Leptothrix ochracea genomes reveal potential for mixotrophic growth on Fe(II) and organic carbon.

Leptothrix ochracea creates distinctive iron-mineralized mats that carpet streams and wetlands. Easily recognized by its iron-mineralized sheaths, L. ochracea was one of the first microorganisms described in the 1800s. Yet it has never been isolated and does not have a complete genome sequence available, so key questions about its physiology remain unresolved. It is debated whether iron oxidation can be used for energy or growth and if L. ochracea is an autotroph, heterotroph, or mixotroph. To address these issues, we sampled L. ochracea-rich mats from three of its typical environments (a stream, wetlands, and a drainage channel) and reconstructed nine high-quality genomes of L. ochracea from metagenomes. These genomes contain iron oxidase genes cyc2 and mtoA, showing that L. ochracea has the potential to conserve energy from iron oxidation. Sox genes confer potential to oxidize sulfur for energy. There are genes for both carbon fixation (RuBisCO) and utilization of sugars and organic acids (acetate, lactate, and formate). In silico stoichiometric metabolic models further demonstrated the potential for growth using sugars and organic acids. Metatranscriptomes showed a high expression of genes for iron oxidation; aerobic respiration; and utilization of lactate, acetate, and sugars, as well as RuBisCO, supporting mixotrophic growth in the environment. In summary, our results suggest that L. ochracea has substantial metabolic flexibility. It is adapted to iron-rich, organic carbon-containing wetland niches, where it can thrive as a mixotrophic iron oxidizer by utilizing both iron oxidation and organics for energy generation and both inorganic and organic carbon for cell and sheath production. IMPORTANCE: Winogradsky's observations of L. ochracea led him to propose autotrophic iron oxidation as a new microbial metabolism, following his work on autotrophic sulfur-oxidizers. While much culture-based research has ensued, isolation proved elusive, so most work on L. ochracea has been based in the environment and in microcosms. Meanwhile, the autotrophic Gallionella became the model for freshwater microbial iron oxidation, while heterotrophic and mixotrophic iron oxidation is not well-studied. Ecological studies have shown that Leptothrix overtakes Gallionella when dissolved organic carbon content increases, demonstrating distinct niches. This study presents the first near-complete genomes of L. ochracea, which share some features with autotrophic iron oxidizers, while also incorporating heterotrophic metabolisms. These genome, metabolic modeling, and transcriptome results give us a detailed metabolic picture of how the organism may combine lithoautotrophy with organoheterotrophy to promote Fe oxidation and C cycling and drive many biogeochemical processes resulting from microbial growth and iron oxyhydroxide formation in wetlands.

Sphaerotilus uruguayifluvii sp. nov., a novel filamentous bacterium isolated from river water.

Strain C29T, a Gram-staining-negative, straight rod occurring singly, in pairs or short chains, was isolated from floating filamentous biomass of the Uruguay River. The strain was catalase and oxidase positive, chemoorganotrophic, strictly aerobic, non-motile, and grew at pH 6.0-9.0, 15-45 °C, and 0-0.5% (w/v) NaCl. Polyhydroxybutyrate was accumulated in nutrient-limited conditions. Phylogenetic analysis based on the 16S rRNA gene revealed that strain C29T had the highest sequence similarity with Leptothrix discophora SS-1T (97.82%), Ideonella livida TBM-1T (97.82%), Vitreoscilla filiformis L1401-2T (97.52%), Sphaerotilus sulfidivorans D-501T (97.50%) and Sphaerotilus natans DSM 6575T (97.46%). Other type strains with validly published names had similarities below 97.46%. Further phylogenomic analysis showed that strain C29T was affiliated to the family Sphaerotilaceae. Average nucleotide identity (ANI) and in silico DNA-DNA hybridization (dDDH) values with its phylogenetic relatives were lower than 91 and 41%, respectively, revealing that strain C29T represented a new species. The DNA G + C content of strain C29T was 70.9%. The annotation of the genome of the novel strain shows it possessed genes for the degradation of aromatic compounds. It also contained genes that encode sigma factors involved in response regulation of stress resistance, which is an important function for adaptation and survival in natural niches. Based on the results of the phylogenetic and phenotypic analyses, we propose that strain C29T represents a novel species, for which the name Sphaerotilus uruguayifluvii sp. nov. is proposed. The type strain is C29T (= CCM 9043T = DSM 113250T).

