Methane emission by terrestrial invertebrates is restricted to millipedes, termites, cockroaches, and scarab beetles. The arthropod-associated archaea known to date belong to the orders Methanobacteriales, Methanomassiliicoccales, Methanomicrobiales, and Methanosarcinales, and in a few cases also to non-methanogenic Nitrososphaerales and Bathyarchaeales. However, all major host groups are severely undersampled, and the taxonomy of existing lineages is not well developed. Full-length 16S rRNA gene sequences and genomes of arthropod-associated archaea are scarce, reference databases lack resolution, and the names of many taxa are either not validly published or under-classified and require revision. Here, we investigated the diversity of archaea in a wide range of methane-emitting arthropods, combining phylogenomic analysis of isolates and metagenome-assembled genomes (MAGs) with amplicon sequencing of full-length 16S rRNA genes. Our results allowed us to describe numerous new species in hitherto undescribed taxa among the orders Methanobacteriales (Methanacia, Methanarmilla, Methanobaculum, Methanobinarius, Methanocatella, Methanoflexus, Methanorudis, and Methanovirga, all gen. nova), Methanomicrobiales (Methanofilum and Methanorbis, both gen. nova), Methanosarcinales (Methanofrustulum and Methanolapillus, both gen. nova), Methanomassiliicoccales (Methanomethylophilaceae fam. nov., Methanarcanum, Methanogranum, Methanomethylophilus, Methanomicula, Methanoplasma, Methanoprimaticola, all gen. nova), and the new family Bathycorpusculaceae (Bathycorpusculum gen. nov.). Reclassification of amplicon libraries from this and previous studies using this new taxonomic framework revealed that arthropods harbor only CO2 and methyl-reducing hydrogenotrophic methanogens. Numerous genus-level lineages appear to be present exclusively in arthropods, suggesting long evolutionary trajectories with their termite, cockroach, and millipede hosts, and a radiation into various microhabitats and ecological niches provided by their digestive tracts (e.g., hindgut compartments, gut wall, or anaerobic protists). The distribution patterns among the different host groups are often complex, indicating a mixed mode of transmission and a parallel evolution of invertebrate and vertebrate-associated lineages.
As the name of the genus Pantoea (“of all sorts and sources”) suggests, this genus includes bacteria with a wide range of provenances, including plants, animals, soils, components of the water cycle, and humans. Some members of the genus are pathogenic to plants, and some are suspected to be opportunistic human pathogens; while others are used as microbial pesticides or show promise in biotechnological applications. During its taxonomic history, the genus and its species have seen many revisions. However, evolutionary and comparative genomics studies have started to provide a solid foundation for a more stable taxonomy. To move further toward this goal, we have built a 2,509-gene core genome tree of 437 public genome sequences representing the currently known diversity of the genus Pantoea. Clades were evaluated for being evolutionarily and ecologically significant by determining bootstrap support, gene content differences, and recent recombination events. These results were then integrated with genome metadata, published literature, descriptions of named species with standing in nomenclature, and circumscriptions of yet-unnamed species clusters, 15 of which we assigned names under the nascent SeqCode. Finally, genome-based circumscriptions and descriptions of each species and each significant genetic lineage within species were uploaded to the LINbase Web server so that newly sequenced genomes of isolates belonging to any of these groups could be precisely and accurately identified.
