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.

Effects of Co-existing Heterotrophs on Physiology of and Nitrogen Metabolism in Autotrophic Nitrite-oxidizing Candidatus Nitrotoga.

Interactions between autotrophic nitrifiers and heterotrophs have attracted considerable attention in microbial ecology. However, the mechanisms by which heterotrophs affect the physiological activity of and nitrogen metabolism in autotrophic nitrite oxidizers remain unclear. We herein focused on nitrite-oxidizing Candidatus Nitrotoga and compared an axenic culture including only Ca. Nitrotoga with a co-culture of both Ca. Nitrotoga and Acidovorax in physiological experiments and transcriptomics. In the co-culture with Acidovorax, nitrite consumption by Ca. Nitrotoga was promoted, and some genes relevant to nitrogen metabolism in Ca. Nitrotoga were highly expressed. These results provide insights into the mechanisms by which co-existing heterotrophs affect autotrophic nitrifiers.

Gallionellaceae in rice root plaque: metabolic roles in iron oxidation, nutrient cycling, and plant interactions.

IMPORTANCE: In waterlogged soils, iron plaque forms a reactive barrier between the root and soil, collecting phosphate and metals such as arsenic and cadmium. It is well established that iron-reducing bacteria solubilize iron, releasing these associated elements. In contrast, microbial roles in plaque formation have not been clear. Here, we show that there is a substantial population of iron oxidizers in plaque, and furthermore, that these organisms (Sideroxydans and Gallionella) are distinguished by genes for plant colonization and nutrient fixation. Our results suggest that iron-oxidizing and iron-reducing bacteria form and remodel iron plaque, making it a dynamic system that represents both a temporary sink for elements (P, As, Cd, C, etc.) as well as a source. In contrast to abiotic iron oxidation, microbial iron oxidation results in coupled Fe-C-N cycling, as well as microbe-microbe and microbe-plant ecological interactions that need to be considered in soil biogeochemistry, ecosystem dynamics, and crop management.

Gallionellaceae pangenomic analysis reveals insight into phylogeny, metabolic flexibility, and iron oxidation mechanisms.

IMPORTANCE: Neutrophilic iron-oxidizing bacteria (FeOB) produce copious iron (oxyhydr)oxides that can profoundly influence biogeochemical cycles, notably the fate of carbon and many metals. To fully understand environmental microbial iron oxidation, we need a thorough accounting of iron oxidation mechanisms. In this study, we show the Gallionellaceae FeOB genomes encode both characterized iron oxidases as well as uncharacterized multiheme cytochromes (MHCs). MHCs are predicted to transfer electrons from extracellular substrates and likely confer metabolic capabilities that help Gallionellaceae occupy a range of different iron- and mineral-rich niches. Gallionellaceae appear to specialize in iron oxidation, so it would be advantageous for them to have multiple mechanisms to oxidize various forms of iron, given the many iron minerals on Earth, as well as the physiological and kinetic challenges faced by FeOB. The multiple iron/mineral oxidation mechanisms may help drive the widespread ecological success of Gallionellaceae.

Copper and iron metal resistant rhizospheric bacteria boost the plant growth and bacoside A content in Bacopa monnieri under stress conditions.

Bacteria that enhance plant growth and development and are found in the vicinity of roots are referred to as plant growth-promoting rhizobacteria. Some beneficial bacteria help plant tolerance to many hazardous chemical elements. In this context, Cupriavidus basilensis , Novosphingobium humi , Bacillus zanthoxyli , Bacillus sp., Paenibacillus alvei , Ancylobacter aquaticus and Ralstonia syzygii metal-tolerant rhizospheric bacteria were isolated from rhizospheric soil associated with Bacopa monnieri . The beneficial effects of rhizospheric bacteria on B. monnieri plant physiology and biochemical responses were investigated under pot conditions at two levels (100µM and 500µM) of CuSO4 or FeCl3 . N. humi , A. aquaticus and R. syzygii bacterial strains were associated with significantly increased height and biomass under normal and stress conditions. An assay for indole acetic acid in isolated rhizospheric bacteria found differential secretion except Bacillus zanthoxyli . Bacoside A is a major phytocompound in B. monnieri with medicinal value; maximum induction was observed in the R. syzygii treatment. High concentration of copper and iron salts negatively influenced height, biomass and photosynthetic pigments; however N. humi , A. aquaticus , Bacilllus sp. and R. syzygii beneficial bacterial helped plants under stress conditions. Moreover, a significant enhancement in chlorophyll a and b was noticed in C. basilensis , B. zanthoxyli , Bacilllus sp., P. alvei and R. syzygii treatments, without much influence on carotenoid levels. Therefore, the present study emphasises the importance of isolating plant growth-promoting rhizobacteria for use in bacopa plants exposed to metals such as copper and iron in soil.

