Formation of Biogenic Manganese Oxide Nodules on Hyphae of a New Fungal Isolate of Periconia That Immobilizes Aqueous Copper.

Mn(II)-oxidizing microorganisms are considered to play significant roles in the natural geochemical cycles of Mn and other heavy metals because the insoluble biogenic Mn oxides (BMOs) that are produced by these microorganisms adsorb other dissolved heavy metals and immobilize them as precipitates. In the present study, a new Mn(II)-oxidizing fungal strain belonging to the ascomycete genus Periconia, a well-studied plant-associating fungal genus with Mn(II)-oxidizing activity that has not yet been exami-ned in detail, was isolated from natural groundwater outflow sediment. This isolate, named strain TS-2, was confirmed to oxidize dissolved Mn(II) and produce insoluble BMOs that formed characteristic, separately-located nodules on their hyphae while leaving major areas of the hyphae free from encrustation. These BMO nodules also adsorbed and immobilized dissolved Cu(II), a model analyte of heavy metals, as evidenced by elemental mapping ana-lyses of fungal hyphae-BMO assemblages using a scanning electron microscope with energy-dispersive X-ray spectroscopy (SEM-EDX). Analyses of functional genes within the whole genome of strain TS-2 further revealed the presence of multiple genes predicted to encode laccases/multicopper oxidases that were potentially responsible for Mn(II) oxidation by this strain. The formation of BMO nodules may have functioned to prevent the complete encrustation of fungal hyphae, thereby enabling the control of heavy metal concentrations in their local microenvironments while maintaining hyphal functionality. The present results will expand our knowledge of the physiological and morphological traits of Mn(II)-oxidizing Periconia, which may affect the natural cycle of heavy metals through their immobilization.

Whole-genome sequence of Periconia sp. strain TS-2, an ascomycete fungus isolated from a freshwater outflow and capable of Mn(II) oxidation.

Members of the genus Periconia are commonly found as plant-associated filamentous fungi. Here, the first draft genome sequence of a new Periconia strain, TS-2, that was isolated from freshwater outflow sediment and possesses the ability to oxidize dissolved Mn(II), was obtained and has an estimated size of 40.7 Mb.

Genomic analysis of a marine alphaproteobacterium Sagittula sp. strain MA-2 that carried eight plasmids.

Bacteria that belong to the family Roseobacteraceae in the Alphaproteobacteria class are widely distributed in marine environments with remarkable physiological diversity, which is considered to be attributed to their genomic plasticity. In this study, a novel isolate of the genus Sagittula within Roseobacteraceae, strain MA-2, was obtained from a coastal marine bacterial consortium enriched with aromatic hydrocarbons, and its complete genome was sequenced. The genome with a total size of 5.69 Mbp was revealed to consist of a 4.67-Mbp circular chromosome and eight circular plasmids ranging in size from 19.5 to 361.5 kbp. Further analyses of functional genes in the strain MA-2 genome identified homologous genes responsible for the biotransformation of gentisic acid, which were located on one of its plasmids and were not found in genomes of other Sagittula strains available from databases. This suggested that strain MA-2 had acquired these genes via horizontal gene transfers that enabled them to degrade and utilize gentisic acid as a growth substrate. This study provided the second complete genome sequence of the genus Sagittula and supports the hypothesis that acquisition of ecologically relevant genes in extrachromosomal replicons allows Roseobacteraceae to be highly adaptable to diverse lifestyles.

Nondesulfurizing benzothiophene biotransformation to hetero and homodimeric ortho-substituted diaryl disulfides by the model PAH-degrading Sphingobium barthaii.

