Persistence and resistance: survival mechanisms of Candidatus Dormibacterota from nutrient-poor Antarctic soils

Candidatus Dormibacterota is an uncultured bacterial phylum found predominantly in soil that is present in high abundances within cold desert soils. Here, we interrogate nine metagenome-assembled genomes (MAGs), including six new MAGs derived from soil metagenomes obtained from two eastern Antarctic sites. Phylogenomic and taxonomic analyses revealed these MAGs represent four genera and five species, representing two order-level clades within Ca. Dormibacterota. Metabolic reconstructions of these MAGs revealed the potential for aerobic metabolism, and versatile adaptations enabling persistence in the ‘extreme’ Antarctic environment. Primary amongst these adaptations were abilities to scavenge atmospheric H₂ and CO as energy sources, as well as using the energy derived from H₂ oxidation to fix atmospheric CO₂ via the Calvin–Bassham–Benson cycle, using a RuBisCO type IE. We propose that these allow Ca. Dormibacterota to persist using H₂ oxidation and grow using atmospheric chemosynthesis in terrestrial Antarctica. Fluorescence in situ hybridization revealed Ca. Dormibacterota to be coccoid cells, 0.3–1.4 μm in diameter, with some cells exhibiting the potential for a symbiotic or syntrophic lifestyle.     

Metagenomic insights into the metabolism and evolution of a new Thermoplasmata order (Candidatus Gimiplasmatales)

Thermoplasmata is a widely distributed and ecologically important archaeal class in the phylum Euryarchaeota. Because few cultures and genomes are available, uncharacterized Thermoplasmata metabolisms remain unexplored. In this study, we obtained four medium- to high-quality archaeal metagenome-assembled genomes (MAGs) from the filamentous fragments of black-odorous aquatic sediments (Foshan, Guangdong, China). Based on their 16S rRNA gene and ribosomal protein phylogenies, the four MAGs belong to the previously unnamed Thermoplasmata UBA10834 clade. We propose that this clade (five reference genomes from the Genome Taxonomy Database (GTDB) and four MAGs from this study) be considered a new order, Candidatus Gimiplasmatales. Metabolic pathway reconstructions indicated that the Ca. Gimiplasmatales MAGs can biosynthesize isoprenoids and nucleotides de novo. Additionally, some taxa have genes for formaldehyde and acetate assimilation, and the Wood–Ljungdahl CO₂-fixation pathway, indicating a mixotrophic lifestyle. Sulfur reduction, hydrogen metabolism, and arsenic detoxification pathways were predicted, indicating sulfur-, hydrogen-, and arsenic-transformation potentials. Comparative genomics indicated that the H₄F Wood–Ljungdahl pathway of both Ca. Gimiplasmatales and Methanomassiliicoccales was likely obtained by the interdomain lateral gene transfer from the Firmicutes. Collectively, this study elucidates the taxonomic and potential metabolic diversity of the new order Ca. Gimiplasmatales and the evolution of this subgroup and its sister lineage Methanomassiliicoccales.     

Genomic Features of a Bumble Bee Symbiont Reflect Its Host Environment

Here, we report the genome of one gammaproteobacterial member of the gut microbiota, for which we propose the name “Candidatus Schmidhempelia bombi,” that was inadvertently sequenced alongside the genome of its host, the bumble bee, Bombus impatiens. This symbiont is a member of the recently described bacterial order Orbales, which has been collected from the guts of diverse insect species; however, “Ca. Schmidhempelia” has been identified exclusively with bumble bees. Metabolic reconstruction reveals that “Ca. Schmidhempelia” lacks many genes for a functioning NADH dehydrogenase I, all genes for the high-oxygen cytochrome o, and most genes in the tricarboxylic acid (TCA) cycle. “Ca. Schmidhempelia” has retained NADH dehydrogenase II, the low-oxygen specific cytochrome bd, anaerobic nitrate respiration, mixed-acid fermentation pathways, and citrate fermentation, which may be important for survival in low-oxygen or anaerobic environments found in the bee hindgut. Additionally, a type 6 secretion system, a Flp pilus, and many antibiotic/multidrug transporters suggest complex interactions with its host and other gut commensals or pathogens. This genome has signatures of reduction (2.0 megabase pairs) and rearrangement, as previously observed for genomes of host-associated bacteria. A survey of wild and laboratory B. impatiens revealed that “Ca. Schmidhempelia” is present in 90% of individuals and, therefore, may provide benefits to its host.     

