Publications

4497

Abstract The CRISPR–Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) systems are adaptive immune mechanisms that play critical roles in defending archaea and bacteria against invading entities. These systems can be divided into two classes, with class 2 comprising three types (II, V, and VI). Because of their ability to cleave double-stranded DNA, many class 2 CRISPR–Cas proteins have been harnessed as genome editing tools. Unlike the well-studied type II Cas9 proteins, the structural studies of the type V-B Cas12b proteins are limited, hindering their engineering and broader application. Here, we report four complex structures of ChCas12b, which reveal many unique structural features. The folding of the single guide RNA (sgRNA) of ChCas12b is distinct from that of AacCas12b and BthCas12b. Notably, many of these unique features are involved in ChCas12b–sgRNA interaction, suggesting that they are co-evolved. While ChCas12b shares a conserved two-cation-assisted catalytic mechanism with its homologs, it recognizes a longer guide:target heteroduplex, potentially offering higher fidelity in DNA editing. Altogether, our studies suggested that Cas12b family proteins exhibit significant diversity in their folding, sgRNA and target DNA binding. In the future, it is worth characterizing more representative proteins to identify CRISPR–Cas proteins with higher gene editing ability and fidelity.

The genus Moraxella (Moraxellaceae, Pseudomonadales) comprises a diverse group of bacteria inhabiting human and animal mucosa, as well as environmental niches such as water, soil and food. While some species are clinically significant pathogens, others play ecological or biotechnological roles. Despite previous taxonomic revisions, Moraxella remains polyphyletic, necessitating a refined classification. In this study, we conducted comprehensive taxogenomic analyses integrating core protein phylogeny, average amino acid identity and the percentage of conserved proteins, along with 16S rRNA similarities and inferred phylogeny. Our results revealed three distinct phylogenetic clusters within Moraxella. The core group, comprising Moraxella lacunata NBRC 102154T and closely related species, exhibited strong genomic cohesion. A second cluster, consisting of Moraxella boevrei DSM 14165T, Moraxella osloensis CCUG 350T and Moraxella atlantae NBRC 14588T, showed greater genetic affinity to Faucicola mancuniensis GVCNT2T, supporting their reassignment to the genus Faucicola. The third group, represented by Moraxella lincolnii CCUG 9405T, was phylogenetically distinct, occupying a basal position relative to Psychrobacter, indicating the need for its classification within a novel genus within the family Moraxellaceae, for which we propose the name Lwoffella lincolnii gen. nov., comb. nov. Phenotypic data compiled from the original published descriptions of the respective species, including fatty acid composition and enzymatic profiles, further corroborated these genomic findings. This study refines Moraxella taxonomy by clarifying genus boundaries and evolutionary relationships, with implications for ecology and clinical microbiology.

The actinobacterial family Microbacteriaceae comprises a diverse group of Gram-positive bacteria with high G+C content and complex taxonomic challenges. Traditional polyphasic approaches, based on 16S rRNA phylogeny and phenotypic traits, have in some instances resulted in polyphyletic taxonomic groupings, necessitating genome-wide methodologies to better resolve evolutionary relationships. This study employs a taxogenomic approach – incorporating 16S rRNA gene sequencing, core protein phylogeny, average amino acid identity and percentage of conserved proteins – to reassess the genera Leifsonia, Salinibacterium, Homoserinimonas, Glaciibacter, Antiquaquibacter and Diaminobutyricibacter. The findings reveal significant polyphyly in Leifsonia, Glaciibacter and Salinibacterium, indicating ecological convergence rather than shared ancestry. Proposed taxonomic revisions include the reclassification of Salinibacterium soli and Salinibacterium metalliresistens into Antiquaquibacter, Salinibacterium hongtaonis and Salinibacterium sedimenticola into Homoserinimonas, and Leifsonia psychrotolerans and Leifsonia kafniensis into Glaciibacter. Diaminobutyricibacter tongyongensis should be incorporated into Leifsonia. Additionally, Leifsonia rubra is reassigned to Salinibacterium. Two new genera were also proposed to encompass “Leifsonia flava” and Glaciibacter flavus, named Orlajensenia gen. nov. and another to encompass Leifsonia bigeumensis, named Leifsonella gen. nov. These genome-based insights provide a refined framework for the taxonomy of Microbacteriaceae, enhancing our understanding of their evolutionary and ecological roles.