Development of a Gene Replacement Method for the Filamentous BacteriumLeptothrix cholodniiSP-6.

Genetic strategies such as gene disruption and fluorescent protein tagging largely contribute to understanding the molecular mechanisms of biological functions in bacteria. However, the methods for gene replacement remain underdeveloped for the filamentous bacteriaLeptothrix cholodniiSP-6. Their cell chains are encased in sheath composed of entangled nanofibrils, which may prevent the conjugation for gene transfer. Here, we describe a protocol optimized for gene disruption through gene transfer mediated by conjugation withEscherichia coliS17-1 with details on cell ratio, sheath removal, and loci validation. The obtained deletion mutants for specific genes can be used to clarify the biological functions of the proteins encoded by the target genes. Graphical overview.

Efficient Degradation for Raffinose and Stachyose of a ß-D-Fructofuranosidase and Its New Function to Improve Gel Properties of Coagulated Fermented-Soymilk.

A novel ß-D-fructofuranosidase gene was identified via database mining from Leptothrix cholodnii. The gene was chemically synthesized and expressed in Escherichia coli, resulting in the production of a highly efficient enzyme known as LcFFase1s. The enzyme exhibited optimal activity at pH 6.5 and a temperature of 50 °C while maintaining stability at pH 5.5-8.0 and a temperature below 50 °C. Furthermore, LcFFase1s exhibited remarkable resistance to commercial proteases and various metal ions that could interfere with its activity. This study also revealed a new hydrolysis function of LcFFase1s, which could completely hydrolyze 2% raffinose and stachyose within 8 h and 24 h, respectively, effectively reducing the flatulence factor in legumes. This discovery expands the potential applications of LcFFase1s. Additionally, the incorporation of LcFFase1s significantly reduced the particle size of coagulated fermented-soymilk gel, resulting in a smoother texture while maintaining the gel hardness and viscosity formed during fermentation. This represents the first report of ß-D-fructofuranosidase enhancing coagulated fermented-soymilk gel properties, highlighting promising possibilities for future applications of LcFFase1s. Overall, the exceptional enzymatic properties and unique functions of LcFFase1s render it a valuable tool for numerous applications.

Identification of lthB, a Gene Encoding a Putative Glycosyltransferase Family 8 Protein Required for Leptothrix Sheath Formation.

The bacterium Leptothrix cholodnii generates cell chains encased in sheaths that are composed of woven nanofibrils. The nanofibrils are mainly composed of glycoconjugate repeats, and several glycosyltransferases (GTs) are required for its biosynthesis. However, only one GT (LthA) has been identified to date. In this study, we screened spontaneous variants of L. cholodnii SP6 to find those that form smooth colonies, which is one of the characteristics of sheathless variants. Genomic DNA sequencing of an isolated variant revealed an insertion in the locus Lcho_0972, which encodes a putative GT family 8 protein. We thus designated this protein LthB and characterized it using deletion mutants and antibodies. LthB localized adjacent to the cell envelope. ΔlthB cell chains were nanofibril free and thus sheathless, indicating that LthB is involved in nanofibril biosynthesis. Unlike the ΔlthA mutant and the wild-type strain, which often generate planktonic cells, most ΔlthB organisms presented as long cell chains under static conditions, resulting in deficient pellicle formation, which requires motile planktonic cells. These results imply that sheaths are not required for elongation of cell chains. Finally, calcium depletion, which induces cell chain breakage due to sheath loss, abrogated the expression of LthA, but not LthB, suggesting that these GTs cooperatively participate in glycoconjugate biosynthesis under different signaling controls. IMPORTANCE In recent years, the regulation of cell chain elongation of filamentous bacteria via extracellular signals has attracted attention as a potential strategy to prevent clogging of water distribution systems and filamentous bulking of activated sludge in industrial settings. However, a fundamental understanding of the ecology of filamentous bacteria remains elusive. Since sheath formation is associated with cell chain elongation in most of these bacteria, the molecular mechanisms underlying nanofibril sheath formation, including the intracellular signaling cascade in response to extracellular stimuli, must be elucidated. Here, we isolated a sheathless variant of L. cholodnii SP6 and thus identified a novel glycosyltransferase, LthB. Although mutants with deletions of lthA, encoding another GT, and lthB were both defective for nanofibril formation, they exhibited different phenotypes of cell chain elongation and pellicle formation. Moreover, LthA expression, but not LthB expression, was influenced by extracellular calcium, which is known to affect nanofibril formation, indicating the functional diversities of LthA and LthB. Such molecular insights are critical for a better understanding of ecology of filamentous bacteria, which, in turn, can be used to improve strategies to control filamentous bacteria in industrial facilities.