Rare Branched Fatty Acids Characterize the Lipid Composition of the Intra-Aerobic Methane Oxidizer “Candidatus Methylomirabilis oxyfera”

The recently described bacterium “Candidatus Methylomirabilis oxyfera” couples the oxidation of the important greenhouse gas methane to the reduction of nitrite. The ecological significance of “Ca. Methylomirabilis oxyfera” is still underexplored, as our ability to identify the presence of this bacterium is thus far limited to DNA-based techniques. Here, we investigated the lipid composition of “Ca. Methylomirabilis oxyfera” to identify new, gene-independent biomarkers for the environmental detection of this bacterium. Multiple “Ca. Methylomirabilis oxyfera” enrichment cultures were investigated. In all cultures, the lipid profile was dominated up to 46% by the fatty acid (FA) 10-methylhexadecanoic acid (10MeC_16:0). Furthermore, a unique FA was identified that has not been reported elsewhere: the monounsaturated 10-methylhexadecenoic acid with a double bond at the Δ7 position (10MeC_16:1Δ7), which comprised up to 10% of the total FA profile. We propose that the typical branched fatty acids 10MeC_16:0 and 10MeC_16:1Δ7 are key and characteristic components of the lipid profile of “Ca. Methylomirabilis oxyfera.” The successful detection of these fatty acids in a peatland from which one of the enrichment cultures originated supports the potential of these unique lipids as biomarkers for the process of nitrite-dependent methane oxidation in the environment.     

Bacterial Community and “Candidatus Accumulibacter” Population Dynamics in Laboratory-Scale Enhanced Biological Phosphorus Removal Reactors

