent NO₂⁻ production at a rate identical to NH₃-driven NO₂⁻ production (19, 20). N. maritimus also demonstrates hydroxylamine-dependent NO₂⁻ production when inactivated with acetylene (32), suggesting that acetylene inhibition is AMO specific, and led those authors to suggest that hydroxylamine is an intermediate in NH₄⁺ oxidation in AOA, as well as AOB. However, we cannot rule out that partial inhibition by octyne occurs at a site other than AMO since we were unable to measure hydroxylamine-dependent NO₂⁻ production in cultures of N. viennensis either partially inhibited by octyne, inactivated with acetylene, or under NH₄⁺-depleted conditions. Much remains to be resolved regarding the mechanism of NH₃/NH₄⁺ oxidation in AOA (33), and further work is needed to determine whether there are fundamental differences among the AOA.