Understanding the biotransformation mechanisms of toxic sulfur-containing polycyclic aromatic hydrocarbon (PASH) pollutants such as benzothiophene (BT) is useful for predicting their environmental fates. In the natural environment, nondesulfurizing hydrocarbon-degrading bacteria are major active contributors to PASH biodegradation at petroleum-contaminated sites; however, BT biotransformation pathways by this group of bacteria are less explored when compared to desulfurizing organisms. When a model nondesulfurizing polycyclic aromatic hydrocarbon-degrading soil bacterium, Sphingobium barthaii KK22, was investigated for its ability to cometabolically biotransform BT by quantitative and qualitative methods, BT was depleted from culture media but was biotransformed into mostly high molar mass (HMM) hetero and homodimeric ortho-substituted diaryl disulfides (diaryl disulfanes). HMM diaryl disulfides have not been reported as biotransformation products of BT. Chemical structures were proposed for the diaryl disulfides by comprehensive mass spectrometry analyses of the chromatographically separated products and were supported by the identification of transient upstream BT biotransformation products, which included benzenethiols. Thiophenic acid products were also identified, and pathways that described BT biotransformation and novel HMM diaryl disulfide formation were constructed. This work shows that nondesulfurizing hydrocarbon-degrading organisms produce HMM diaryl disulfides from low molar mass polyaromatic sulfur heterocycles, and this may be taken into consideration when predicting the environmental fates of BT pollutants.

Comprehensive Genomic Characterization of Marine Bacteria Thalassospira spp. Provides Insights into Their Ecological Roles in Aromatic Hydrocarbon-Exposed Environments.

The marine bacterial genus Thalassospira has often been identified as an abundant member of polycyclic aromatic hydrocarbon (PAH)-exposed microbial communities. However, despite their potential usability for biotechnological applications, functional genes that are conserved in their genomes have barely been investigated. Thus, the goal of this study was to comprehensively examine the functional genes that were potentially responsible for aromatic hydrocarbon biodegradation in the Thalassospira genomes available from databases, including a new isolate of Thalassospira, strain GO-4, isolated from a phenanthrene-enriched marine bacterial consortium. Strain GO-4 was used in this study as a model organism to link the genomic data and their metabolic functions. Strain GO-4 growth assays confirmed that it utilized a common phenanthrene biodegradation intermediate 2-carboxybenzaldehyde (CBA) as the sole source of carbon and energy, but did not utilize phenanthrene. Consistently, strain GO-4 was found to possess homologous genes of phdK, pht, and pca that encode enzymes for biodegradation of CBA, phthalic acid, and protocatechuic acid, respectively. Further comprehensive genomic analyses for 33 Thalassospira genomes from databases showed that a gene cluster that consisted of phdK and pht homologs was conserved in 13 of the 33 strains. pca gene homologs were found in all examined genomes; however, homologs of the known PAH-degrading genes, such as the pah, phn, or nah genes, were not found. Possibly Thalassospira spp. co-occupy niches with other PAH-degrading bacteria that provide them with PAH degradation intermediates and facilitated their inhabitation in PAH-exposed microbial ecosystems. IMPORTANCE Comprehensive investigation of multiple genomic data sets from targeted microbial taxa deposited in databases may provide substantial information to predict metabolic capabilities and ecological roles in different environments. This study is the first report that details the functional profiling of Thalassospira spp. that have repeatedly been found in polycyclic aromatic hydrocarbon (PAH)-exposed marine bacterial communities by using genomic data from a new isolate, Thalassospira strain GO-4, and other strains in databases. Through screening of functional genes potentially involved in aromatic hydrocarbon biodegradation across 33 Thalassospira genomes and growth assays for strain GO-4, it was suggested that Thalassospira spp. unexceptionally conserved the ability to metabolize single-ring, PAH biodegradation intermediates, while being incapable of utilizing PAHs. This expanded our understanding of this potentially important contributing member to PAH-degrading microbial ecosystems; such species are considered to be specialized in driving downstream reactions of PAH biodegradation.

Complete Genome Sequence of Thalassospira sp. Strain GO-4, a Marine Bacterium Isolated from a Phenanthrene-Enriched Bacterial Consortium.

The genus Thalassospira has often been studied as a potential major contributing member of aromatic hydrocarbon-exposed microbial communities. Here, the complete genome sequence of a new isolate of Thalassospira, strain GO-4, was obtained and was confirmed to possess functional genes that are responsible for its metabolism of phthalic acid.

Alum Addition Triggers Hypoxia in an Engineered Pit Lake.

