Meta-omics Reveal Gallionellaceae and Rhodanobacter Species as Interdependent Key Players for Fe(II) Oxidation and Nitrate Reduction in the Autotrophic Enrichment Culture KS.

Nitrate reduction coupled to Fe(II) oxidation (NRFO) has been recognized as an environmentally important microbial process in many freshwater ecosystems. However, well-characterized examples of autotrophic nitrate-reducing Fe(II)-oxidizing bacteria are rare, and their pathway of electron transfer as well as their interaction with flanking community members remain largely unknown. Here, we applied meta-omics (i.e., metagenomics, metatranscriptomics, and metaproteomics) to the nitrate-reducing Fe(II)-oxidizing enrichment culture KS growing under autotrophic or heterotrophic conditions and originating from freshwater sediment. We constructed four metagenome-assembled genomes with an estimated completeness of ≥95%, including the key players of NRFO in culture KS, identified as Gallionellaceae sp. and Rhodanobacter sp. The Gallionellaceae sp. and Rhodanobacter sp. transcripts and proteins likely involved in Fe(II) oxidation (e.g., mtoAB, cyc2, and mofA), denitrification (e.g., napGHI), and oxidative phosphorylation (e.g., respiratory chain complexes I to V) along with Gallionellaceae sp. transcripts and proteins for carbon fixation (e.g., rbcL) were detected. Overall, our results indicate that in culture KS, the Gallionellaceae sp. and Rhodanobacter sp. are interdependent: while Gallionellaceae sp. fixes CO2 and provides organic compounds for Rhodanobacter sp., Rhodanobacter sp. likely detoxifies NO through NO reduction and completes denitrification, which cannot be performed by Gallionellaceae sp. alone. Additionally, the transcripts and partial proteins of cbb3- and aa3-type cytochrome c suggest the possibility for a microaerophilic lifestyle of the Gallionellaceae sp., yet culture KS grows under anoxic conditions. Our findings demonstrate that autotrophic NRFO is performed through cooperation among denitrifying and Fe(II)-oxidizing bacteria, which might resemble microbial interactions in freshwater environments. IMPORTANCE Nitrate-reducing Fe(II)-oxidizing bacteria are widespread in the environment, contribute to nitrate removal, and influence the fate of the greenhouse gases nitrous oxide and carbon dioxide. The autotrophic growth of nitrate-reducing Fe(II)-oxidizing bacteria is rarely investigated and not fully understood. The most prominent model system for this type of study is the enrichment culture KS. To gain insights into the metabolism of nitrate reduction coupled to Fe(II) oxidation in the absence of organic carbon and oxygen, we performed metagenomic, metatranscriptomic, and metaproteomic analyses of culture KS and identified Gallionellaceae sp. and Rhodanobacter sp. as interdependent key Fe(II) oxidizers in culture KS. Our work demonstrates that autotrophic nitrate reduction coupled to Fe(II) oxidation is not performed by an individual strain but is a cooperation of at least two members of the bacterial community in culture KS. These findings serve as a foundation for our understanding of nitrate-reducing Fe(II)-oxidizing bacteria in the environment.

Sulfurimicrobium lacus gen. nov., sp. nov., a sulfur oxidizer isolated from lake water, and review of the family Sulfuricellaceae to show that it is not a later synonym of Gallionellaceae.

A facultatively anaerobic sulfur-oxidizing bacterium, strain skT11T, was isolated from anoxic lake water of a stratified freshwater lake. As electron donor for chemolithoautotrophic growth, strain skT11T oxidized thiosulfate, tetrathionate, and elemental sulfur under nitrate-reducing conditions. Oxygen-dependent growth was observed under microoxic conditions, but not under fully oxygenated conditions. Growth was observed at a temperature range of 5-37 °C, with optimum growth at 28 °C. Strain skT11T grew at a pH range of 5.1-7.5, with optimum growth at pH 6.5-6.9. Heterotrophic growth was not observed. Major components in the cellular fatty acid profile were C16:1 and C16:0. The complete genome of strain skT11T consisted of a circular chromosome with a size of 3.8 Mbp and G + C content of 60.2 mol%. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the strain skT11T is related to sulfur-oxidizing bacteria of the genera Sulfuricella, Sulfurirhabdus, and Sulfuriferula, with sequence identities of 95.4% or lower. The analysis also indicated that these three genera should be excluded from the family Gallionellaceae, as members of another family. On the basis of its genomic and phenotypic properties, strain skT11T (= DSM 110711 T = NBRC 114323 T) is proposed as the type strain of a new species in a new genus, Sulfurimicrobium lacus gen. nov., sp. nov. In addition, emended descriptions of the families Gallionellaceae and Sulfuricellaceae are proposed to declare that Sulfuricellaceae is not a later synonym of Gallionellaceae.