Porous Pellicle Formation of a Filamentous Bacterium, Leptothrix.

The bacterium Leptothrix cholodnii generates filaments encased in a sheath comprised of woven nanofibrils. In static liquid culture, L. cholodnii moves toward the air-liquid interface, where it forms porous pellicles. Observations of aggregation at the interface reveal that clusters consisting of only a few bacteria primarily grow by netting free cells. These growing clusters hierarchically enlarge through the random docking of other small clusters. We find that the bacteria swim using their polar flagellum toward the interface, where their sheath assists them in intertwining with others and thereby promotes the formation of small clusters. In contrast, sheathless hydrophobic mutant cells get stuck to the interface. We find that the nanofibril sheath is vital for robust pellicle formation as it lowers cell surface hydrophobicity by 60%, thereby reducing their adsorption and enabling cells to move toward and stick together at the air-liquid interface. IMPORTANCE Efficient and sustainable management of water resources is becoming a fundamental issue for supporting growing populations and for developing economic activity. Fundamental to this management is the treatment of wastewater. Microorganisms are the active component of activated sludge that is employed in the biodegradation process of many wastewater treatment facilities. However, uncontrolled growth of filamentous bacteria such as Sphaerotilus often results in filamentous bulking, lowering the efficiency of water treatment systems. To prevent this undesirable condition, strategies based on a fundamental understanding of the ecology of filamentous bacteria are required. Although the filamentous bacterium Leptothrix cholodnii, which is closely related to Sphaerotilus, is a minor inhabitant of activated sludge, its complete genome sequence is known, making gene manipulation relatively easy. Moreover, L. cholodnii generates porous pellicles under static conditions, which may be a characteristic of filamentous bulking. We show that both swimming motility and nanofibril-mediated air-liquid interface attachment are required for porous pellicle formation. These insights are critical for a better understanding of the characteristics of filamentous bulking and might improve strategies to control activated sludge.

Vaginal Leptothrix: An Innocent Bystander?

Leptothrix are long bacteria of rare occurrence; although these bacteria have been implicated in causing vaginal symptoms identical to candidiasis, studies on prevalence and effect on overall vaginal health are lacking. In this study, we evaluated data of women referred to a private clinic for treating vulvovaginal symptoms (n = 1847) and reassessed data of our previous and ongoing studies (n = 1773). The overall rate of leptothrix was 2.8% (102/3620), and the mean age of affected women was 38.8 ± 10.65 years (range 18-76). The majority of the women with leptothrix had normal vaginal flora (63.7% [65/102]). Leptothrix was associated with a higher risk of candidiasis (relative risk (RR) 1.90, 95% confidence interval (CI) 1.1600-3.1013; p = 0.010) and a lower risk of bacterial vaginosis (RR 0.55, 95% CI, 0.3221-0.9398; p = 0.029) and cytolytic vaginosis (RR 0.11, 95% CI, 0.0294-0.4643; p = 0.002). No cases of trichomoniasis were observed. Human immunodeficiency virus infection increased the risk of leptothrix (RR 3.0, 95% CI, 1.6335-5.7245; p = 0.000). Among the women evaluated for vulvovaginal symptoms, 2.4% (45/1847) had leptothrix, and in 26.7% (12/45), leptothrix was considered the causative entity. This study suggests that leptothrix occurrence is rare; it remains unresolved if it can be a cause of vulvar symptoms.

Green inhibitors reduce unwanted calcium carbonate precipitation: Implications for technical settings.

Mineral scale deposits in water drainage and supply systems are a common and challenging issue, especially by clogging the water flow. The removal of such unwanted deposits is cost intensive arguing for case-specific and sustainable prevention strategies. In the present study, a novel on-site approach to prevent calcium carbonate (CaCO3) scale formation was assessed in two road tunnel drainages: Application of the eco-friendly green inhibitor polyaspartate (PASP) caused (i) a significant inhibition of CaCO3 precipitation, (ii) a more porous or even unconsolidated consistence of the deposits, and (iii) a shift from calcite to the metastable aragonite and vaterite polymorphs. Even relatively low PASP concentrations (1-33 mg/l) can significantly decrease CaCO3 scale deposition, removing up to ∼7 t CaCO3/year at an efficiency up to 84%. Application of PASP for water conditioning should also consider case-specific microbial activity effects, where consumption of PASP, e.g. by Leptothrix ochracea, can limit inhibition effects.