Highly purified cultures of alkaliphilic aceticlastic methanogens were collected for the first time using methanogenic enrichments with acetate from a soda lake and a terrestrial mud volcano. The cells of two strains were non-motile rods forming filaments. The mud volcano strain M04Ac was alkalitolerant, with the pH range for growth from 7.5 to 10.0 (optimum at 9.0), while the soda lake strain Mx was an obligate alkaliphile growing in the pH range 7.7–10.2 (optimum 9.3–9.5) in the presence of optimally 0.2–0.3 M total Na+. Genomes of both strains encoded all enzymes required for aceticlastic methanogenesis and different mechanisms of (halo)alkaline adaptations, including ectoine biosynthesis, which is the first evidence for the formation of this osmoprotectant in archaea. According to 16S rRNA gene phylogeny, the strains possessed 98.3–98.9% sequence identity and belonged to the obligately aceticlastic genus Methanothrix with M. harundinaceae as the most closely related species. However, a more advanced phylogenomic reconstruction based on 122 conserved single-copy archaeal protein-coding marker genes clearly indicated a polyphyletic origin of the species included in the genus Methanothrix. We propose to reclassify Methanothrix harrundinacea (type strain 8AcT) into a new genus, Methanocrinis gen. nov., with the type species Methanocrinis harrundinaceus comb. nov. We also propose under SeqCode the complete genome sequences of strain MxTs (GCA_029167045.1) and strain M04AcTs (GCA_029167205.1) as nomenclatural types of Methanocrinis natronophilus sp. nov. and Methanocrinis alkalitolerans sp. nov., respectively, which represent other species of the novel genus. This work demonstrates that the low energy aceticlastic methanogenesis may function at extreme conditions present in (halo)alkaline habitats.
Phytoplasmas are phloem-limited plant pathogens, such as sugarcane white leaf (SCWL) phytoplasma, which are responsible for heavy economic losses to the sugarcane industry. Characterization of phytoplasmas has been limited because they cannot be cultured in vitro. However, with the advent of genome sequencing, different aspects of phytoplasmas are being investigated. In this study, we developed a DNA enrichment method for sugarcane white leaf (SCWL) phytoplasma, evaluated the effect of DNA enrichment via Illumina sequencing technologies, and utilized Illumina and Nanopore sequencing technologies to obtain the complete genome sequence of the “Candidatus Phytoplasma sacchari” isolate SCWL1 that is associated with sugarcane white leaf in China. Illumina sequencing analysis elucidated that only 1.21% of the sequencing reads from total leaf DNA were mapped to the SCWL1 genome, whereas 40.97% of the sequencing reads from the enriched DNA were mapped to the SCWL1 genome. The genome of isolate SCWL1 consists of a 538,951 bp and 2976 bp long circular chromosome and plasmid, respectively. We identified 459 protein-encoding genes, 2 complete 5S-23S-16S rRNA gene operons, 27 tRNA genes, and an incomplete potential mobile unit (PMU) in the circular chromosome. Phylogenetic analyses and average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values based on the sequenced genome revealed that SCWL phytoplasma and sugarcane grassy shoot (SCGS) phytoplasma belonged to the same phytoplasma species. This study provides a genomic DNA enrichment method for phytoplasma sequencing. Moreover, we report the first complete genome of a “Ca. Phytoplasma sacchari” isolate, thus contributing to future studies on the evolutionary relationships and pathogenic mechanisms of “Ca. Phytoplasma sacchari” isolates.
Members of the archaeal order Caldarchaeales (previously the phylum Aigarchaeota) are poorly sampled and are represented in public databases by relatively few genomes. Additional representative genomes will help resolve their placement among all known members of Archaea and provide insights into their roles in the environment. In this study, we analyzed 16S rRNA gene amplicons belonging to the Caldarchaeales that are available in public databases, which demonstrated that archaea of the order Caldarchaeales are diverse, widespread, and most abundant in geothermal habitats. We also constructed five metagenome-assembled genomes (MAGs) of Caldarchaeales from two geothermal features to investigate their metabolic potential and phylogenomic position in the domain Archaea. Two of the MAGs were assembled from microbial community DNA extracted from fumarolic lava rocks from Mauna Ulu, Hawai‘i, and three were assembled from DNA obtained from hot spring sinters from the El Tatio geothermal field in Chile. MAGs from Hawai‘i are high quality bins with completeness &gt;95% and contamination &lt;1%, and one likely belongs to a novel species in a new genus recently discovered at a submarine volcano off New Zealand. MAGs from Chile have lower completeness levels ranging from 27 to 70%. Gene content of the MAGs revealed that these members of Caldarchaeales are likely metabolically versatile and exhibit the potential for both chemoorganotrophic and chemolithotrophic lifestyles. The wide array of metabolic capabilities exhibited by these members of Caldarchaeales might help them thrive under diverse harsh environmental conditions. All the MAGs except one from Chile harbor putative prophage regions encoding several auxiliary metabolic genes (AMGs) that may confer a fitness advantage on their Caldarchaeales hosts by increasing their metabolic potential and make them better adapted to new environmental conditions. Phylogenomic analysis of the five MAGs and over 3,000 representative archaeal genomes showed the order Caldarchaeales forms a monophyletic group that is sister to the clade comprising the orders Geothermarchaeales (previously Candidatus Geothermarchaeota), Conexivisphaerales and Nitrososphaerales (formerly known as Thaumarchaeota), supporting the status of Caldarchaeales members as a clade distinct from the Thaumarchaeota.