Here, we examine the geobiological response to a whole-lake alum (aluminum sulfate) treatment (2016) of Base Mine Lake (BML), the first pilot-scale pit lake established in the Alberta oil sands region. The rationale for trialing this management amendment was based on its successful use to reduce internal phosphorus loading to eutrophying lakes. Modest increases in water cap epilimnetic oxygen concentrations, associated with increased Secchi depths and chlorophyll-a concentrations, were co-incident with anoxic waters immediately above the fluid fine tailings (FFT) layer post alum. Decreased water cap nitrate and detectable sulfide concentrations, as well as increased hypolimnetic phospholipid fatty acid abundances, signaled greater anaerobic heterotrophic activity. Shifts in microbial community to groups associated with greater organic carbon degradation (i.e., SAR11-LD12 subclade) and the SRB group Desulfuromonodales emerged post alum and the loss of specialist groups associated with carbon-limited, ammonia-rich restricted niches (i.e., MBAE14) also occurred. Alum treatment resulted in additional oxygen consumption associated with increased autochthonous carbon production, watercap anoxia and sulfide generation, which further exacerbate oxygen consumption associated with on-going FFT mobilized reductants. The results illustrate the importance of understanding the broader biogeochemical implications of adaptive management interventions to avoid unanticipated outcomes that pose greater risks and improve tailings reclamation for oil sands operations and, more broadly, the global mining sector.

Natural Chromosome-Chromid Fusion across rRNA Operons in a Burkholderiaceae Bacterium.

Chromids (secondary chromosomes) in bacterial genomes that are present in addition to the main chromosome appear to be evolutionarily conserved in some specific bacterial groups. In rare cases among these groups, a small number of strains from Rhizobiales and Vibrionales were shown to possess a naturally fused single chromosome that was reported to have been generated through intragenomic homologous recombination between repeated sequences on the chromosome and chromid. Similar examples have never been reported in the family Burkholderiaceae, a well-documented group that conserves chromids. Here, an in-depth genomic characterization was performed on a Burkholderiaceae bacterium that was isolated from a soil bacterial consortium maintained on diesel fuel and mutagenic benzo[a]pyrene. This organism, Cupriavidus necator strain KK10, was revealed to carry a single chromosome with unexpectedly large size (>6.6 Mb), and results of comparative genomics with the genome of C. necator N-1T indicated that the single chromosome of KK10 was generated through fusion of the prototypical chromosome and chromid at the rRNA operons. This fusion hypothetically occurred through homologous recombination with a crossover between repeated rRNA operons on the chromosome and chromid. Some metabolic functions that were likely expressed from genes on the prototypical chromid region were indicated to be retained. If this phenomenon-the bacterial chromosome-chromid fusion across the rRNA operons through homologous recombination-occurs universally in prokaryotes, the multiple rRNA operons in bacterial genomes may not only contribute to the robustness of ribosome function, but also provide more opportunities for genomic rearrangements through frequent recombination. IMPORTANCE A bacterial chromosome that was naturally fused with the secondary chromosome, or "chromid," and presented as an unexpectedly large single replicon was discovered in the genome of Cupriavidus necator strain KK10, a biotechnologically useful member of the family Burkholderiaceae. Although Burkholderiaceae is a well-documented group that conserves chromids in their genomes, this chromosomal fusion event has not been previously reported for this family. This fusion has hypothetically occurred through intragenomic homologous recombination between repeated rRNA operons and, if so, provides novel insight into the potential of multiple rRNA operons in bacterial genomes to lead to chromosome-chromid fusion. The harsh conditions under which strain KK10 was maintained-a genotoxic hydrocarbon-enriched milieu-may have provided this genotype with a niche in which to survive.

Complete Genome Sequence of Cupriavidus necator KK10, an Azaarene-Degrading and Polyhydroxyalkanoate-Producing Soil Bacterium.

Cupriavidus necator KK10 has been investigated in azaarene and diesel fuel biodegradation studies and is capable of polyhydroxyalkanoate (PHA) production. Its complete genome sequence revealed two closed circular sequences, the chromosome (6.68 Mb) and plasmid (1.67 Mb). The KK10 genome carries functional genes potentially responsible for azaarene biodegradation and polyhydroxyalkanoate biosynthesis.

Complete Genome Sequence of Sphingobium barthaii KK22, a High-Molecular-Weight Polycyclic Aromatic Hydrocarbon-Degrading Soil Bacterium.