Leptothrix cholodnii Response to Nutrient Limitation.

Microorganisms are widely utilized for the treatment of wastewater in activated sludge systems. However, the uncontrolled growth of filamentous bacteria leads to bulking and adversely affects wastewater treatment efficiency. To clarify the nutrient requirements for filament formation, we track the growth of a filamentous bacterium, Leptothrix cholodnii SP-6 in different nutrient-limited conditions using a high aspect-ratio microfluidic chamber to follow cell-chain elongation and sheath formation. We find that limitations in Na+, K+, and Fe2+ yield no observable changes in the elongation of cell chains and sheath formation, whereas limitations of C, N, P, or vitamins lead to more pronounced changes in filament morphology; here we observe the appearance of partially empty filaments with wide intercellular gaps. We observe more dramatic differences when SP-6 cells are transferred to media lacking Mg2+ and Ca2+. Loss of Mg2+ results in cell autolysis, while removal of Ca2+ results in the catastrophic disintegration of the filaments. By simultaneously limiting both carbon and Ca2+ sources, we are able to stimulate planktonic cell generation. These findings paint a detailed picture of the ecophysiology of Leptothrix, which may lead to improved control over the unchecked growth of deleterious filamentous bacteria in water purification systems.

Ideonella livida sp. nov., isolated from a freshwater lake.

A novel bacterial strain, designated TBM-1T, isolated from a freshwater lake in Taiwan, was characterized using a polyphasic taxonomic approach. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 92 protein clusters indicated that strain TBM-1T formed a phylogenetic lineage in the genus Ideonella. Analysis of 16S rRNA gene sequences showed that strain TBM-1T was most closely related to Ideonella dechloratans CCUG 30898T with 98.4 % sequence similarity. The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between strain TBM-1T and closely related strains of the genus Ideonella were 74.4-77.5 %, 69.7-75.4 % and 19.8-21.8 %, respectively, supporting that strain TBM-1T represents a novel species of the genus Ideonella. Cells were Gram-stain-negative, motile by means of a single polar flagellum, rod-shaped and formed blue colonies. Optimal growth occurred at 30 °C, pH 6 and 0 % NaCl. The predominant fatty acids of strain TBM-1T were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C18 : 1 ω7c and C16 : 0. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, two uncharacterized aminophospholipids and two uncharacterized phospholipids. The main polyamine was putrescine. The major isoprenoid quinone was Q-8. The estimated genome size was 5.26 Mb, with an average G+C content of 70.0 mol%. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain TBM-1T should be classified in a novel species of the genus Ideonella, for which the name Ideonella livida sp. nov. is proposed. The type strain is TBM-1T (=BCRC 81199T =LMG 31339T).

Aquabacterium lacunae sp. nov., isolated from a freshwater pond.

A novel bacterial strain, designated KMB7T, isolated from a freshwater pond in Taiwan, was characterized using a polyphasic taxonomic approach. Cells were Gram-stain-negative, motile by means of a single polar flagellum, rod-shaped and formed cream colonies. Optimal growth occurred at 25 °C, pH 7, and in the absence of NaCl. Phylogenetic analyses based on 16S rRNA gene sequences and an up-to-date bacterial core gene set (92 protein clusters) indicated that strain KMB7T is affiliated with species in the genus Aquabacterium. The 16S rRNA gene sequence similarity indicated that strain KMB7T is closely related to species within the genus Aquabacterium (95.2-97.6 % sequence similarity) and is most similar to A. fontiphilum CS-6T (97.6 %), followed by A. parvum B6T (97.5 %). The average nucleotide identity and digital DNA-DNA hybridization identity between strain KMB7T and the closely related strains were 74.6-78.0 % and 19.0-21.2 %, respectively. The major fatty acids of strain KMB7T were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C18 : 1 ω7c and C16 : 0. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, diphosphatidylglycerol and four unidentified phospholipids. The sole isoprenoid quinone was ubiquinone-8 (Q-8). Genomic DNA G+C content of strain KMB7T was 65.4 %. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain KMB7T should be classified in a novel species of the genus Aquabacterium, for which the name Aquabacterium lacunae sp. nov. is proposed. The type strain is KMB7T (=BCRC 81156T=LMG 30924T=KCTC 62867T).

