Characterization of pH dependent Mn(II) oxidation strategies and formation of a bixbyite-like phase by Mesorhizobium australicum T-G1

Abstract

Despite the ubiquity of Mn oxides in natural environments, there are only a few observations of biological Mn(II) oxidation at pH < 6. The lack of low pH Mn-oxidizing bacteria (MOB) isolates limits our understanding of how pH influences biological Mn(II) oxidation in extreme environments. Here, we report that a novel MOB isolate, Mesorhizobium australicum strain T-G1, isolated from an acidic and metalliferous uranium mining area, can oxidize Mn(II) at both acidic and neutral pH using different enzymatic pathways. X-ray diffraction, Raman spectroscopy, and scanning electron microscopy with energy dispersive X-ray spectroscopy revealed that T-G1 initiated bixbyite-like Mn oxide formation at pH 5.5 which coincided with multi-copper oxidase expression from early exponential phase to late stationary phase. In contrast, reactive oxygen species (ROS), particularly superoxide, appeared to be more important for T-G1 mediated Mn(II) oxidation at neutral pH. ROS was produced in parallel with the occurrence of Mn(II) oxidation at pH 7.2 from early stationary phase. Solid phase Mn oxides did not precipitate, which is consistent with the presence of a high amount of H2O2 and lower activity of catalase in the liquid culture at pH 7.2. Our results show that M. australicum T-G1, an acid tolerant MOB, can initiate Mn(II) oxidation by varying its oxidation mechanisms depending on the pH and may play an important role in low pH manganese biogeochemical cycling.

Keywords: Mn(II) oxidation; catalase; low pH; multi-copper oxidase; reactive oxygen species.

FIGURE 1

Morphology and Mn(II) oxidation by strain T-G1: (A) scanning electron micrograph of T-G1 cells, showing abundant extracellular polymeric substances; (B) Mn(II) oxidation by T-G1 as seen by a color change in PYG medium with 10 mM MnSO₄ (right) and without Mn (left); and (C) leucoberbelin blue (LBB) spot test on colony of T-G1 showing a blue color change indicative of Mn oxide production.

FIGURE 2

Phylogenetic tree showing the evolutionary placement of Mesorhizobium australicum T-G1 (in rectangle) relative to known MOB based on 16S rRNA gene sequences. black squares indicate non-Mn(II)-oxidizing strains. The tree was built using a neighbor joining algorithm with bootstrap values, displayed as percentages, based on 1000 replicates. Accession numbers for published sequences are shown in parentheses. The scale bar represents 0.02 substitutions per nucleotide position.

FIGURE 3

Characterization of biogenic Mn oxides produced by strain T-G1 in PYG medium at pH 5.5. (A) Scanning electron micrograph showing spherical clusters of micron to sub-micron sheets of Mn oxide minerals. (B) An enlargement of the area indicated by a red box in (A). (C) Confocal laser scanning microscopy (CLSM) of a mineral aggregate in association with T-G1. Bacterial cells stained by FM 1-43 probe shown in green, reflection of minerals was indicated in gray. (D) Energy-dispersive X-ray (EDS) spectra showing elemental composition of biogenic Mn oxide particles. (E) Raman spectroscopy of biogenic Mn oxides. (F) XRD pattern of biogenic Mn-oxide minerals with vertical lines indicating strong (red) and weak peak positions (green) of bixbyite.

FIGURE 4

Mn(II) oxidation and growth of T-G1 at different pH values. Growth (OD₆₀₀) and Mn(II) oxidation at pH 5.5 and pH 7.2. The data for the Mn(II) oxidation control at pH 7.2 overlapped with that at pH 5.5, therefore, only the data at pH 5.5 is shown. Error bars represent SD.

FIGURE 5

Mn(III) trapping by 100 μM pyrophosphate during incubation of cell-free filtrate with 100 μM MnSO₄ at pH 5.5, pH 7.2 and with the addition of superoxide dismutase (SOD, 5 μM) or proteinase-K (100 μg ml^-1). Error bars represent SD and are smaller than symbols.

FIGURE 6

In-gel activity of Mn(II) oxidation enzymes. (A) Gel was stained with Coomassie. Lane m-1, Precision Plus Protein Dual Xtra Standards; Lane a, whole cell protein (pH 7.2, exponential phase); Lane b, whole cell protein (pH 5.5, exponential phase); Lane c, whole cell protein (pH 7.2, stationary phase); Lane d, whole cell protein (pH 5.5, stationary phase). Triangles 2 and 3 indicated the putative Mn(II) oxidizing enzymes with molecular masses of 140 and 120 KDa, respectively. (B) Gel was stained with 200 μM MnSO₄. The color of the gel was modified to blue in Adobe Photoshop CC to enhance the clarity of the bands. Lane m-2 and m-3, Precision Plus Protein Dual Xtra Standards; Lane e, whole cell protein (pH 7.2, stationary phase); Lane f, whole cell protein (pH 5.5, stationary phase); Lane g, whole cell protein (pH 7.2, exponential phase); Lane h, whole cell protein (pH 5.5, exponential phase). Triangle 1 indicated the putative Mn(II) oxidizing enzyme with a molecular mass between 120 and 140 KDa.

FIGURE 7

Reactive oxygen species, MCO, H₂O₂, and catalase quantification during exponential (E) or stationary (S) growth at pH 5.5 or 7.2. (A) Superoxide quantification during different growth phases; (B) Superoxide quantification in the presence of NAD, NADH, NADPH, and DPI at pH 7.2; (C) Quantification of MCO gene copies using qRT-PCR; (D) MCO activity; (E) H₂O₂ quantification during different growth phases; (F) Catalase quantification during different growth phases. Error bars represent SD.