Publications

4865

ABSTRACT Biogas, a mix of CO 2 , CH 4 and small proportions of other gases, is a biofuel obtained by anaerobic digestion (AD). Biogas production is often considered a black box process, as the role and dynamics of some of the microorganisms involved remain undisclosed. Previous metataxonomic studies in the frame of the MICRO4BIOGAS project ( www.micro4biogas.eu ) revealed that MBA03, an uncharacterised and uncultured bacterial taxon belonging to phylum Bacillota , was very prevalent and abundant in industrial full‐scale AD plants. Despite the efforts, this taxon has not yet been cultivated, which makes the analysis of its taxonomy, ecology and metabolism even more challenging. In the present work, 30 samples derived from anaerobic digesters were sequenced, allowing the reconstruction of 108 metagenome‐assembled genomes (MAGs) potentially belonging to MBA03. According to phylogenetic analyses and genomic similarity indices, MBA03 was classified as a new bacterial order, proposed as ‘ Candidatus Darwinibacteriales’ ord. nov., which includes ‘ Candidatus Darwinibacter acetoxidans’ gen. nov., sp. nov. of ‘ Candidatus Darwinibacteriaceae’ fam. nov., along with ‘ Candidatus Wallacebacter cryptica’ gen. nov., sp. nov. of the ‘ Candidatus Wallacebacteriaceae’ fam. nov. Ecotaxonomic studies determined that AD processes are the main ecological niche of ‘ Candidatus Darwinibacteriales’. Moreover, metabolic predictions identified Darwinibacteraceae members as putative syntrophic acetate‐oxidising bacteria (SAOB), as they encode for the reversed Wood–Ljungdahl (W–L) pathway coupled to the glycine cleavage system. This suggests that Darwinibacteraceae members could work in collaboration with hydrogenotrophic methanogenic archaea to produce methane in industrial biogas plants. Overall, our findings present ‘ Candidatus Darwinibacteriales’ as a potential key player in anaerobic digestion and pave the way towards the complete characterisation of this newly described bacterial taxon, which has not yet been cultured.

ABSTRACT Candidatus Saccharimonadia is a class‐level lineage of ultrasmall bacteria within the phylum Minisyncoccota (formerly Candidate Phyla Radiation or Ca . Patescibacteria), commonly found in activated sludge processes treating municipal wastewater. In this study, we aimed to elucidate the metabolic potential of Ca . Saccharimonadia by using shotgun metagenomic sequencing combined with a filtration‐based size‐fractionation approach for activated sludge from five wastewater treatment plants. A total of 65 high‐quality metagenomic bins were recovered, belonging to four orders and 19 families of Ca . Saccharimonadia, including previously unreported lineages in activated sludge. These bins had small genomes (approximately 0.46–1.73 Mbp) with limited metabolic capabilities, indicating dependency on other microorganisms. Notably, the order Ca . Saccharimonadales retained a type IV secretion system and effector gene cluster for parasitic interactions with the hosts, suggesting that Ca . Saccharimonadales bacteria may exhibit a parasitic lifestyle. Co‐occurrence network analysis showed that members of the order Ca . Saccharimonadales were significantly correlated with multiple lineages, including Actinobacteriota , for which a parasitic relationship has been previously demonstrated. Our results shed light on the potential ecophysiology of the diverse members of Ca . Saccharimonadia, providing a comprehensive understanding of Ca . Saccharimonadia in activated sludge.

Abstract Pistachio ( Pistacia vera L.) is a vital crop for Türkiye's economy, occupying over 420 000 ha, primarily in south‐eastern Anatolia. In 2023, severe symptoms, such as curling, chlorosis and needle‐like leaf deformation, were observed on pistachio trees in Şanlıurfa Province. This prompted a molecular study of phytoplasma pathogens. A real‐time PCR targeting the 16SrDNA gene was used to analyse DNA from eight symptomatic and two asymptomatic trees. A phytoplasma was found in two symptomatic samples but not in asymptomatic controls. Positive samples were further analysed using nested PCR. One sequence with 99% identity to ‘ Candidatus Phytoplasma trifolii’ (16SrVI‐A subgroup) was found by bidirectional sequencing of PCR products. The identification of the phytoplasma was confirmed by a phylogenetic analysis showing the clustering of the UP 1 isolate with ‘ Ca. Phytoplasma trifolii’ reference strain and by a virtual RFLP analysis using 17 restrictions enzymes. This first report of ‘ Ca. Phytoplasma trifolii’ in pistachio in Türkiye highlights the presence of this phytoplasma in a key agricultural sector. Although no yield losses have been observed to date, broader surveys and monitoring of leafhopper vectors are necessary to assess the potential risk and implement timely management strategies.

ABSTRACT Six strains of a novel anaerobic bacterial taxa exhibiting bile acid-transforming activity were isolated from fecal samples of clinically healthy cats living in Oakland, California. 16S rRNA sequencing and phylogenetic analysis indicate that strain AB800 T belongs to the genus Peptacetobacter , with Peptacetobacter hiranonis strain JCM 10541 (98% coverage, 99.7% identity) as its closest relative. Whole genome sequencing shows an average nucleotide identity (ANI) of less than 96% (95.73% ANI, 90.53% AF) with the closest validly named species being Peptacetobacter hiranonis DGF055142 . The size is about 2.58 Mb, containing 2,355 predicted coding sequences and 2,450 annotated genes. Phenotypic characterisation, and comprehensive genomic analysis, including ANI, digital DNA-DNA hybridization (dDDH) and core genome phylogenetic analysis, placing the strain AB800 T on a separate branch, supported the classification of a novel species, strain AB800 T (DSM 120482 = LMG 34035) with the proposed name ‘ Candidatus Peptacetobacter felis’ sp. nov., derived from “ felis” the latin name of cat felis catus . These findings expand our understanding of host-associated bile acid converters and provide promising candidates for probiotic development.