“Candidatus Accumulibacter” and total bacterial community dynamics were studied in two lab-scale enhanced biological phosphorus removal (EBPR) reactors by using a community fingerprint technique, automated ribosomal intergenic spacer analysis (ARISA). We first evaluated the quantitative capability of ARISA compared to quantitative real-time PCR (qPCR). ARISA and qPCR provided comparable relative quantification of the two dominant “Ca. Accumulibacter” clades (IA and IIA) detected in our reactors. The quantification of total “Ca. Accumulibacter” 16S rRNA genes relative to that from the total bacterial community was highly correlated, with ARISA systematically underestimating “Ca. Accumulibacter” abundance, probably due to the different normalization techniques applied. During 6 months of normal (undisturbed) operation, the distribution of the two clades within the total “Ca. Accumulibacter” population was quite stable in one reactor while comparatively dynamic in the other reactor. However, the variance in the clade distribution did not appear to affect reactor performance. Instead, good EBPR activity was positively associated with the abundance of total “Ca. Accumulibacter.” Therefore, we concluded that the different clades in the system provided functional redundancy. We disturbed the reactor operation by adding nitrate together with acetate feeding in the anaerobic phase to reach initial reactor concentrations of 10 mg/liter NO₃-N for 35 days. The reactor performance deteriorated with a concomitant decrease in the total “Ca. Accumulibacter” population, suggesting that a population shift was the cause of performance upset after a long exposure to nitrate in the anaerobic phase.Enhanced biological phosphorus removal (EBPR) has been widely applied to reduce phosphorus (P) levels in wastewater treatment effluents, through the transformation of soluble inorganic phosphate (P_i) to intracellular polyphosphate [poly(P)] by poly(P)-accumulating organisms (PAOs) under alternating anaerobic/aerobic conditions. Anaerobically, PAOs take up organic substrates such as acetate, coupled to P release, as a result of intracellular poly(P) degradation. Lacking an external electron acceptor, acetate is converted to polyhydroxybutyrate (PHB), which is depolymerized and oxidized under subsequent aerobic conditions, leading to ATP generation and poly(P) regeneration (21).A currently uncultured bacterial group in Betaproteobacteria, named “Candidatus Accumulibacter phosphatis” (13), was found to be the primary PAO in lab-scale and some full-scale EBPR systems (6, 37). Based on the phylogeny of polyphosphate kinase genes (ppk1), the “Ca. Accumulibacter” lineage is comprised of two major types, and each type contains a number of coherent clades (11, 23). Several studies suggested that these clades differ in their ability to reduce nitrate (5, 9) and the involvement of the tricarboxylic acid cycle in EBPR metabolism (33).Although “Ca. Accumulibacter” clades other than IA and IIA have been found in several lab-scale sequencing batch reactors (SBRs) (33), in our previous study we only detected IA and IIA in two acetate-fed SBRs operated under similar conditions but at different geographical locations with different inoculation sludge sources (11). In addition, we found that the identity of the dominant clade switched between two sampling events (i.e., changed from IIA to IA). This raises intriguing questions, such as, how frequently the population shift occurs, how the clade dynamics influences the reactor performance, and how reactor operating conditions affect the clade composition.To answer these questions, we studied “Ca. Accumulibacter” population composition and dynamics on a fine time scale. Since “Ca. Accumulibacter” clades may interact positively or negatively with each other, or with other bacterial groups, we searched for bacterial community composition patterns associated with shifts in the relative abundances of the two clades. For this purpose, we applied a community fingerprint method, automated ribosomal intergenic spacer analysis (ARISA) (8), which had been used to study bacterial community composition and dynamics in freshwater lakes (22, 27), activated sludge (34), and even environments with comparatively more complex microbial communities, such as soils (24). ARISA relies on the length heterogeneity of the internal transcribed spacer (ITS) region between 16S and 23S rRNA to distinguish different operational taxonomic units (OTUs). “Ca. Accumulibacter” clades IA and IIA detected in our reactors have distinct ITS lengths (12), thus allowing their unique detection by ARISA.In this study, we first evaluated the quantitative capability of ARISA, compared to quantitative real-time PCR (qPCR) assays previously developed (11), and then used ARISA to monitor bacterial community composition dynamics. We analyzed samples collected weekly from two lab-scale SBRs during a 6-month period, when both reactors were operated under undisturbed and nearly identical conditions. We also evaluated samples obtained under disturbed conditions, when nitrate was introduced in the anaerobic phase for a period of 35 days, as well as those collected from time points when the reactors experienced “Ca. Accumulibacter” clade shift or poor performance, in order to explore potential relationships between “Ca. Accumulibacter” clade dynamics, total bacterial community composition patterns, operating conditions, and reactor performance. An understanding of such relationships should bring us closer to a mechanistic understanding of EBPR ecology and therefore more rational process design and operation.     

Characterization of the Denitrification-Associated Phosphorus Uptake Properties of “Candidatus Accumulibacter phosphatis” Clades in Sludge Subjected to Enhanced Biological Phosphorus Removal

To characterize the denitrifying phosphorus (P) uptake properties of “Candidatus Accumulibacter phosphatis,” a sequencing batch reactor (SBR) was operated with acetate. The SBR operation was gradually acclimated from anaerobic-oxic (AO) to anaerobic-anoxic-oxic (A2O) conditions by stepwise increases of nitrate concentration and the anoxic time. The communities of “Ca. Accumulibacter” and associated bacteria at the initial (AO) and final (A2O) stages were compared using 16S rRNA and polyphosphate kinase genes and using fluorescence in situ hybridization (FISH). The acclimation process led to a clear shift in the relative abundances of recognized “Ca. Accumulibacter” subpopulations from clades IIA > IA > IIF to clades IIC > IA > IIF, as well as to increases in the abundance of other associated bacteria (Dechloromonas [from 1.2% to 19.2%] and “Candidatus Competibacter phosphatis” [from 16.4% to 20.0%]), while the overall “Ca. Accumulibacter” abundance decreased (from 55.1% to 29.2%). A series of batch experiments combined with FISH/microautoradiography (MAR) analyses was performed to characterize the denitrifying P uptake properties of the “Ca. Accumulibacter” clades. In FISH/MAR experiments using slightly diluted sludge (∼0.5 g/liter), all “Ca. Accumulibacter” clades successfully took up phosphorus in the presence of nitrate. However, the “Ca. Accumulibacter” clades showed no P uptake in the presence of nitrate when the sludge was highly diluted (∼0.005 g/liter); under these conditions, reduction of nitrate to nitrite did not occur, whereas P uptake by “Ca. Accumulibacter” clades occurred when nitrite was added. These results suggest that the “Ca. Accumulibacter” cells lack nitrate reduction capabilities and that P uptake by “Ca. Accumulibacter” is dependent upon nitrite generated by associated nitrate-reducing bacteria such as Dechloromonas and “Ca. Competibacter.”     

