Publications

4851

The anaerobic oxidation of methane coupled to sulfate reduction is a microbially mediated process requiring a syntrophic partnership between anaerobic methanotrophic (ANME) archaea and sulfate-reducing bacteria (SRB). Based on genome taxonomy, ANME lineages are polyphyletic within the phylumHalobacterota, none of which have been isolated in pure culture. Here, we reconstruct 28 ANME genomes from environmental metagenomes and flow sorted syntrophic consortia. Together with a reanalysis of previously published datasets, these genomes enable a comparative analysis of all marine ANME clades. We review the genomic features that separate ANME from their methanogenic relatives and identify what differentiates ANME clades. Large multiheme cytochromes and bioenergetic complexes predicted to be involved in novel electron bifurcation reactions are well distributed and conserved in the ANME archaea, while significant variations in the anabolic C1 pathways exists between clades. Our analysis raises the possibility that methylotrophic methanogenesis may have evolved from a methanotrophic ancestor.

Summary Halorhodospira halophila , one of the most‐xerophilic halophiles, inhabits biophysically stressful and energetically expensive, salt‐saturated alkaline brines. Here, we report an additional stress factor that is biotic: a diminutive Candidate‐Phyla‐Radiation bacterium, that we named ‘ Ca . Absconditicoccus praedator’ M39‐6, which predates H . halophila M39‐5, an obligately photosynthetic, anaerobic purple‐sulfur bacterium. We cultivated this association (isolated from the hypersaline alkaline Lake Hotontyn Nur, Mongolia) and characterized their biology. ‘Ca . Absconditicoccus praedator’ is the first stably cultivated species from the candidate class‐level lineage Gracilibacteria (order‐level lineage Absconditabacterales). Its closed‐and‐curated genome lacks genes for the glycolytic, pentose phosphate‐ and Entner–Doudoroff pathways which would generate energy/reducing equivalents and produce central carbon currencies. Therefore, ‘Ca . Absconditicoccus praedator’ is dependent on host‐derived building blocks for nucleic acid‐, protein‐, and peptidoglycan synthesis. It shares traits with (the uncultured) ‘ Ca . Vampirococcus lugosii’, which is also of the Gracilibacteria lineage. These are obligate parasitic lifestyle, feeding on photosynthetic anoxygenic Gammaproteobacteria, and absorption of host cytoplasm. Commonalities in their genomic composition and structure suggest that the entire Absconditabacterales lineage consists of predatory species which act to cull the populations of their respective host bacteria. Cultivation of vampire : host associations can shed light on unresolved aspects of their metabolism and ecosystem dynamics at life‐limiting extremes.

The phloem-restricted, insect-transmitted bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) is associated with huanglongbing (HLB), the most devastating disease of citrus worldwide. The inability to culture CLas impairs the understanding of its virulence mechanisms and the development of effective management strategies to control this incurable disease. Previously, our research group used commercial grapefruit juice (GJ) to prolong the viability of CLas in vitro. In the present study, GJ was amended with a wide range of compounds and incubated under different conditions to optimize CLas growth. Remarkably, results showed that CLas growth ratios were inversely proportional to the initial inoculum concentration. This correlation is probably regulated by a cell density-dependent mechanism, because diluting samples between subcultures allowed CLas to resume growth. Moreover, strategies to reduce the cell density of CLas, such as subculturing at short intervals and incubating samples under flow conditions, allowed this bacterium to multiply and reach maximum growth as early as 3 days after inoculation, although no sustained exponential growth was observed under any tested condition. Unfortunately, cultures were only transient, because CLas lost viability over time; nevertheless, we obtained populations of about 105genome equivalents/ml repeatedly. Finally, we established an ex vivo system to grow CLas within periwinkle calli that could be used to propagate bacterial inoculum in the lab. In this study we determined the influence of a comprehensive set of conditions and compounds on CLas growth in culture. We hope our results will help guide future efforts toward the long-sought goal of culturing CLas axenically.

Huanglongbing, or citrus greening disease, is the most serious disease of citrus worldwide and is associated with plant infection by ‘Candidatus Liberibacter asiaticus’ (CLas) and other Liberibacter species. CLas is transmitted by Diaphorina citri, the Asian citrus psyllid, in a circulative propagative manner. Circulative propagative transmission is a complex process comprising at least three steps: movement of the pathogen into vector tissues, translocation and replication of the pathogen within the vector host, and pathogen inoculation of a new host by the vector. In this work, we describe an excised leaf CLas acquisition assay, which enables precise measurements of CLas acquisition by D. citri in a streamlined laboratory assay. Briefly, healthy fourth and fifth instar D. citri nymphs acquire CLas from excised CLas-positive leaves, where the insects also complete their developmental cycle. CLas titer in the resulting adults is measured using quantitative PCR and CLas-specific 16S rRNA gene primers. We observed positive correlations between CLas titer in each leaf replicate and the CLas titer that developed in the insects after acquisition (rs = 0.78; P = 0.0002). This simple assay could be used to detect CLas acquisition phenotypes and their underlying genotypes, facilitate assessment of plant factors that impact acquisition, and screen for compounds that interfere with CLas acquisition by delivering these compounds through the excised leaf.

Grafting a scion onto a rootstock results in physical and physiological changes in plant growth and development, which can affect tree vigor, productivity, and tolerance to stress and disease. Huanglongbing (HLB) is one of the most destructive citrus diseases and has become endemic in Florida since its introduction in 2005. It is associated with the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas), which cause severe metabolic disruptions in affected plants. Although most scion cultivars are highly susceptible, some rootstock cultivars are tolerant and allow the grafted tree to cope better with the disease. The objectives of this study were to identify rootstock traits that can be used to assess cultivars under controlled greenhouse conditions in advance of longer-term field trials. We used 10 commercially important rootstocks with different genetic backgrounds and known field performance in graft combination with ‘Valencia’ sweet orange scion. Trees were graft-inoculated with CLas and compared against mock-inoculated trees. Tree health and CLas populations were assessed regularly, and root growth was monitored using a minirhizotron imaging system. Plants were excavated and destructively sampled 21 months after inoculation to assess biomass distributions and other CLas-induced effects. We found significant differences between healthy and infected trees for most variables measured, regardless of the rootstock. In contrast to leaf CLas titers, root titers were significantly influenced by the rootstock, and highest levels were measured for ‘Ridge’ sweet orange and sour orange. Root growth and root biomasses were reduced upon infection but differences among rootstocks did not always agree with reported field performances. Despite severe biomass reductions plants maintained their relative distribution of biomass among different components of the root system, and no dead roots were observed. Root respiration was reduced by CLas infection and was overall higher in tolerant cultivars suggesting its potential as a physiological marker. This study improves our knowledge about the strengths and weaknesses of assessing rootstock traits of grafted trees in a controlled greenhouse setting. Results from the study suggest that in addition to HLB tolerance, other rootstock traits will ultimately have major contributions to field survival and productivity of the grafted trees in an HLB endemic production environment.