Publications

3558

Phosphatidylcholine (PtdCho) is an unusual membrane phospholipid present in some endosymbiotic and intracellular pathogenic prokaryotes. ‘ Candidatus Liberibacter asiaticus’ (CLas) is a phloem-limited, uncultured, fastidious α-Proteobacterium associated with the devastating citrus “greening” disease (huanglongbing). Phylogenetically related but nonpathogenic Liberibacter crescens (Lcr) was used as a culturable surrogate to examine PtdCho biosynthesis in pathogenic CLas. Genes encoding key enzymes for two alternative PtdCho biosynthetic routes are present in the Lcr genome: the one-step cytidine diphosphate (CDP)-choline ( pcs-encoding phosphatidylcholine synthase) and the three-step methyl-transferase pathway ( pmt-encoding phospholipid N-methyltransferase). However, only the CDP-choline pathway genes for incorporating exogenous Cho were identified in the CLas genome. Exogenous Cho enhanced growth and alleviated osmotic stress in wild-type Lcr and in the pmt insertion mutant strains when cultured in a sugar-rich medium. Quantitative RT-PCR analyses confirmed active uptake and condensation of nutritional Cho into PtdCho by CLas in both its plant host and psyllid vector. CLas-infected grapefruit leaves showed transcriptional activation of Cho biosynthesis genes and 2.8-fold higher levels of Cho. In plant cells, the compatible osmolyte glycine-betaine (GlyBet) is also derived from Cho. Expression of GlyBet biosynthesis genes and the GlyBet content were similar in both CLas-infected and healthy leaf tissue. The data presented here suggest that CLas likely exploits the Cho biosynthetic pathway in citrus hosts to expand the nutritional Cho pool. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Two phloem-limited pathogens, ‘Candidatus Arsenophonus phytopathogenicus’ and ‘Candidatus Phytoplasma solani’, threaten sugar beet production in France, Switzerland, and Germany. Previous studies of these pathogens in Germany had focused on its western and southern regions, leaving a knowledge gap about eastern Germany. Despite their importance, this study is the first to investigate phytoplasmas in sugar beet in Saxony-Anhalt, Germany. A phytoplasma strain related to ‘Ca. P. solani’ is found predominant in Saxony-Anhalt, unlike in France, where ‘Ca. P. solani’ has a minor role compared with ‘Ca. A. phytopathogenicus’. The phytoplasma strain infecting sugar beet in Saxony-Anhalt was classified into a new subgroup designated as 16SrXII-P. The multilocus sequence analysis (MLSA) of nonribosomal genes of the novel phytoplasma strain showed that it is significantly different from the reference and all previously reported ‘Ca. P. solani’ strains including the strain from western Germany. Analyses of sugar beet samples from previous years confirmed the presence of the 16SrXII-P strain in sugar beet as early as 2020 and also in Bavaria in southern Germany. Based on 16S rDNA analysis, ‘Ca. A. phytopathogenicus’ in Saxony-Anhalt is identical to strains in sugar beet in other parts of Germany and France, as well as to a strain in potato from Germany. The presence and prevalence of two phytoplasmas in sugar beet in Germany suggest that more attention should be directed toward understanding phytoplasma infection in sugar beet in this country.

AbstractThe bacterium Candidatus Liberibacter asiaticus (CLas) causes citrus Huanglongbing disease. Our understanding of the pathogenicity and biology of this microorganism remains limited because CLas has not yet been cultivated in artificial media. Its genome is relatively small and encodes approximately 1136 proteins, of which 415 have unknown functions. Here, we use a high-throughput yeast-two-hybrid (Y2H) screen to identify interactions between CLas proteins, thus providing insights into their potential functions. We identify 4245 interactions between 542 proteins, after screening 916 bait and 936 prey proteins. The false positive rate of the Y2H assay is estimated to be 2.9%. Pull-down assays for nine protein-protein interactions (PPIs) likely involved in flagellar function support the robustness of the Y2H results. The average number of PPIs per node in the CLas interactome is 15.6, which is higher than the numbers previously reported for interactomes of free-living bacteria, suggesting that CLas genome reduction has been accompanied by increased protein multi-functionality. We propose potential functions for 171 uncharacterized proteins, based on the PPI results, guilt-by-association analyses, and comparison with data from other bacterial species. We identify 40 hub-node proteins, including quinone oxidoreductase and LysR, which are known to protect other bacteria against oxidative stress and might be important for CLas survival in the phloem. We expect our PPI database to facilitate research on CLas biology and pathogenicity mechanisms.