Infection Dynamics of the Tick-Borne Pathogen “Candidatus Neoehrlichia mikurensis” and Coinfections with Borrelia afzelii in Bank Voles in Southern Sweden

The tick-borne bacterium “Candidatus Neoehrlichia mikurensis” has recently been recognized as a human pathogen. Together with Borrelia afzelii, it is one of the most common pathogens found in the tick Ixodes ricinus. Here, we compared the epidemiologies of “Ca. Neoehrlichia mikurensis” and B. afzelii by longitudinal sampling from May to September in one of their most abundant vertebrate hosts, the bank vole (Myodes glareolus), using real-time PCR for detection and quantification. The prevalences of “Ca. Neoehrlichia mikurensis” and B. afzelii were determined to be 19% (50/261) and 22% (56/261), respectively. The prevalence of “Ca. Neoehrlichia mikurensis” increased significantly during the sampling season. The clearance rate of “Ca. Neoehrlichia mikurensis” was significantly higher than that of B. afzelii. We found a high frequency of double infections; 46% of all samples infected with “Ca. Neoehrlichia mikurensis” also had a coinfection with B. afzelii. The frequency of coinfections was significantly higher than expected from the prevalence of each pathogen. The high level of coinfections can be caused by interactions between the pathogens or might reflect variation in general susceptibility among voles.     

Into the darkness: the ecologies of novel ‘microbial dark matter’ phyla in an Antarctic lake

Uncultivated microbial clades (‘microbial dark matter’) are inferred to play important but uncharacterized roles in nutrient cycling. Using Antarctic lake (Ace Lake, Vestfold Hills) metagenomes, 12 metagenome-assembled genomes (MAGs; 88%–100% complete) were generated for four ‘dark matter’ phyla: six MAGs from Candidatus Auribacterota (=Aureabacteria, SURF-CP-2), inferred to be hydrogen- and sulfide-producing fermentative heterotrophs, with individual MAGs encoding bacterial microcompartments (BMCs), gas vesicles, and type IV pili; one MAG (100% complete) from Candidatus Hinthialibacterota (=OLB16), inferred to be a facultative anaerobe capable of dissimilatory nitrate reduction to ammonia, specialized for mineralization of complex organic matter (e.g. sulfated polysaccharides), and encoding BMCs, flagella, and Tad pili; three MAGs from Candidatus Electryoneota (=AABM5-125-24), previously reported to include facultative anaerobes capable of dissimilatory sulfate reduction, and here inferred to perform sulfite oxidation, reverse tricarboxylic acid cycle for autotrophy, and possess numerous proteolytic enzymes; two MAGs from Candidatus Lernaellota (=FEN-1099), inferred to be capable of formate oxidation, amino acid fermentation, and possess numerous enzymes for protein and polysaccharide degradation. The presence of 16S rRNA gene sequences in public metagenome datasets (88%–100% identity) suggests these ‘dark matter’ phyla contribute to sulfur cycling, degradation of complex organic matter, ammonification and/or chemolithoautotrophic CO₂ fixation in diverse global environments.     

