Publications

4478

Diaphorina citri serves as the primary vector for ‘Candidatus Liberibacter asiaticus (CLas),’ the bacterium associated with the severe Asian form of huanglongbing. CLas-positive D. citri are more fecund than their CLas-negative counterparts and require extra energy expenditure. Therefore, understanding the molecular mechanisms linking metabolism and reproduction is of particular importance. In this study, we found adipokinetic hormone (DcAKH) and its receptor (DcAKHR) were essential for increasing lipid metabolism and fecundity in response to CLas infection in D. citri. Knockdown of DcAKH and DcAKHR not only resulted in the accumulation of triacylglycerol and a decline of glycogen, but also significantly decreased fecundity and CLas titer in ovaries. Combined in vivo and in vitro experiments showed that miR-34 suppresses DcAKHR expression by binding to its 3’ untranslated region, whilst overexpression of miR-34 resulted in a decline of DcAKHR expression and CLas titer in ovaries and caused defects that mimicked DcAKHR knockdown phenotypes. Additionally, knockdown of DcAKH and DcAKHR significantly reduced juvenile hormone (JH) titer and JH signaling pathway genes in fat bodies and ovaries, including the JH receptor, methoprene-tolerant (DcMet), and the transcription factor, Krüppel homolog 1 (DcKr-h1), that acts downstream of it, as well as the egg development related genes vitellogenin 1-like (DcVg-1-like), vitellogenin A1-like (DcVg-A1-like) and the vitellogenin receptor (DcVgR). As a result, CLas hijacks AKH/AKHR-miR-34-JH signaling to improve D. citri lipid metabolism and fecundity, while simultaneously increasing the replication of CLas, suggesting a mutualistic interaction between CLas and D. citri ovaries.

Objective/Background. Bocconia frutescens (Papaveraceae) is a small tree distributed naturally from Mexico to Argentina and the Caribbean Bassin. Bocconia trees showing symptoms resembling phytoplasmas infection, such as little leaves and witches´-broom, were found in Cartago province, Costa Rica. Detection and identification of the potential phytoplasmas associated with B. frutescens little leaf symptoms was the objective out of this study. Materials and Methods. Evaluation of leaves tissue using transmission electron microscopy (TEM), nested PCR using universal and specific primers to amplify phytoplasmas 16S rRNA and secA genes. Nucleotidic sequences (Sanger method) were obtained from amplicons, and used for BLASTn, phylogenetic analyses, and in silico RFLP’s. Results. Presence of phytoplasmas into phloem tissue, only in symptomatic trees, was evidenced by TEM. Comparison of partial sequences (16Sr and secA genes) by BLASTn, in silico RFLP´s and phylogenetic analyses, showed the occurrence of a ´Candidatus Phytoplasma pruni´ related strain in the samples evaluated. Conclusion. Phytoplasmas were found only in the symptomatic B. frutescens trees evaluated. The phytoplasmas were identified as a ´Ca. Phytoplasma pruni´ related strain. This is the first report of B. frutescens as a natural host of ´Ca. Phytoplasma pruni´.

Diaphorina citri serves as the primary vector for ‘Candidatus Liberibacter asiaticus (CLas),’ the bacterium associated with the severe Asian form of huanglongbing. CLas-positive D. citri are more fecund than their CLas-negative counterparts and require extra energy expenditure. Therefore, understanding the molecular mechanisms linking metabolism and reproduction is of particular importance. In this study, we found adipokinetic hormone (DcAKH) and its receptor (DcAKHR) were essential for increasing lipid metabolism and fecundity in response to CLas infection in D. citri. Knockdown of DcAKH and DcAKHR not only resulted in the accumulation of triacylglycerol and a decline of glycogen, but also significantly decreased fecundity and CLas titer in ovaries. Combined in vivo and in vitro experiments showed that miR-34 suppresses DcAKHR expression by binding to its 3’ untranslated region, whilst overexpression of miR-34 resulted in a decline of DcAKHR expression and CLas titer in ovaries and caused defects that mimicked DcAKHR knockdown phenotypes. Additionally, knockdown of DcAKH and DcAKHR significantly reduced juvenile hormone (JH) titer and JH signaling pathway genes in fat bodies and ovaries, including the JH receptor, methoprene-tolerant (DcMet), and the transcription factor, Krüppel homolog 1 (DcKr-h1), that acts downstream of it, as well as the egg development related genes vitellogenin 1-like (DcVg-1-like), vitellogenin A1-like (DcVg-A1-like) and the vitellogenin receptor (DcVgR). As a result, CLas hijacks AKH/AKHR-miR-34-JH signaling to improve D. citri lipid metabolism and fecundity, while simultaneously increasing the replication of CLas, suggesting a mutualistic interaction between CLas and D. citri ovaries.

