Frontiers in Microbiology

Insect–microbe endosymbiotic associations are omnipresent in nature, wherein the symbiotic microbes often play pivotal biological roles for their host insects. In particular, insects utilizing nutritionally imbalanced food sources are dependent on specific microbial symbionts to compensate for the nutritional deficiency via provisioning of B vitamins in blood-feeding insects, such as tsetse flies, lice, and bedbugs. Bat flies of the family Nycteribiidae (Diptera) are blood-sucking ectoparasites of bats and shown to be associated with co-speciating bacterial endosymbiont “Candidatus Aschnera chinzeii,” although functional aspects of the microbial symbiosis have been totally unknown. In this study, we report the first complete genome sequence of Aschnera from the bristled bat fly Penicillidia jenynsii. The Aschnera genome consisted of a 748,020 bp circular chromosome and a 18,747 bp circular plasmid. The chromosome encoded 603 protein coding genes (including 3 pseudogenes), 33 transfer RNAs, and 1 copy of 16S/23S/5S ribosomal RNA operon. The plasmid contained 10 protein coding genes, whose biological function was elusive. The genome size, 0.77 Mbp, was drastically reduced in comparison with 4–6 Mbp genomes of free-living γ-proteobacteria. Accordingly, the Aschnera genome was devoid of many important functional genes, such as synthetic pathway genes for purines, pyrimidines, and essential amino acids. On the other hand, the Aschnera genome retained complete or near-complete synthetic pathway genes for biotin (vitamin B7), tetrahydrofolate (vitamin B9), riboflavin (vitamin B2), and pyridoxal 5'-phosphate (vitamin B6), suggesting that Aschnera provides these vitamins and cofactors that are deficient in the blood meal of the host bat fly. Similar retention patterns of the synthetic pathway genes for vitamins and cofactors were also observed in the endosymbiont genomes of other blood-sucking insects, such as Riesia of human lice, Arsenophonus of louse flies, and Wigglesworthia of tsetse flies, which may be either due to convergent evolution in the blood-sucking host insects or reflecting the genomic architecture of Arsenophonus-allied bacteria.

Genus Pseudomonas is a large assemblage of diverse microorganisms, not sharing a common evolutionary history. To clarify their evolutionary relationships and classification, we have conducted comprehensive phylogenomic and comparative analyses on 388 Pseudomonadaceae genomes. In phylogenomic trees, Pseudomonas species formed 12 main clusters, apart from the “Aeruginosa clade” containing its type species, P. aeruginosa. In parallel, our detailed analyses on protein sequences from Pseudomonadaceae genomes have identified 98 novel conserved signature indels (CSIs), which are uniquely shared by the species from different observed clades/groups. Six CSIs, which are exclusively shared by species from the “Aeruginosa clade,” provide reliable demarcation of this clade corresponding to the genus Pseudomonas sensu stricto in molecular terms. The remaining 92 identified CSIs are specific for nine other Pseudomonas species clades and the genera Azomonas and Azotobacter which branch in between them. The identified CSIs provide strong independent evidence of the genetic cohesiveness of these species clades and offer reliable means for their demarcation/circumscription. Based on the robust phylogenetic and molecular evidence presented here supporting the distinctness of the observed Pseudomonas species clades, we are proposing the transfer of species from the following clades into the indicated novel genera: Alcaligenes clade – Aquipseudomonas gen. nov.; Fluvialis clade – Caenipseudomonas gen. nov.; Linyingensis clade – Geopseudomonas gen. nov.; Oleovorans clade – Ectopseudomonas gen. nov.; Resinovorans clade – Metapseudomonas gen. nov.; Straminea clade – Phytopseudomonas gen. nov.; and Thermotolerans clade – Zestomonas gen. nov. In addition, descriptions of the genera Azomonas, Azotobacter, Chryseomonas, Serpens, and Stutzerimonas are emended to include information for the CSIs specific for them. The results presented here should aid in the development of a more reliable classification scheme for Pseudomonas species.

