Diversity studies and molecular analyses with single cells and filaments of large, colorless sulfur bacteria

Large sulfur bacteria feature conspicuous morphologies that are usually visible with the naked eye. Most representatives were already described in the 19th and early 20th century and it needed nearly another 100 years until a new morphotype of large sulfur bacteria was discovered. This discovery encouraged a search for other yet unknown types in this group of bacteria and indeed led to the finding of a new type in two marine seep settings. This novel morphotype is presented in Chapter 2 and is the first non-filamentous member in the family Beggiatoaceae that shows a dimorphic life cycle, exhibiting alternation between sessile and free-living forms. In Chapter 3, another three novel morphotypes are presented, which were discovered in Namibian sediments. The detection of these novel morphotypes of large sulfur bacteria led to the necessity for an improved phylogenetic classification of the entire group. Earlier attempts to sequence the 16S rRNA genes of the large sulfur bacteria resulted in only few nearly full-length sequences, whereas some genera were even represented by only partial sequences. Accordingly, differentiation of genera in this group is still based on morphological features. Now, Chapter 3 presents the sequencing of 16S rRNA genes and ITS regions of more than 100 individual cells and filaments of large sulfur bacteria, revealing a major insight into the phylogeny of these extraordinary bacteria. It is demonstrated that the traditional, morphology-based classification does not correlate with the phylogeny derived from 16S rRNA gene sequences. Consequently, a reclassification of the entire family is proposed, being completely independent from former morphological categories. Sequencing single cells and filaments of large sulfur bacteria furthermore revealed that they represent the first group of bacteria, which commonly contain large and numerous introns in their 16S rRNA genes. In Chapter 4, it is demonstrated that the introns are removed efficiently from the rRNA precursor, thereby ligating the two exons, and are not present in the native ribosome. Furthermore, it was experimentally verified that a commonly applied PCR approach introduces a length heterogeneity bias and systematically discriminates against enlarged genes and favors the amplification of shorter homologues. Consequently, this fact questions the universality of 16S rRNA-based clone libraries for diversity studies. At least the group of large sulfur bacteria is systematically discriminated against in such universal PCR approaches and it can be assumed that this PCR bias also affects a yet unknown amount of other microorganisms

How Clonal Is Clonal? Genome Plasticity across Multicellular Segments of a “Candidatus Marithrix sp.” Filament from Sulfidic, Briny Seafloor Sediments in the Gulf of Mexico

"Candidatus Marithrix" is a recently described lineage within the group of large sulfur bacteria (Beggiatoaceae, Gammaproteobacteria). This group of bacteria comprises vacuolated, attached-living filaments that inhabit the sediment surface around vent and seep sites in the marine environment. A single filament is ca. 100 µm in diameter, several millimeters long, and consists of hundreds of clonal cells, which are considered highly polyploid. Based on these characteristics, "Candidatus Marithrix" was used as a model organism for the assessment of genomic plasticity along segments of a single filament using next generation sequencing to possibly identify hotspots of microevolution. Using six consecutive segments of a single filament sampled from a mud volcano in the Gulf of Mexico, we recovered ca. 90% of the "Candidatus Marithrix" genome in each segment. There was a high level of genome conservation along the filament with average nucleotide identities between 99.98-100%. Different approaches to assemble all reads into a complete consensus genome could not fill the gaps. Each of the six segment datasets encoded merely a few hundred unique nucleotides and 5 or less unique genes - the residual content was redundant in all datasets. Besides the overall high genomic identity, we identified a similar number of single nucleotide polymorphisms (SNPs) between the clonal segments, which are comparable to numbers reported for other clonal organisms. An increase of SNPs with greater distance of filament segments was not observed. The polyploidy of the cells was apparent when analyzing the heterogeneity of reads within a segment. Here, a strong increase in single nucleotide variants, or 'intrasegmental sequence heterogeneity' (ISH) events, was observed. These sites may represent hotspots for genome plasticity, and possibly microevolution, since two thirds of these variants were not co-localized across the genome copies of the multicellular filament

Response of Bacterial Communities to Different Detritus Compositions in Arctic Deep-Sea Sediments