Ferrigenium kumadai gen. nov., sp. nov., a microaerophilic iron-oxidizing bacterium isolated from a paddy field soil.

An iron-oxidizing bacterium, designated strain An22T, which was isolated from a paddy field soil in Anjo, Japan, was described taxonomically. Strain An22T was motile by a single polar flagellum, curved-rod, Gram-negative bacterium that was able to grow at 12-37 °C (optimally at 25-30 °C) and at pH 5.2-6.8 (pH 5.9-6.1). The strain grew microaerobically and autotrophically by oxidizing ferrous iron, but did not form stalks, a unique structure of iron oxides. The major cellular fatty acids were C16 : 0 and C16 : 1ω7c/C16 : 1ω6c. The major respiratory quinones were UQ-10 and UQ-8. The strain possessed ribulose-1,5-bisphosphate carboxylase/oxygenase indicating an autotrophic nature via the Calvin-Benson-Bassham cycle. The total DNA G+C content was 61.4 mol%. 16S rRNA gene sequence analysis revealed that strain An22T was affiliated with the class Betaproteobacteria and clustered with iron-oxidizing bacteria, Gallionella ferrugineaJohan (94.8 % similarity) and Ferriphaselus amnicola OYT1T (94.4 %) in the family Gallionellaceae. Based on the low 16S rRNA gene sequence similarity to the phylogenetically closest genera and the combination of unique morphological, physiological and biochemical characteristics, strain An22T represents a novel genus and species within the family Gallionellaceae, for which the name Ferrigenium kumadai gen. nov., sp. nov. is proposed. The type strain is An22T (=JCM 30584T=NBRC 112974T=ATCC TSD-51T).

Metagenomic Analyses of the Autotrophic Fe(II)-Oxidizing, Nitrate-Reducing Enrichment Culture KS.

Nitrate-dependent ferrous iron [Fe(II)] oxidation (NDFO) is a well-recognized chemolithotrophic pathway in anoxic sediments. The neutrophilic chemolithoautotrophic enrichment culture KS originally obtained from a freshwater sediment (K. L. Straub, M. Benz, B. Schink, and F. Widdel, Appl Environ Microbiol 62:1458-1460, 1996) has been used as a model system to study NDFO. However, the primary Fe(II) oxidizer in this culture has not been isolated, despite extensive efforts to do so. Here, we present a metagenomic analysis of this enrichment culture in order to gain insight into electron transfer pathways and the roles of different bacteria in the culture. We obtained a near-complete genome of the primary Fe(II) oxidizer, a species in the family Gallionellaceae, and draft genomes from its flanking community members. A search of the putative extracellular electron transfer pathways in these genomes led to the identification of a homolog of the MtoAB complex [a porin-multiheme cytochromec system identified in neutrophilic microaerobic Fe(II)-oxidizing Sideroxydans lithotrophicus ES-1] in a Gallionellaceae sp., and findings of other putative genes involving cytochromecand multicopper oxidases, such as Cyc2 and OmpB. Genome-enabled metabolic reconstruction revealed that this Gallionellaceae sp. lacks nitric oxide and nitrous oxide reductase genes and may partner with flanking populations capable of complete denitrification to avoid toxic metabolite accumulation, which may explain its resistance to growth in pure culture. This and other revealed interspecies interactions and metabolic interdependencies in nitrogen and carbon metabolisms may allow these organisms to cooperate effectively to achieve robust chemolithoautotrophic NDFO. Overall, the results significantly expand our knowledge of NDFO and suggest a range of genetic targets for further exploration.