The taxonomic relationships of 10 strains isolated from seaweeds collected from two beaches in Republic of Korea were studied by sequencing and analyses of 16S rRNA genes and whole genomes. For the construction of a more reliable and robust 16S rRNA gene phylogeny, the authentic and nearly complete 16S rRNA gene sequences of all the Microbacterium type strains were selected through pairwise comparison of the sequences contained in several public databases including the List of Prokaryotic names with Standing in Nomenclature (LPSN). The clustering of the ten study strains into five distinct groups was apparent in this single gene-based phylogenetic tree. In addition, the 16S rRNA gene sequences of a few type strains were shown to be incorrectly listed in LPSN. An overall phylogenomic clustering of the genus Microbacterium was performed with a total of 113 genomes by core genome analysis. As a result, nine major (≥ three type strains) and eight minor (two type strains) clusters were defined mostly at gene support index of 92 and mean intra-cluster OrthoANIu of >80.00%. All of the study strains were assigned to a Microbacterium liquefaciens clade and distributed further into four subclusters in the core genome-based phylogenetic tree. In vitro phenotypic assays for physiological, biochemical, and chemotaxonomic characteristics were also carried out with the ten study strains and seven closely related type strains. Comparison of the overall genomic relatedness indices (OGRI) including OrthoANIu and digital DNA–DNA hybridization supported that the study strains constituted four new species of the genus Microbacterium. In addition, some Microbacterium type strains were reclassified as members of preexisting species. Moreover, some of them were embedded in a new genus of the family Microbacteriaceae based on their distinct separation in the core genome-based phylogenetic tree and amino acid identity matrices. Based on the results here, four new species, namely, Microbacterium aurugineum sp. nov., Microbacterium croceum sp. nov., Microbacterium galbinum sp. nov., and Microbacterium sufflavum sp. nov., are described, along with the proposal of Paramicrobacterium gen. nov. containing five reclassified Microbacterium species from the “Microbacterium agarici clade”, with Paramicrobacterium agarici gen. nov., comb. nov. as the type species.

The familyHalomonadaceaeis the largest family composed of halophilic bacteria, with more than 160 species with validly published names as of July 2023. Several classifications to circumscribe this family are available in major resources, such as those provided by the List of Prokaryotic names with Standing in Nomenclature (LPSN), NCBI Taxonomy, Genome Taxonomy Database (GTDB), and Bergey’s Manual of Systematics of Archaea and Bacteria (BMSAB), with some degree of disagreement between them. Moreover, regardless of the classification adopted, the genusHalomonasis not phylogenetically consistent, likely because it has been used as a catch-all for newly described species within the familyHalomonadaceaethat could not be clearly accommodated in otherHalomonadaceaegenera. In the past decade, some taxonomic rearrangements have been conducted on theHalomonadaceaebased on ribosomal and alternative single-copy housekeeping gene sequence analysis. High-throughput technologies have enabled access to the genome sequences of many type strains belonging to the familyHalomonadaceae; however, genome-based studies specifically addressing its taxonomic status have not been performed to date. In this study, we accomplished the genome sequencing of 17 missing type strains ofHalomonadaceaespecies that, together with other publicly available genome sequences, allowed us to re-evaluate the genetic relationship, phylogeny, and taxonomy of the species and genera within this family. The approach followed included the estimate of the Overall Genome Relatedness Indexes (OGRIs) such as the average amino acid identity (AAI), phylogenomic reconstructions using amino acid substitution matrices customized for the familyHalomonadaceae, and the analysis of clade-specific signature genes. Based on our results, we conclude that the genusHalovibriois obviously out of place within the familyHalomonadaceae,and, on the other hand, we propose a division of the genusHalomonasinto seven separate genera and the transfer of seven species fromHalomonasto the genusModicisalibacter, together with the emendation of the latter. Additionally, data from this study demonstrate the existence of various synonym species names in this family.

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 >95% and contamination <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.