XoxF-Type Methanol Dehydrogenase from the Anaerobic Methanotroph “Candidatus Methylomirabilis oxyfera”

“Candidatus Methylomirabilis oxyfera” is a newly discovered anaerobic methanotroph that, surprisingly, oxidizes methane through an aerobic methane oxidation pathway. The second step in this aerobic pathway is the oxidation of methanol. In Gram-negative bacteria, the reaction is catalyzed by pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH). The genome of “Ca. Methylomirabilis oxyfera” putatively encodes three different MDHs that are localized in one large gene cluster: one so-called MxaFI-type MDH and two XoxF-type MDHs (XoxF1 and XoxF2). MxaFI MDHs represent the canonical enzymes, which are composed of two PQQ-containing large (α) subunits (MxaF) and two small (β) subunits (MxaI). XoxF MDHs are novel, ecologically widespread, but poorly investigated types of MDHs that can be phylogenetically divided into at least five different clades. The XoxF MDHs described thus far are homodimeric proteins containing a large subunit only. Here, we purified a heterotetrameric MDH from “Ca. Methylomirabilis oxyfera” that consisted of two XoxF and two MxaI subunits. The enzyme was localized in the periplasm of “Ca. Methylomirabilis oxyfera” cells and catalyzed methanol oxidation with appreciable specific activity and affinity (V_max of 10 μmol min⁻¹ mg⁻¹ protein, K_m of 17 μM). PQQ was present as the prosthetic group, which has to be taken up from the environment since the known gene inventory required for the synthesis of this cofactor is lacking. The MDH from “Ca. Methylomirabilis oxyfera” is the first representative of type 1 XoxF proteins to be described.     

Enrichment and Genome Sequence of the Group I.1a Ammonia-Oxidizing Archaeon “Ca. Nitrosotenuis uzonensis” Representing a Clade Globally Distributed in Thermal Habitats

The discovery of ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota and the high abundance of archaeal ammonia monooxygenase subunit A encoding gene sequences in many environments have extended our perception of nitrifying microbial communities. Moreover, AOA are the only aerobic ammonia oxidizers known to be active in geothermal environments. Molecular data indicate that in many globally distributed terrestrial high-temperature habits a thaumarchaeotal lineage within the Nitrosopumilus cluster (also called “marine” group I.1a) thrives, but these microbes have neither been isolated from these systems nor functionally characterized in situ yet. In this study, we report on the enrichment and genomic characterization of a representative of this lineage from a thermal spring in Kamchatka. This thaumarchaeote, provisionally classified as “Candidatus Nitrosotenuis uzonensis”, is a moderately thermophilic, non-halophilic, chemolithoautotrophic ammonia oxidizer. The nearly complete genome sequence (assembled into a single scaffold) of this AOA confirmed the presence of the typical thaumarchaeotal pathways for ammonia oxidation and carbon fixation, and indicated its ability to produce coenzyme F₄₂₀ and to chemotactically react to its environment. Interestingly, like members of the genus Nitrosoarchaeum, “Candidatus N. uzonensis” also possesses a putative artubulin-encoding gene. Genome comparisons to related AOA with available genome sequences confirmed that the newly cultured AOA has an average nucleotide identity far below the species threshold and revealed a substantial degree of genomic plasticity with unique genomic regions in “Ca. N. uzonensis”, which potentially include genetic determinants of ecological niche differentiation.     

Infection Density Dynamics of the Citrus Greening Bacterium “Candidatus Liberibacter asiaticus” in Field Populations of the Psyllid Diaphorina citri and Its Relevance to the Efficiency of Pathogen Transmission to Citrus Plants