High spatial resolution of distribution and interconnections between Fe- and N-redox processes in profundal lake sediments.

The Fe and N biogeochemical cycles play key roles in freshwater environments. We aimed to determine the spatial positioning and interconnections of the N and Fe cycles in profundal lake sediments. The gradients of O2, NO3(-), NH4(+), pH, Eh, Fe(II) and Fe(III) were determined and the distribution of microorganisms was assessed by most probable numbers and quantitative polymerase chain reaction. The redox zones could be divided into an oxic zone (0-8 mm), where microaerophiles (Gallionellaceae) were most abundant at a depth of 7 mm. This was followed by a denitrification zone (6-12 mm), where NO3(-)-dependent Fe(II) oxidizers and organoheterotrophic denitrifiers both reduce nitrate. Lastly, an iron redox transition zone was identified at 12.5-22.5 mm. Fe(III) was most abundant above this zone while Fe(II) was most abundant beneath. The high abundance of poorly crystalline iron suggested iron cycling. The Fe and N cycles are biologically connected through nitrate-reducing Fe(II) oxidizers and chemically by NOx(-) species formed during denitrification, which can chemically oxidize Fe(II). This study combines high resolution chemical, molecular and microbiological data to pinpoint sedimentary redox zones in which Fe is cycled between Fe(II) and Fe(III) and where Fe and N-redox processes interact.

Chemolithotrophic primary production in a subglacial ecosystem.

Glacial comminution of bedrock generates fresh mineral surfaces capable of sustaining chemotrophic microbial communities under the dark conditions that pervade subglacial habitats. Geochemical and isotopic evidence suggests that pyrite oxidation is a dominant weathering process generating protons that drive mineral dissolution in many subglacial systems. Here, we provide evidence correlating pyrite oxidation with chemosynthetic primary productivity and carbonate dissolution in subglacial sediments sampled from Robertson Glacier (RG), Alberta, Canada. Quantification and sequencing of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) transcripts suggest that populations closely affiliated with Sideroxydans lithotrophicus, an iron sulfide-oxidizing autotrophic bacterium, are abundant constituents of microbial communities at RG. Microcosm experiments indicate sulfate production during biological assimilation of radiolabeled bicarbonate. Geochemical analyses of subglacial meltwater indicate that increases in sulfate levels are associated with increased calcite and dolomite dissolution. Collectively, these data suggest a role for biological pyrite oxidation in driving primary productivity and mineral dissolution in a subglacial environment and provide the first rate estimate for bicarbonate assimilation in these ecosystems. Evidence for lithotrophic primary production in this contemporary subglacial environment provides a plausible mechanism to explain how subglacial communities could be sustained in near-isolation from the atmosphere during glacial-interglacial cycles.

Ferriphaselus amnicola gen. nov., sp. nov., a neutrophilic, stalk-forming, iron-oxidizing bacterium isolated from an iron-rich groundwater seep.

A neutrophilic, stalk-forming, iron-oxidizing bacterium, strain OYT1(T), which was isolated from a groundwater seep in Ohyato Park, Tokyo, Japan, was subjected to taxonomic analysis. OYT1(T) was a motile, bean-shaped, Gram-negative bacterium that was able to grow at 8-30 °C (optimally at 25-30 °C) and at pH 5.6-7.3 (optimally at pH 6.1-6.5). The strain grew microaerobically and autotrophically. Major cellular fatty acids detected were C16 : 1ω7c/C16 : 1ω6c and C16 : 0. The total DNA G+C content was 57.6 mol%. 16S rRNA gene sequence analysis revealed that strain OYT1(T) was affiliated with the class Betaproteobacteria and clustered with iron-oxidizing bacteria isolated from groundwater seeps and wetlands and with uncultured clones detected in freshwater iron-rich environments. Based on the phenotypic and phylogenetic characteristics of strain OYT1(T), we propose that the strain represents a novel species in a new genus, for which the name Ferriphaselus amnicola gen. nov., sp. nov. is proposed; the type strain of Ferriphaselus amnicola is OYT1(T) ( = JCM 18545(T) = DSM 26810(T)).

