Microbiology

The naming of prokaryotes is governed by the International Code of Nomenclature of Prokaryotes (ICNP) and partially by the International Code of Nomenclature for Algae, Fungi and Plants (ICN). Such codes must be able to determine names of taxa in a universal and unambiguous manner, thus serving as a common language across different fields and activities. This unity is undermined when a new code of nomenclature emerges that overlaps in scope with an established, time-tested code and uses the same format of names but assigns different nomenclatural status values to the names. The resulting nomenclatural confusion is not beneficial to the wider scientific community. Such ambiguity is expected to result from the establishment of the ‘Code of Nomenclature of Prokaryotes Described from DNA Sequence Data’ (‘SeqCode’), which is in general and specific conflict with the ICNP and the ICN. Shortcomings in the interpretation of the ICNP may have exacerbated the incompatibility between the codes. It is reiterated as to why proposals to accept sequences as nomenclatural types of species and subspecies with validly published names, now implemented in the SeqCode, have not been implemented by the International Committee on Systematics of Prokaryotes (ICSP), which oversees the ICNP. The absence of certain regulations from the ICNP for the naming of as yet uncultivated prokaryotes is an acceptable scientific argument, although it does not justify the establishment of a separate code. Moreover, the proposals rejected by the ICSP are unnecessary to adequately regulate the naming of uncultivated prokaryotes. To provide a better service to the wider scientific community, an alternative proposal to emend the ICNP is presented, which would result in Candidatus names being regulated analogously to validly published names. This proposal is fully consistent with previous ICSP decisions, preserves the essential unity of nomenclature and avoids the expected nomenclatural confusion.

ABSTRACT Filamentous Chloroflexota are abundant in activated sludge wastewater treatment plants (WWTPs) worldwide and are occasionally associated with poor solid-liquid separation or foaming, but most of the abundant lineages remain undescribed. Here, we present a comprehensive overview of Chloroflexota abundant in WWTPs worldwide, using high-quality metagenome-assembled genomes (MAGs) and 16S rRNA amplicon data from 740 Danish and global WWTPs. Many novel taxa were described, encompassing 4 families, 13 genera, and 29 novel species. These were widely distributed across most continents, influenced by factors such as climate zone and WWTP process design. Visualization by fluorescence in situ hybridization (FISH) confirmed their high abundances in many WWTPs based on the amplicon data and showed a filamentous morphology for nearly all species. Most formed thin and short trichomes integrated into the floc structure, unlikely to form the typical inter-floc bridging that hinders activated sludge floc settling. Metabolic reconstruction of 53 high-quality MAGs, representing most of the novel genera, offered further insights into their versatile metabolisms and suggested a primary role in carbon removal and involvement in nitrogen cycling. The presence of glycogen reserves, detected by FISH-Raman microspectroscopy, seemed widespread across the phylum, demonstrating that these bacteria likely utilize glycogen as energy storage to survive periods with limited resources. This study gives a broad overview of the Chloroflexota community in global activated sludge WWTPs and improves our understanding of their roles in these engineered ecosystems. IMPORTANCE Chloroflexota are often abundant members of the biomass in wastewater treatment plants (WWTPs) worldwide, typically with a filamentous morphology, forming the backbones of the activated sludge floc. However, their overgrowth can often cause operational issues connected to poor settling or foaming, impairing effluent quality and increasing operational costs. Despite their importance, few Chloroflexota genera have been characterized so far. Here, we present a comprehensive overview of Chloroflexota abundant in WWTPs worldwide and an in-depth characterization of their morphology, phylogeny, and ecophysiology, obtaining a broad understanding of their ecological role in activated sludge.