Polyfunctional Nanofibril Appendages Mediate Attachment, Filamentation, and Filament Adaptability in Leptothrix cholodnii.

Leptothrix is a species of Fe/Mn-oxidizing bacteria known to form long filaments composed of chains of cells that eventually produce a rigid tube surrounding the filament. Prior to the formation of this brittle microtube, Leptothrix cells secrete hair-like structures from the cell surface, called nanofibrils, which develop into a soft sheath that surrounds the filament. To clarify the role of nanofibrils in filament formation in L. cholodnii SP-6, we analyze the behavior of individual cells and multicellular filaments in high-aspect ratio microfluidic chambers using time-lapse and intermittent in situ fluorescent staining of nanofibrils, complemented with atmospheric scanning electron microscopy. We show that in SP-6 nanofibrils are important for attachment and their distribution on young filaments post-attachment is correlated to the directionality of filament elongation. Elongating filaments demonstrate a surprising ability to adapt to their physical environment by changing direction when they encounter obstacles: they bend or reverse direction depending on the angle of the collision. We show that the forces involved in the collision can be used to predict the behavior of filament. Finally, we show that as filaments grow in length, the older region becomes confined by the sheath, while the newly secreted nanofibrils at the leading edge of the filament form a loose, divergent, structure from which cells periodically escape.

Rubrivivax albus sp. nov., isolated from a freshwater pond.

Strain ICH-3T, isolated from a freshwater pond in Taiwan 9ROC), was characterized using a polyphasic taxonomy approach. Phylogenetic analyses based on 16S rRNA gene sequences and an up-to-date bacterial core gene set (92 protein clusters) indicated that ICH-3T is affiliated with the species in the genus Rubrivivax. ICH-3T was most closely related to Rubrivivax benzoatilyticus JA2T and Rubrivivax gelatinosus NCIB 8290T with 97.5 and 97.4 % 16S rRNA gene sequence similarity. The average nucleotide identity and digital DNA-DNA hybridization identity between ICH-3T and the two closely related strains were 77.3 and 20.9-21.0 %, respectively, indicating that ICH-3T represents a novel species of the genus Rubrivivax. Cells were Gram-stain-negative, aerobic, non-motile, rod-shaped and formed white colonies. Optimal growth occurred at 30 °C, pH 7.5-8.0 and with 0.5 % NaCl. The major fatty acids (>20 %) of ICH-3T were summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an uncharacterized aminophospholipid and two uncharacterized phospholipids. The major isoprenoid quinone was Q-8. The genomic DNA G+C content of ICH-3T was 70.3 mol%. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain ICH-3T should be classified as representing a novel species of the genus Rubrivivax, for which the name Rubrivivax albus sp. nov. is proposed. The type strain is ICH-3T (=BCRC 81155T=LMG 30930T=KCTC 62866T).

Biogenic iron oxides for phosphate removal.

Biogenic iron oxides (BioFeO) formed by Leptothrix sp. and Gallionella sp. were compared with chemically formed iron oxides (ChFeO) for their suitability to remove and recover phosphate from solutions. The ChFeO used for comparison included a commercial iron-based adsorbent (GEH) and chemically oxidized iron precipitates from groundwater. Despite contrary observations in earlier studies, the batch experiments showed that BioFeO do not have superior phosphate adsorption capacities compared to ChFeO. However, it seems multiple mechanisms are involved in phosphate removal by BioFeO which make their overall phosphate removal capacity higher than that of ChFeO. The overall phosphate removal capacity of Leptothrix sp. deposits was 26.3 mg P/g d.s., which could be attributed to multiple mechanisms. This included adsorption on the solid phase (6.4 mg P/g d.s.) as well as removal via precipitation and/or adsorption onto suspended complexes released from the BioFeO of Leptothrix sp. (19.6 mg P/g d.s.). Only a very small part of phosphorus (0.3 mg P/g d.s.) was retained in the Leptothrix sp. sheats during bacterial growth. Deposits of Gallionella sp. had an overall phosphate removal capacity of 39.6 mg P/g d.s. Significant amounts of phosphate were apparently incorporated into the Gallionella sp. stalks during their growth (31.0 mg P/g d.s.) and only one-fifth of the total phosphate removal can be related to adsorption (8.6 mg P/g d.s.). Their overall ability to immobilize large quantities of phosphate from solutions indicates that BioFeO could play an important role in environmental and engineered systems for removal of contaminants.