Diversity and distribution of iron-oxidising bacteria belonging to Gallionellaceae in different sites of a hydroelectric power plant.

Iron (Fe) is the fourth most abundant element on the planet, and iron-oxidising bacteria (FeOB) play an important role in the biogeochemical cycle of this metal in nature. FeOB stands out as Fe oxidisers in microaerophilic environments, and new members of this group have been increasingly discussed in the literature, even though their isolation can still be challenging. Among these bacteria is the Gallionellaceae family, mainly composed of neutrophilic FeOB, highlighting Gallionella ferruginea, and nitrite-oxidiser genera. In the previous metagenomic study of the biofilm and sediments of the cooling system from the Irapé hydroelectric power plant (HPP-Irapé), 5% of the total bacteria sequences were related to Gallionellaceae, being 99% unclassified at genus level. Thus, in the present study, a phylogenetic tree based on this family was constructed, in order to search for shared and unique Gallionellaceae signatures in a deep phylogenetic level affiliation and correlated them with geomorphologic characteristics. The results revealed that Gallionella and Ferrigenium were ubiquitous reflecting their ability to adapt to various locations in the power plant. The cave was considered a hotspot for neutrophilic FeOB since it harboured most of the Gallionellaceae diversity. Microscopic biosignatures were detected only in the CS1 sample, which presented abundance of the stalk-forming Ferriphaselus and of the sheath-forming Crenothrix. Further studies are required to provide more detailed insights on Gallionellaceae distribution and diversity patterns in hydroelectric power plants, particularly its biotechnological potential in this industry.

Functional gene analysis of freshwater iron-rich flocs at circumneutral pH and isolation of a stalk-forming microaerophilic iron-oxidizing bacterium.

Iron-rich flocs often occur where anoxic water containing ferrous iron encounters oxygenated environments. Culture-independent molecular analyses have revealed the presence of 16S rRNA gene sequences related to diverse bacteria, including autotrophic iron oxidizers and methanotrophs in iron-rich flocs; however, the metabolic functions of the microbial communities remain poorly characterized, particularly regarding carbon cycling. In the present study, we cultivated iron-oxidizing bacteria (FeOB) and performed clone library analyses of functional genes related to carbon fixation and methane oxidization (cbbM and pmoA, respectively), in addition to bacterial and archaeal 16S rRNA genes, in freshwater iron-rich flocs at groundwater discharge points. The analyses of 16S rRNA, cbbM, and pmoA genes strongly suggested the coexistence of autotrophic iron oxidizers and methanotrophs in the flocs. Furthermore, a novel stalk-forming microaerophilic FeOB, strain OYT1, was isolated and characterized phylogenetically and physiologically. The 16S rRNA and cbbM gene sequences of OYT1 are related to those of other microaerophilic FeOB in the family Gallionellaceae, of the Betaproteobacteria, isolated from freshwater environments at circumneutral pH. The physiological characteristics of OYT1 will help elucidate the ecophysiology of microaerophilic FeOB. Overall, this study demonstrates functional roles of microorganisms in iron flocs, suggesting several possible linkages between Fe and C cycling.

Isolation and characterization of a novel biomineral stalk-forming iron-oxidizing bacterium from a circumneutral groundwater seep.

The Fe-depositing microorganism Gallionella ferruginea was first described in 1836 based on its association with Fe-rich environments and its distinctive morphology. Since then, this morphology has been widely used to identify G. ferruginea. Researchers have isolated several Fe-oxidizing bacteria (FeOB) related to Gallionella; however, few isolates have produced organized extracellular biomineral structures, and of these, only one stalk former has a sequenced 16S rRNA gene, listed as G. ferruginea in the GenBank database. Here we report the isolation and characterization of a novel stalk-forming Fe-oxidizing bacterium, strain R-1, from a freshwater Fe seep. Despite a strong morphological similarity to G. ferruginea, this isolate has only 93.55% 16S rRNA gene sequence similarity with the previously determined sequence. R-1 only grows on Fe(II) substrates, at pH 5.6 to 7.0 and from 10°C to 35°C, with a doubling time of ∼15 h at pH 6.3 and 22°C. It is a Betaproteobacterium, most closely related to uncultured bacteria from microaerobic Fe(II)-rich groundwater springs. The most closely related isolates are Sideroxydans spp. (94.05-94.42% sequence similarity), FeOB that are not known to produce morphologically distinct minerals. To our knowledge, this is the first reported stalk-forming freshwater FeOB isolate distinct from Gallionella.

