Publications

3975

Background Candidate Phyla Radiation (CPR) is a large monophyletic group encompassing about 25% of bacterial diversity. Among CPR, “Candidatus Saccharibacteria” is one of the most clinically relevant phyla. Indeed, it is enriched in the oral microbiota of subjects suffering from immune-mediated disorders and it has been found to have immunomodulatory activities. For these reasons, it is crucial to have reliable methods to detect and quantify this bacterial lineage in human samples, including saliva. Methods and results Four qPCR protocols for quantifying “Ca. Saccharibacteria” (one targeting the 23S rRNA gene and three the 16S) were tested and compared. The efficiency and coverage of these four protocols were evaluated in silico on large genomic datasets, and in vitro on salivary DNA samples, already characterized by amplicon sequencing on the V3-V4 regions of the 16S rRNA. In silico PCR analyses showed that all qPCR primers lose part of the “Ca. Saccharibacteria” genetic variability, even if the 23S qPCR primers matched more lineages than the 16S qPCR primers. In vitro qPCR experiments confirmed that all 16S-based protocols strongly underestimated “Ca. Saccharibacteria” in salivary DNA, while the 23S qPCR protocol gave quantifications more comparable to 16S amplicon sequencing. Conclusion Overall, our results show that the 23S-based qPCR protocol is more precise than the 16S-based ones in quantifying “Ca. Saccharibacteria”, although all protocols probably underestimate specific lineages. These results underline the current limits in quantifying “Ca. Saccharibacteria”, highlighting the needs for novel experimental strategies or methods. Indeed, the underestimation of “Ca. Saccharibacteria” in clinical samples could hide its role in human health and in the development of immune-mediated diseases.

Recirculating aquaculture systems (RAS) hold significant potential for sustainable aquaculture by providing a stable, controlled environment that supports optimal fish growth and welfare. In RAS, ammonium (NH4+) is biologically converted into nitrate (NO3−) via nitrite (NO2−) by nitrifying bacteria. As a result, NO3− usually accumulates in RAS and must subsequently be removed through denitrification in full RAS, or by regular water exchanges in partial RAS. The marine anammox bacteria Candidatus Scalindua can directly convert toxic NH4+ and NO2− into harmless nitrogen gas (N2) and has previously been identified as a promising alternative to the complex denitrification process or unsustainable frequent water exchanges in marine RAS. In this study, we evaluated the impact of high NO3− levels typically encountered in RAS on the performance and abundance of Ca. Scalindua in a laboratory-scale bioreactor. The bacterial composition of the granules, including the relative abundance of key nitrogen-cycling taxa, was analyzed along with the functional profile (i.e., NH4+ and NO2− removal efficiencies). For this purpose, a bioreactor was inoculated and fed a synthetic feed, enriched in NH4+, NO2−, minerals and trace elements until stabilization (Phase 1, 52 days). NO3− concentrations were then gradually increased to 400 mg·L−1 NO3−-N (Phase 2, 52 days), after which the reactor was followed for another 262 days (Phase 3). The reactor maintained high removal efficiencies; 88.0 ± 8.6% for NH4+ and 97.4 ± 1.7% for NO2− in Phase 2, and 95.0 ± 6.5% for NH4+ and 98.6 ± 2.7% for NO2− in Phase 3. The relative abundance of Ca. Scalindua decreased from 22.7% to 10.2% by the end of Phase 3. This was likely due to slower growth of Ca. Scalindua compared to heterotrophic bacteria present in the granule, which could use NO3− as a nitrogen source. Fluorescence in situ hybridization confirmed the presence of a stable population of Ca. Scalindua, which maintained high and stable NH4+ and NO2− removal efficiencies. These findings support the potential of Ca. Scalindua as an alternative filtering technology in marine RAS. Future studies should investigate pilot-scale applications under real-world conditions.

