Hitoshi Kunoh

Nanometer-Scale Visualization and Structural Analysis of the Inorganic/Organic Hybrid Structure of Gallionella ferruginea Twisted Stalks†

ABSTRACT The so-called Fe/Mn-oxidizing bacteria have long been recognized for their potential to form extracellular iron hydroxide or manganese oxide structures in aquatic environments. Bacterial species belonging to the genus Gallionella, one type of such bacteria, oxidize iron and produce uniquely twisted extracellular stalks consisting of iron oxide-encrusted inorganic/organic fibers. This paper describes the ultrastructure of Gallionella cells and stalks and the visualized structural and spatial localization of constitutive elements within the stalks. Electron microscopy with energy-dispersive X-ray microanalysis showed the export site of the stalk fibers from the cell and the uniform distribution of iron, silicon, and phosphorous in the stalks. Electron energy-loss spectroscopy revealed that the stalk fibers had a central carbon core of bacterial exopolymers and that aquatic iron interacted with oxygen at the surface of the carbon core, resulting in deposition of iron oxides at the surface. This new knowledge of the structural and spatial associations of iron with oxygen and carbon provides deeper insights into the unique inorganic/organic hybrid structure of the stalks.

Structural and spatial associations between Fe, O, and C in the network structure of the Leptothrix ochracea sheath surface.

ABSTRACT The structural and spatial associations of Fe with O and C in the outer coat fibers of the Leptothrix ochracea sheath were shown to be substantially similar to the stalk fibers of Gallionella ferruginea, i.e., a central C core, probably of bacterial origin, and aquatic Fe interacting with O at the surface of the core.

Isolation of a Leptothrix Strain, OUMS1, from Ocherous Deposits in Groundwater

Leptothrix species in aquatic environments produce uniquely shaped hollow microtubules composed of aquatic inorganic and bacterium-derived organic hybrids. Our group termed this biologically derived iron oxide as “biogenous iron oxide (BIOX)”. The artificial synthesis of most industrial iron oxides requires massive energy and is costly while BIOX from natural environments is energy and cost effective. The BIOX microtubules could potentially be used as novel industrial functional resources for catalysts, adsorbents and pigments, among others if effective and efficient applications are developed. For these purposes, a reproducible system to regulate bacteria and their BIOX productivity must be established to supply a sufficient amount of BIOX upon industrial demand. However, the bacterial species and the mechanism of BIOX microtubule formation are currently poorly understood. In this study, a novel Leptothrix sp. strain designated OUMS1 was successfully isolated from ocherous deposits in groundwater by testing various culture media and conditions. Morphological and physiological characters and elemental composition were compared with those of the known strain L. cholodnii SP-6 and the differences between these two strains were shown. The successful isolation of OUMS1 led us to establish a basic system to accumulate biological knowledge of Leptothrix and to promote the understanding of the mechanism of microtubule formation. Additional geochemical studies of the OUMS1-related microstructures are expected provide an attractive approach to study the broad industrial application of bacteria-derived iron oxides.

Silicon and phosphorus linkage with iron via oxygen in the amorphous matrix of Gallionella ferruginea stalks.

ABSTRACT Bacterial species belonging to the genus Gallionella are Fe-oxidizing bacteria that produce uniquely twisted extracellular stalks consisting of iron-oxide-encrusted inorganic/organic fibers in aquatic environments. This paper describes the degree of crystallinity of Gallionella stalks and the chemical linkages of constituent elements in the stalk fibers. Transmission electron microscopy revealed that the matrix of the fiber edge consisted of an assembly of primary particles of approximately 3 nm in diameter. Scanning transmission electron microscopy revealed the rough granular surfaces of the fibers, which reflect the disordered assembly of the primary particles, indicating a high porosity and large specific surface area of the fibers. This may provide the surface with broader reactive properties. X-ray diffractometry, selected-area electron diffraction, and high-resolution transmission electron microscopy together showed that the primary particles had an amorphous structure. Furthermore, energy-dispersive X-ray analysis and Fourier transform infrared spectroscopy detected the bands characteristic of the vibrational modes assigned to O-H, Fe-O-H, P-O-H, Si-O-H, Si-O-Fe, and P-O-Fe bonds in the stalks, suggesting that the minor constituent elements P and Si could affect the degree of crystallinity of the fibers by linking with Fe via O. This knowledge about the mutual associations of these elements provides deeper insights into the unique inorganic/organic hybrid structure of the stalks.

Abiotic Deposition of Fe Complexes onto Leptothrix Sheaths.

Bacteria classified in species of the genus Leptothrix produce extracellular, microtubular, Fe-encrusted sheaths. The encrustation has been previously linked to bacterial Fe oxidases, which oxidize Fe(II) to Fe(III) and/or active groups of bacterial exopolymers within sheaths to attract and bind aqueous-phase inorganics. When L. cholodnii SP-6 cells were cultured in media amended with high Fe(II) concentrations, Fe(III) precipitates visibly formed immediately after addition of Fe(II) to the medium, suggesting prompt abiotic oxidation of Fe(II) to Fe(III). Intriguingly, these precipitates were deposited onto the sheath surface of bacterial cells as the population was actively growing. When Fe(III) was added to the medium, similar precipitates formed in the medium first and were abiotically deposited onto the sheath surfaces. The precipitates in the Fe(II) medium were composed of assemblies of globular, amorphous particles (ca. 50 nm diameter), while those in the Fe(III) medium were composed of large, aggregated particles (≥3 µm diameter) with a similar amorphous structure. These precipitates also adhered to cell-free sheaths. We thus concluded that direct abiotic deposition of Fe complexes onto the sheath surface occurs independently of cellular activity in liquid media containing Fe salts, although it remains unclear how this deposition is associated with the previously proposed mechanisms (oxidation enzyme- and/or active group of organic components-involved) of Fe encrustation of the Leptothrix sheaths.

