Publications

4595

AbstractThe continental bedrock contains groundwater-bearing fractures that are home to microbial populations that are vital in mediating the Earth’s biogeochemical cycles. However, their diversity is poorly understood due to the difficulty of obtaining samples from this environment. Here, a groundwater-bearing fracture at 975 m depth was isolated by employing packers in order to characterize the microbial community via metagenomes combined with prokaryotic and eukaryotic marker genes (16S and 18S ribosomal RNA gene). Genome-resolved analyses revealed a community dominated by sulfate-reducing Bacillota, predominantly represented by Candidatus Desulforudis audaxviator and with Wood-Ljungdahl as the most prevalent pathway for inorganic carbon fixation. Moreover, the eukaryotic community had a considerable diversity and was comprised of mainly flatworms, chlorophytes, crustaceans, ochrophytes, and fungi. These findings support the important role of the Bacillota, with the sulfate reducer Candidatus Desulforudis audaxviator as its main representative, as primary producers in the often energy-limited groundwaters of the continental subsurface.

Phytoplasmas are obligate intracellular pathogens that profoundly modify the development, physiology and behavior of their hosts by secreting effector proteins that disturb signal pathways and interactions both in plant and insect hosts. The characterization of effectors and their host-cell targets was performed for only a few phytoplasma species where it was shown that the SAP11 effector alters plant morphology by destabilizing plant transcription factors: TEOSINTE BRANCHED 1-CYCLOIDEA-PROLIFERATING CELL FACTOR (TCPs). To explore the possible role of the SAP11-like effector from ‘Ca. P. solani’, we used Arabidopsis thaliana as a model plant. The SAP11-like effector gene from ‘Ca. P. solani’ was introduced into arabidopsis by floral dip and transgenic lines were regenerated. In planta bimolecular fluorescence complementation (BIFC) assays in agroinfiltrated Nicotiana benthamiana leaf cells were conducted to detect interactions between SAP11-like and AtTCP2 and AtTCP4 using confocal microscopy. SAP11-like from ‘Ca. P. solani’ induced significant phenotypic changes in arabidopsis, including crinkled leaves with reduced size, lower biomass, more axillary branches, changes in root morphology, and crinkled and smaller siliques. The BIFC assays proved in planta interaction of SAP11-like effector with AtTCP2 and AtTCP4. To our knowledge, this is the first characterization of the interaction between the ‘Ca. P. solani’ effector and plant transcription factors, suggesting a potential mechanism of modulating plant development and induction of characteristic symptoms in ‘Ca. P. solani’-infected plants.

‘Candidatus Phytoplasma brasiliense’ (CPB) is a phytoplasma originally discovered in South America and is known to infect a wide variety of economically important crops. It is most prevalent in Hibiscus spp., where it causes witches broom symptoms, and papaya, where it causes bunchy top. Recently, CPB was documented for the first time in North America in a new host, globe sedge. In this study, two quantitative PCR assays are developed: one using high-resolution melt curve analysis (HRMA) based on the secA gene and the other a TaqMan assay based on the dnaK gene. The secA/HRMA and dnaK/TaqMan assay successfully amplified two of the three isolates of CPB. Both assays were screened against available isolates of 16SrI, 16SrII, and 16SrIV phytoplasmas. The secA/HRMA assay failed to amplify 16SrI and 16SrIV phytoplasmas but successfully amplified 16SrII phytoplasmas. The resulting melting point (Tm) products of CPB and 16SrII phytoplasmas displayed a difference of 0.5°C, easily distinguishing them by melt curves. The dnaK/TaqMan assay failed to amplify all non-CPB phytoplasma isolates in the study. The development of these assays provides a valuable tool that will significantly improve monitoring programs in Florida and will aid in developing a better fundamental understanding of the epidemiology of this phytoplasma.

AbstractMetagenomic analysis was conducted to assess the microbial community in the high‐altitude Tamdykul geothermal spring in Tajikistan. This analysis yielded six high‐quality bins from the members of Thermaceae, Aquificaceae, and Halothiobacillaceae, with a 41.2%, 19.7%, and 18.1% share in the total metagenome, respectively. Minor components included Schleiferia thermophila (1.6%) and members of the archaeal taxa Pyrobaculum (1.2%) and Desulfurococcaceae (0.7%). Further analysis of the metagenome‐assembled genome (MAG) from the Desulfurococcaceae family (MAG002) revealed novel taxonomy with only 80.95% closest placement average nucleotide identity value to its most closely related member of the Desulfurococcaceae family, which is part of the Thermoproteota phylum comprising hyperthermophilic members widespread in geothermal environments. MAG002 consisted of 1.3 Mbp, distributed into 48 contigs with 1504 predicted coding sequences, had an average GC content of 41.3%, a completeness and contamination rate of 98.7% and 2.6%, respectively, and branched phylogenetically between the Ignisphaera and Zestosphaera lineages. Digital DNA‐DNA hybridization values compared with Ignisphaera aggregans and Zestosphaera tikiterensis were 33.7% and 19.4%, respectively, suggesting that this MAG represented a novel species and genus. Its 16S rRNA gene contained a large 421 bp intron. It encodes a complete gluconeogenesis pathway involving a bifunctional fructose‐1,6‐bisphosphate phosphatase/aldolase; however, the glycolysis pathway is incomplete. The ribulose monophosphate pathway enzymes could be used for pentose synthesis. MAG002 encodes several hydrogen‐evolving hydrogenases, with possible roles as hydrogen sinks during fermentation. We propose the name Zestomicrobium tamdykulense gen. nov. sp. nov. for this organism; it is the first thermophilic genome reported from Tajikistan.

Abstract Candidatus Patescibacteria, also known as candidate phyla radiation (CPR), including the class‐level uncultured clade JAEDAM01 (formerly a subclass of Gracilibacteria/GN02/BD1‐5), are ubiquitous in activated sludge. However, their characteristics and relationships with other organisms are largely unknown. They are believed to be episymbiotic, endosymbiotic or predatory. Despite our understanding of their limited metabolic capacity, their precise roles remain elusive due to the difficulty in cultivating and identifying them. In previous research, we successfully recovered high‐quality metagenome‐assembled genomes (MAGs), including a member of JAEDAM01 from activated sludge flocs. In this study, we designed new probes to visualize the targeted JAEDAM01‐associated MAG HHAS10 and identified its host using fluorescence in situ hybridization (FISH). The FISH observations revealed that JAEDAM01 HHAS10‐like cells were located within dense clusters of Zoogloea , and the fluorescence brightness of zoogloeal cells decreased in the vicinity of the CPR cells. The Zoogloea MAGs possessed genes related to extracellular polymeric substance biosynthesis, floc formation and nutrient removal, including a polyhydroxyalkanoate (PHA) accumulation pathway. The JAEDAM01 MAG HHAS10 possessed genes associated with type IV pili, competence protein EC and PHA degradation, suggesting a Zoogloea ‐dependent lifestyle in activated sludge flocs. These findings indicate a new symbiotic relationship between JAEDAM01 and Zoogloea .