Methane and Trichloroethylene Degradation by Methylosinus trichosporium OB3b Expressing Particulate Methane Monooxygenase

Abstract

Whole-cell assays of methane and trichloroethylene (TCE) consumption have been performed on Methylosinus trichosporium OB3b expressing particulate methane monooxygenase (pMMO). From these assays it is apparent that varying the growth concentration of copper causes a change in the kinetics of methane and TCE degradation. For M. trichosporium OB3b, increasing the copper growth concentration from 2.5 to 20 μM caused the maximal degradation rate of methane (V max) to decrease from 300 to 82 nmol of methane/min/mg of protein. The methane concentration at half the maximal degradation rate (Ks ) also decreased from 62 to 8.3 μM. The pseudo-first-order rate constant for methane,V max/Ks , doubled from 4.9 × 10−3 to 9.9 × 10−3liters/min/mg of protein, however, as the growth concentration of copper increased from 2.5 to 20 μM. TCE degradation by M. trichosporium OB3b was also examined with varying copper and formate concentrations. M. trichosporium OB3b grown with 2.5 μM copper was unable to degrade TCE in both the absence and presence of an exogenous source of reducing equivalents in the form of formate. Cells grown with 20 μM copper, however, were able to degrade TCE regardless of whether formate was provided. Without formate theV max for TCE was 2.5 nmol/min/mg of protein, while providing formate increased the V max to 4.1 nmol/min/mg of protein. The affinity for TCE also increased with increasing copper, as seen by a change in Ks from 36 to 7.9 μM.V max/Ks for TCE degradation by pMMO also increased from 6.9 × 10−5to 5.2 × 10−4 liters/min/mg of protein with the addition of formate. From these whole-cell studies it is apparent that the amount of copper available is critical in determining the oxidation of substrates in methanotrophs that are expressing only pMMO.

Effects of copper on expression of methane monooxygenases, trichloroethylene degradation, and community structure in methanotrophic consortia

“…In prior studies, Hylckama et al [36] used Methylosinus trichosporium OB3b to assess the transformation kinetics of chlorinated ethenes and obtained a k 1,TCE of <0.03 mL/(mg cells min) when Cu 2+ was omitted from the medium. Similarly, Lontoh et al [19] evaluated the cometabolic degradation of TCE using the same methanotroph and found that the k 1,TCE was 0.035 mL/(mg cells min) when expression of sMMO was repressed by adjusting the Cu 2+ concentration to 2.5 μM. Subsequently, Aziz et al [37] assessed the cometabolism of chlorinated solvents using a mutant methanotroph Methylosinus trichosporium OB3b PP358 and determined the k 1,TCE was 0.014 mL/(mg cells min).…”

“…Prior results suggest that a high copper-tobiomass ratio stimulates pMMO and represses sMMO expression in pure cultures [13,16,17]. Furthermore, Cu 2+ has been shown to alter pMMO expression up to 55-fold and to change its substrate affinity and specificity [18][19][20]. It has also been reported that sMMO activity is undetectable at a copper-tobiomass ratio of 45.64 mmol Cu·g −1 protein in the culture medium of Methylosinus trichosporium OB3b [21].…”

“…The results suggested that cells grown on low-copper medium did not oxidize TCE and had a variable rate of CH 4 oxidation. Similarly, Sonny Lontoh et al [19] explored the effects of Cu 2+ on CH 4 and TCE degradation and enzyme expression using the model methanotroph Methylosinus trichosporium OB3B. The results indicated that the kinetic parameters of CH 4 oxidation decreased along with Cu 2+ concentrations (2.5-20 μM) and the affinity for TCE increased with increasing Cu 2+ by providing formate only when expressing pMMO.…”

Response and adaptation of different methanotrophic bacteria to low methane mixing ratios

“…f. Joergensen (1985). g. Lontoh and Semrau (1998); four measurements at different copper concentrations (2.5-20 mM). h. Calhoun and King (1997); values from eight root-associated methanotrophic isolates.…”

Methanobactin and MmoD work in concert to act as the ‘copper‐switch’ in methanotrophs

“…The sMMO, an ironcontaining enzyme, is only expressed by a small number of methanotrophs and typically then only under conditions of copper deficiency (Dedysh et al, 2000;Dunfield et al, 2003;Semrau et al, 2010;Vorobev et al, 2011). Most extant methanotrophs, including those that have the ability to synthesize sMMO, also express pMMO, a copper containing enzyme, and such expression and activity increases with increasing copper availability (Lontoh and Semrau, 1998;Choi et al, 2003), i.e. there is a 'copperswitch' that regulates the expression of the two forms of MMO.…”