Huanglongbing, or citrus greening, is a devastating disease of citrus plants recently spreading worldwide, which is caused by an uncultivable bacterial pathogen, “Candidatus Liberibacter asiaticus,” and vectored by a phloem-sucking insect, Diaphorina citri. We investigated the infection density dynamics of “Ca. Liberibacter asiaticus” in field populations of D. citri with experiments using field-collected insects to address how “Ca. Liberibacter asiaticus” infection density in the vector insect is relevant to pathogen transmission to citrus plants. Of 500 insects continuously collected from “Ca. Liberibacter asiaticus”-infected citrus trees with pathological symptoms in the spring and autumn of 2009, 497 (99.4%) were “Ca. Liberibacter asiaticus” positive. The infections were systemic across head-thorax and abdomen, ranging from 10³ to 10⁷ bacteria per insect. In spring, the infection densities were low in March, at ∼10³ bacteria per insect, increasing up to 10⁶ to 10⁷ bacteria per insect in April and May, and decreasing to 10⁵ to 10⁶ bacteria per insect in late May, whereas the infection densities were constantly ∼10⁶ to 10⁷ bacteria per insect in autumn. Statistical analysis suggested that several factors, such as insect sex, host trees, and collection dates, may be correlated with “Ca. Liberibacter asiaticus” infection densities in field D. citri populations. Inoculation experiments with citrus seedlings using field-collected “Ca. Liberibacter asiaticus”-infected insects suggested that (i) “Ca. Liberibacter asiaticus”-transmitting insects tend to exhibit higher infection densities than do nontransmitting insects, (ii) a threshold level (∼10⁶ bacteria per insect) of “Ca. Liberibacter asiaticus” density in D. citri is required for successful transmission to citrus plants, and (iii) D. citri attaining the threshold infection level transmits “Ca. Liberibacter asiaticus” to citrus plants in a stochastic manner. These findings provide valuable insights into understanding, predicting, and controlling this notorious citrus pathogen.     

High Incidence of Preharvest Colonization of Huanglongbing-Symptomatic Citrus sinensis Fruit by Lasiodiplodia theobromae (Diplodia natalensis) and Exacerbation of Postharvest Fruit Decay by That Fungus

Huanglongbing (HLB), presumably caused by the bacterium “Candidatus Liberibacter asiaticus,” is a devastating citrus disease associated with excessive preharvest fruit drop. Lasiodiplodia theobromae (diplodia) is the causal organism of citrus stem end rot (SER). The pathogen infects citrus fruit under the calyx abscission zone (AZ-C) and is associated with cell wall hydrolytic enzymes similar to plant enzymes involved in abscission. By means of DNA sequencing, diplodia was found in “Ca. Liberibacter asiaticus”-positive juice from HLB-symptomatic fruit (S) but not in “Ca. Liberibacter asiaticus”-negative juice. Therefore, the incidence of diplodia in fruit tissues, the impact on HLB-related postharvest decay, and the implications for HLB-related preharvest fruit drop were investigated in Hamlin and Valencia oranges. Quantitative PCR results (qPCR) revealed a significantly (P < 0.001) greater incidence of diplodia in the AZ-C of HLB-symptomatic (S; “Ca. Liberibacter asiaticus” threshold cycle [C_T] of <30) than in the AZ-C of in asymptomatic (AS; “Ca. Liberibacter asiaticus” C_T of ≥30) fruit. In agreement with the qPCR results, 2 weeks after exposure to ethylene, the incidences of SER in S fruit were 66.7% (Hamlin) and 58.7% (Valencia), whereas for AS fruit the decay rates were 6.7% (Hamlin) and 5.3% (Valencia). Diplodia colonization of S fruit AZ-C was observed by scanning electron microscopy and confirmed by PCR test and morphology of conidia in isolates from the AZ-C after surface sterilization. Diplodia C_T values were negatively correlated with ethylene production (R = −0.838 for Hamlin; R = −0.858 for Valencia) in S fruit, and positively correlated with fruit detachment force (R = 0.855 for Hamlin; R = 0.850 for Valencia), suggesting that diplodia colonization in AZ-C may exacerbate HLB-associated preharvest fruit drop.     

Diversity of “Candidatus Liberibacter asiaticus,” Based on the omp Gene Sequence

Huanglongbing (yellow dragon disease) is a destructive disease of citrus. The etiological agent is a noncultured, phloem-restricted alpha-proteobacterium, “Candidatus Liberibacter africanus” in Africa and “Candidatus Liberibacter asiaticus” in Asia. In this study, we used an omp-based PCR-restriction fragment length polymorphism (RFLP) approach to analyze the genetic variability of “Ca. Liberibacter asiaticus” isolates. By using five different enzymes, each the 10 isolates tested could be associated with a specific combination of restriction profiles. The results indicate that the species “Ca. Liberibacter asiaticus,” even within a given region, may comprise several different variants. Thus, omp-based PCR-RFLP analysis is a simple method for detecting and differentiating “Ca. Liberibacter asiaticus” isolates.     