Abstract Background Globally, the disease ecology of reptiles remains understudied, even for threatened and iconic species such as the Galápagos marine iguana ( Amblyrhynchus cristatus ). Although marine iguanas are parasitized by distinct species of ticks and mites, research on vector-borne diseases for this species is limited. Methods In this study, we detected 16S ribosomal RNA (rRNA) sequences of Candidatus Allocryptoplasma in transcriptomic data from marine iguana blood samples. These 16S rRNA sequences were further characterized through phylogenetic analysis and a haplotype network. Results Our analysis revealed the first molecular evidence for the infection of marine iguanas with Candidatus Allocryptoplasma, a candidate genus in the family Anaplasmataceae with unknown pathogenic potential, likely transmitted by ticks. Phylogenetic analysis of the novel 16S rRNA sequences together with available Anaplasmataceae sequences confirmed their assignment to this candidate genus. A haplotype network analysis indicated that the agent infecting the marine iguana represents a distinct lineage within the known Ca . Allocryptoplasma diversity. Conclusions Candidatus Allocryptoplasma had a high prevalence within marine iguanas, infecting individuals across most of the geographical range of this species. To elucidate the transmission dynamics of this bacterium in the Galápagos ecosystem, ectoparasites of the marine iguana and shared vertebrate hosts should be screened for infection with Ca . Allocryptoplasma. Graphical Abstract

We report the presence of the emerging plant pathogen ‘ Xanthomonas cannabis ’ in Australia through a comprehensive analysis of five historical isolates and all publicly available genomes of the species. Using comparative genomics, we characterized four isolates collected from Zinnia spp. and one from Cucurbita pepo . Our findings show that the Zinnia isolates form a distinct phylogroup with the pathotype strain of ‘ X. cannabis ’ pv. zinniae . This group possesses genes for the type 3 secretion system (T3SS) and effectors, a variety of genes unique within the species, and nine genomic islands associated with virulence and drug resistance. In contrast, the C. pepo isolate is genetically distinct and lacks the T3SS but contains its own genes unique within the species. Hypersensitivity response assays confirmed the pathogenic potential of all five isolates in black bean, eggplant, green bean, tomato, sunflower, zinnia and zucchini plants. These results highlight the genetic diversity and evolving threat of this pathogen in Australia, underscoring the critical need for ongoing biosecurity surveillance.

Abstract The DPANN superphylum is a deep-branching radiation of Archaea with small cell and genome sizes. Most DPANN lineages are predicted or validated to be host-dependent. However, certain lineages have substantial biosynthetic capacities and are potentially less dependent on hosts or even free-living. Here, we reconstructed 163 Micrarchaeota genomes, comprising 48 assigned to previously undescribed orders and 115 affiliated with known orders. Investigation of their genetic repertoire revealed substantial metabolic capacity in Norongarragalinales, Anstonellales, and the newly proposed Wunengiarchaeales-associated lineages, including complete or near-complete glycolysis and de novo biosynthetic pathways for nucleotides, amino acids, co-factors, and cell envelopes. We classified these genes related to central metabolism, but which are uncommon in DPANN archaea as putative free-living associated genes (pFLAGs). The extensive presence of pFLAGs in Norongarragalinales suggests a potential host-independent lifestyle. Reconstruction of evolutionary history revealed that these pFLAGs were not ancestral within the DPANN superphylum. Instead, we suggest that less host-dependent organisms evolved from symbionts through the gradual acquisition of pFLAGs through horizontal gene transfer, whereas other Micrarchaeota lineages with streamlined genomes experienced reductive evolution due to thermal adaptation. Our analyses demonstrate that host dependency is not always an evolutionary dead end, but can be reversed through acquisition of new metabolic capabilities by horizontal transfer.

Phloem feeders, such as the psyllid Diaphorina citri ( D. citri ), feed on plants by inserting their stylet bundle followed by probing the apoplast before reaching the phloem. The psyllids secrete watery saliva containing various proteins into the phloem, which can act as effectors to facilitate their feeding or modulate host defense responses. Concomitantly, feeding is the main mode of transmitting the Candidatus Liberibacter asiaticus ( C Las) bacteria to the phloem. C Las produces several effectors that have been hypothesized to contribute to Huanglongbing (HLB) virulence. Here, we aimed to identify putative effector proteins from both D. citri and C Las. To achieve this, we used different omics techniques on different tissues and organs from both plants and insects. More specifically, we performed transcriptomics on the heads of healthy and C Las-infected D. citri and proteomics of artificial diet and of phloem of four different plant species fed on by healthy and C Las-infected D. citri. Subsequently, we used various criteria and bioinformatics tools to predict putative effectors. This resulted in the identification of four proteins from D. citri [ferritin, prisilkin, CG31997-PA, and pterin-4-alpha-carbinolamine dehydratase-like protein (PCBD)] and two Sec-dependent effectors from C Las, CLIBASIA_04560 and CLIBASIA_05320, that were used for further functional studies. The expression of these six proteins in Nicotiana benthamiana modified the ROS burst triggered by flagellin, indicating that they can indeed function as effector proteins in planta . In addition, expression of the psyllid effectors in planta significantly reduced the growth of Pseudomonas syringae pv. tabaci ( Pta ).