Genes expressed in tissue-cultured seedlings of mountain laurel (Kalmia latifolia L.) with colonizing Streptomyces padanus AOK30

An endophytic actinomycete, Streptomyces padanus AOK30, is capable of protecting mountain laurel against infection by Pestalotiopsis sydowiana, a causal agent of Pestalotia disease, when applied to the seedling of the plant. In this study, suppression subtractive hybridization was used to identify genes differentially expressed in seedlings of mountain laurel after application of S. padanus AOK30. Subsequent dot hybridization with independent RNA from S. padanus-colonized and control plants identified nonredundant 180 cDNAs involving 71 and 109 clones, which were up- and downregulated after inoculation with the bacteria, respectively. Comparison of the sequences with databases revealed that a number of transcripts encoding proteins or enzymes that function directly in defense or stress response and regulatory proteins were regulated differentially in the seedlings with colonizing S. padanus AOK30. Semi-quantitative RT-PCR analysis for the selected genes demonstrated that inoculation of mountain laurel seedlings with S. padanus AOK30 increased expression of defense-related genes as well as distinct classes of glutathione S-transferase, although endochitinases were exclusively suppressed. These results clearly indicate that the S. padanus-colonizing seedlings likely initiate or prime plant defense responses toward pathogen infection. Selected genes were also differentially expressed in S. padanus-colonized seedlings, compared to those solely challenged with the fungal pathogen P. sydowiana. This approach will assist in efforts not only to understand the molecular basis of the enhanced tolerance and/or enhanced disease resistance of mountain laurel, but also to define a core set of genes during colonization or association with S. padanus AOK30.

Treatment of Leptothrix Cells with Ultrapure Water Poses a Threat to Their Viability

The genus Leptothrix, a type of Fe/Mn-oxidizing bacteria, is characterized by its formation of an extracellular and microtubular sheath. Although almost all sheaths harvested from natural aquatic environments are hollow, a few chained bacterial cells are occasionally seen within some sheaths of young stage. We previously reported that sheaths of Leptothrix sp. strain OUMS1 cultured in artificial media became hollow with aging due to spontaneous autolysis within the sheaths. In this study, we investigated environmental conditions that lead the OUMS1 cells to die. Treatment of the cells with ultrapure water or acidic buffers (pH 6.0) caused autolysis of the cells. Under these conditions, the plasma membrane and cytoplasm of cells were drastically damaged, resulting in leakage of intracellular electrolytes and relaxation of genomic DNA. The autolysis was suppressed by the presence of Ca2+. The hydrolysis of peptidoglycan by the lysozyme treatment similarly caused autolysis of the cells and was suppressed also by the presence of Ca2+. However, it remains unclear whether the acidic pH-dependent autolysis is attributable to damage of peptidoglycan. It was observed that L. discophora strain SP-6 cells also underwent autolysis when suspended in ultrapure water; it is however, uncertain whether this phenomenon is common among other members of the genus Leptothrix.

Dissociation and re-aggregation of multicell-ensheathed fragments responsible for rapid production of massive clumps of Leptothrix sheaths

Species of the Fe/Mn-oxidizing bacteria Leptothrix produce tremendous amounts of microtubular, Fe/Mn-encrusted sheaths within a few days in outwells of groundwater that can rapidly clog water systems. To understand this mode of rapid sheath production and define the timescales involved, behaviors of sheath-forming Leptothrix sp. strain OUMS1 were examined using time-lapse video at the initial stage of sheath formation. OUMS1 formed clumps of tangled sheaths. Electron microscopy confirmed the presence of a thin layer of bacterial exopolymer fibrils around catenulate cells (corresponding to the immature sheath). In time-lapse videos, numerous sheath filaments that extended from the periphery of sheath clumps repeatedly fragmented at the apex of the same fragment, the fragments then aggregated and again elongated, eventually forming a large sheath clump comprising tangled sheaths within two days. In this study, we found that fast microscopic fragmentation, dissociation, re-aggregation and re-elongation events are the basis of the rapid, massive production of Leptothrix sheaths typically observed at macroscopic scales.

Amino group in Leptothrix sheath skeleton is responsible for direct deposition of Fe(III) minerals onto the sheaths

Leptothrix species produce microtubular organic–inorganic materials that encase the bacterial cells. The skeleton of an immature sheath, consisting of organic exopolymer fibrils of bacterial origin, is formed first, then the sheath becomes encrusted with inorganic material. Functional carboxyl groups of polysaccharides in these fibrils are considered to attract and bind metal cations, including Fe(III) and Fe(III)-mineral phases onto the fibrils, but the detailed mechanism remains elusive. Here we show that NH2 of the amino-sugar-enriched exopolymer fibrils is involved in interactions with abiotically generated Fe(III) minerals. NH2-specific staining of L. cholodnii OUMS1 detected a terminal NH2 on its sheath skeleton. Masking NH2 with specific reagents abrogated deposition of Fe(III) minerals onto fibrils. Fe(III) minerals were adsorbed on chitosan and NH2-coated polystyrene beads but not on cellulose and beads coated with an acetamide group. X-ray photoelectron spectroscopy at the N1s edge revealed that the terminal NH2 of OUMS1 sheaths, chitosan and NH2-coated beads binds to Fe(III)-mineral phases, indicating interaction between the Fe(III) minerals and terminal NH2. Thus, the terminal NH2 in the exopolymer fibrils seems critical for Fe encrustation of Leptothrix sheaths. These insights should inform artificial synthesis of highly reactive NH2-rich polymers for use as absorbents, catalysts and so on.