Microbiology of diverse acidic and non-acidic microhabitats within a sulfidic ore mine.

A wide variety of microhabitats within the extremely acidic abandoned underground copper mine Zlaté Hory (Czech Republic) was investigated. SSU rDNA libraries were analyzed from 15 samples representing gossan, sulfide-leaching environments in the oxidation zone, and acidic water springs in the mine galleries. Microbial analyses were extended by analyses of chemical composition of water and solid phases and identification of arising secondary minerals. The microbial communities of the three main classes of microenvironments differed in almost every aspect. Among others, ecological partitioning of Acidithiobacillus ferrooxidans and the recently described A. ferrivorans was observed. Distinct types of communities inhabiting the water springs were detected. The more extreme springs (pH <3, conductivity >2 mS/cm) were inhabited by "Ferrovum" spp. and A. ferrivorans, whereas Gallionella sp. dominated the less extreme ones. A new role for gossan in the extremely acidic ecosystem is proposed. This zone was inhabited by a large diversity of neutrophilic heterotrophs that appeared to be continuously washed out to the acidic environments localized downstream. Five species originating in gossan were found in several acidic habitats. Here they can survive and probably serve as scavengers of dead biomass, particularly from chemoautotrophic growths. No such process has been described from acidic mine environments so far.

'Candidatus ferrigenium straubiae' sp. nov., 'Candidatus ferrigenium bremense' sp. nov., 'Candidatus ferrigenium altingense' sp. nov., are autotrophic Fe(II)-oxidizing bacteria of the family Gallionellaceae.

Iron(II) [Fe(II)] oxidation coupled to denitrification is recognized as an environmentally important process in many ecosystems. However, the Fe(II)-oxidizing bacteria (FeOB) dominating autotrophic nitrate-reducing Fe(II)-oxidizing enrichment cultures, affiliated with the family Gallionellaceae, remain poorly taxonomically defined due to lack of representative isolates. We describe the taxonomic classification of three novel FeOB based on metagenome-assembled genomes (MAGs) acquired from the autotrophic nitrate-reducing enrichment cultures KS, BP and AG. Phylogenetic analysis of nearly full-length 16S rRNA gene sequences demonstrated that these three FeOB were most closely affiliated to the genera Ferrigenium, Sideroxydans and Gallionella, with up to 96.5%, 95.4% and 96.2% 16S rRNA gene sequence identities to representative isolates of these genera, respectively. In addition, average amino acid identities (AAI) of the genomes compared to the most closely related genera revealed highest AAI with Ferrigenium kumadai An22 (76.35-76.74%), suggesting that the three FeOB are members of this genus. Phylogenetic analysis of conserved functional genes further supported that these FeOB represent three novel species of the genus Ferrigenium. Moreover, the three novel FeOB likely have characteristic features, performing partial denitrification coupled to Fe(II) oxidation and carbon fixation. Scanning electron microscopy of the enrichment cultures showed slightly curved rod-shaped cells, ranging from 0.2-0.7 µm in width and 0.5-2.3 µm in length. Based on the phylogenetic, genomic and physiological characteristics, we propose that these FeOB represent three novel species, 'Candidatus Ferrigenium straubiae' sp. nov., 'Candidatus Ferrigenium bremense' sp. nov. and 'Candidatus Ferrigenium altingense' sp. nov. that might have unique metabolic features among the genus Ferrigenium.

Distribution and diversity of Gallionella-like neutrophilic iron oxidizers in a tidal freshwater marsh.