AbstractGenomes are fundamental to understanding microbial ecology and evolution. The emergence of high-throughput, long-read DNA sequencing has enabled recovery of microbial genomes from environmental samples at scale. However, expanding the microbial genome catalogue of soils and sediments has been challenging due to the enormous complexity of these environments. Here, we performed deep, long-read Nanopore sequencing of 154 soil and sediment samples collected across Denmark and through an optimised bioinformatics pipeline, we recovered genomes of 15,314 novel microbial species, including 4,757 high-quality genomes. The recovered microbial genomes span 1,086 novel genera and provide the first high-quality reference genomes for 612 previously known genera, expanding the phylogenetic diversity of the prokaryotic tree of life by 8 %. The long-read assemblies also enabled the recovery of thousands of complete rRNA operons, biosynthetic gene clusters and CRISPR-Cas systems, all of which were underrepresented and highly fragmented in previous terrestrial genome catalogues. Furthermore, the incorporation of the recovered MAGs into public genome databases significantly improved species-level classification rates for soil and sediment metagenomic datasets, thereby enhancing terrestrial microbiome characterization. With this study, we demonstrate that long-read sequencing and optimised bioinformatics, allows cost-effective recovery of high-quality microbial genomes from highly complex ecosystems, which remain the largest untapped source of biodiversity for expanding genome databases and filling in the gaps of the tree of life.

ABSTRACT “ Candidatus Liberibacter asiaticus” (CLas) is associated with citrus huanglongbing, a severe disease with global importance that affects citrus production in Brazil. This study reports the first complete genome of a Brazilian strain of CLas. The genomic structure comparison of strain 9PA with those of 13 complete CLas genomes revealed 9,091 mismatches and 992 gaps/insertions, highlighting eight locally colinear blocks, among which six are in the prophage region. Phylogenetic analysis categorized 13 CLas genomes into two clusters with 9PA clustered with strains from China and the United States. Whole-genomic comparison identified diverse hypervariable genomic regions (HGRs). Three HGRs in the chromosomal region and three in the prophage region were selected and investigated by polymerase chain reaction. HGRs assessed from 68 samples, from medium- to high-huanglongbing incidence areas in Sao Paulo state, were grouped into haplotypes A to P. Haplotype A, which includes strain 9PA, is the second most prevalent, representing 19.1% of the samples. Haplotype B, the most common, accounts for 42.6%. Together with haplotype C, these make up 72% of the evaluated samples. The 9PA strain has prophage P-9PA-1, both integrated and circularized, and P-9PA-3, only found in a circularized form. Prophages show high identity with SC1 (83%) and P-JXGC-3 (98%). Co-occurrence of both type 1 and 3 prophages was observed in field samples. The approach employed provides insights into the Brazilian CLas population, providing markers for population studies and highlighting the prevalence of type 1 and 3 prophages in the population. IMPORTANCE CLas is a destructive pathogen responsible for causing the severe citrus disease known as huanglongbing. Our study presents the first fully sequenced Brazilian strain of CLas, designated as 9PA, and includes an analysis of two prophages occurring in this strain. The main objective of our research was to compare the genome features of this Brazilian strain with other fully sequenced genomes and to identify its hypervariable genetic regions. These regions were subsequently used to assess genomic variability within both the chromosomal and prophage regions in Brazilian isolates of CLas. Our findings offer valuable insights into the diversified adaptation of CLas.

ABSTRACTThe Oil Refinery (OR) consortium is a model methanogenic enrichment culture for studying anaerobic benzene degradation. Over 80% of the culture’s bacterial community is comprised of two closely related strains of benzene-fermentingDesulfobacterota(designated ORM2a and ORM2b) whose mechanism of benzene degradation is unknown. Two new metagenomes, including a fully closed metagenome-assembled genome (MAG) for ORM2a, enabled a thorough investigation of this culture’s proteome. Among the proteins identified were Bam-like subunits of an ATP-independent benzoyl-CoA degradation pathway and associated downstream beta-oxidation proteins producing acetyl-CoA. The most abundant proteins identified mapped to two ORM2 gene clusters of unknown function. Syntenic gene clusters were identified in one other known benzene degrader,Pelotomaculumcandidate BPL, as well as a handful of contigs assembled from hydrothermal vent metagenomes. Extensive searches against reference sequence and structural databases indicate that the first (“Magic”) gene cluster likely catalyzes the chemically difficult benzene activation step. The second (“Nanopod”) gene cluster is predicted to code for an efflux system that pumps excess benzene out of cells, mitigating some of its toxigenic effects. Phylogenomic analyses place ORM2a and ORM2b within a novel genus of benzene-degrading specialists which we propose naming “CandidatusBenzenivorax”. We hope to engage the research community to help in confirming the roles of the proteins in the “Magic” and “Nanopod” gene clusters, and to search through their own cultures for these features.