The bacterial strain WB46 was isolated from the rhizosphere of willow plants (Salix purpurea L.) growing in soil contaminated with petroleum hydrocarbons. The strain was subjected to whole-genome shotgun sequencing using Illumina HiSeq. Its draft genome is 7.15 Mb, with a 69.55% GC content, containing 6387 protein-coding genes and 51 tRNA and 15 rRNA sequences. The quality and reliability of the genome were assessed using CheckM, attaining an estimated genome completeness of 98.75% and an estimated contamination of 1.68%. These results indicate a high-quality genome (>95%) and low contamination (<5%). Many of these genes are responsible for petroleum hydrocarbon degradation, such as alkane 1-monooxygenase (alkB) and naphthalene dioxygenase (ndo). 16S rRNA gene analysis, including in silico DNA–DNA hybridization (DDH) and average nucleotide identity (ANI), showed that strain WB46 belongs to the genus Nocardia, and the most closely related species is Nocardia asteroides. The strain WB46 showed a distance of 63.4% and sequence identity of 88.63%, respectively. These values fall below the threshold levels of 70% and 95%, respectively, suggesting that the strain WB46 is a new species. We propose the name of Nocardia canadensis sp. nov. for this new species. Interestingly, the sequence divergence of the 16S rRNA gene showed that the divergence only occurred in the V2 region. Therefore, the conventional V3–V4, V5–V7, or V8–V9 targeting metabarcoding, among others, would not be able to assess the diversity related to this new species.

Abstract Symbiotic bacteria can alter host biology by providing protection from natural enemies, or alter reproduction or vectoral competence. Symbiont‐linked control of vector‐borne disease in Anopheles has been hampered by a lack of symbioses that can establish stable vertical transmission in the host. Previous screening found the symbiont ‘ Candidatus Tisiphia’ in Anopheles plumbeus , an aggressive biter and potential secondary vector of malaria parasites and West Nile virus. We screened samples collected over 10‐years across Germany and used climate databases to assess environmental influence on incidence. We observed a 95% infection rate, and that the frequency of infection did not fluctuate with broad environmental factors. Maternal inheritance is indicated by presence in the ovaries through FISH microscopy. Finally, we assembled a high‐quality 1.6 Mbp draft genome of ‘ Ca . Tisiphia’ to explore its phylogeny and potential metabolic competence. The infection is closely related to strains found in Culicoides biting midges and shows similar patterns of metabolism, providing no evidence of the capacity to synthesize B‐vitamins. This infection offers avenues for onward research in anopheline mosquito symbioses. Additionally, it provides future opportunity to study the impact of ‘ Ca . Tisiphia’ on natural and transinfected hosts, especially in relation to reproductive fitness and vectorial competence and capacity.

The methanogenic strain Mx-05T was isolated from the human fecal microbiome. A phylogenetic analysis based on the 16S rRNA gene and protein marker genes indicated that the strain is affiliated with the order Methanomassiliicoccales. It shares 86.9% 16S rRNA gene sequence identity with Methanomassiliicoccus luminyensis, the only member of this order previously isolated. The cells of Mx-05T were non-motile cocci, with a diameter range of 0.4–0.7 μm. They grew anaerobically and reduced methanol, monomethylamine, dimethylamine, and trimethylamine into methane, using H2 as an electron donor. H2/CO2, formate, ethanol, and acetate were not used as energy sources. The growth of Mx-05T required an unknown medium factor(s) provided by Eggerthella lenta and present in rumen fluid. Mx-05T grew between 30 °C and 40 °C (optimum 37 °C), over a pH range of 6.9–8.3 (optimum pH 7.5), and between 0.02 and 0.34 mol.L−1 NaCl (optimum 0.12 mol.L−1 NaCl). The genome is 1.67 Mbp with a G+C content of 55.5 mol%. Genome sequence annotation confirmed the absence of the methyl branch of the H4MPT Wood–Ljungdahl pathway, as described for other Methanomassiliicoccales members. Based on an average nucleotide identity analysis, we propose strain Mx-05T as being a novel representative of the order Methanomassiliicoccales, within the novel family Methanomethylophilaceae, for which the name Methanomethylophilus alvi gen. nov, sp. nov. is proposed. The type strain is Mx-05T (JCM 31474T).

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.