ABSTRACT Ca . Babelota is a phylum of strictly intracellular bacteria whose representatives are commonly detected in various environments through metagenomics, though their presence, ecology, and biology have never been addressed so far. As a group of strict intracellular, we hypothesize that their presence, occurrence, and abundance heavily depend on their hosts, which are known as heterotrophic protists, based on few described isolates. Here, we conducted a sampling campaign allowing to characterize protists and associated bacterial communities, using high-throughput sequencing. In parallel, a systematic enrichment of protists from samples was performed to attempt characterization and isolation of new Ca . Babelota within native hosts. We found that Ca . Babelota are among the most widespread phylum among the rare ones. Protist enrichments are allowed in certain cases to enrich as well for Ca . Babelota, which could be visualized in vivo infecting protist cells. Though cosmopolitan, Ca . Babelota diversity was highly site-specific. Cooccurrence analyses allowed to retrieve well-known as well as new putative associations involving numerous protists of various trophic regimes. The combination of approaches developed in this study enhances our understanding of Ca . Babelota ecology and biology, while paving the way for future isolation of new members of this elusive phylum, which could have huge impact on protists—and ecosystems—functioning. IMPORTANCE Our understanding of microbial diversity surrounding us and colonizing the environment has been dramatically impacted by the advent of DNA-based analyses. Such progress helped shine a new light on numerous lineages of yet-to-be-characterized microbes, whose ecology and biology are basically unknown. Among those uncharacterized clades is the Candidatus Babelota, a bacterial phylum for which parasitism seems to be an ancestral trait. All known Ca. Babelota thrive by infecting phagotrophic protist hosts, thereby impacting this basal link of the trophic chain. The Ca. Babelota constitutes a model that stands out, as phylum-wide conserved parasitism has only been described in one previous occurrence for Bacteria, with the Chlamydiota. Thus, exploring the intricate interplay between Ca. Babelota and their protist hosts will advance our knowledge of bacterial diversity, their ecology, and global impact on ecosystem functioning.

Abstract Methanol, the simplest alcohol, has long been recognized as a key energy and carbon source for soil and plant-associated bacteria and fungi, and is increasingly recognized as an important substrate for marine bacteria. Lanthanide-dependent methanol dehydrogenases (encoded by the gene xoxF) are now recognized as the key catalysts for methylotrophy in many environments, yet the identity of the most transcriptionally active methylotrophs in open ocean waters (“Clade X”) has remained elusive. Here we show that “Clade X” methylotrophs belong to the deep-branching alphaproteobacterial order TMED127, which we propose be renamed ‘Methylaequorales’; ‘methyl’ for ‘methylotrophic metabolism’ and ‘aequor’ for ‘ocean surface’, as these bacteria are most transcriptionally active near the sea surface. TMED127/Methylaequorales are present in surface waters of tropical and subtropical oceans throughout the global ocean. They have small, streamlined genomes (∼1.5 Mb), and appear to be obligate methylotrophs that use the serine cycle for carbon assimilation. They display a diel pattern of xoxF5 and glucose dehydrogenase (gdh) transcription, peaking in the late afternoon, in oligotrophic surface water of the Sargasso Sea. Several other highly transcribed genes of unknown function had no homologs outside of TMED127/Methylaequorales genomes. Our findings illuminate an overlooked marine methylotrophic bacterium and predict a diel cycle of methanol production in surface seawater by an unknown pathway. Importance Methanol metabolism is increasingly recognized as an important process in the marine carbon cycle, yet the identity and metabolism of the microorganisms mediating methylotrophy in the open ocean have remained unknown. This study reveals that bacteria in the TMED127 order of Alphaproteobacteria, proposed new name of “Methylaequorales”, abundantly transcribe the key gene for lanthanide-dependent methylotrophy in oligotrophic surface waters of the world’s oceans. TMED127/Methylaequorales likely require methanol as a carbon and energy source and display a diel pattern of transcription of key genes for methylotrophy that peaks in the late afternoon. These findings motivate future studies on the mechanisms of methanol production in surface seawater.

AbstractRecent discoveries of aerobic methanotrophs in non-seep carbonate-rich environments in the deep sea suggest that these organisms may persist as part of the rare biosphere. Recovering rare, active methanotrophs through targeted culturing is essential for understanding their persistence under the oligotrophic non-seep conditions, and for uncovering their genomic adaptations related to the survival in energy-limited ecosystems. In our study, using metagenomic analysis of enrichment cultures from the Alpha Crucis Carbonate Ridge, we discoveredMethylotuvimicrobium crucissp. nov., a novel methanotroph representing the rare biosphere in native sediments. Phylogenomic analysis revealed <95% ANI to described species, with genomic evidence of deep-sea specialization including: (1) stress adaptation through cold-shock proteins (CspA) and DNA repair systems (UvrD/LexA), (2) metabolic versatility via complete methane oxidation(pmoABC), nitrogen fixation (nifHDK), and sulfur cycling (sox/sqr) pathways, and (3) niche partitioning through biofilm formation (GGDEF/EAL) and heavy metal resistance (CopZ/CzcD). Comparative genomics identified a 1,234-gene deep-sea core shared withM. sp. wino1, enriched in mobile elements (TnpA, prophages) suggesting horizontal gene transfer drives adaptation. While undetectedin situamplicon surveys,M. crucisexhibited rapid enrichment under methane availability, demonstrating its role as a latent methane filter. These findings contribute for the understanding of the ecological significance of aerobic methanotrophs in deep-sea systems, revealing how rare microbial taxa with genomic plasticity have the potential to influence biogeochemical cycling in deep carbonate-rich environments.