Microbial iron oxidation is an integral part of the iron redox cycle in wetlands. Nonetheless, relatively little is known about the composition and ecology of iron-oxidizing communities in the soils and sediments of wetlands. In this study, sediment cores were collected across a freshwater tidal marsh in order to characterize the iron-oxidizing bacteria (FeOB) and to link their distributions to the geochemical properties of the sediments. We applied recently designed 16S rRNA primers targeting Gallionella-related FeOB by using a nested PCR-denaturing gradient gel electrophoresis (DGGE) approach combined with a novel quantitative PCR (qPCR) assay. Gallionella-related FeOB were detected in most of the samples. The diversity and abundance of the putative FeOB were generally higher in the upper 5 to 12 cm of sediment than in deeper sediment and higher in samples collected in April than in those collected in July and October. Oxygen supply by macrofauna appears to be a major force in controlling the spatial and temporal variations in FeOB communities. The higher abundance of Gallionella-related FeOB in April coincided with elevated concentrations of extractable Fe(III) in the sediments. Despite this coincidence, the distributions of FeOB did not exhibit a simple relationship to the redox zonation inferred from the geochemical depth profiles.

Presence of Fe(II) and nitrate shapes aquifer-originating communities leading to an autotrophic enrichment dominated by an Fe(II)-oxidizing Gallionellaceae sp.

Autotrophic nitrate reduction coupled to Fe(II) oxidation is an important nitrate removal process in anoxic aquifers. However, it remains unknown how changes of O2 and carbon availability influence the community structure of nitrate-reducing Fe(II)-oxidizing (NRFeOx) microbial assemblages and what the genomic traits of these NRFeOx key players are. We compared three metabolically distinct denitrifying assemblages, supplemented with acetate, acetate/Fe(II) or Fe(II), enriched from an organic-poor, pyrite-rich aquifer. The presence of Fe(II) promoted the growth of denitrifying Burkholderiaceae spp. and an unclassified Gallionellaceae sp. This Gallionellaceae sp. was related to microaerophilic Fe(II) oxidizers; however, it did not grow under microoxic conditions. Furthermore, we explored a metagenome and 15 metagenome-assembled genomes from an aquifer-originating, autotrophic NRFeOx culture. The dominant Gallionellaceae sp. revealed the potential to oxidize Fe(II) (e.g. cyc2), fix CO2 (e.g. rbcL) and perform near-complete denitrification leading to N2O formation (e.g. narGHJI,nirK/S and norBC). In addition, Curvibacter spp.,Methyloversatilis sp. and Thermomonas spp. were identified as novel putative NRFeOx taxa. Our findings provide first insights into the genetic traits of the so far only known autotrophic NRFeOx culture originating from an organic-poor aquifer, providing the genomic basis to study mechanisms of nitrate removal in organic-poor subsurface ecosystems.

Characterization of bacterial community structure in a drinking water distribution system during an occurrence of red water.

The role of bacteria in the occasional emergence of red water, which has been documented worldwide, has yet to be determined. To better understand the mechanisms that drive occurrences of red water, the bacterial community composition and the relative abundance of several functional bacterial groups in a water distribution system of Beijing during a large-scale red water event were determined using several molecular methods. Individual clone libraries of the 16S rRNA gene were constructed for three red water samples and one sample of normal water. Beta-, Alpha-, and Gammaproteobacteria comprised the major bacterial communities in both red water and normal water samples, in agreement with previous reports. A high percentage of red water clones (25.2 to 57.1%) were affiliated with or closely related to a diverse array of iron-oxidizing bacteria, including the neutrophilic microaerobic genera Gallionella and Sideroxydans, the acidophilic species Acidothiobacillus ferrooxidans, and the anaerobic denitrifying Thermomonas bacteria. The genus Gallionella comprised 18.7 to 28.6% of all clones in the three red water libraries. Quantitative real-time PCR analysis showed that the 16S rRNA gene copy concentration of Gallionella spp. was between (4.1 ± 0.9) × 107 (mean ± standard deviation) and (1.6 ± 0.3) × 108 per liter in red water, accounting for 13.1% ± 2.9% to 17.2% ± 3.6% of the total Bacteria spp. in these samples. By comparison, the percentages of Gallionella spp. in the normal water samples were 0.1% or lower (below the limit of detection), suggesting an important role of Gallionella spp. in the formation of red water.

Genome reconstruction reveals distinct assemblages of Gallionellaceae in surface and subsurface redox transition zones.