Microbial Diversity in Actively Forming Iron Oxides from Weathered Banded Iron Formation Systems.

The surface crust that caps highly weathered banded iron formations (BIFs) supports a unique ecosystem that is a post-mining restoration priority in iron ore areas. Geochemical evidence indicates that biological processes drive the dissolution of iron oxide minerals and contribute to the ongoing evolution of this duricrust. However, limited information is available on present-day biogeochemical processes in these systems, particularly those that contribute to the precipitation of iron oxides and, thus, the cementation and stabilization of duricrusts. Freshly formed iron precipitates in water bodies perched on cangas in Karijini National Park, Western Australia, were sampled for microscopic and molecular analyses to understand currently active microbial contributions to iron precipitation in these areas. Microscopy revealed sheaths and stalks associated with iron-oxidizing bacteria. The iron-oxidizing lineages Sphaerotilus, Sideroxydans, and Pedomicrobium were identified in various samples and Leptothrix was common in four out of five samples. The iron-reducing bacteria Anaeromyxobacter dehalogens and Geobacter lovleyi were identified in the same four samples, with various heterotrophs and diverse cyanobacteria. Given this arid, deeply weathered environment, the driver of contemporary iron cycling in Karijini National Park appears to be iron-reducing bacteria, which may exist in anaerobic niches through associations with aerobic heterotrophs. Overall oxidizing conditions and Leptothrix iron-oxidizers contribute to net iron oxide precipitation in our sampes, rather than a closed biogeochemical cycle, which would result in net iron oxide dissolution as has been suggested for canga caves in Brazil. Enhancements in microbial iron oxide dissolution and subsequent reprecipitation have potential as a surface-crust-ecosystem remediation strategy at mine sites.

Insights into the Fundamental Physiology of the Uncultured Fe-Oxidizing Bacterium Leptothrix ochracea.

Leptothrix ochracea is known for producing large volumes of iron oxyhydroxide sheaths that alter wetland biogeochemistry. For over a century, these delicate structures have fascinated microbiologists and geoscientists. Because L. ochracea still resists long-term in vitro culture, the debate regarding its metabolic classification dates back to 1885. We developed a novel culturing technique for L. ochracea using in situ natural waters and coupled this with single-cell genomics and nanoscale secondary-ion mass spectrophotometry (nanoSIMS) to probe L. ochracea's physiology. In microslide cultures L. ochracea doubled every 5.7 h and had an absolute growth requirement for ferrous iron, the genomic capacity for iron oxidation, and a branched electron transport chain with cytochromes putatively involved in lithotrophic iron oxidation. Additionally, its genome encoded several electron transport chain proteins, including a molybdopterin alternative complex III (ACIII), a cytochrome bd oxidase reductase, and several terminal oxidase genes. L. ochracea contained two key autotrophic proteins in the Calvin-Benson-Bassham cycle, a form II ribulose bisphosphate carboxylase, and a phosphoribulose kinase. L. ochracea also assimilated bicarbonate, although calculations suggest that bicarbonate assimilation is a small fraction of its total carbon assimilation. Finally, L. ochracea's fundamental physiology is a hybrid of those of the chemolithotrophic Gallionella-type iron-oxidizing bacteria and the sheathed, heterotrophic filamentous metal-oxidizing bacteria of the Leptothrix-Sphaerotilus genera. This allows L. ochracea to inhabit a unique niche within the neutrophilic iron seeps.IMPORTANCELeptothrix ochracea was one of three groups of organisms that Sergei Winogradsky used in the 1880s to develop his hypothesis on chemolithotrophy. L. ochracea continues to resist cultivation and appears to have an absolute requirement for organic-rich waters, suggesting that its true physiology remains unknown. Further, L. ochracea is an ecological engineer; a few L. ochracea cells can generate prodigious volumes of iron oxyhydroxides, changing the ecosystem's geochemistry and ecology. Therefore, to determine L. ochracea's basic physiology, we employed new single-cell techniques to demonstrate that L. ochracea oxidizes iron to generate energy and, despite having predicted genes for autotrophic growth, assimilates a fraction of the total CO2 that autotrophs do. Although not a true chemolithoautotroph, L. ochracea's physiological strategy allows it to be flexible and to extensively colonize iron-rich wetlands.

