Starvation Alters the Apparent Half-Saturation Constant for Methane in the Type II Methanotroph Methylocystis Strain LR1

Abstract

When cells of a type II methanotrophic bacterium (Methylocystis strain LR1) were starved of methane, both the K_m(app) and the V_max(app) for methane decreased. The specific affinity (a^o_s) remained nearly constant. Therefore, the decreased K_m(app) in starved cells was probably not an adjustment to better utilize low-methane concentrations.

Microbial oxidation of atmospheric methane (CH₄) takes place in most aerobic upland soils (5, 10). Because these soils exhibit a lower half-saturation constant [K_m(app)] for CH₄ than do pure cultures of methanotrophic bacteria, it has been postulated that the active bacteria are unknown species. These have been popularly dubbed “high-affinity” methane oxidizers (5, 10). Recently, a novel group of pmoA-like sequences was detected in several soils which oxidize atmospheric CH₄ (11, 12), and incubation of soils under ¹⁴CH₄ resulted in the labeling of signature phospholipid fatty acids which differed from those of known type II methanotrophs (18). It is therefore likely that as-yet-uncultured species are involved in atmospheric CH₄ uptake. However, it remains unknown whether atmospheric CH₄ oxidation is limited to particular species and whether these possess a specialized high-affinity CH₄ oxidation enzyme.

We previously demonstrated that high-affinity CH₄ oxidation is probably not limited to uncultured methanotrophic groups. We enriched a methane-oxidizing bacterium (strain LR1) from an organic soil and identified it based on 16S rDNA, pmoA, and mxaF gene sequences as a type II methanotrophic species of the Methylosinus/Methylocystis cluster (8). Mixed cultures containing strain LR1, when grown under <275 ppm volume CH₄, had a low K_m(app) for CH₄ (56 to 188 nM) similar to the value measured in soil. This increased to >1 μM when cells were grown under >1% CH₄. In the present study, we investigated the kinetics of the isolated bacterium (culture is available upon request). Instead of the time-consuming process of growing the organism under low CH₄ mixing ratios, we tested the effect of starving cells of CH₄.

Kinetics of strain LR1.

Culture was grown in liquid nitrate-mineral salts (NMS) medium (8) under 10% CH₄. Purity was controlled microscopically and by plating onto NMS agar, R2A agar, 10% strength Nutrient Agar, and 10% strength AC Broth (Difco). After 1 to 2 months, the culture was diluted to about 10⁹ cells ml⁻¹ with 0.5 mM phosphate buffer (pH 6.0), and 7.5-ml amounts were added to 13-ml serum vials. The vials were capped with sterile butyl rubber stoppers and incubated with gentle shaking (6 rpm) at 25°C without added CH₄. After 1 to 2 weeks, some vials were injected with CH₄ to a final mixing ratio of 1% and incubated for a further 24 h (“unstarved”). Others remained without CH₄ (“starved”). Cell counts were made using a Neubauer chamber and showed that no population growth occurred during the 24-h incubation with 1% CH₄ (data not shown).

For determination of kinetic properties, CH₄ was injected into these vials to final mixing ratios ranging from 5 to 1,500 ppm volume. The unstarved vials still contained >0.5% CH₄ after 24 h and were first flushed well with air. Vials were shaken at 280 rpm. Starting 5 min after CH₄ addition and at 30-min intervals thereafter, CH₄ was measured by gas chromatography-flame ionization detection (8). Methane oxidation rates and kinetic parameters were estimated as previously described (8).

Unstarved cells had both a higher K_m(app) and a higher V_max(app) for CH₄ than did starved cells (Table 1). Addition of KCl may also have decreased the K_m(app). The increase of both K_m(app) and V_max(app) in unstarved culture was about 1 order of magnitude, and the specific affinity (a^o_s) (V_max(app)/K_m(app)) therefore remained nearly constant. The specific affinity is the initial slope of the hyperbolic curve, or the pseudo first-order rate constant, and directly indicates how rapidly the culture metabolized limiting substrate (4). At low CH₄ concentrations, the rate of CH₄ uptake was therefore similar in starved and unstarved culture.