Nitrobacterstrain NHB1 is a nitrite-oxidising bacterium previously co-enriched with the neutrophilic ammonia-oxidising bacteriumNitrosospiraAHB1, a consortium that nitrifies in acidic conditions in co-culture. Here we characterise the growth of the isolateNitrobacterstrain NHB1 as a function of pH and nitrite (NO2-) concentration, and its influence on the activity of acidophilic soil ammonia-oxidising archaea (AOA). NHB1 is acidotolerant and grows optimally at pH 6.0 (range 5.0 - 7.5) at initial NO2-concentrations of 500 μM. However, the optimum decreases to pH 5.0 at lower initial NO2-concentrations typically found in soil, with detectable growth down to pH 3.5. NHB1 has a comparatively high affinity for NO2-with an apparent-half-saturation constant (54 μM) one order of magnitude lower than its closest relative, the neutrophilic strainNitrobacter hamburgensisX14. In co-culture, NHB1 enhances the growth of acidophilic AOA. Specifically,Nitrosotalea devaniterraeNd1 andNitrosotalea sinensisNd2 are sensitive to NO2--derived compounds and only oxidise ~200-300 μM ammonia (NH3) in batch cultures. However, in co-culture with NHB1, pH ranges were lowered by ~0.5 pH units and both strains could oxidise up to 3 mM NH3, only limited by buffering capacity. NHB1 possesses a cyanase facilitating reciprocal cross-feeding via generating cyanate-derived NH3and utilising AOA-derived NO2-. Removal of NO2-is likely crucial for the growth of nitrifiers in acidic soils and this study highlights the importance of considering substrate and metabolic product concentrations when characterising physiology. Genome analysis reveals NHB1 is distinct from validated species and the name "Nitrobacter laanbroekii" is proposed.

Abstract Background The Atribacterota are widely distributed in the subsurface biosphere. Recently, the first Atribacterota isolate was described and the number of Atribacterota genome sequences retrieved from environmental samples has increased significantly; however, their diversity, physiology, ecology, and evolution remain poorly understood. Results We report the isolation of the second member of Atribacterota, Thermatribacter velox gen. nov., sp. nov., within a new family Thermatribacteraceae fam. nov., and the short-term laboratory cultivation of a member of the JS1 lineage, Phoenicimicrobium oleiphilum HX-OS.bin.34TS, both from a terrestrial oil reservoir. Physiological and metatranscriptomics analyses showed that Thermatribacter velox B11T and Phoenicimicrobium oleiphilum HX-OS.bin.34TS ferment sugars and n-alkanes, respectively, producing H2, CO2, and acetate as common products. Comparative genomics showed that all members of the Atribacterota lack a complete Wood-Ljungdahl Pathway (WLP), but that the Reductive Glycine Pathway (RGP) is widespread, indicating that the RGP, rather than WLP, is a central hub in Atribacterota metabolism. Ancestral character state reconstructions and phylogenetic analyses showed that key genes encoding the RGP (fdhA, fhs, folD, glyA, gcvT, gcvPAB, pdhD) and other central functions were gained independently in the two classes, Atribacteria (OP9) and Phoenicimicrobiia (JS1), after which they were inherited vertically; these genes included fumarate-adding enzymes (faeA; Phoenicimicrobiia only), the CODH/ACS complex (acsABCDE), and diverse hydrogenases (NiFe group 3b, 4b and FeFe group A3, C). Finally, we present genome-resolved community metabolic models showing the central roles of Atribacteria (OP9) and Phoenicimicrobiia (JS1) in acetate- and hydrocarbon-rich environments. Conclusion Our findings expand the knowledge of the diversity, physiology, ecology, and evolution of the phylum Atribacterota. This study is a starting point for promoting more incisive studies of their syntrophic biology and may guide the rational design of strategies to cultivate them in the laboratory.

