Manganese Stress Adaptation Mechanisms of Bacillus safensis Strain ST7 From Mine Soil

Abstract

The mechanism of bacterial adaption to manganese-polluted environments was explored using 50 manganese-tolerant strains of bacteria isolated from soil of the largest manganese mine in China. Efficiency of manganese removal by the isolated strains was investigated using atomic absorption spectrophotometry. Bacillus safensis strain ST7 was the most effective manganese-oxidizing bacteria among the tested isolates, achieving up to 82% removal at a Mn(II) concentration of 2,200 mg/L. Bacteria-mediated manganese oxide precipitates and high motility were observed, and the growth of strain ST7 was inhibited while its biofilm formation was promoted by the presence of Mn(II). In addition, strain ST7 could grow in the presence of high concentrations of Al(III), Cr(VI), and Fe(III). Genome-wide analysis of the gene expression profile of strain ST7 using the RNA-seq method revealed that 2,580 genes were differently expressed under Mn(II) exposure, and there were more downregulated genes (n = 2,021) than upregulated genes (n = 559) induced by Mn stress. KAAS analysis indicated that these differently expressed genes were mainly enriched in material metabolisms, cellular processes, organism systems, and genetic and environmental information processing pathways. A total of twenty-six genes from the transcriptome of strain ST7 were involved in lignocellulosic degradation. Furthermore, after 15 genes were knocked out by homologous recombination technology, it was observed that the transporters, multicopper oxidase, and proteins involved in sporulation and flagellogenesis contributed to the removal of Mn(II) in strain ST7. In summary, B. safensis ST7 adapted to Mn exposure by changing its metabolism, upregulating cation transporters, inhibiting sporulation and flagellogenesis, and activating an alternative stress-related sigB pathway. This bacterial strain could potentially be used to restore soil polluted by multiple heavy metals and is a candidate to support the consolidated bioprocessing community.

Keywords: Bacillus safensis; adaptation; manganese oxidation; soil; transcriptome.

FIGURE 1

Gene expression patterns of strain ST7 in the presence of Mn(II) at exponential (stage 1) and stationary (stage 2) growth phases.

FIGURE 2

Venn diagram showing the overlap of numbers of expressed genes in strain ST7 in the presence or absence of 250 mg/L Mn(II). Numbers of expressed genes in (A) exponential growth phase (EGP) and (B) stationary growth phase (SGP) are indicated.

FIGURE 3

Enriched pathways of genes affected by manganese exposure. (A) Enriched pathways of genes expressed during the exponential growth phase (EGP) of strain ST7 in the presence or absence of Mn(II). (B) Enriched pathways of genes expressed during stationary growth phase (SGP) of strain ST7 in the presence or absence of Mn(II).

FIGURE 4

Enrichment hierarchies of differentially expressed genes (DEGs) in strain ST7 under manganese stress. The horizontal ordinate represents the number of enriched genes in each hierarchy. Numbers of enriched DEGs that were upregulated, downregulated, and without difference in exponential growth phase (EGP) are indicated as EGP-up (the upregulated DEGs at EGP), EGP-down (the downregulated DEGs at EGP), and EGP-no diff (genes with no significance at EGP), respectively. Numbers of DEGs that were upregulated and downregulated in stationary growth phase (SGP) are shown as SGP-up (the upregulated DEGs at SGP) and SGP-down (the downregulated DEGs at SGP), respectively.

FIGURE 5

Volcano plots of DEGs in strain ST7 under manganese stress. (A) DEGs in mid-exponential growth phase. (B) DEGs in the onset of stationary growth phase. Green and blue dots represent the downregulated genes, while red and orange dots represent the upregulated genes.

FIGURE 6

RT-qPCR validation of expressed genes in strain ST7 cultured in media supplemented with 250 mg/L Mn(II).

FIGURE 7

Manganese oxidation ability of mutants. Mutants with deleted genes and the wild-type strain were cultivated in PYCM liquid media supplemented with 250 mg/L Mn(II) for 7 days. (A) Manganese removal rates of mutants detected by ICP-OES method. (B) Manganese oxidation abilities of mutants detected by adding 150 μL of 0.04% LBB solution into the culture supernatants.