Fe-oxidizing bacteria of the family Gallionellaceae are major players in the Fe biogeochemical cycle in freshwater. These bacteria thrive in redox transition zones where they benefit from both high Fe concentrations and microaerobic conditions. We analysed the Gallionellaceae genomic diversity in an artesian hard-rock aquifer where redox transition zones develop (i) in the subsurface, where ancient, reduced groundwater mixes with recent oxygenated groundwater, and (ii) at the surface, where groundwater reaches the open air. A total of 15 new draft genomes of Gallionellaceae representing to 11 candidate genera were recovered from the two redox transition zones. Sulfur oxidation genes were encoded in most genomes while denitrification genes were much less represented. One genus dominated microbial communities belowground and we propose to name it 'Candidatus Houarnoksidobacter'. The two transition zones were populated by completely different assemblages of Gallionellaceae despite the almost constant upward circulation of groundwater between the two zones. The processes leading to redox transition zones, oxygen diffusion at the surface or groundwater mixing in subsurface, appear to be a major driver of the Gallionellaceae diversity.

Opportunistic pathogens and large microbial diversity detected in source-to-distribution drinking water of three remote communities in Northern Australia.

In the wet-dry tropics of Northern Australia, drinking water in remote communities is mostly sourced from bores accessing groundwater. Many aquifers contain naturally high levels of iron and some are shallow with surface water intrusion in the wet season. Therefore, environmental bacteria such as iron-cycling bacteria promoting biofilm formation in pipes or opportunistic pathogens can occur in these waters. An opportunistic pathogen endemic to northern Australia and Southeast Asia and emerging worldwide is Burkholderia pseudomallei. It causes the frequently fatal disease melioidosis in humans and animals. As we know very little about the microbial composition of drinking water in remote communities, this study aimed to provide a first snapshot of the microbiota and occurrence of opportunistic pathogens in bulk water and biofilms from the source and through the distribution system of three remote water supplies with varying iron levels. Using 16s-rRNA gene sequencing, we found that the geochemistry of the groundwater had a substantial impact on the untreated microbiota. Different iron-cycling bacteria reflected differences in redox status and nutrients. We cultured and sequenced B. pseudomallei from bores with elevated iron and from a multi-species biofilm which also contained iron-oxidizing Gallionella, nitrifying Nitrospira and amoebae. Gallionella are increasingly used in iron-removal filters in water supplies and more research is needed to examine these interactions. Similar to other opportunistic pathogens, B. pseudomallei occurred in water with low organic carbon levels and with low heterotrophic microbial growth. No B. pseudomallei were detected in treated water; however, abundant DNA of another opportunistic pathogen group, non-tuberculous mycobacteria was recovered from treated parts of one supply. Results from this study will inform future studies to ultimately improve management guidelines for water supplies in the wet-dry tropics.

Genomic profiling of four cultivated Candidatus Nitrotoga spp. predicts broad metabolic potential and environmental distribution.

Nitrite-oxidizing bacteria (NOB) play a critical role in the mitigation of nitrogen pollution by metabolizing nitrite to nitrate, which is removed via assimilation, denitrification, or anammox. Recent studies showed that NOB are phylogenetically and metabolically diverse, yet most of our knowledge of NOB comes from only a few cultured representatives. Using cultivation and genomic sequencing, we identified four putative Candidatus Nitrotoga NOB species from freshwater sediments and water column samples in Colorado, USA. Genome analyses indicated highly conserved 16S rRNA gene sequences, but broad metabolic potential including genes for nitrogen, sulfur, hydrogen, and organic carbon metabolism. Genomic predictions suggested that Ca. Nitrotoga can metabolize in low oxygen or anoxic conditions, which may support an expanded environmental niche for Ca. Nitrotoga similar to other NOB. An array of antibiotic and metal resistance genes likely allows Ca. Nitrotoga to withstand environmental pressures in impacted systems. Phylogenetic analyses highlighted a deeply divergent nitrite oxidoreductase alpha subunit (NxrA), suggesting a novel evolutionary trajectory for Ca. Nitrotoga separate from any other NOB and further revealing the complex evolutionary history of nitrite oxidation in the bacterial domain. Ca. Nitrotoga-like 16S rRNA gene sequences were prevalent in globally distributed environments over a range of reported temperatures. This work considerably expands our knowledge of the Ca. Nitrotoga genus and suggests that their contribution to nitrogen cycling should be considered alongside other NOB in wide variety of habitats.