TABLE 1.

Kinetic coefficients of starved and unstarved cultures of Methylocystis strain LR1 in several trials^a

Trial	Measurement time (h)	Km(app) (nM CH4)	Vmax(app) (10−9 nmol cell−1 h−1)	aos (10−9 ml cell−1 h−1)
				Vmax(app)/Km(app)	Regression
Trial 1
Cells starved 12 days	1	298	2.61	8.75	4.65
Starved + KCla	1	138	1.22	8.84	5.27
Unstarved	1	3,170	18.7	5.90	4.33
Trial 2
Cells starved 34 days	2	281	0.935	3.32	2.09
Starved + KClb	2	226	0.380	1.68	0.95
Unstarved	1.5	12,600	27.8	2.21	2.12
Trial 3
Cells starved 10 days	2	336	2.97	8.88	4.73
Freshc	2	2,190	13.27	6.08	4.39
Trial 4
Cells starved 19 days	2	329	0.779	2.36	1.39
Unstarved	1.5	3,400	21.7	6.38	4.81

^aEach calculation was based on data from 12 to 54 individual vials. Michaelis-Menten kinetic coefficients are shown, as well as the specific affinity (a^o_s) either calculated (V_max(app)/K_m(app)) or estimated by linear regression of the CH₄ oxidation rate versus the dissolved CH₄ concentration for all data points less than K_m(app). 

^bKCl was added to a final concentration of 250 mM immediately before test. 

^cCells were not preshaken and unstarved, but were taken directly from growing stock culture containing >1% CH₄. 

These results extend our previous observations on strain LR1 by demonstrating that (i) the K_m(app) can vary in pure culture, (ii) starvation of CH₄ decreases the K_m(app), (iii) the specific affinity (a^o_s) changes little with K_m(app), and (iv) K_m(app) varies in rapid (<2 h) tests without added chloramphenicol (previous tests were run over several days and chloramphenicol was necessary to prevent enzyme production). As previously discussed (8), the variable K_m(app) could have resulted because type II methanotrophs possesses different forms of methane monooxygenase (MMO): a particulate (pMMO) and a soluble (sMMO) form. Multiple catalytic forms of pMMO also exist, depending on Cu availability (15, 17). However, while the K_m(app) values of these MMOs are different (10, 15, 21), all measured values in pure culture are above 0.8 μM. The values measured in strain LR1 are as low as 56 to 336 nM (present results and reference 8).

Multiple enzymes may be responsible for the variable K_m(app) in LR1, but because the a^o_s per cell remained constant, it cannot be concluded that a high-affinity enzyme was induced to better utilize limiting CH₄. This possibility is consistent with the data only if reactivation of inactive cells and expression of a lower-affinity enzyme compensated for each other in the unstarved culture and caused the a^o_s to remain constant. However, if this were so, a biphasic kinetic curve should have been evident, and this was never observed. A better explanation for our results is that measured K_m(app) values do not always represent true, constant enzyme properties. Because of diffusion limitation in experimental systems, many reported K_m(app) values are gross overestimates (14). In order to control for this in our experiments, the CH₄ oxidation rate constants in the linear portion of the hyperbolic curve were also often measured with culture diluted 50%. These rate constants were close to 50% of the rate constants in undiluted culture (in six cases, rate constants were 42, 54, 61, 73, 74, and 81%), indicating that there was only a slight limitation in CH₄ movement from the gas phase to the liquid phase. We also used the minimum incubation times which yielded reproducible data (<2 h) to estimate CH₄ oxidation rates. To illustrate the effect of longer incubation times, we measured an initial 0 to 2 h rate at five CH₄ concentrations in a 10-day-starved culture, and then after 4 h (during which the CH₄ declined <40%) reinjected enough CH₄ to bring each vial back to its initial CH₄ concentration and measured a 4 to 6 h rate (Fig. 1). The CH₄ oxidation rate increased with time, most strikingly at the higher CH₄ concentrations. This trend leads to an overestimation of the K_m(app) with increasing incubation times.