Sphingobium barthaii KK22T is a high-molecular-weight polycyclic aromatic hydrocarbon-degrading soil bacterium that has been investigated in biotransformation, microbial ecology, and DNA damage studies. The complete genome sequence of S. barthaii revealed four closed circular sequences, including two chromosomes, a megaplasmid, and a smaller plasmid, by hybrid assembly using short- and long-read sequencing technologies.

Multispecies Diesel Fuel Biodegradation and Niche Formation Are Ignited by Pioneer Hydrocarbon-Utilizing Proteobacteria in a Soil Bacterial Consortium.

A soil bacterial consortium that was grown on diesel fuel and consisted of more than 10 members from different genera was maintained through repetitive subculturing and was utilized as a practical model to investigate a bacterial community that was continuously exposed to petroleum hydrocarbons. Through metagenomics analyses, consortium member isolation, growth assays, and metabolite identification which supported the linkage of genomic data and functionality, two pioneering genera, Sphingobium and Pseudomonas, whose catabolic capabilities were differentiated, were found to be responsible for the creation of specialized ecological niches that were apparently occupied by other bacterial members for survival within the consortium. Coexisting genera Achromobacter and Cupriavidus maintained their existence in the consortium through metabolic dependencies by utilizing hydrocarbon biotransformation products of pioneer metabolism, which was confirmed through growth tests and identification of biotransformation products of the isolated strains. Pioneering Sphingobium and Pseudomonas spp. utilized relatively water-insoluble hydrocarbon parent compounds and facilitated the development of a consortium community structure that resulted in the creation of niches in response to diesel fuel exposure which were created through the production of more-water-soluble biotransformation products available to cocolonizers. That these and other organisms were still present in the consortium after multiple transfers spanning 15 years provided evidence for these ecological niches. Member survival through occupation of these niches led to robustness of each group within the multispecies bacterial community. Overall, these results contribute to our understanding of the complex ecological relationships that may evolve during prokaryotic hydrocarbon pollutant biodegradation.IMPORTANCE There are few metagenome studies that have explored soil consortia maintained on a complex hydrocarbon substrate after the community interrelationships were formed. A soil bacterial consortium maintained on diesel fuel was utilized as a practical model to investigate bacterial community relationships through metagenomics analyses, consortium member isolation, growth assays, and metabolite identification, which supported the linkage of genomic data and functionality. Two pioneering genera were responsible for the biodegradation of aromatics and alkanes by initiating biotransformation and thereby created specialized niches that were populated by other members. A model that represents these relationships was constructed, which contributes to our understanding of the complex ecological relationships that evolve during prokaryotic hydrocarbon pollutant biodegradation.

Putative Mixotrophic Nitrifying-Denitrifying Gammaproteobacteria Implicated in Nitrogen Cycling Within the Ammonia/Oxygen Transition Zone of an Oil Sands Pit Lake.

Anthropogenically-impacted environments offer the opportunity to discover novel microbial species and metabolisms, which may be undetectable in natural systems. Here, a combined metagenomic and geochemical study in Base Mine Lake, Alberta, Canada, which is the only oil sands end pit lake to date, revealed that nitrification was performed by members from Nitrosomonadaceae, Chloroflexi and unclassified Gammaproteobacteria "MBAE14." While Nitrosomonadaceae and Chloroflexi groups were relatively abundant in the upper oxygenated zones, MBAE14 dominated the hypoxic hypolimnetic zones (approximately 30% of total microbial communities); MBAE14 was not detected in the underlying anoxic tailings. Replication rate analyses indicate that MBAE14 grew in metalimnetic and hypolimnetic water cap regions, most actively at the metalimnetic, ammonia/oxygen transition zone consistent with it putatively conducting nitrification. Detailed genomic analyses of MBAE14 evidenced both ammonia oxidation and denitrification into dinitrogen capabilities. However, the absence of known CO2-fixation genes suggests a heterotrophic denitrifying metabolism. Functional marker genes of ammonia oxidation (amo and hao) in the MBAE14 genome are homologous with those conserved in autotrophic nitrifiers, but not with those of known heterotrophic nitrifiers. We propose that this novel MBAE14 inhabits the specific ammonia-rich, oxygen and labile organic matter-limited conditions occurring in Base Mine Lake which selectively favors mixotrophic coupled nitrifier denitrification metabolism. Our results highlight the opportunities to better constrain biogeochemical cycles from the application of metagenomics to engineered systems associated with extractive resource sectors.