GroE is a chaperonin folding system consisting of GroEL (Cpn60, a 60 kDa chaperonin), and the smaller co-chaperonin GroES (Cpn10). Many “client” proteins require GroE to fold properly, including several that are essential for cell viability. Unsurprisingly then, GroE is found in nearly all bacteria and eukaryotes. Mollicutes are the only microorganisms that lack GroE in almost all cases. Only two clades of Mollicutes have retained the ancestral GroE system, or perhaps reacquired one; these exceptions include the family Acholeplasmataceae (consisting of the genera Acholeplasma and Phytoplasma). The role of GroEL in these “exceptional” Mollicutes is a source of speculation, given how many non-canonical “moonlighting” roles have been ascribed to this protein. GroEL has been suggested to play a role in pathogenesis in plant and animal pathogenic Mollicutes, by binding to host cells and facilitating invasion. However, in one further layer of exception, the phytopathogenic taxon ‘Candidatus Phytoplasma pruni’ (ribosomal group 16SrIII), was reported to lack a GroE system. This study confirms the lack of a functional GroE system in 16SrIII by providing two new, high quality, non-fragmented genome assemblies, as well as a thorough survey of other 16SrIII genomes for genes encoding GroEL/GroES, including those that may not resemble phytoplasma groEL (ie. acquired by horizontal gene transfer, HGT). We discuss the implications of a clearly phytopathogenic, invasive group of Mollicutes that nevertheless lacks GroE, in light of the presumed role of GroEL for these microorganisms. We determined that three groups of genomes of 16SrIII contain short, non-functional groEL pseudogenes, while most of the reported genomes lack any semblance of a GroE system. Examination of the new assemblies allowed us to rule out HGT as a means of GroE acquisition.

Abstract The presence of fastidious phytopathogenic bacteria ‘Candidatus Arsenophonus phytopathogenicus’ and ‘Candidatus Phytoplasma solani’ challenges sugar beet cultivation in Austria. Reptalus quinquecostatus sensu Holzinger et al. 2023 is the principal vector of ‘Ca. P. solani’ to sugar beet in Serbia, while Pentastiridius leporinus is the primary vector of ‘Ca. A. phytopathogenicus’ in western Europe and can also transmit ‘Ca. P. solani’. This study investigates the epidemiology of the associated diseases in Austrian sugar beet fields. An extensive survey in 2024 at 33 locations revealed significant spatial and temporal variability in cixiid populations, with R. quinquecostatus emerging as the predominant species. Transmission trials and field surveys confirmed R. quinquecostatus as a vector of ‘Ca. A. phytopathogenicus’ and ‘Ca. P. solani’ 16SrXII-P driving a disease outbreak in Nikitsch. The predominance of R. quinquecostatus in nearly all monitored fields suggests its significant role in the pathogens' epidemiology on a broader scale in the Pannonian plain. Spatial and temporal variations in pathogen incidence in Austrian sugar beet mirror patterns in Serbia, driven by R. quinquecostatus. The co-occurrence of ‘Ca. A. phytopathogenicus’ and ‘Ca. P. solani’ 16SrXII-A and 16SrXII-P and their transmission by both R. quinquecostatus and P. leporinus complicate disease management efforts.

The genus Lactococcus was proposed by Schleifer et al. by separating Lactococcus lactis from the genus Streptococcus. Although the family Streptococcaceae consists of four genera, each genus contains a relatively small number of species, with the exception of the genus Streptococcus, which contains more than 100 species. The genera Lactococcus and Lactovum currently comprise 26 species and a single species, respectively. This study evaluated the taxonomy of the genus Lactococcus based on the 16S rRNA gene phylogeny, core-genome phylogeny and (conserved) pairwise average amino acid identity. These evaluations clearly indicated that the genus Lactococcus could be divided into two genus-level clusters, and we propose to reclassify this genus into two; the authentic Lactococcus, which includes the L. lactis group, and a novel genus for which the name Pseudolactococcus is proposed. Three lactic acid bacterial strains, RyT2T, OfM1T and OfM2, were isolated from the gut of termites in Okinawa, Japan. Based on the combination of genetic and phenotypic data, we conclude that these isolates represent two novel species of the genera Pseudolactococcus and Lactovum, respectively, for which we propose the names Pseudolactococcus yaeyamensis sp. nov. (RyT2T=JCM 36015T=DSM 118067T) and Lactovum odontotermitis sp. nov. (OfM1T=JCM 34431T=DSM 118066T, OfM2=JCM 34432), respectively.