'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.

Some like it cold: the cellular organization and physiological limits of cold-tolerant nitrite-oxidizing Nitrotoga.

Chemolithoautotrophic production of nitrate is accomplished by the polyphyletic functional group of nitrite-oxidizing bacteria (NOB). A widely distributed and important NOB clade in nitrogen removal processes at low temperatures is Nitrotoga, which however remains understudied due to the scarcity of cultivated representatives. Here, we present physiological, ultrastructural and genomic features of Nitrotoga strains from various habitats, including the first marine species enriched from an aquaculture system. Immunocytochemical analyses localized the nitrite-oxidizing enzyme machinery in the wide irregularly shaped periplasm, apparently without contact to the cytoplasmic membrane, confirming previous genomic data suggesting a soluble nature. Interestingly, in two strains we also observed multicellular complexes with a shared periplasmic space, which seem to form through incomplete cell division and might enhance fitness or survival. Physiological tests revealed differing tolerance limits towards dissolved inorganic nitrogen concentrations and confirmed the generally psychrotolerant nature of the genus. Moreover, comparative analysis of 15 Nitrotoga genomes showed, e.g. a unique gene repertoire of the marine strain that could be advantageous in its natural habitat and confirmed the lack of genes for assimilatory nitrite reduction in a strain found to require ammonium for growth. Overall, these novel insights largely broaden our knowledge of Nitrotoga and elucidate the metabolic variability, physiological limits and thus potential ecological roles of this group of nitrite oxidizers.

Unraveling Fe(II)-Oxidizing Mechanisms in a Facultative Fe(II) Oxidizer, Sideroxydans lithotrophicus Strain ES-1, via Culturing, Transcriptomics, and Reverse Transcription-Quantitative PCR.

Sideroxydans lithotrophicus ES-1 grows autotrophically either by Fe(II) oxidation or by thiosulfate oxidation, in contrast to most other isolates of neutrophilic Fe(II)-oxidizing bacteria (FeOB). This provides a unique opportunity to explore the physiology of a facultative FeOB and constrain the genes specific to Fe(II) oxidation. We compared the growth of S. lithotrophicus ES-1 on Fe(II), thiosulfate, and both substrates together. While initial growth rates were similar, thiosulfate-grown cultures had higher yield with or without Fe(II) present, which may give ES-1 an advantage over obligate FeOB. To investigate the Fe(II) and S oxidation pathways, we conducted transcriptomics experiments, validated with reverse transcription-quantitative PCR (RT-qPCR). We explored the long-term gene expression response at different growth phases (over days to a week) and expression changes during a short-term switch from thiosulfate to Fe(II) (90 min). The dsr and sox sulfur oxidation genes were upregulated in thiosulfate cultures. The Fe(II) oxidase gene cyc2 was among the top expressed genes during both Fe(II) and thiosulfate oxidation, and addition of Fe(II) to thiosulfate-grown cells caused an increase in cyc2 expression. These results support the role of Cyc2 as the Fe(II) oxidase and suggest that ES-1 maintains readiness to oxidize Fe(II), even in the absence of Fe(II). We used gene expression profiles to further constrain the ES-1 Fe(II) oxidation pathway. Notably, among the most highly upregulated genes during Fe(II) oxidation were genes for alternative complex III, reverse electron transport, and carbon fixation. This implies a direct connection between Fe(II) oxidation and carbon fixation, suggesting that CO2 is an important electron sink for Fe(II) oxidation. IMPORTANCE Neutrophilic FeOB are increasingly observed in various environments, but knowledge of their ecophysiology and Fe(II) oxidation mechanisms is still relatively limited. Sideroxydans isolates are widely observed in aquifers, wetlands, and sediments, and genome analysis suggests metabolic flexibility contributes to their success. The type strain ES-1 is unusual among neutrophilic FeOB isolates, as it can grow on either Fe(II) or a non-Fe(II) substrate, thiosulfate. Almost all our knowledge of neutrophilic Fe(II) oxidation pathways comes from genome analyses, with some work on metatranscriptomes. This study used culture-based experiments to test the genes specific to Fe(II) oxidation in a facultative FeOB and refine our model of the Fe(II) oxidation pathway. We gained insight into how facultative FeOB like ES-1 connect Fe, S, and C biogeochemical cycling in the environment and suggest a multigene indicator would improve understanding of Fe(II) oxidation activity in environments with facultative FeOB.