Wide Distribution of Phage That Infect Freshwater SAR11 Bacteria.

Fonsibacter (LD12 subclade) is among the most abundant bacterioplankton in freshwater ecosystems. These bacteria belong to the order Pelagibacterales (SAR11) and are related to Pelagibacter (marine SAR11), which dominates many marine habitats. Although a few Pelagibacter phage (Pelagiphage) have been described, no phage that infect Fonsibacter have been reported. In this study, we describe two groups of Podoviridae phage that infect Fonsibacter A complete Fonsibacter genome containing a prophage was reconstructed from metagenomic data. A circularized and complete genome related to the prophage, referred to as uv-Fonsiphage-EPL (lysogenic strategy), shows high similarity to marine Pelagiphage HTVC025P. Additionally, we reconstructed three complete genomes and one draft genome of phage related to marine Pelagiphage HTVC010P and predicted a lytic strategy. The similarity in codon usage and cooccurrence patterns of HTVC010P-related phage and Fonsibacter suggested that these phage infect Fonsibacter Similar phage were detected in Lake Mendota, Wisconsin, where Fonsibacter is also present. A search of related phage revealed the worldwide distribution of some genotypes in freshwater ecosystems, suggesting their substantial role in shaping indigenous microbial assemblages and influence on biogeochemical cycling. However, the uv-Fonsiphage-EPL and one group of HTVC010P-related phage have a more limited distribution in freshwater ecosystems. Overall, the findings provide insights into the genomic features of phage that infect Fonsibacter and expand understanding of the ecology and evolution of these important bacteria.IMPORTANCE Fonsibacter represents a significant microbial group of freshwater ecosystems. Although the genomic and metabolic features of these bacteria have been well studied, no phage infecting them has been reported. In this study, we reconstructed complete genomes of Fonsibacter and infecting phage and revealed their close relatedness to the phage infecting marine SAR11 members. Also, we illustrated that phage that infect Fonsibacter are widely distributed in freshwater habitats. In summary, the results contribute new insights into the ecology and evolution of Fonsibacter and phage.

The Potential Role of Halothiobacillus spp. in Sulfur Oxidation and Acid Generation in Circum-Neutral Mine Tailings Reservoirs.

The biogeochemistry of acid mine drainage (AMD) derived from waste rock associated sulfide mineral oxidation is relatively well-characterized and linked to Acidithiobacillus spp.. However, little is understood about the microbial communities and sulfur cycling before AMD develops, a key component of its prevention. This study aimed to examine circum-neutral mining impacted water (MIW) communities and its laboratory enrichments for sulfur oxidizing bacteria (SoxBac). MIW in situ microbial communities differed in diversity, structure and relative abundance consistent with site specific variations in total aqueous sulfur concentrations (TotS; ~2-17 mM), pH (3.67-7.34), and oxygen (22-93% saturation). However, the sulfur oxidizer, Halothiobacillus spp. dominated seven of the nine total SoxBac enrichment communities (~76-100% relative abundance), spanning three of the four mines. The presence and relative abundance of the identified sixteen known and five unclassified Halothiobacillus spp. here, were the important clustering determinants across parent MIW and enrichment communities. Further, the presence of Halothiobacillus spp. was associated with driving the pH <4 in enrichment experiments, and the combination of specific Halothiobacillus spp. in the enrichments affected the observed acid to sulfate ratios indicating differential sulfur cycling. Halothiobacillus spp. also dominated the parent communities of the two acidic MIWs providing corroborating evidence for its active role in net acid generation within these waters. These results identify a putative indicator organism specific to mine tailings reservoirs and highlight the need for further study of tailings associated sulfur cycling for better mine management and environmental stewardship.

Physiological and ecological implications of an iron- or hydrogen-oxidizing member of the Zetaproteobacteria, Ghiorsea bivora, gen. nov., sp. nov.

