Characterization of Neutrophilic Fe(II)-Oxidizing Bacteria Isolated from the Rhizosphere of Wetland Plants and Description of Ferritrophicum radicicola gen. nov. sp. nov., and Sideroxydans paludicola sp. nov.

Abstract

Iron deposits (Fe plaque) on wetland plant roots contain abundant microbial populations, including Fe(II)-oxidizing bacteria (FeOB) that have not been cultured previously. In this study, 4 strains of Fe plaque-associated FeOB were isolated from 4 species of wetland plants. All 4 isolates grew in tight association with Fe-oxides, but did not form any identifiable Fe-oxide structures. All strains were obligate lithotrophic Fe(II)-oxidizers that were microaerobic, and were unable to use other inorganic or organic energy sources. One strain, BrT, was shown to fix ¹⁴ CO ₂ at a rate consistent with its requirement for total cell carbon. The doubling times for the strains varied between 9.5 and 15.8 hours. The fatty acid methyl ester (FAME) profiles of 2 strains, BrT and CCJ, revealed that 16:0, 15:1 isoG, and 14:0 were dominant fatty acids. Phylogenetic analysis of the 16S rRNA gene indicated that all the strains were Betaproteobacteria. Two of the strains, BrT and Br-1 belong to a new species, Sideroxydans paludicola; a third strain, LD-1, is related to Sideroxydans lithotrophicus, a recently described species of FeOB. The fourth isolate, Ferritrophicum radicicola, represented a new genus in a new order of Betaproteobacteria, the Ferritrophicales. There are no other cultured isolates in this order. A small subunit rRNA gene-based, cultivation-independent analysis of Typha latifolia collected from a wetland revealed terminal restriction fragment profiles (tRFLP) consistent with the presence of these bacteria in the rhizosphere. These novel organisms likely play an important role in Fe(II) oxidation kinetics and Fe cycling within many terrestrial and freshwater environments.

Keywords:

• iron-oxidizing bacteria
• rhizosphere
• wetlands
• lithotrophs
• Sideroxydans
• Ferritrophicum

ACKNOWLEDGMENTS

We thank Paul Krader (ATCC) for the FAME analysis, and Jeff Benson and Jason Cooper of the molecular authentication resource center at the ATCC for assistance with tRFLP analysis. This work was funded by grants from the NASA Astrobiology Institute and the National Science Foundation (DEB-9985922 and DEB-0516197), the Jeffress Memorial Trust, an NSF Dissertation Improvement Grant to JW (DEB-0073080), and an American Association of University Women American Fellowship to JW.

Notes

¹Fe(II) was added a ferrous sulfide or ferrous carbonate in gradient tubes. Ferrous chloride was used in growing larger batch cultures.

²Substrates include H_2, ammonium, sulfide, thiosulfate, H₂, Mn(II), and tetrathionate.

³Substrates include galactose, pyruvate, acetate, succinate, glycerol, glucose, formate, maltose, ribose, and aspartate.

⁴Calculated at log phase growth on FeS in gradient tubes.

⁵Tested pH range = 4.5–8.0.

⁶Tested temperature range = 4⁵ tested temperature range = 4–43°C.