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.

Ligand Effects on Biotic and Abiotic Fe(II) Oxidation by the Microaerophile Sideroxydans lithotrophicus.

Organic ligands are widely distributed and can affect microbially driven Fe biogeochemical cycles, but effects on microbial iron oxidation have not been well quantified. Our work used a model microaerophilic Fe(II)-oxidizing bacterium Sideroxydans lithotrophicus ES-1 to quantify biotic Fe(II) oxidation rates in the presence of organic ligands at 0.02 atm O2 and pH 6.0. We used two common Fe chelators with different binding strengths: citrate (log KFe(II)-citrate = 3.20) and nitrilotriacetic acid (NTA) (log KFe(II)-NTA = 8.09) and two standard humic substances, Pahokee peat humic acid (PPHA) and Suwannee River fulvic acid (SRFA). Our results provide rate constants for biotic and abiotic Fe(II) oxidation over different ligand concentrations and furthermore demonstrate that various models and natural iron-binding ligands each have distinct effects on abiotic versus biotic Fe(II) oxidation rates. We show that NTA accelerates abiotic oxidation and citrate has negligible effects, making it a better laboratory chelator. The humic substances only affect biotic Fe(II) oxidation, via a combination of chelation and electron transfer. PPHA accelerates biotic Fe(II) oxidation, while SRFA decelerates or accelerates the rate depending on concentration. The specific nature of organic-Fe microbe interactions may play key roles in environmental Fe(II) oxidation, which have cascading influences on cycling of nutrients and contaminants that associate with Fe oxide minerals.

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.

Uranium and neptunium retention mechanisms in Gallionella ferruginea/ferrihydrite systems for remediation purposes.

The ubiquitous ß-Proteobacterium Gallionella ferruginea is known as stalk-forming, microaerophilic iron(II) oxidizer, which rapidly produces iron oxyhydroxide precipitates. Uranium and neptunium sorption on the resulting intermixes of G. ferruginea cells, stalks, extracellular exudates, and precipitated iron oxyhydroxides (BIOS) was compared to sorption to abiotically formed iron oxides and oxyhydroxides. The results show a high sorption capacity of BIOS towards radionuclides at circumneutral pH values with an apparent bulk distribution coefficient (Kd) of 1.23 × 104 L kg-1 for uranium and 3.07 × 105 L kg-1 for neptunium. The spectroscopic approach by X-ray absorption spectroscopy (XAS) and ATR FT-IR spectroscopy, which was applied on BIOS samples, showed the formation of inner-sphere complexes. The structural data obtained at the uranium LIII-edge and the neptunium LIII-edge indicate the formation of bidentate edge-sharing surface complexes, which are known as the main sorption species on abiotic ferrihydrite. Since the rate of iron precipitation in G. ferruginea-dominated systems is 60 times faster than in abiotic systems, more ferrihydrite will be available for immobilization processes of heavy metals and radionuclides in contaminated environments and even in the far-field of high-level nuclear waste repositories.

Iron is not everything: unexpected complex metabolic responses between iron-cycling microorganisms.