Mitochondrial Acclimation Capacities to Ocean Warming and Acidification Are Limited in the Antarctic Nototheniid Fish,Notothenia rossii and Lepidonotothen squamifrons

Antarctic notothenioid fish are characterized by their evolutionary adaptation to the cold, thermostable Southern Ocean, which is associated with unique physiological adaptations to withstand the cold and reduce energetic requirements but also entails limited compensation capacities to environmental change. This study compares the capacities of mitochondrial acclimation to ocean warming and acidification between the Antarctic nototheniid Notothenia rossii and the sub-Antarctic Lepidonotothen squamifrons, which share a similar ecology, but different habitat temperatures. After acclimation of L. squamifrons to 9°C and N. rossii to 7°C (normocapnic/hypercapnic, 0.2 kPa CO₂/2000 ppm CO₂) for 4–6 weeks, we compared the capacities of their mitochondrial respiratory complexes I (CI) and II (CII), their P/O ratios (phosphorylation efficiency), proton leak capacities and mitochondrial membrane fatty acid compositions. Our results reveal reduced CII respiration rates in warm-acclimated L. squamifrons and cold hypercapnia-acclimated N. rossii. Generally, L. squamifrons displayed a greater ability to increase CI contribution during acute warming and after warm-acclimation than N. rossii. Membrane unsaturation was not altered by warm or hypercapnia-acclimation in both species, but membrane fatty acids of warm-acclimated L. squamifrons were less saturated than in warm normocapnia−/hypercapnia-acclimated N. rossii. Proton leak capacities were not affected by warm or hypercapnia-acclimation of N. rossii. We conclude that an acclimatory response of mitochondrial capacities may include higher thermal plasticity of CI supported by enhanced utilization of anaplerotic substrates (via oxidative decarboxylation reactions) feeding into the citrate cycle. L. squamifrons possesses higher relative CI plasticities than N. rossii, which may facilitate the usage of energy efficient NADH-related substrates under conditions of elevated energy demand, possibly induced by ocean warming and acidification. The observed adjustments of electron transport system complexes with a higher flux through CI under warming and acidification suggest a metabolic acclimation potential of the sub-Antarctic L. squamifrons, but only limited acclimation capacities for N. rossii.     

Evolution and Diversity of Facultative Symbionts from the Aphid Subfamily Lachninae

