Methane monooxygenase gene expression mediated by methanobactin in the presence of mineral copper sources.

Methane is a major greenhouse gas linked to global warming; however, patterns of in situ methane oxidation by methane-oxidizing bacteria (methanotrophs), nature's main biological mechanism for methane suppression, are often inconsistent with laboratory predictions. For example, one would expect a strong relationship between methanotroph ecology and Cu level because methanotrophs require Cu to sustain particulate methane monooxygenase (pMMO), the most efficient enzyme for methane oxidation. However, no correlation has been observed in nature, which is surprising because methane monooxygenase (MMO) gene expression has been unequivocally linked to Cu availability. Here we provide a fundamental explanation for this lack of correlation. We propose that MMO expression in nature is largely controlled by solid-phase Cu geochemistry and the relative ability of Cu acquisition systems in methanotrophs, such as methanobactins (mb), to obtain Cu from mineral sources. To test this hypothesis, RT-PCR expression assays were developed for Methylosinus trichosporium OB3b (which produces mb) to quantify pMMO, soluble MMO (the alternate MMO expressed when Cu is "unavailable"), and 16S-rRNA gene expression under progressively more stringent Cu supply conditions. When Cu was provided as CuCl(2), pMMO transcript levels increased significantly consistent with laboratory work. However, when Cu was provided as Cu-doped iron oxide, pMMO transcript levels increased only when mb was also present. Finally, when Cu was provided as Cu-doped borosilicate glass, pMMO transcription patterns varied depending on the ambient mb:Cu supply ratio. Cu geochemistry clearly influences MMO expression in terrestrial systems, and, as such, local Cu mineralogy might provide an explanation for methane oxidation patterns in the natural environment.

Determination of intrinsic bacterial surface acidity constants using a donnan shell model and a continuous pK(a) distribution method.

Intrinsic acidity constants (pK(a)(int)) for Bacillus subtilis (Gram+) and Escherichia coli (Gram-) cells were calculated from potentiometric titration data at different salt concentrations. Master curves were generated by replotting charge excess data as a function of pH(S) (pH at the location of surface reactive sites) where pH(S) was determined as a function of Donnan potential, Psi(DON). This potential decreased in magnitude with increasing ionic strength, from -48.5+/-0.2 to -3.5+/-0.0 mV for B. subtilis and -47.9+/-0.3 to -3.5+/-0.0 mV for E. coli at 0.01 and 0.5 M K(+), respectively, indicating an efficient surface charge neutralization by counterions. A fully optimized continuous (FOCUS) pK(a) distribution method revealed four binding sites on B. subtilis and E. coli surfaces from the master curves with pK(a)(int) values of 3.59+/-0.38, 4.33+/-0.57, 5.94+/-0.66, and 8.64+/-0.57 for B. subtilis and 3.73+/-0.44, 4.85+/-0.71, 6.56+/-0.64, and 8.79+/-0.62 for E. coli. These were assigned to functional groups according to reported pK(a) ranges of 2.0-6.0 (carboxylic acid), 3.2-3.5 (phosphodiesters), 5.6-7.2 (phosphoric acid), and 9.0-11.0 (amine groups). Average points of zero salt effect (pH(pzse)) for B. subtilis experiments were 6.63+/-0.21 and 6.42+/-0.08 as a function of pH(bulk) and pH(S), respectively. Under the same criteria, E. coli calculations yielded 5.73+/-0.23 and 5.45+/-0.05. An understanding of metal and proton reactivity on bacterial cell surfaces can be addressed quantitatively through the use of electrostatic and chemical equilibrium modeling techniques proposed in this study. The results are consistent with those of electrical force microscopy studies used to document the intrinsic electrochemical heterogeneity of bacterial cell surfaces.