Coexistence of microaerophilic Fe(II)-oxidizers and anaerobic Fe(III)-reducers in environments with fluctuating redox conditions is a prime example of mutualism, in which both partners benefit from the sustained Fe-pool. Consequently, the Fe-cycling machineries (i.e., metal-reducing or -oxidizing pathways) should be most affected during co-cultivation. However, contrasting growth requirements impeded systematic elucidation of their interactions. To disentangle underlying interaction mechanisms, we established a suboxic co-culture system of Sideroxydans sp. CL21 and Shewanella oneidensis. We showed that addition of the partner's cell-free supernatant enhanced both growth and Fe(II)-oxidizing or Fe(III)-reducing activity of each partner. Metabolites of the exometabolome of Sideroxydans sp. CL21 are generally upregulated if stimulated with the partner´s spent medium, while S. oneidensis exhibits a mixed metabolic response in accordance with a balanced response to the partner. Surprisingly, RNA-seq analysis revealed genes involved in Fe-cycling were not differentially expressed during co-cultivation. Instead, the most differentially upregulated genes included those encoding for biopolymer production, lipoprotein transport, putrescine biosynthesis, and amino acid degradation suggesting a regulated inter-species biofilm formation. Furthermore, the upregulation of hydrogenases in Sideroxydans sp. CL21 points to competition for H2 as electron donor. Our findings reveal that a complex metabolic and transcriptomic response, but not accelerated formation of Fe-end products, drive interactions of Fe-cycling microorganisms.

Sulfuriferula nivalis sp. nov., a sulfur oxidizer isolated from snow and emended description of Sulfuriferula plumbiphila.

A chemolithoautotrophic sulfur-oxidizing bacterium, strain SGTMT was isolated from snow collected in Japan. As electron donors for growth, SGTMT oxidized thiosulfate, tetrathionate and elemental sulfur. Heterotrophic growth was not observed. Growth of the novel isolate was observed at a temperature range of 5-28 °C, with optimum growth at 18 °C. SGTMT grew at a pH range of 4.3-7.4, with optimum growth at pH 6.1-7.1. Major components in the cellular fatty acid profile were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0. The complete genome of SGTMT consisted of a circular chromosome of approximately 3.4 Mbp and two plasmids. Phylogenetic analysis based on the 16S rRNA gene indicated that SGTMT represented a member of the genus Sulfuriferula, and its closest relative is Sulfuriferula thiophila mst6T with a sequence identity of 98 %. A comparative genome analysis showed dissimilarity between the genomes of SGTMT and S. thiophila mst6T, as low values of average nucleotide identity (74.9 %) and digital DNA-DNA hybridization (20.4%). On the basis of its genomic and phenotypic properties, SGTMT (=DSM 109609T=BCRC 81185T) is proposed as the type strain of a novel species, Sulfuriferula nivalis sp. nov. Some characteristics of another species in the same genus, Sulfuriferula plumbiphila, were also investigated to revise and supplement its description. The type strain of S. plumbiphila can grow on thiosulfate, tetrathionate and elemental sulfur. The strain showed optimum growth at pH 6.3-7.0 and shared major cellular fatty acids with the other species of the genus Sulfuriferula.

Physiological and genomic characterization of a new 'Candidatus Nitrotoga' isolate.

Oxidation of nitrite to nitrate is an important process in the global nitrogen cycle. Recent molecular biology-based studies have revealed that the widespread nitrite-oxidizing bacteria (NOB) belonging to the genus 'Candidatus Nitrotoga' may be highly important for the environment. However, the insufficient availability of pure Nitrotoga cultures has limited our understanding of their physiological and genomic characteristics. Here, we isolated the 'Ca. Nitrotoga' sp. strain AM1P, from a previously enriched Nitrotoga culture, using an improved isolation strategy. Although 'Ca. Nitrotoga' have been recognized as cold-adapted NOB, the strain AM1P had a slightly higher optimum growth temperature at 23°C. Strain AM1P showed a pH optimum of 8.3 and was not inhibited even at high nitrite concentrations (20 mM). We obtained the complete genome of the strain and compared the genome profile to five previously sequenced 'Ca. Nitrotoga' strains. Comparative genomics suggested that lactate dehydrogenase may be only encoded in the strain AM1P and closely related genomes. While the growth yield of AM1P did not change, we observed faster growth in the presence of lactate in comparison to purely chemolithoautotrophic growth. The characterization of the new strain AM1P sheds light on the physiological adaptation of this environmentally important, but understudied genus 'Ca. Nitrotoga'.