Benthic deep-sea communities are largely dependent on particle flux from surface waters. In the Arctic Ocean, environmental changes occur more rapidly than in other ocean regions, and have major effects on the export of organic matter to the deep sea. Because bacteria constitute the majority of deep-sea benthic biomass and influence global element cycles, it is important to better understand how changes in organic matter input will affect bacterial communities at the Arctic seafloor. In a multidisciplinary ex situ experiment, benthic bacterial deep-sea communities from the Long-Term Ecological Research Observatory HAUSGARTEN were supplemented with different types of habitat-related detritus (chitin, Arctic algae) and incubated for 23 days under in situ conditions. Chitin addition caused strong changes in community activity, while community structure remained similar to unfed control incubations. In contrast, the addition of phytodetritus resulted in strong changes in community composition, accompanied by increased community activity, indicating the need for adaptation in these treatments. High-throughput sequencing of the 16S rRNA gene and 16S rRNA revealed distinct taxonomic groups of potentially fast-growing, opportunistic bacteria in the different detritus treatments. Compared to the unfed control, Colwelliaceae, Psychromonadaceae, and Oceanospirillaceae increased in relative abundance in the chitin treatment, whereas Flavobacteriaceae, Marinilabiaceae, and Pseudoalteromonadaceae increased in the phytodetritus treatments. Hence, these groups may constitute indicator taxa for the different organic matter sources at this study site. In summary, differences in community structure and in the uptake and remineralization of carbon in the different treatments suggest an effect of organic matter quality on bacterial diversity as well as on carbon turnover at the seafloor, an important feedback mechanism to be considered in future climate change scenarios

Spatial Distribution of Arctic Bacterioplankton Abundance Is Linked to Distinct Water Masses and Summertime Phytoplankton Bloom Dynamics (Fram Strait, 79°N)

The Arctic is impacted by climate warming faster than any other oceanic region on Earth. Assessing the baseline of microbial communities in this rapidly changing ecosystem is vital for understanding the implications of ocean warming and sea ice retreat on ecosystem functioning. Using CARD-FISH and semi-automated counting, we quantified 14 ecologically relevant taxonomic groups of bacterioplankton (Bacteria and Archaea) from surface (0-30 m) down to deep waters (2,500 m) in summer ice-covered and ice-free regions of the Fram Strait, the main gateway for Atlantic inflow into the Arctic Ocean. Cell abundances of the bacterioplankton communities in surface waters varied from 10(5) cells mL(-1) in ice-covered regions to 10(6) cells mL(-1) in the ice-free regions. Observations suggest that these were overall driven by variations in phytoplankton bloom conditions across the Strait. The bacterial groups Bacteroidetes and Gammaproteobacteria showed several-fold higher cell abundances under late phytoplankton bloom conditions of the ice-free regions. Other taxonomic groups, such as the Rhodobacteraceae, revealed a distinct association of cell abundances with the surface Atlantic waters. With increasing depth (\u3e500 m), the total cell abundances of the bacterioplankton communities decreased by up to two orders of magnitude, while largely unknown taxonomic groups (e.g., SAR324 and SAR202 clades) maintained constant cell abundances throughout the entire water column (ca. 10(3) cells mL(-1)). This suggests that these enigmatic groups may occupy a specific ecological niche in the entire water column. Our results provide the first quantitative spatial variations assessment of bacterioplankton in the summer ice-covered and ice-free Arctic water column, and suggest that further shift toward ice-free Arctic summers with longer phytoplankton blooms can lead to major changes in the associated standing stock of the bacterioplankton communities

Intracellular calcite and sulfur dynamics of Achromatium cells observed in a lab-based enrichment and aerobic incubation experiment

We investigated the intracellular dynamics of calcite and sulfur in the large sulfur-oxidizing, calcite-accumulating bacterium Achromatium, with an emphasis on oxygen exposure as a physiological control. For this purpose, morphological changes and possible accretion mechanisms of calcite granules in cells that were freshly collected from natural Achromatium-containing sediment were compared to cells from the same source after prolonged exposure to atmospheric oxygen. Intracellular sulfur is oxidized and removed in response to oxygen exposure. Calcite granules also undergo distinct oxygen-related dynamics; they alternate between tightly packaged, smooth granules with narrow but sharply defined interstitial spaces in atmospheric oxygen-exposed cells, and more loosely packaged granules with irregular, bumpy surface texture and larger interstitial spaces in cells that were not artificially exposed to oxygen. These results suggest that morphological changes of the calcite granules reflect their changing physiological role inside the cell. Sulfur oxidation and calcite dissolution appear to be linked in that proton generation during sulfur oxidation is buffered by gradual calcite erosion, visible in the smooth, rounded surface morphology observed after oxygen exposure. Our results support the hypothesis that calcite dynamics buffer the intracellular pH fluctuations linked to electron acceptor limitation during proton-consuming sulfide oxidation to sulfur, and electron acceptor abundance during proton-generating sulfur oxidation to sulfate