An anaerobic, mesophilic, syntrophic, archaeon strain MK-D1T, was isolated as a pure co-culture with Methanogenium sp. strain MK-MG from deep-sea methane seep sediment. This organism is, to our knowledge, the first cultured representative of ‘Asgard’ archaea, an archaeal group closely related to eukaryotes. Here, we describe the detailed physiology and phylogeny of MK-D1T and propose Promethearchaeum syntrophicum gen. nov., sp. nov. to accommodate this strain. Cells were non-motile, small cocci, approximately 300–750 nm in diameter and produced membrane vesicles, chains of blebs and membrane-based protrusions. MK-D1T grew at 4–30 °C with optimum growth at 20 °C. The strain grew chemoorganotrophically with amino acids, peptides and yeast extract with obligate dependence on syntrophy with H2-/formate-utilizing organisms. MK-D1T showed the fastest growth and highest maximum cell yield when grown with yeast extract as the substrate: approximately 3 months to full growth, reaching up to 6.7×106 16S rRNA gene copies ml−1. MK-D1T had a circular 4.32 Mb chromosome with a DNA G+C content of 31.1 mol%. The results of phylogenetic analyses of the 16S rRNA gene and conserved marker proteins indicated that the strain is affiliated with ‘Asgard’ archaea and more specifically DHVC1/DSAG/MBG-B and ‘Lokiarchaeota’/‘Lokiarchaeia’. On the basis of the results of 16S rRNA gene sequence analysis, the most closely related isolated relatives were Infirmifilum lucidum 3507LTT (76.09 %) and Methanothermobacter tenebrarum RMAST (77.45 %) and the closest relative in enrichment culture was Candidatus ‘Lokiarchaeum ossiferum’ (95.39 %). The type strain of the type species is MK-D1T (JCM 39240T and JAMSTEC no. 115508). We propose the associated family, order, class, phylum, and kingdom as Promethearchaeaceae fam. nov., Promethearchaeales ord. nov., Promethearchaeia class. nov., Promethearchaeota phyl. nov., and Promethearchaeati regn. nov., respectively. These are in accordance with ICNP Rules 8 and 22 for nomenclature, Rule 30(3)(b) for validation and maintenance of the type strain, and Rule 31a for description as a member of an unambiguous syntrophic association.

Abstract Asgard archaea were pivotal in the origin of complex cellular life. Hodarchaeales (Asgardarchaeota class Heimdallarchaeia) were recently shown to be the closest relatives of eukaryotes. However, limited sampling of these archaea constrains our understanding of their ecology and evolution 1–3 , including their anticipated role in eukaryogenesis. Here, we nearly double the number of Asgardarchaeota metagenome-assembled genomes (MAGs) to 869, including 136 new Heimdallarchaeia (49 Hodarchaeales) and several novel lineages. Examining global distribution revealed Hodarcheales are primarily found in coastal marine sediments. Detailed analysis of their metabolic capabilities revealed guilds of Heimdallarchaeia are distinct from other Asgardarchaeota. These archaea encode hallmarks of aerobic eukaryotes, including electron transport chain complexes (III and IV), biosynthesis of heme, and response to reactive oxygen species (ROS). The predicted structural architecture of Heimdallarchaeia membrane-bound hydrogenases includes additional Complex-I-like subunits potentially increasing the proton motive force and ATP synthesis. Heimdallarchaeia genomes encode CoxD, which regulates the electron transport chain (ETC) in eukaryotes. Thus, key hallmarks for aerobic respiration may have been present in the Asgard-eukaryotic ancestor. Moreover, we found that Heimdallarchaeia is present in a variety of oxic marine environments. This expanded diversity reveals these Archaea likely conferred energetic advantages during early stages of eukaryogenesis, fueling cellular complexity.