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.

Long Range Correlation in Redox Potential Fluctuations Signals Energetic Efficiency of Bacterial Fe(II) Oxidation.

Differentiating biotic and abiotic processes in nature remains a persistent challenge, specifically in evaluating microbial contributions to geochemical processes through time. Building on previous work reporting that biologically-influenced systems exhibit stronger long-range correlation than abiotic systems, this study evaluated the relationship between long-range correlation of redox potential and oxidation rates of circumneutral microaerophilic bacterial Fe(II) oxidation using a series of batch microcosms with bacteriogenic iron oxides (BIOS). Initial detrended fluctuation analysis (DFA) scaling exponents of the abiotic microcosms were lower (ca. 1.20) than those of the biotic microcosms (ca. 1.80). As Fe(II) oxidation proceeded, correlation strength decayed as a logistic function of elapsed reaction time, exhibiting direct dependence on the free energy of reaction. Correlation strength for all microcosms decayed sharply from strong correlation to uncorrelated fluctuations. The decay rates are greater for abiotic microcosms than biotic microcosms. The ΔGm relaxation edges for biotic microcosms were lower, indicating less remaining free energy for Fe(II) oxidation than abiotic systems, with the implication that biologically-catalyzed reactions are likely more energetically efficient than abiotic reactions. These results strengthen the case for employing novel DFA techniques to distinguish in situ microbial metabolic activity from abiotic processes, as well as to potentially differentiate metabolisms among different chemoautotrophs.

Low Temperature and Neutral pH Define "Candidatus Nitrotoga sp." as a Competitive Nitrite Oxidizer in Coculture with Nitrospira defluvii.

Nitrification is an essential process for N removal in activated sludge to avoid toxicity of ammonium and nitrite. Besides Nitrospira, "Candidatus Nitrotoga" has been identified as a key nitrite-oxidizing bacterium (NOB) performing the second step of nitrification, nitrite oxidation to nitrate, in wastewater treatment plants (WWTPs). However, the driving forces for the dominance of Nitrotoga in certain plants have often remained unclear and could not be explained solely by temperature effects. In this study, we characterized the physiology of the ammonium-dependent Nitrotoga sp. BS with regard to temperature and pH variations and evaluated its competitiveness against Nitrospira defluvii Both NOB originated from the same WWTP and shared a comparable pH optimum of 7.3. Based on these results, coculturing experiments with these NOB were performed in batch reactors operated at either 17°C or 22°C to compare their abundances under optimal (pH 7.4) or suboptimal (pH 6.4) conditions using 1 mM nitrite. As revealed by quantitative PCR (qPCR), fluorescence in situ hybridization (FISH), and 16S amplicon sequencing, Nitrotoga sp. BS was clearly favored by its optimal growth parameters and dominated over Ns. defluvii at pH 7.4 and 17°C, whereas a pH of 6.4 was more selective for Ns. defluvii Our synthetic communities revealed that niche differentiation of NOB is influenced by a complex interaction of environmental parameters and has to be evaluated for single species.IMPORTANCE "Ca. Nitrotoga" is a NOB of high environmental relevance, but physiological data exist for only a few representatives. Initially, it was detected in specialized niches of low temperature and low nitrite concentrations, but later on, its ubiquitous distribution revealed its critical role for N removal in engineered systems like WWTPs. In this study, we analyzed the competition between Nitrotoga and Nitrospira in bioreactors and identified conditions where the K strategist Ns. defluvii was almost replaced by Nitrotoga sp. BS. We show that the pH value is an important factor that regulates the composition of the nitrite-oxidizing enrichment with a dominance of Nitrotoga sp. BS versus Ns. defluvii at a neutral pH of 7.4 in combination with a temperature of 17°C. The physiological diversity of novel Nitrotoga cultures improves our knowledge about niche differentiation of NOB with regard to functional nitrification under suboptimal conditions.