Chemosynthetic Fe-oxidizing communities are common at diffuse-flow hydrothermal vents throughout the world's oceans. The foundational members of these communities are the Zetaproteobacteria, a class of Proteobacteria that is primarily associated with ecosystems fueled by ferrous iron, Fe(II). We report here the discovery of two new isolates of Zetaproteobacteria isolated from the Mid-Atlantic Ridge (TAG-1), and the Mariana back-arc (SV-108), that are unique in that they can utilize either Fe(II) or molecular hydrogen (H2) as sole electron donor and oxygen as terminal electron acceptor for growth. Both strains precipitated Fe-oxyhydroxides as amorphous particulates. The cell doubling time on H2 vs Fe(II) for TAG-1 was 14.1 vs 21.8 h, and for SV-108 it was 16.3 vs 20 h, and it appeared both strains could use either H2 or Fe(II) simultaneously. The strains were close relatives, based on genomic analysis, and both possessed genes for the uptake NiFe-hydrogenase required for growth on H2. These two strains belong to Zetaproteobacteria operational taxonomic unit 9 (ZetaOTU9). A meta-analysis of public databases found ZetaOTU9 was only associated with Fe(II)-rich habitats, and not in other environments where known H2-oxidizers exist. These results expand the metabolic repertoire of the Zetaproteobacteria, yet confirm that Fe(II) metabolism is the primary driver of their physiology and ecology.

Sticking together: inter-species aggregation of bacteria isolated from iron snow is controlled by chemical signaling.

Marine and lake snow is a continuous shower of mixed organic and inorganic aggregates falling from the upper water where primary production is substantial. These pelagic aggregates provide a niche for microbes that can exploit these physical structures and resources for growth, thus are local hot spots for microbial activity. However, processes underlying their formation remain unknown. Here, we investigated the role of chemical signaling between two co-occurring bacteria that each make up more than 10% of the community in iron-rich lakes aggregates (iron snow). The filamentous iron-oxidizing Acidithrix strain showed increased rates of Fe(II) oxidation when incubated with cell-free supernatant of the heterotrophic iron-reducing Acidiphilium strain. Amendment of Acidithrix supernatant to motile cells of Acidiphilium triggered formation of cell aggregates displaying similar morphology to those of iron snow. Comparative metabolomics enabled the identification of the aggregation-inducing signal, 2-phenethylamine, which also induced faster growth of Acidiphilium. We propose a model that shows rapid iron snow formation, and ultimately energy transfer from the photic zone to deeper water layers, is controlled via a chemically mediated interplay.

Schwertmannite formation at cell junctions by a new filament-forming Fe(II)-oxidizing isolate affiliated with the novel genus Acidithrix.

A new acidophilic iron-oxidizing strain (C25) belonging to the novel genus Acidithrix was isolated from pelagic iron-rich aggregates ('iron snow') collected below the redoxcline of an acidic lignite mine lake. Strain C25 catalysed the oxidation of ferrous iron [Fe(II)] under oxic conditions at 25 °C at a rate of 3.8 mM Fe(II) day(-1) in synthetic medium and 3.0 mM Fe(II) day(-1) in sterilized lake water in the presence of yeast extract, producing the rust-coloured, poorly crystalline mineral schwertmannite [Fe(III) oxyhydroxylsulfate]. During growth, rod-shaped cells of strain C25 formed long filaments, and then aggregated and degraded into shorter fragments, building large cell-mineral aggregates in the late stationary phase. Scanning electron microscopy analysis of cells during the early growth phase revealed that Fe(III)-minerals were formed as single needles on the cell surface, whereas the typical pincushion-like schwertmannite was observed during later growth phases at junctions between the cells, leaving major parts of the cell not encrusted. This directed mechanism of biomineralization at specific locations on the cell surface has not been reported from other acidophilic iron-oxidizing bacteria. Strain C25 was also capable of reducing Fe(III) under micro-oxic conditions which led to a dissolution of the Fe(III)-minerals. Thus, strain C25 appeared to have ecological relevance for both the formation and transformation of the pelagic iron-rich aggregates at oxic/anoxic transition zones in the acidic lignite mine lake.