Psyllids are major vectors of plant diseases, including Candidatus Liberibacter solanacearum (CLso), the bacterial agent associated with 'zebra chip' disease in potatoes and 'carrot yellows' disease in carrot. Despite their agricultural significance, there is limited knowledge on the genome structure and genetic diversity of psyllids. In this study, we provide chromosome-level genome assemblies for three psyllid species known to transmit CLso: Dyspersa apicalis (carrot psyllid), Dyspersa pallida, and Trioza urticae (nettle psyllid). As D. apicalis is recognised as the primary vector of CLso by carrot growers in Northern Europe, we also resequenced populations of this species from Finland, Norway, and Austria. Genome assemblies were constructed using PacBio HiFi and Hi-C sequencing data, yielding genome sizes of: 594.01 Mbp for D. apicalis; 587.80 Mbp for D. pallida; and 655.58 Mbp for T. urticae. Over 90% of sequences anchored into 13 pseudo-chromosomes per species. The assemblies for D. apicalis and D. pallida exhibited high completeness, capturing over 92% of conserved Hemiptera single-copy orthologues, as assessed by Benchmarking Universal Single-Copy Orthologues (BUSCO) analysis. Furthermore, we identified sequences of the primary psyllid symbiont, Candidatus Carsonella ruddii, in all three species. Comparative genomic analyses demonstrated synteny with other psyllid species. Notably, we observed significant expansions in gene families, particularly those linked to potential insecticide detoxification, within the Dyspersa lineage. Resequencing efforts also revealed the existence of multiple subpopulations of D. apicalis across Europe. These high-quality genome resources will support future research on genome evolution, insect-plant-pest interactions, and strategies for disease management.

ABSTRACTThe Coxiellaceae bacterial family, within the order Legionellales, is defined by a collection of poorly characterized obligate intracellular bacteria. The zoonotic pathogen and causative agent of human Q fever,Coxiella burnetii, represents the best characterized member of this family. Coxiellaceae establish replicative niches within diverse host cells and rely on their host for survival, making them challenging to isolate and cultivate within a laboratory setting. Here we describe a new genus within the Coxiellaceae family that has been previously shown to infect economically significant freshwater crayfish. Using culture-independent long-read metagenomics, we reconstructed the complete genome of this novel organism and demonstrate that the previously referred to as CandidatusCoxiella cheraxirepresents a novel genus within this family, herein denoted CandidatusParacoxiella cheracis. Interestingly, we demonstrate that CandidatusP. cheracisencodes for a complete, putatively functional Dot/Icm type 4 secretion system that likely mediates the intracellular success of this pathogen. In silico analysis defined a unique repertoire of Dot/Icm effector proteins and highlighted homologues of several importantC. burnetiieffectors including a homologue of CpeB that was demonstrated to be a Dot/Icm substrate inC. burnetii.IMPORTANCEUsing long-read sequencing technology we have uncovered the full genome sequence of CandidatusParacoxiellacheracis, a pathogen of economic importance in aquaculture. Analysis of this sequence has revealed new insight into this novel member of the Coxiellaceae family, demonstrating that it represents a new genus within this poorly characterized family of intracellular organisms. Importantly, the genome sequence reveals invaluable information that will support diagnostics and potentially both preventative and treatment strategies within crayfish breeding facilities. CandidatusP. cheracisalso represents a new member of Dot/Icm pathogens that rely on this system to establish an intracellular niche. CandidatusP. cheracispossesses a unique cohort of putative Dot/Icm substrates that constitute a collection of new eukaryotic cell biology manipulating effector proteins.