Microscale sulfur cycling in the phototrophic pink berry consortia of the Sippewissett Salt Marsh

Microbial metabolism is the engine that drives global biogeochemical cycles, yet many key transformations are carried out by microbial consortia over short spatiotemporal scales that elude detection by traditional analytical approaches. We investigate syntrophic sulfur cycling in the ‘pink berry’ consortia of the Sippewissett Salt Marsh through an integrative study at the microbial scale. The pink berries are macroscopic, photosynthetic microbial aggregates composed primarily of two closely associated species: sulfide-oxidizing purple sulfur bacteria (PB-PSB1) and sulfate-reducing bacteria (PB-SRB1). Using metagenomic sequencing and 34S-enriched sulfate stable isotope probing coupled with nanoSIMS, we demonstrate interspecies transfer of reduced sulfur metabolites from PB-SRB1 to PB-PSB1. The pink berries catalyse net sulfide oxidation and maintain internal sulfide concentrations of 0–500 μm. Sulfide within the berries, captured on silver wires and analysed using secondary ion mass spectrometer, increased in abundance towards the berry interior, while δ34S-sulfide decreased from 6‰ to −31‰ from the exterior to interior of the berry. These values correspond to sulfate–sulfide isotopic fractionations (15–53‰) consistent with either sulfate reduction or a mixture of reductive and oxidative metabolisms. Together this combined metagenomic and high-resolution isotopic analysis demonstrates active sulfur cycling at the microscale within well-structured macroscopic consortia consisting of sulfide-oxidizing anoxygenic phototrophs and sulfate-reducing bacteria

Filamentous Giant Beggiatoaceae from the Guaymas Basin Are Capable of both Denitrification and Dissimilatory Nitrate Reduction to Ammonium

Filamentous large sulfur-oxidizing bacteria (FLSB) of the family Beggiatoaceae are globally distributed aquatic bacteria that can control geochemical fluxes from the sediment to the water column through their metabolic activity. FLSB mats from hydrothermal sediments of Guaymas Basin, Mexico, typically have a “fried-egg” appearance, with orange filaments dominating near the center and wider white filaments at the periphery, likely reflecting areas of higher and lower sulfide fluxes, respectively. These FLSB store large quantities of intracellular nitrate that they use to oxidize sulfide. By applying a combination of 15N-labeling techniques and genome sequence analysis, we demonstrate that the white FLSB filaments were capable of reducing their intracellular nitrate stores to both nitrogen gas and ammonium by denitrification and dissimilatory nitrate reduction to ammonium (DNRA), respectively. On the other hand, our combined results show that the orange filaments were primarily capable of DNRA. Microsensor profiles through a laboratory-incubated white FLSB mat revealed a 2- to 3-mm vertical separation between the oxic and sulfidic zones. Denitrification was most intense just below the oxic zone, as shown by the production of nitrous oxide following exposure to acetylene, which blocks nitrous oxide reduction to nitrogen gas. Below this zone, a local pH maximum coincided with sulfide oxidation, consistent with nitrate reduction by DNRA. The balance between internally and externally available electron acceptors (nitrate) and electron donors (reduced sulfur) likely controlled the end product of nitrate reduction both between orange and white FLSB mats and between different spatial and geochemical niches within the white FLSB mat

Single-cell Sequencing of Thiomargarita Reveals Genomic Flexibility for Adaptation to Dynamic Redox Conditions