Bathyarchaeia are widespread in various anoxic ecosystems and are considered one of the most abundant microbial groups on the earth. There are only a few reports of laboratory cultivation of Bathyarchaeia, and none of the representatives of this class has been isolated in pure culture. Here, we report a sustainable cultivation of the Bathyarchaeia archaeon (strain M17CTs) enriched from anaerobic sediment of a coastal lake. The cells of strain M17CTs were small non-motile cocci, 0.4–0.7 μm in diameter. The cytoplasmic membrane was surrounded by an S-layer and covered with an outermost electron-dense sheath. Strain M17CTs is strictly anaerobic mesophile. It grows at 10–45°C (optimum 37°C), at pH 6.0–10.0 (optimum 8.0), and at NaCl concentrations of 0–60 g l−1 (optimum 20 g l−1). Growth occurred in the presence of methoxylated aromatic compounds (3,4-dimethoxybenzoate and vanillate) together with complex proteinaceous substrates. Dimethyl sulfoxide and nitrate stimulated growth. The phylogenomic analysis placed strain M17CTs to BIN-L-1 genus-level lineage from the BA1 family-level lineage and B26-1 order-level lineage (former subgroup-8) within the class Bathyarchaeia. The complete genome of strain M17CTs had a size of 2.15 Mb with a DNA G + C content of 38.1%. Based on phylogenomic position and phenotypic and genomic properties, we propose to assign strain M17CTs to a new species of a novel genus Bathyarchaeum tardum gen. nov., sp. nov. within the class Bathyarchaeia. This is the first sustainably cultivated representative of Bathyarchaeia. We propose under SeqCode the complete genome sequence of strain M17CTs (CP122380) as a nomenclatural type of Bathyarchaeum tardum, which should be considered as a type for the genus Bathyarchaeum, which is proposed as a type for the family Bathyarchaeaceae, order Bathyarchaeales, and of the class Bathyarchaeia.
‘Candidatus Phytoplasma solani’ (stolbur phytoplasma) is associated with rubbery taproot disease (RTD) of sugar beet (Beta vulgaris L.), while Macrophomina phaseolina is considered the most important root rot pathogen of this plant in Serbia. The high prevalence of M. phaseolina root rot reported on sugar beet in Serbia, unmatched elsewhere in the world, coupled with the notorious tendency of RTD-affected sugar beet to rot, has prompted research into the relationship between the two diseases. This study investigates the correlation between the occurrence of sugar beet RTD and the presence of root rot fungal pathogens in a semi-field ‘Ca. P. solani’ transmission experiment with the cixiid vector Reptalus quinquecostatus (Dufour), in addition to naturally infected sugar beet in the open field. Our results showed that: (i) Reptalus quinquecostatus transmitted ‘Ca. P. solani’ to sugar beet which induced typical RTD root symptoms; (ii) Macrophomina phaseolina root rot was exclusively present in ‘Ca. P. solani’-infected sugar beet in both the semi-field experiment and naturally infected sugar beet; and that (iii) even under environmental conditions favorable to the pathogen, M. phaseolina did not infect sugar beet, unless the plants had been previously infected with phytoplasma.
