Publications

3843

Abstract Background Lake Baikal, the world’s deepest freshwater lake, contains important numbers of Candidatus Patescibacteria (formerly CPR) in its deepest reaches. However, previously obtained CPR metagenome-assembled genomes recruited very poorly indicating the potential of other groups being present. Here, we have applied for the first time a long-read (PacBio CCS) metagenomic approach to analyze in depth the Ca. Patescibacteria living in the bathypelagic water column of Lake Baikal at 1600 m. Results The retrieval of nearly complete 16S rRNA genes before assembly has allowed us to detect the presence of a novel and a likely endemic group of Ca. Patescibacteria inhabiting bathypelagic Lake Baikal. This novel group seems to possess extremely high intra-clade diversity, precluding complete genomes' assembly. However, read binning and scaffolding indicate that these microbes are similar to other Ca. Patescibacteria (i.e. parasites or symbionts), although they seem to carry more anabolic pathways, likely reflecting the extremely oligotrophic habitat they inhabit. The novel bins have not been found anywhere, but one of the groups appears in small amounts in an oligotrophic and deep alpine Lake Thun. We propose this novel group be named Baikalibacteria. Conclusion The recovery of 16S rRNA genes via long-read metagenomics plus the use of long-read binning to uncover highly diverse “hidden” groups of prokaryotes are key strategies to move forward in ecogenomic microbiology. The novel group possesses enormous intraclade diversity akin to what happens with Ca. Patescibacteria at the interclade level, which is remarkable in an environment that has changed little in the last 25 million years.

Terrestrial hydrothermal springs and aquifers are excellent sites to study microbial biogeography because of their high physicochemical heterogeneity across relatively limited geographic regions. In this study, we performed 16S rRNA gene sequencing and metagenomic analyses of the microbial diversity of 11 different geothermal aquifers and springs across the tectonically active Biga Peninsula (Turkey). Across geothermal settings ranging in temperature from 43 to 79°C, one of the most highly represented groups in both 16S rRNA gene and metagenomic datasets was affiliated with the uncultivated phylum “Candidatus Bipolaricaulota” (former “Ca. Acetothermia” and OP1 division). The highest relative abundance of “Ca. Bipolaricaulota” was observed in a 68°C geothermal brine sediment, where it dominated the microbial community, representing 91% of all detectable 16S rRNA genes. Correlation analysis of “Ca. Bipolaricaulota” operational taxonomic units (OTUs) with physicochemical parameters indicated that salinity was the strongest environmental factor measured associated with the distribution of this novel group in geothermal fluids. Correspondingly, analysis of 23 metagenome-assembled genomes (MAGs) revealed two distinct groups of “Ca. Bipolaricaulota” MAGs based on the differences in carbon metabolism: one group encoding the bacterial Wood-Ljungdahl pathway (WLP) for H2 dependent CO2 fixation is selected for at lower salinities, and a second heterotrophic clade that lacks the WLP that was selected for under hypersaline conditions in the geothermal brine sediment. In conclusion, our results highlight that the biogeography of “Ca. Bipolaricaulota” taxa is strongly correlated with salinity in hydrothermal ecosystems, which coincides with key differences in carbon acquisition strategies. The exceptionally high relative abundance of apparently heterotrophic representatives of this novel candidate Phylum in geothermal brine sediment observed here may help to guide future enrichment experiments to obtain representatives in pure culture.

Long non-coding RNAs (lncRNAs) serve as crucial regulators in plant response to various diseases, while none have been systematically identified and characterized in response to citrus Huanglongbing (HLB) caused by Candidatus Liberibacter asiaticus (CLas) bacteria. Here, we comprehensively investigated the transcriptional and regulatory dynamics of the lncRNAs in response to CLas. Samples were collected from leaf midribs of CLas- and mock-inoculated HLB-tolerant rough lemon (Citrus jambhiri) and HLB-sensitive sweet orange (C. sinensis) at week 0, 7, 17, and 34 following inoculation using CLas+ budwood of three biological replicates in the greenhouse. A total of 8,742 lncRNAs, including 2,529 novel lncRNAs, were identified from RNA-seq data with rRNA-removed from strand-specific libraries. Genomic variation analyses of conserved lncRNAs from 38 citrus accessions showed that 26 single nucleotide polymorphisms (SNPs) were significantly correlated with HLB. In addition, lncRNA-mRNA weighted gene co-expression network analysis (WGCNA) showed a significant module correlated with CLas-inoculation in rough lemon. Notably, the most significant LNC_28805 and multiple co-expressed genes related to plant defense in the module were targeted by miRNA5021, suggesting that LNC28805 might compete with endogenous miR5021 to maintain the homeostasis of immune gene expression levels. Candidate WRKY33 and SYP121 genes targeted by miRNA5021 were identified as two key hub genes interacting with bacteria pathogen response genes based on the prediction of protein-protein interaction (PPI) network. These two genes were also found within HLB-associated QTL in linkage group 6. Overall, our findings provide a reference for a better understanding of the role of lncRNAs involved in citrus HLB regulation.

