Siderophore for Lanthanide and Iron Uptake for Methylotrophy and Plant Growth Promotion in Methylobacterium aquaticum Strain 22A

Abstract

Methylobacterium and Methylorubrum species are facultative methylotrophic bacteria that are abundant in the plant phyllosphere. They have two methanol dehydrogenases, MxaF and XoxF, which are dependent on either calcium or lanthanides (Lns), respectively. Lns exist as insoluble minerals in nature, and their solubilization and uptake require a siderophore-like substance (lanthanophore). Methylobacterium species have also been identified as plant growth-promoting bacteria although the actual mechanism has not been well-investigated. This study aimed to reveal the roles of siderophore in Methylobacterium aquaticum strain 22A in Ln uptake, bacterial physiology, and plant growth promotion. The strain 22A genome contains an eight-gene cluster encoding the staphyloferrin B-like (sbn) siderophore. We demonstrate that the sbn siderophore gene cluster is necessary for growth under low iron conditions and was complemented by supplementation with citrate or spent medium of the wild type or other strains of the genera. The siderophore exhibited adaptive features, including tolerance to oxidative and nitrosative stress, biofilm formation, and heavy metal sequestration. The contribution of the siderophore to plant growth was shown by the repressive growth of duckweed treated with siderophore mutant under iron-limited conditions; however, the siderophore was dispensable for strain 22A to colonize the phyllosphere. Importantly, the siderophore mutant could not grow on methanol, but the siderophore could solubilize insoluble Ln oxide, suggesting its critical role in methylotrophy. We also identified TonB-dependent receptors (TBDRs) for the siderophore-iron complex, iron citrate, and Ln, among 12 TBDRs in strain 22A. Analysis of the siderophore synthesis gene clusters and TBDR genes in Methylobacterium genomes revealed the existence of diverse types of siderophores and TBDRs. Methylorubrum species have an exclusive TBDR for Ln uptake that has been identified as LutH. Collectively, the results of this study provide insight into the importance of the sbn siderophore in Ln chelation, bacterial physiology, and the diversity of siderophore and TBDRs in Methylobacterium species.

Keywords: Methylobacterium species; heavy metal sequestration; lanthanide; lanthanophore; plant growth promoter; siderophore.

FIGURE 1

(A) Growth of M. aquaticum strain 22A wild type (WT), siderophore sbn gene mutants on succinate with FeSO₄. (B) Growth of WT and ΔsbnCF on succinate with/without the addition of a spent medium (SM) of the WT. (C) Growth of WT and ΔsbnCF on succinate in the presence of 100 μM FeSO₄ or 100 μM iron citrate. (D) Growth of WT and ΔsbnCF on succinate in the presence of 50 μM FeSO₄ and different concentrations of citrate. Bars, specific growth rate (μ, h^–1); circles, cell yield (OD₆₀₀). All results are presented as the average ± standard deviation (SD) (biological triplicates).

FIGURE 2

sbnA promoter activity assay with luciferase as a reporter in the wild type grown on succinate in the presence of 5 and 100 μM FeSO₄ or iron citrate. Bars, sbnA promoter activity (arbitrary unit, luminescence/OD₆₀₀); circles, cell yield (OD₆₀₀). The results were presented as the average ± standard deviation (SD) (biological triplicates).

FIGURE 3

(A) Growth of duckweed plants in liquid 1/2 MS medium inoculated with none (non-inoculated control, NIC), strain 22A wild type (WT), and ΔsbnCF, under 10 and 1 mg/l FeSO₄ for 28 days. Representative pictures are shown for each treatment. (B) Dry weight of plants after cultivation. (C) Iron content in the plants and bacterial cells. (D) Bacterial mass measured as wet weight after cultivation. All the data are reported as the mean of three replicates ± standard deviation (SD).

FIGURE 4

(A) Solubilization of La₂O₃ by the spent media (SM) of the wild-type strain 22A (WT) and ΔsbnCF, and non-inoculated medium control. (B) Solubilization of La₂O₃ by citrate (5–500 μM). (C) P_sbnA activity in ΔmxaF in the presence of varied concentrations of LaCl₃ under 5 and 50 μM FeSO₄. (D) Growth of the WT, ΔmxaF, ΔsbnCF, and ΔmxaFΔsbnCF on methanol in the presence of 100 μM FeSO₄, 100 μM iron citrate, 9 mg/l La₂O₃, and 30 μM LaCl₃, and their mixtures. (E) Growth of the same set of mutants on methanol in the presence/absence of the spent medium of the WT, under 100 μM FeSO₄ and 30 μM LaCl₃. (F) Growth of the same set of mutants on methanol in the presence of different concentrations of citrate, under 50 μM FeSO₄ and 30 μM LaCl₃. (G) P_mxaF and P_xoxF activity in WT and ΔsbnCF grown on methanol plus succinate in the absence/presence of 30 μM LaCl₃. All data represent the mean of three replicates ± standard deviation (SD).

FIGURE 5

(A) Growth of the wild-type strain 22A and ΔsbnCF on succinate in the presence of 100 μM of ZnSO₄, CuSO₄, NiSO₄, and MnSO₄ under 50 μM iron citrate, growth of ΔsbnCF under 50 μM under 100 μM iron citrate. Growth of ΔsbnCF on succinate in the presence of 100 μM heavy metals under 50 μM iron citrate supplemented with 22A wild-type spent media. (B) P_sbnA activity in the wild-type strain 22A in the presence of 100 μM of each heavy metal under 5 and 100 μM FeSO₄. (C) Heavy metal accumulation in the wild-type strain 22A and ΔsbnCF cells supplemented with 25 μM heavy metals (HM) or without (NT). (D) Iron accumulation in the cells of wild-type strain 22A and ΔsbnCF grown on succinate in the presence of 25 μM of ZnSO₄, CuSO₄, NiSO₄, and MnSO₄, under 100 μM iron citrate. Data represent the mean of three replicates ± standard deviation (SD).

FIGURE 6

(A) Growth of the mutants of each TBDR gene generated under the wild-type strain 22A (WT) and ΔSbnCF backgrounds on succinate supplemented with 100 μM FeSO₄ 10% WT spent medium (SM), respectively. (B) Growth of the mutants of each TBDR gene generated under the background of ΔsbnCF on succinate under 100 μM iron citrate. (C) Same experiment as (B) but with 50 μM citric acid and 50 μM FeSO₄. (D) Growth of the mutants of each TBDR gene generated under the ΔmxaF background on methanol under 100 μM iron citrate and 30 μM LaCl₃. (E) P_{tonB_Ln} activity in WT grown on methanol in the presence of different concentrations of LaCl₃. The gene disruption mutants were grown in the presence of kanamycin throughout the experiment. All data represent the mean of three replicates ± standard deviation (SD).

FIGURE 7

Phylogenetic tree and gene counts of TBDRs found in the genomes of different subclades of Methylobacterium type strains. The phylogenetic tree is constructed based on the amino acid sequences of TBDR genes. The Methylobacterium subclade definition is based on our previous report (Alessa et al., 2021). A total of 25 clades are labeled A-Y. The counts of TBDRs and description of characterized TBDRs are indicated in brackets. Details are presented in Supplementary Table 3.