Abstract Background The RCA (Roseobacter clade affiliated) cluster belongs to the family Roseobacteracea and represents a major Roseobacter lineage in temperate to polar oceans. Despite its prevalence and abundance, only a few genomes and one described species, Planktomarina temperata, exist. To gain more insights into our limited understanding of this cluster and its taxonomic and functional diversity and biogeography, we screened metagenomic datasets from the global oceans and reconstructed metagenome-assembled genomes (MAG) affiliated to this cluster. Results The total of 82 MAGs, plus five genomes of isolates, reveal an unexpected diversity and novel insights into the genomic features, the functional diversity, and greatly refined biogeographic patterns of the RCA cluster. This cluster is subdivided into three genera: Planktomarina, Pseudoplanktomarina, and the most deeply branching Candidatus Paraplanktomarina. Six of the eight Planktomarina species have larger genome sizes (2.44–3.12 Mbp) and higher G + C contents (46.36–53.70%) than the four Pseudoplanktomarina species (2.26–2.72 Mbp, 42.22–43.72 G + C%). Cand. Paraplanktomarina is represented only by one species with a genome size of 2.40 Mbp and a G + C content of 45.85%. Three novel species of the genera Planktomarina and Pseudoplanktomarina are validly described according to the SeqCode nomenclature for prokaryotic genomes. Aerobic anoxygenic photosynthesis (AAP) is encoded in three Planktomarina species. Unexpectedly, proteorhodopsin (PR) is encoded in the other Planktomarina and all Pseudoplanktomarina species, suggesting that this light-driven proton pump is the most important mode of acquiring complementary energy of the RCA cluster. The Pseudoplanktomarina species exhibit differences in functional traits compared to Planktomarina species and adaptations to more resource-limited conditions. An assessment of the global biogeography of the different species greatly expands the range of occurrence and shows that the different species exhibit distinct biogeographic patterns. They partially reflect the genomic features of the species. Conclusions Our detailed MAG-based analyses shed new light on the diversification, environmental adaptation, and global biogeography of a major lineage of pelagic bacteria. The taxonomic delineation and validation by the SeqCode nomenclature of prominent genera and species of the RCA cluster may be a promising way for a refined taxonomic identification of major prokaryotic lineages and sublineages in marine and other prokaryotic communities assessed by metagenomics approaches.

Huanglongbing (HLB) is a devastating citrus disease caused by Candidatus Liberibacter asiaticus (CLas). Since its initial outbreak in Guangdong Province, China, it has spread to 10 provinces and caused significant economic losses. Hence, assessing CLas genetic diversity and demographic history is crucial for HLB epidemic prevention and control. In this study, we collected 500 leaf samples of CLas-infected plants from 10 provinces. We performed multi-loci sequence analysis on four gene fragments (omp, DnaA, GroEL, and SDE1) to explore the genetic differentiation and diversity of CLas in China. Our results indicated low nucleotide diversity (0.00005 ± 0.00001) in CLas, with the absence of significant systematic geographic structure in its distribution. Molecular variance analysis revealed predominant (81.7%) genetic variations within the population, with a minor variation (18.3%) occurring between populations as well as Yunnan provinces. In the Fujian population, significant gene exchange occurred with the other nine populations. Significant negative values in Tajima’s D and Fu’s FS neutrality tests indicated historical population expansions. The nucleotide mismatch distribution curve exhibits a single peak pattern, further supporting the expansion events. Our findings hold potential for advancing epidemiological research and providing suggestions for effective strategies to mitigate the spread of CLas and control HLB.

AbstractThe roles of Asgard archaea in eukaryogenesis and marine biogeochemical cycles are well studied, yet their contributions in soil ecosystems are unknown. Of particular interest are Asgard archaeal contributions to methane cycling in wetland soils. To investigate this, we reconstructed two complete genomes for soil-associated Atabeyarchaeia, a new Asgard lineage, and the first complete genome of Freyarchaeia, and defined their metabolismin situ. Metatranscriptomics highlights high expression of [NiFe]-hydrogenases, pyruvate oxidation and carbon fixation via the Wood-Ljungdahl pathway genes. Also highly expressed are genes encoding enzymes for amino acid metabolism, anaerobic aldehyde oxidation, hydrogen peroxide detoxification and glycerol and carbohydrate breakdown to acetate and formate. Overall, soil-associated Asgard archaea are predicted to be non-methanogenic acetogens, likely impacting reservoirs of substrates for methane production in terrestrial ecosystems.One-Sentence SummaryComplete genomes of Asgard archaea, coupled with metatranscriptomic data, indicate roles in production and consumption of carbon compounds that are known to serve as substrates for methane production in wetlands.