Metatranscriptomic evidence of pervasive and diverse chemolithoautotrophy relevant to C, S, N and Fe cycling in a shallow alluvial aquifer.

Groundwater ecosystems are conventionally thought to be fueled by surface-derived allochthonous organic matter and dominated by heterotrophic microbes living under often-oligotrophic conditions. However, in a 2-month study of nitrate amendment to a perennially suboxic aquifer in Rifle (CO), strain-resolved metatranscriptomic analysis revealed pervasive and diverse chemolithoautotrophic bacterial activity relevant to C, S, N and Fe cycling. Before nitrate injection, anaerobic ammonia-oxidizing (anammox) bacteria accounted for 16% of overall microbial community gene expression, whereas during the nitrate injection, two other groups of chemolithoautotrophic bacteria collectively accounted for 80% of the metatranscriptome: (1) members of the Fe(II)-oxidizing Gallionellaceae family and (2) strains of the S-oxidizing species, Sulfurimonas denitrificans. Notably, the proportion of the metatranscriptome accounted for by these three groups was considerably greater than the proportion of the metagenome coverage that they represented. Transcriptional analysis revealed some unexpected metabolic couplings, in particular, putative nitrate-dependent Fe(II) and S oxidation among nominally microaerophilic Gallionellaceae strains, including expression of periplasmic (NapAB) and membrane-bound (NarGHI) nitrate reductases. The three most active groups of chemolithoautotrophic bacteria in this study had overlapping metabolisms that allowed them to occupy different yet related metabolic niches throughout the study. Overall, these results highlight the important role that chemolithoautotrophy can have in aquifer biogeochemical cycling, a finding that has broad implications for understanding terrestrial carbon cycling and is supported by recent studies of geochemically diverse aquifers.

Dominance of 'Gallionella capsiferriformans' and heavy metal association with Gallionella-like stalks in metal-rich pH 6 mine water discharge.

Heavy metal-contaminated, pH 6 mine water discharge created new streams and iron-rich terraces at a creek bank in a former uranium-mining area near Ronneburg, Germany. The transition from microoxic groundwater with ~5 mm Fe(II) to oxic surface water may provide a suitable habitat for microaerobic iron-oxidizing bacteria (FeOB). In this study, we investigated the potential contribution of these FeOB to iron oxidation and metal retention in this high-metal environment. We (i) identified and quantified FeOB in water and sediment at the outflow, terraces, and creek, (ii) studied the composition of biogenic iron oxides (Gallionella-like twisted stalks) with scanning and transmission electron microscopy (SEM, TEM) as well as confocal laser scanning microscopy (CLSM), and (iii) examined the metal distribution in sediments. Using quantitative PCR, a very high abundance of FeOB was demonstrated at all sites over a 6-month study period. Gallionella spp. clearly dominated the communities, accounting for up to 88% of Bacteria, with a minor contribution of other FeOB such as Sideroxydans spp. and 'Ferrovum myxofaciens'. Classical 16S rRNA gene cloning showed that 96% of the Gallionella-related sequences had ≥ 97% identity to the putatively metal-tolerant 'Gallionella capsiferriformans ES-2', in addition to known stalk formers such as Gallionella ferruginea and Gallionellaceae strain R-1. Twisted stalks from glass slides incubated in water and sediment were composed of the Fe(III) oxyhydroxide ferrihydrite, as well as polysaccharides. SEM and scanning TEM-energy-dispersive X-ray spectroscopy revealed that stalk material contained Cu and Sn, demonstrating the association of heavy metals with biogenic iron oxides and the potential for metal retention by these stalks. Sequential extraction of sediments suggested that Cu (52-61% of total sediment Cu) and other heavy metals were primarily bound to the iron oxide fractions. These results show the importance of 'G. capsiferriformans' and biogenic iron oxides in slightly acidic but highly metal-contaminated freshwater environments.