Comparative genomic analysis of metal-tolerant bacteria reveals significant differences in metal adaptation strategies

Abstract

Metal-tolerant bacteria have been commercially used in wastewater treatment, bio-fertilizer, and soil remediation, etc. However, the mechanisms underlying their actions are not yet fully understood. We isolated metal-tolerant bacteria from the rhizosphere soil samples with metal-enriched media containing Cu, Fe, or Mn, sequenced and compared the genomes, and analyzed their metal adaptation strategies at genomic levels to better understand their action mechanisms. Totally, 32 metal-tolerant isolates were identified and classified into 12 genera based on phylogenetic analysis. The determination of maximum tolerance concentration and the effect of metal ions on the isolates indicated that Serratia marcescens X1 (CuSO₄: 1,000 mg/L, FeSO₄: 1,000 mg/L, and MnSO₄.4H₂O: 2,000 mg/L), Mammaliicoccus sciuri X26 (FeSO₄: 600 mg/L and MnSO₄.4H₂O: 2,000 mg/L), and Rummeliibacillus pycnus X33 (CuSO₄: 400 mg/L, FeSO₄: 1,000 mg/L, and MnSO₄.4H₂O: 800 mg/L) showed significant differences in metal tolerance to Cu, Fe, and Mn with other isolates. They possess quite different genomic features that enable them to adapt to various metal ions. S. marcescens X1 possesses abundant genes required for Cu, Fe, and Mn homeostasis. M. sciuri X26 has a number of genes involved in Mn and Zn homeostasis but with no genes responsible for Cu and Ca transport. R. pycnus X33 is rich in Fe, Zn, and Mg transport systems but poor in Cu and Mn transport systems. It is thus inferred that the combined use of them would compensate for their differences and enhance their ability in accumulating a wider range of heavy metals for promoting their applications in industry, agriculture, and ecology.

Importance: Metal-tolerant bacteria have wide applications in environmental, agricultural, and ecological fields, but their action strategies are not yet fully understood. We isolated 32 metal-tolerant bacteria from the rhizosphere soil samples. Among them, Serratia marcescens X1, Mammaliicoccus sciuri X26, and Rummeliibacillus pycnus X33 showed significant differences in metal tolerance to Cu, Fe, and Mn with other isolates. Comparative genomic analysis revealed that they have abundant and different genomic features to adapt to various metal ions. It is thus inferred that the combined use of them would compensate for their differences and enhance their ability to accumulate heavy metal ions, widening their applications in industry, agriculture, and ecology.

Keywords: application; combined use; genomic analysis; metal-stress-response; metal-tolerant bacteria.

Fig 1

MTC of 16 metal-tolerant bacterial isolates. Sixteen isolates were grown on Luria-Bertani (LB) medium (A). Sixteen isolates were grown on LB medium supplemented with various concentrations of CuSO₄ (B), FeSO₄ (C), and MnSO₄.4H₂O (D). Photographs were taken 3 days after inoculation at 30°C.

Fig 2

Phylogenetic relationships of 16 metal-tolerant bacterial isolates and the related species. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic positions of 16 metal-tolerant bacterial isolates (indicated with solid red diamond). The bootstrap values are shown at branch nodes. Bar, 0.02 nucleotide position.

Fig 3

Effect of metals on bacterial growth. S. marcescens X1 (A), M. sciuri X26 (B), and R. pycnus X33 (C) in the presence of Cu, Fe, and Mn.

Fig 4

Circular maps of S. marcescens X1 (A), R. pycnus X33 (B), and M. sciuri X26 (C) genomes. The numbers 1 to 7 are the circles of circular maps from inner to outer: (1) GC skew. Dark gray represents areas with G content greater than C, while red represents areas with C content greater than G. (2) GC content. The value is plotted as the deviation from the average GC content of the entire sequence. (3) tRNA (blue) and rRNA (purple). (4) Repetitive sequences. (5, 6) CDS colored according to COG functional categories, 5 is negative strand, 6 is positive strand. (7) Marker of genome size, with each scale measuring 5 kb.

Fig 5

Schematic of the proteins identified as playing a putative role in the adaptation of three metal-tolerant bacterial isolates to metal environments. Strains: S. marcescens X1 (blue solid circle), M. sciuri X26 (red solid square), and R. pycnus X33 (green solid triangle). The putative function of genes is shown in Table S2.