ABSTRACTMembers of the “CandidatusAccumulibacter” genus are widely studied as key polyphosphate-accumulating organisms (PAOs) in biological nutrient removal (BNR) facilities performing enhanced biological phosphorus removal (EBPR). This diverse lineage includes 18 “Ca. Accumulibacter” species, which have been proposed based on the phylogenetic divergence of the polyphosphate kinase 1 (ppk1) gene and genome-scale comparisons of metagenome-assembled genomes (MAGs). Phylogenetic classification based on the 16S rRNA genetic marker has been difficult to attain because most “Ca. Accumulibacter” MAGs are incomplete and often do not include the rRNA operon. Here, we investigate the “Ca. Accumulibacter” diversity in pilot-scale treatment trains performing BNR under low dissolved oxygen (DO) conditions using genome-resolved metagenomics. Using long-read sequencing, we recovered medium and high-quality MAGs for 5 of the 18 “Ca. Accumulibacter” species, all with rRNA operons assembled, which allowed a reassessment of the 16S rRNA-based phylogeny of this genus and an analysis of phylogeny based on the 23S rRNA gene. In addition, we recovered a cluster of MAGs that based on 16S rRNA, 23S rRNA,ppk1, and genome-scale phylogenetic analyses do not belong to any of the currently recognized “Ca. Accumulibacter” species for which we propose the new species designation “Ca. Accumulibacter jenkinsii” sp. nov. Relative abundance evaluations of the genus across all pilot plant operations revealed that regardless of the operational mode, “Ca. A. necessarius” and “Ca. A. propinquus” accounted for more than 40% of the “Ca. Accumulibacter” community, whereas the newly proposed “Ca. A. jenkinsii” accounted for about 5% of the “Ca. Accumulibacter” community.IMPORTANCEOne of the main drivers of energy use and operational costs in activated sludge processes is the amount of oxygen provided to enable biological phosphorus and nitrogen removal. Wastewater treatment facilities are increasingly considering reduced aeration to decrease energy consumption, and whereas successful BNR has been demonstrated in systems with minimal aeration, an adequate understanding of the microbial communities that facilitate nutrient removal under these conditions is still lacking. In this study, we used genome-resolved metagenomics to evaluate the diversity of the “CandidatusAccumulibacter” genus in pilot-scale plants operating with minimal aeration. We identified the “Ca.Accumulibacter” species enriched under these conditions, including one novel species for which we propose “Ca.Accumulibacter jenkinsii” sp. nov. as its designation. Furthermore, the MAGs obtained for 5 additional “Ca.Accumulibacter” species further refine the phylogeny of the “Ca.Accumulibacter” genus and provide new insight into its diversity within unconventional biological nutrient removal systems.

AbstractCandidatusAltiarchaea are widespread across aquatic subsurface ecosystems and possess a highly conserved core genome, yet adaptations of this core genome to different biotic and abiotic factors based on gene expression remain unknown. Here, we investigated the metatranscriptome of twoCa. Altiarchaeum populations that thrive in two substantially different subsurface ecosystems. In Crystal Geyser, a high-CO2groundwater system in the USA,Ca. Altiarchaeum crystalense co-occurs with the symbiontCa. Huberiarchaeum crystalense, while in the Muehlbacher sulfidic spring in Germany, an artesian spring high in sulfide concentration,Ca. A. hamiconexum is heavily infected with viruses. We here mapped metatranscriptome reads against their genomes to analyze thein situexpression profile of their core genomes. Out of 537 shared gene clusters, 331 were functionally annotated and 130 differed significantly in expression between the two sites. Main differences were related to genes involved in cell defense like CRISPR-Cas, virus defense, replication, and transcription as well as energy and carbon metabolism. Our results demonstrate that altiarchaeal populations in the subsurface are likely adapted to their environment while influenced by other biological entities that tamper with their core metabolism. We consequently posit that viruses and symbiotic interactions can be major energy sinks for organisms in the deep biosphere.(Originality-Significance StatementOrganisms of the uncultivated phylumCa. Altiarchaeota are globally widespread and fulfill essential roles in carbon cycling,e.g., carbon fixation in the continental subsurface. Here, we show that the transcriptional activity of organisms in the continental subsurface differ significantly depending on the geological and microbial setting of the ecosystem explaining many of the previously observed physiological traits of this organism group.)