Huanglongbing (HLB) is a global devastating citrus disease that is mainly caused by “Candidatus Liberibacter asiaticus” (CLas). It is mostly transmitted by the insect Asian citrus psyllid (ACP, Diaphorina citri) in a persistent and proliferative manner. CLas traverses multiple barriers to complete an infection cycle and is likely involved in multiple interactions with D. citri. However, the protein–protein interactions between CLas and D. citri are largely unknown. Here, we report on a vitellogenin-like protein (Vg_VWD) in D. citri that interacts with a CLas flagellum (flaA) protein. We found that Vg_VWD was upregulated in CLas-infected D. citri. Silencing of Vg_VWD in D. citri via RNAi silencing significantly increased the CLas titer, suggesting that Vg_VWD plays an important role in the CLas–D. citri interaction. Agrobacterium-mediated transient expression assays indicated that Vg_VWD inhibits BAX- and INF1-triggered necrosis and suppresses the callose deposition induced by flaA in Nicotiana benthamiana. These findings provide new insights into the molecular interaction between CLas and D. citri.
Production of organic molecules is largely depending on fossil fuels. A sustainable alternative would be the synthesis of these compounds from CO2 and a cheap energy source, such as H2, CH4, NH3, CO, sulfur compounds or iron(II). Volcanic and geothermal areas are rich in CO2 and reduced inorganic gasses and therefore habitats where novel chemolithoautotrophic microorganisms for the synthesis of organic compounds could be discovered. Here we describe “Candidatus Hydrogenisulfobacillus filiaventi” R50 gen. nov., sp. nov., a thermoacidophilic, autotrophic H2-oxidizing microorganism, that fixed CO2 and excreted no less than 0.54 mol organic carbon per mole fixed CO2. Extensive metabolomics and NMR analyses revealed that Val, Ala and Ile are the most dominant form of excreted organic carbon while the aromatic amino acids Tyr and Phe, and Glu and Lys were present at much lower concentrations. In addition to these proteinogenic amino acids, the excreted carbon consisted of homoserine lactone, homoserine and an unidentified amino acid. The biological role of the excretion remains uncertain. In the laboratory, we noticed the production under high growth rates (0.034 h−1, doubling time of 20 h) in combination with O2-limitation, which will most likely not occur in the natural habitat of this strain. Nevertheless, this large production of extracellular organic molecules from CO2 may open possibilities to use chemolithoautotrophic microorganisms for the sustainable production of important biomolecules.
Cable bacteria are centimeters-long filamentous bacteria that oxidize sulfide in anoxic sediment layers and reduce oxygen at the oxic-anoxic interface, connecting these reactions via electron transport. The ubiquitous cable bacteria have a major impact on sediment geochemistry and microbial communities. This includes diverse bacteria swimming around cable bacteria as dense flocks in the anoxic zone, where the cable bacteria act as chemotactic attractant. We hypothesized that flocking only appears when cable bacteria are highly abundant and active. We set out to discern the timing and drivers of flocking over 81 days in an enrichment culture of the freshwater cable bacterium Candidatus Electronema aureum GS by measuring sediment microprofiles of pH, oxygen, and electric potential as a proxy of cable bacteria activity. Cable bacterial relative abundance was quantified by 16S rRNA amplicon sequencing, and microscopy observations to determine presence of flocking. Flocking was always observed at some cable bacteria, irrespective of overall cable bacteria rRNA abundance, activity, or sediment pH. Diverse cell morphologies of flockers were observed, suggesting that flocking is not restricted to a specific, single bacterial associate. This, coupled with their consistent presence supports a common mechanism of interaction, likely interspecies electron transfer via electron shuttles. Flocking appears exclusively linked to the electron conducting activity of the individual cable bacteria.