Lithotrophic iron-oxidizing bacteria produce organic stalks to control mineral growth: implications for biosignature formation

Abstract

Neutrophilic Fe-oxidizing bacteria (FeOB) are often identified by their distinctive morphologies, such as the extracellular twisted ribbon-like stalks formed by Gallionella ferruginea or Mariprofundus ferrooxydans. Similar filaments preserved in silica are often identified as FeOB fossils in rocks. Although it is assumed that twisted iron stalks are indicative of FeOB, the stalk's metabolic role has not been established. To this end, we studied the marine FeOB M. ferrooxydans by light, X-ray and electron microscopy. Using time-lapse light microscopy, we observed cells excreting stalks during growth (averaging 2.2 μm h(-1)). Scanning transmission X-ray microscopy and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy show that stalks are Fe(III)-rich, whereas cells are low in Fe. Transmission electron microscopy reveals that stalks are composed of several fibrils, which contain few-nanometer-sized iron oxyhydroxide crystals. Lepidocrocite crystals that nucleated on the fibril surface are much larger (∼100 nm), suggesting that mineral growth within fibrils is retarded, relative to sites surrounding fibrils. C and N 1s NEXAFS spectroscopy and fluorescence probing show that stalks primarily contain carboxyl-rich polysaccharides. On the basis of these results, we suggest a physiological model for Fe oxidation in which cells excrete oxidized Fe bound to organic polymers. These organic molecules retard mineral growth, preventing cell encrustation. This model describes an essential role for stalk formation in FeOB growth. We suggest that stalk-like morphologies observed in modern and ancient samples may be correlated confidently with the Fe-oxidizing metabolism as a robust biosignature.

Figure 1

Light micrographs of M. ferrooxydans. (a) Time-lapse series of phase contrast images of a cell creating a twisted stalk in a microslide culture; time interval=10 min. The image series shows that the cell (white arrow) rotates and moves upward as the stalk is formed. Each image is 6.4 μm wide (see Supplementary Video S1). (b) Differential interference contrast (DIC)-extended focus light micrograph showing change in stalk width along the length of the stalk. The white arrow points to a curved rod-shaped cell; the black arrow indicates direction of stalk growth. Scale bar=10 μm.

Figure 2

TEM images of M. ferrooxydans. (a) Cell attached to stalk, which is composed of individual filaments. Inset: smaller cell and stalk, displayed at the same scale showing that smaller cells produce narrower stalks with fewer filaments. Scale bar=500 nm. (b) TEM image of the cell–stalk interface, showing that the fibrils taper toward the cell, and are composed of an electron dense core and a lighter coating. Scale bar=100 nm.

Figure 3

High-resolution TEM images of mineralized stalks. (a) Whole mount of stalk fragment. Scale bar=1 μm. (b) Ultrathin section oriented perpendicular to stalk, showing electron-dense core filaments surrounded by radiating crystals of lepidocrocite. Scale bar=0.5 μm. (c) Oblique section showing filaments containing few-nanometer-sized, granular iron oxyhydroxides surrounded by long, blade-like lepidocrocite (see Supplementary Figure S4 for electron diffraction). Scale bar=100 nm.

Figure 4

STXM-derived elemental maps of cells, a stalk and surrounding mineral particles. Intensity scale is in OD units. Scale bars=1 μm (top four panels), 200 nm (bottom two panels).

Figure 5

C and O 1s NEXAFS spectra of M. ferrooxydans culture (stalk, cells and surrounding minerals), Loihi microbial mat stalk, and standards. Dashed lines indicate the following energies: 285.2, 287.3, 288.2, 288.6 and 289.3 e (see Table 1); 529.9, 531.3 and 532.1 e (O 1s). C standards include E. coli lipopolysaccharide (LPS), bovine serum albumin (protein), alginate (acidic polysaccharide), agarose (neutral polysaccharide), DNA and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (phospholipid). FeOOH standards (phase confirmed by X-ray diffraction) include lepidocrocite, two-line ferrihydrite, akaganeite and goethite. Iron(III) oxyhydroxides exhibit transitions occurring from both O 1s (peaks a and b) and OH 1s (peak c) core orbitals.

Figure 6

Model of stalk formation and mineralization process. Iron oxidation is coupled to O₂ reduction (exact location of iron oxidation is not known). (1) Fe(III)-polysaccharide (EPS) is excreted from the cell as fibrils. (2) Over time, Fe(III) precipitates as Fe oxyhydroxides. (3) As stalks age, lepidocrocite nucleates on fibril surfaces.