Carbon Metabolism of a Soilborne Mn(II)-Oxidizing Escherichia coli Isolate Implicated as a Pronounced Modulator of Bacterial Mn Oxidation

Abstract

Mn(II)-oxidizing microorganisms are generally considered the primary driving forces in the biological formation of Mn oxides. However, the mechanistic elucidation of the actuation and regulation of Mn oxidation in soilborne bacteria remains elusive. Here, we performed joint multiple gene-knockout analyses and comparative morphological and physiological determinations to characterize the influence of carbon metabolism on the Mn oxide deposit amount (MnODA) and the Mn oxide formation of a soilborne bacterium, Escherichia coli MB266. Different carbon source substances exhibited significantly varied effects on the MnODA of MB266. A total of 16 carbon metabolism-related genes with significant variant expression levels under Mn supplementation conditions were knocked out in the MB266 genome accordingly, but only little effect on the MnODA of each mutant strain was accounted for. However, a simultaneous four-gene-knockout mutant (namely, MB801) showed an overall remarkable MnODA reduction and an initially delayed Mn oxide formation compared with the wild-type MB266. The assays using scanning/transmission electron microscopy verified that MB801 exhibited not only a delayed Mn-oxide aggregate processing, but also relatively smaller microspherical agglomerations, and presented flocculent deposit Mn oxides compared with normal fibrous and crystalline Mn oxides formed by MB266. Moreover, the Mn oxide aggregate formation was highly related to the intracellular ROS level. Thus, this study demonstrates that carbon metabolism acts as a pronounced modulator of MnODA in MB266, which will provide new insights into the occurrence of Mn oxidation and Mn oxide formation by soilborne bacteria in habitats where Mn(II) naturally occurs.

Keywords: ROS; carbon metabolism; manganese oxidation; manganese oxide; manganese oxidizing bacteria.

Figure 1

Carbon metabolic pathways potentially associated with Mn oxidation by proteomics analysis (A) and the effects of different carbon source substrates on the MnODA of E. coli MB266 suspension (B). Red/blue arrows in A indicate significantly upregulated/downregulated metabolic reactions, respectively. Abbreviations are listed as follows: CysE, Serine acetyltransferase; SdaA, L-Serine deaminase I; IlvA, Threonine deaminase; PkyF, Pyruvate kinase I; Pps, Phosphoenolpyruvate synthase; PtsI, PEP-protein phosphotransferase; PtsP, Fused PEP-protein phosphotransferase (enzyme I); KatE, Hydroperoxidase HPII (III); SfcA, Malate dehydrogenase; AceE, Pyruvate dehydrogenase; Ydij, FAD-linked oxidoreductase; Dld, a NADH independent D-lactate dehydrogenase; LdhA, D-Lactate dehydrogenase; YqeA, Acyltransferase; GlcB, Malate synthase G. In B, MB266 cells were cultured in LB broth with different additionally added carbon source substances and 5 mmol/L MnCl₂ for 120 h at 37 °C, then MnOA was determined accordingly. The dose used and abbreviations are listed as follows: LB, 0 addition; CYT-C, 10 µmol/L cytochrome C; Glc, 10 mmol/L glucose; Oxa, 10 mmol/L sodium oxalate; Ace, 10 mmol/L sodium acetate; Man, 10 mmol/L mannose; KGA, 10 mmol/L α-ketoglutarate; Frc, 10 mmol/L fructose; Ben, 10 mmol/L sodium benzoate. No detectable MnODA was recorded in each negative control (without inoculation of MB266 cells) after 30 days of incubation. Means followed by * in a column were significantly different (n = at least 3; p < 0.05) according to Fisher’s protected LSD test.

Figure 2

Measurement of MnODA of cell suspension in various gene-knockout mutant strains. The cells were cultured for 5 days under the supplement of 5 mmol/L MnCl₂ at the final concentration prior to the standard LBB assay. Means followed by * in a column were significantly different (n = at least 3; p < 0.05).

Figure 3

Measurements of the growth curve and MnODA of various gene-knockout mutant strains. (A,C,E,G) Growth curve; (B,D,F,H) MnODA (refers to concentration of Mn oxides) of (A,C,E,G). (A,B), pyruvate metabolic pathway; (C,D), glucogenic amino acid metabolic pathway; (E,F), glyoxylic acid metabolic pathway; (G,H), lactic acid metabolic pathway. The wild-type strain MB266 was used as the control for all measurements. Means followed by * in a column were significantly different (n = at least 3; p < 0.05).

Figure 4

Growth curve (A) and MnODA (B,D) measurements and LBB test (C) on culture fractions of the mutant strain MB801. In (A), the wild-type MB266 was used as the control. The OD₆₀₀ values were comparatively determined based on the approximately equivalent inoculum size of both strains. In (B), MnODA (refers to concentration of Mn oxides) of the deposit fraction of the MB801 culture was determined following the culturing of the cells for 5 days in LB broth containing 5 mmol/L MnCl₂ at the final concentration. In (C), LBB test results are shown for the culture fractions of MB801 in 96-cell microtiter plates after culturing for 5 days in LB broth containing 5 mmol/L MnCl₂ at the final concentration. In (D), MnODA (refers to concentration of Mn oxides) was determined over a culture time course of 5 days in Lept medium containing 100 μmol/L Mn(III) complex at the final concentration.

Figure 5

SEM (A) and TEM (B) micrographs of Mn oxide aggregate formation during the culture time course. The cells were cultured in LB broth containing 5 mmol/L MnCl₂ at the final concentration for 11 days. In (A), the samples were statically suspended for 30 min at room temperature, the precipitates were collected by pipetting out the supernatants, and 2.5% glutaraldehyde was added then placed at 4 °C for 12 h, followed by sample drying with ethanol dehydration and freeze-drying processing for SEM observation. In (B), the samples were vigorously stirred and 2.5% glutaraldehyde was added and placed at 4 °C for 12 h.

Figure 6

Effect of ROS on the MnODA of MB801. In (A), the cells were cultured for 24 h prior to the addition of H₂O₂ at various concentrations. The specific DCF fluorescence intensity was recorded. In (B), the MnODA of the cells was determined after culturing the cells for 5 days with H₂O₂ at different concentrations.

Figure 7

H₂O₂ inhibition zone on the growth of MB801. In (A), sterile 6-mm-diameter filter papers were immersed in 10 µL of 1 mol/L H₂O₂, and 5 mmol/M MnCl₂ was used as appropriate. In (B), the plotted inhibition zone diameters are shown with/without MnCl₂ supplementation. Means followed by * in a column were significantly different (n = at least 3; p < 0.05).