‘Candidatus Liberibacter solanacearum’ (Lso) is a phloem-limited bacterial plant pathogen infecting solanaceous plants in the Americas and New Zealand and is associated with diseases of apiaceous crops in Europe, Northern Africa, and the Middle East. This pathogen is also related to other Liberibacter species that infect other crops. In the USA, two haplotypes of Lso, LsoA and LsoB, are predominant and responsible for diseases in potato and tomato. Tobacco, Nicotiana tabacum, a model species to study plant defenses, is a host for Lso; therefore, the interaction between Lso and this host plant could be used to study Liberibacter−plant interactions. In this study, we characterized the infection associated with LsoA and LsoB in tobacco. Under laboratory conditions, LsoB caused more severe symptoms than LsoA, and LsoA and LsoB titers were dynamic during the 7 weeks of the experiment. We also measured SA and other metabolites, including oxylipins, at an early point of infection and found that SA was accumulated in plants infected with LsoB but not with LsoA; whereas ABA levels were reduced in LsoA- but not in LsoB-infected plants.

Intestinal microbiota has been involved in several processes that benefit the host, such as digestion, nutrient metabolism, resistance to pathogens colonization and immune function. In this study, we investigated the diversity, composition and functional prediction of microbiota of reared Seriolella violacea (palm ruff) in the same cohort sampled at different times (7-, 8- and 9-month-old). Microbial community structure analyses, using 16S rRNA amplicon sequencing, revealed that the intestinal microbiota was dominated by the phyla Firmicutes, Proteobacteria, Fusobacteria and Tenericutes. At the genus level, Candidatus Arthromitus was the most abundant in all sampled timepoints, representing in average 78% of the bacterial community (ranging from 18 to 98%), corresponding to segmented filamentous bacteria, which are interesting because they have been associated with the maturation of immune responses in the gut and protecting the host from bacterial infections. The comparisons of the intestinal microbiota among the three groups showed differences in abundance of bacterial taxa and also in alpha diversity indexes (Shannon and Simpson), as well as beta diversity metrics (weighted and unweighted UniFrac). Potential functions of the intestinal microbiota of palm ruff were retrieved using Philipin and Tax4fun and these analyses revealed high levels of genes for sugar metabolism. To our knowledge, this study represents the first description of the intestinal microbiota of S. violacea.

Abstract Huanglongbing (HLB), or citrus greening, is the most destructive disease of cultivated citrus worldwide. Candidatus Liberibacter asiaticus (CLas), the putative causal agent of HLB, is transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). In Florida, D. citri was first reported in 1998, and CLas was confirmed in 2005. Management of HLB relies on the use of insecticides to reduce vector populations. In 2016, antibiotics were approved to manage CLas infection in citrus. Diaphorina citri is host to several bacterial endosymbionts and reducing endosymbiont abundance is known to cause a corresponding reduction in host fitness. We hypothesized that applications of oxytetracycline and streptomycin would reduce: CLas populations in young and mature citrus trees, CLas acquisition by D. citri, and D. citri abundance. Our results indicate that treatment of citrus with oxytetracycline and streptomycin reduced acquisition of CLas by D. citri adults and emerging F1 nymphs as compared with that observed in trees treated only with insecticides, but not with antibiotics. However, under field conditions, neither antibiotic treatment frequency tested affected CLas infection of young or mature trees as compared with insecticide treatment alone (negative control); whereas trees enveloped with mesh screening that excluded vectors did prevent bacterial infection (positive control). Populations of D. citri were not consistently affected by antibiotic treatment under field conditions, as compared with an insecticide only comparison. Collectively, our results suggest that while foliar application of oxytetracycline and streptomycin to citrus reduces acquisition of CLas bacteria by the vector, even high frequency applications of these formulations under field conditions do not prevent or reduce tree infection.

A novel sulphur-reducing bacterium was isolated from a pyrite-forming enrichment culture inoculated with sewage sludge from a wastewater treatment plant. Based on phylogenetic data, strain J.5.4.2-T.3.5.2T could be affiliated with the phylum Synergistota . Among type strains of species with validly published names, the highest 16S rRNA gene sequence identity value was found with Aminiphilus circumscriptus ILE-2T (89.2 %). Cells of the new isolate were Gram-negative, non-spore-forming, straight to slightly curved rods with tapered ends. Motility was conferred by lateral flagella. True branching of cells was frequently observed. The strain had a strictly anaerobic, asaccharolytic, fermentative metabolism with peptides and amino acids as preferred substrates. Sulphur was required as an external electron acceptor during fermentative growth and was reduced to sulphide, whereas it was dispensable during syntrophic growth with a Methanospirillum species. Major fermentation products were acetate and propionate. The cellular fatty acid composition was dominated by unsaturated and branched fatty acids, especially iso-C15 : 0. Its major polar lipids were phosphatidylglycerol, phosphatidylethanolamine and distinct unidentified polar lipids. Respiratory lipoquinones were not detected. Based on the obtained data we propose the novel species and genus Aminithiophilus ramosus, represented by the type strain J.5.4.2-T.3.5.2T (=DSM 107166T=NBRC 114655T) and the novel family Aminithiophilaceae fam. nov. to accommodate the genus Aminithiophilus. In addition, we suggest reclassifying certain members of the Synergistaceae into new families to comply with current standards for the classification of higher taxa. Based on phylogenomic data, the novel families Acetomicrobiaceae fam. nov., Aminiphilaceae fam. nov., Aminobacteriaceae fam. nov., Dethiosulfovibrionaceae fam. nov. and Thermovirgaceae fam. nov. are proposed.