Large, colorless sulfur-oxidizing bacteria (LSB) of the family Beggiatoaceae form thick mats at sulfidic sediment surfaces, where they efficiently detoxify sulfide before it enters the water column. The genus Thiomargarita harbors the largest known freeliving bacteria with cell sizes of up to 750 mm in diameter. In addition to their ability to oxidize reduced sulfur compounds, some Thiomargarita spp. are known to store large amounts of nitrate, phosphate and elemental sulfur internally. To date little is known about their energy yielding metabolic pathways, and how these pathways compare to other Beggiatoaceae. Here, we present a draft single-cell genome of a chain-forming “Candidatus Thiomargarita nelsonii Thio36”, and conduct a comparative analysis to five draft and one full genome of other members of the Beggiatoaceae. “Ca. T. nelsonii Thio36” is able to respire nitrate to both ammonium and dinitrogen, which allows them to flexibly respond to environmental changes. Genes for sulfur oxidation and inorganic carbon fixation confirmed that “Ca. T. nelsonii Thio36” can function as a chemolithoautotroph. Carbon can be fixed via the Calvin–Benson–Bassham cycle, which is common among the Beggiatoaceae. In addition we found key genes of the reductive tricarboxylic acid cycle that point toward an alternative CO2 fixation pathway. Surprisingly, “Ca. T. nelsonii Thio36” also encodes key genes of the C2- cycle that convert 2-phosphoglycolate to 3-phosphoglycerate during photorespiration in higher plants and cyanobacteria. Moreover, we identified a novel trait of a flavin-based energy bifurcation pathway coupled to a NaC-translocating membrane complex (Rnf). The coupling of these pathways may be key to surviving long periods of anoxia. As other Beggiatoaceae “Ca. T. nelsonii Thio36” encodes many genes similar to those of (filamentous) cyanobacteria. In summary, the genome of “Ca. T. nelsonii Thio36” provides additional insight into the ecology of giant sulfur-oxidizing bacteria, and reveals unique genomic features for the Thiomargarita lineage within the Beggiatoaceae

Microscale sulfur cycling in the phototrophic pink berry consortia of the Sippewissett Salt Marsh

Microbial metabolism is the engine that drives global biogeochemical cycles, yet many key transformations are carried out by microbial consortia over short spatiotemporal scales that elude detection by traditional analytical approaches. We investigate syntrophic sulfur cycling in the ‘pink berry’ consortia of the Sippewissett Salt Marsh through an integrative study at the microbial scale. The pink berries are macroscopic, photosynthetic microbial aggregates composed primarily of two closely associated species: sulfide-oxidizing purple sulfur bacteria (PB-PSB1) and sulfate-reducing bacteria (PB-SRB1). Using metagenomic sequencing and 34S-enriched sulfate stable isotope probing coupled with nanoSIMS, we demonstrate interspecies transfer of reduced sulfur metabolites from PB-SRB1 to PB-PSB1. The pink berries catalyse net sulfide oxidation and maintain internal sulfide concentrations of 0–500 μm. Sulfide within the berries, captured on silver wires and analysed using secondary ion mass spectrometer, increased in abundance towards the berry interior, while δ34S-sulfide decreased from 6‰ to −31‰ from the exterior to interior of the berry. These values correspond to sulfate–sulfide isotopic fractionations (15–53‰) consistent with either sulfate reduction or a mixture of reductive and oxidative metabolisms. Together this combined metagenomic and high-resolution isotopic analysis demonstrates active sulfur cycling at the microscale within well-structured macroscopic consortia consisting of sulfide-oxidizing anoxygenic phototrophs and sulfate-reducing bacteria

The Brain Tumor Segmentation (BraTS) Challenge 2023: Focus on Pediatrics (CBTN-CONNECT-DIPGR-ASNR-MICCAI BraTS-PEDs)

Pediatric tumors of the central nervous system are the most common cause of cancer-related death in children. The five-year survival rate for high-grade gliomas in children is less than 20\%. Due to their rarity, the diagnosis of these entities is often delayed, their treatment is mainly based on historic treatment concepts, and clinical trials require multi-institutional collaborations. The MICCAI Brain Tumor Segmentation (BraTS) Challenge is a landmark community benchmark event with a successful history of 12 years of resource creation for the segmentation and analysis of adult glioma. Here we present the CBTN-CONNECT-DIPGR-ASNR-MICCAI BraTS-PEDs 2023 challenge, which represents the first BraTS challenge focused on pediatric brain tumors with data acquired across multiple international consortia dedicated to pediatric neuro-oncology and clinical trials. The BraTS-PEDs 2023 challenge focuses on benchmarking the development of volumentric segmentation algorithms for pediatric brain glioma through standardized quantitative performance evaluation metrics utilized across the BraTS 2023 cluster of challenges. Models gaining knowledge from the BraTS-PEDs multi-parametric structural MRI (mpMRI) training data will be evaluated on separate validation and unseen test mpMRI dataof high-grade pediatric glioma. The CBTN-CONNECT-DIPGR-ASNR-MICCAI BraTS-PEDs 2023 challenge brings together clinicians and AI/imaging scientists to lead to faster development of automated segmentation techniques that could benefit clinical trials, and ultimately the care of children with brain tumors