Aquincola amnicola sp. nov., isolated from a freshwater river.

Strain TTM-94T, isolated from a water sample taken from the Caohu River in Taiwan, was characterized using a polyphasic taxonomic approach. Cells of strain TTM-94T were Gram-staining-negative, aerobic, poly-ß-hydroxybutyrate-accumulating, motile by a single polar flagellum, short rod-shaped and surrounded by a thick capsule and it formed cream colored colonies. Growth occurred at 20-30 °C (optimum, 30 °C), at pH 6.0-8.0 (optimum, pH 6.0), and in the presence of 0-2% NaCl (optimum 0.5%). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain TTM-94T belonged to the genus Aquincola in the Rubrivivax-Roseateles-Leptothrix-Ideonella-Aquabacterium branch of the class Betaproteobacteria and its most closely related neighbour was Aquincola tertiaricarbonis L10T with sequence similarity of 97.0%. Strain TTM-94T contained summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c), C16:0 and C18:1ω7c as the predominant fatty acids. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several uncharacterized lipids. The major respiratory quinone was Q-8. Genomic DNA G + C content of strain TTM-94T was 70.7 mol%. Strain TTM-94T exhibited less than 30% DNA-DNA relatedness with A. tertiaricarbonis L10T. Differential phenotypic properties, together with the phylogenetic inference, demonstrate that strain TTM-94T should be classified as a novel species of the genus Aquincola, for which the name Aquincola amnicola sp. nov. is presented. The type strain is TTM-94T (= BCRC 80890T = LMG 28709T).

Biosorption of metal elements by exopolymer nanofibrils excreted from Leptothrix cells.

Leptothrix species, aquatic Fe-oxidizing bacteria, excrete nano-scaled exopolymer fibrils. Once excreted, the fibrils weave together and coalesce to form extracellular, microtubular, immature sheaths encasing catenulate cells of Leptothrix. The immature sheaths, composed of aggregated nanofibrils with a homogeneous-looking matrix, attract and bind aqueous-phase inorganics, especially Fe, P, and Si, to form seemingly solid, mature sheaths of a hybrid organic-inorganic nature. To verify our assumption that the organic skeleton of the sheaths might sorb a broad range of other metallic and nonmetallic elements, we examined the sorption potential of chemically and enzymatically prepared protein-free organic sheath remnants for 47 available elements. The sheath remnants were found by XRF to sorb each of the 47 elements, although their sorption degree varied among the elements: >35% atomic percentages for Ti, Y, Zr, Ru, Rh, Ag, and Au. Electron microscopy, energy dispersive x-ray spectroscopy, electron and x-ray diffractions, and Fourier transform infrared spectroscopy analyses of sheath remnants that had sorbed Ag, Cu, and Pt revealed that (i) the sheath remnants comprised a 5-10 nm thick aggregation of fibrils, (ii) the test elements were distributed almost homogeneously throughout the fibrillar aggregate, (iii) the nanofibril matrix sorbing the elements was nearly amorphous, and (iv) these elements plausibly were bound to the matrix by ionic binding, especially via OH. The present results show that the constitutive protein-free exopolymer nanofibrils of the sheaths can contribute to creating novel filtering materials for recovering and recycling useful and/or hazardous elements from the environment.

Leptothrix sp. sheaths modified with iron oxide particles: Magnetically responsive, high aspect ratio functional material.

Smart materials of biological origin are attracting a lot of attention nowadays, especially as catalysts, carriers or adsorbents. Among them, magnetically modified biomaterials are especially important due to their response to external magnetic field. This report demonstrates that naturally occurring micrometer sized, high aspect ratio material (native and autoclaved Leptothrix sp. sheaths) efficiently bind synthetically prepared magnetite and maghemite nanoparticles and their aggregates. Magnetic modification of Leptothrix sheaths enables to prepare a promising material for advanced biotechnology and environmental technology applications. The prepared magnetically responsive sheaths were tested as inexpensive adsorbent for crystal violet removal from aqueous solutions. The observed maximum adsorption capacity was 243.1mg of dye per 1g of adsorbent.

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.