Endosymbionts play crucial roles in various physiological activities within insect hosts. The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is an important vector for Candidatus Liberibacter asiaticus (CLas), a fatal pathogenic bacterial agent causing the disease Huanglongbing in the citrus industry. This study combines high-throughput sequencing of 16S ribosomal RNA amplicons to explore how CLas affects the bacterial community in different color morphs (blue, gray), genders, and tissues (cuticle, gut, mycetome, Malpighian tubule, ovary, and testis) of ACP. We found that there was no significant differences in the bacterial community diversity and CLas acquired ratio between the different color morphs and genders of ACP adults. However, acquiring CLas could promote the adult bacterial community’s diversity and richness more than in the uninfected condition. The presence of CLas could increase the Wolbachia and unclassified_Enterobacteriaceae proportions more than in the uninfected condition. The bacterial community diversity in the CLas infected tissues of ovary and cuticle, was lower than the uninfected condition, but the richness of all tissues was not different between the infected and uninfected conditions. CLas could also change the bacterial structure in different tissues and make the bacterial relationship network simpler than it is in an uninfected condition. Furthermore, we used quantitative real-time PCR to assess the dynamic changes of Wolbachia in CLas uninfected and infected color morphs and tissues of ACP. The results showed that Wolbachia titers were significantly higher in CLas infected adults than in uninfected adults. In different tissues, the Wolbachia titers in the testis, ovary, and Malpighian tubule were higher than their uninfected counterparts. Our results provide essential knowledge for understanding the symbionts of the ACP and how CLas affects the bacterial community of the ACP.

AbstractBenthic microbial mats dominated by Cyanobacteria are important features of polar lakes. Although culture-independent studies have provided important insights into their diversity, only a handful of genomes of polar Cyanobacteria have been sequenced to date. Here, we applied a genome-resolved metagenomics approach to data obtained from Arctic, sub-Antarctic, and Antarctic microbial mats. We recovered 22 unique metagenome-assembled genomes (MAGs) of Cyanobacteria, most of which are only distantly related to genomes that have been sequenced so far. These include i) lineages that are common in polar microbial mats such as the filamentous taxaPseudanabaena, Leptolyngbya, Microcoleus/Tychonema, andPhormidium; ii) the less common taxaCrinaliumandChamaesiphon; iii) an enigmatic Chroococcales lineage only distantly related toMicrocystis; and iv) an early branching lineage in the order Gloeobacterales that is almost exclusively restricted to the cold biosphere, for which we propose the nameCandidatusSivonenia alaskensis. Our results show that genome-resolved metagenomics is a powerful tool for expanding our understanding of the diversity of Cyanobacteria, especially in understudied remote and extreme environments.Data summaryThe sequencing data generated in this study have been submitted to the European Nucleotide Archive (ENA) under the BioProject PRJEB59431. Individual accession numbers for raw reads and genomic bins are listed inTable S1andTable S3, respectively. Genomic bins can also be downloaded from doi.org/10.6084/m9.figshare.22003967. The commands used throughout this study are available ingithub.com/igorspp/polar-cyanobacteria-MAGs.Impact statementCyanobacteria are photosynthetic microorganisms that play important roles in polar lacustrine ecosystems. Many Cyanobacteria are difficult to grow in the laboratory, particularly in isolation from other organisms, which makes it challenging to sequence their genomes. As such, considerably fewer genomes of Cyanobacteria have been sequenced so far compared to other bacteria. In this study, we used a metagenomics approach to recover novel genomes of Cyanobacteria from Arctic and Antarctic microbial mats without the need to isolate the organisms. The community DNA was extracted and sequenced, and the genomes of individual populations were separated using bioinformatics tools. We recovered 22 different genomes of Cyanobacteria, many of which have not been sequenced before. We describe in more detail an interesting lineage of ancestral Cyanobacteria in the order Gloeobacterales, for which we propose the nameCandidatusSivonenia alaskensis. Our study shows that genome-resolved metagenomics is a valuable approach for obtaining novel genomes of Cyanobacteria, which are needed to improve our understanding of life in the polar regions and the planet at large.