FIG. 1.

CH₄ oxidation rates at five CH₄ concentrations in a 10-day-starved culture of Methylocystis strain LR1, measured 0 to 2 h (□) and 4 to 6 h (○) after adding CH₄.

Although we minimized these methodological problems, there are further ways in which an apparent K_m in a complex system can vary. The K_m(app) of MMO for CH₄ is affected by the CH₄ association and dissociation constants, the rate of CH₄ diffusion across the cell envelope, and the concentrations of cosubstrates (O₂ and reductant). One explanation for the lower K_m(app) in starved cells is low reductant availability. The mechanism of pMMO is not well elucidated, but sMMO follows a catalytic sequence in which O₂, CH₄, and NADH sequentially bind (9, 20). If in starved cells CH₄ binds normally but the overall catalytic cycle is slowed by NADH limitation, the rate-limiting step may cease to be the association and dissociation of CH₄ to the enzyme (i.e., the affinity constant) and instead become the reaction rate (i.e., the kinetic constant). In such a case, both the V_max(app) and the K_m(app) for CH₄ decrease (19). To illustrate this, the following reaction diagram has been simplified to consider only CH₄ as a reactant. The binding of O₂ and NADH and the formation of methanol are considered by the rate constant k_p:

Here, K_m(app) = [k_(−s) + k_p]/[k_s + k_(−p)]. Limitation of reductant will decrease the reaction rate constant k_p and in turn decrease K_m(app) (i.e., cause a higher apparent affinity). The above model is a very simple explanation of the observed variability. The truth may of course be more complex.

Bender and Conrad (2) observed that incubation of various soils under 20% CH₄ increased methanotrophic K_m(app) and V_max(app) values by 1 to 3 orders of magnitude, but increased methanotrophic cell counts only 3- to 10-fold. When specific affinities were calculated (Table 2), two of these soils (a meadow cambisol and a cultivated cambisol) had a similar pattern as LR1—that a^o_s varied little despite large changes (30- to 100-fold) of K_m(app). A third soil (forest luvisol) had a much lower a^o_s after enrichment than before, suggesting that a different population had become active in CH₄-enriched soil. Comparisons must be cautiously made, but these data suggest that the pattern noted in LR1 is applicable to some, but not all, soils.

TABLE 2.

Michaelis-Menten apparent half-saturation constants for CH₄ oxidation in three soils, and calculated specific affinities from data given in reference 2^a

Sample	Km(app) (nM CH4)	aos (10−9 ml cell−1 h−1)
Cultivated cambisol
Fresh	50.6	0.0039
+20% CH4	91	0.017
	1,740	0.016
Meadow cambisol
Fresh	49.9	0.050
+20% CH4	12.6	0.051
	4,560	0.027
Forest luvisol
Fresh	29.7	0.505
+20% CH4	470	0.136
	27,900	0.025

^aData are given for fresh soil and soils preincubated 3 weeks under 20% CH₄. In preincubated soils, a dual kinetic was evident, with both a high-affinity (second row for each soil) and a low-affinity (third row for each soil) activity. 

It is clear from this and other work (3, 8) that the K_m(app) for CH₄ changes with culture conditions. Nevertheless, our lowest measured K_m(app) is still higher than the lowest measured values in soil of about 10 nM (8), so we hesitate to conclude that no true high-affinity MMO exists. Calculations based on maintenance energy requirements suggest that methanotrophic bacteria cannot survive on atmospheric CH₄ without a more efficient CH₄-oxidizing system (6). However, soil methanotrophs may not consume only atmospheric methane but also alternate substrates, such as methanol (3, 13), or CH₄ produced in anaerobic soil microsites (1, 7, 16, 22). Our present results show that the observed high-affinity activity in soil cannot in itself be taken as proof that the responsible bacteria are novel oligotrophic species with a specialized form of MMO.