Many aphids harbor a variety of endosymbiotic bacteria. The functions of these symbionts can range from an obligate nutritional role to a facultative role in protecting their hosts against environmental stresses. One such symbiont is “Candidatus Serratia symbiotica,” which is involved in defense against heat and potentially also in aphid nutrition. Lachnid aphids have been the focus of several recent studies investigating the transition of this symbiont from a facultative symbiont to an obligate symbiont. In a phylogenetic analysis of Serratia symbionts from 51 lachnid hosts, we found that diversity in symbiont morphology, distribution, and function is due to multiple independent origins of symbiosis from ancestors belonging to Serratia and possibly also to evolution within distinct symbiont clades. Our results do not support cocladogenesis of “Ca. Serratia symbiotica” with Cinara subgenus Cinara species and weigh against an obligate nutritional role. Finally, we show that species belonging to the subfamily Lachninae have a high incidence of facultative symbiont infection.Many insect species harbor heritable endosymbiotic bacteria. Among the best studied of these species are aphids. Almost all aphids are infected with the obligate nutritional symbiont Buchnera aphidicola, which is generally required for the survival of aphids and provides essential amino acids that are rare in their phloem sap diet (32). Many aphids also possess additional symbionts that may be facultative from the host''s perspective and that coexist with Buchnera (20).Three lineages of facultative symbionts that are prevalent in aphids belong to the Enterobacteriaceae. Two of these lineages (“Candidatus Hamiltonella defensa” and “Candidatus Regiella insecticola”) form well-defined clades distinct from free-living bacterial species (4, 20) and confer clear advantages to their hosts by protecting them against natural enemies. “Ca. Hamiltonella defensa” prevents wasp parasitism by arresting development of wasp larvae in pea aphids, and “Ca. Regiella insecticola” provides resistance against the fungal pathogen Pandora neoaphidis (24, 31). The third lineage, “Candidatus Serratia symbiotica,” is closely related to free-living members of the genus Serratia. This symbiont is distributed sporadically among aphid species and has been proposed to have a variety of effects on hosts. In pea aphids (Acyrthosiphon pisum; Macrosiphini), “Ca. Serratia symbiotica” ameliorates the deleterious fitness effects of heat shock by protecting symbiont-harboring bacteriocyte cells (2, 19, 29). Additionally, a strain of “Ca. Serratia symbiotica” provided some resistance to parasitoid wasp attack (24). “Ca. Serratia symbiotica” has been proposed to play a role in nutrition by producing amino acids for its aphid host and by decreasing its host''s reliance on Buchnera (10, 15, 16, 26). In contrast to most Buchnera strains, Buchnera strains from Cinara cedri (Lachnini) have lost the genes for biosynthesis of the essential amino acid tryptophan, while “Ca. Serratia symbiotica” in the same host possesses at least part of the pathway, suggesting that it has a mutualistic role in the nutrition of aphids (26).In A. pisum, “Ca. Serratia symbiotica” cells are rod-shaped bacteria that are present in the sheath cells, hemolymph, and bacteriocytes of some individuals. In contrast, in C. cedri “Ca. Serratia symbiotica” occurs in all individuals, and its cells are large, round, and pleomorphic, similar to the cells of many obligate bacterial aphid endosymbionts, including Buchnera (10, 26). Furthermore, “Ca. Serratia symbiotica” has consistently been present in other Cinara species sampled (28). Both the rod-shaped and pleomorphic forms are assigned to “Ca. Serratia symbiotica” based on phylogenetic analyses of several gene sequences, but they fall into two distinct sister clades of symbiont lineages that seem to coincide with bacterial morphology (17, 20).This diversity in “Ca. Serratia symbiotica” morphology, distribution, and functions may represent evolution of different features within lineages of a single symbiont clade. If “Ca. Serratia symbiotica” is an obligate nutritional symbiont in Cinara hosts, it is expected that Cinara-associated symbionts would form a clade in which the intraclade relationships mirror those of the hosts (cocladogenesis), as observed for Buchnera and other obligate nutritional symbionts of insects (13, 21, 38). Indeed, Lamelas et al. postulated that, based on their similar phylogenies, Serratia symbionts from aphids belonging to the subgenus Cinara have had a long-term relationship with their hosts (17).In addition to the three most common facultative symbiont types found in aphids described above, several other symbiont lineages with unknown functions have been identified by amplification of bacterial 16S rRNA gene sequences from various aphid species (10, 28, 39). Here we examine the diversity of Serratia and other facultative symbionts in aphids belonging to the subfamily Lachninae. We investigated the distribution of symbionts in aphid species and geographic locations and looked for coevolutionary patterns that may correspond to the functions of facultative symbionts within their hosts.     

Metabolic shifts in the Antarctic fish <Emphasis Type="Italic">Notothenia rossii</Emphasis> in response to rising temperature and <Emphasis Type="Italic">P</Emphasis> CO<Subscript>2</Subscript>

Introduction

Ongoing ocean warming and acidification increasingly affect marine ecosystems, in particular around the Antarctic Peninsula. Yet little is known about the capability of Antarctic notothenioid fish to cope with rising temperature in acidifying seawater. While the whole animal level is expected to be more sensitive towards hypercapnia and temperature, the basis of thermal tolerance is set at the cellular level, with a putative key role for mitochondria. This study therefore investigates the physiological responses of the Antarctic Notothenia rossii after long-term acclimation to increased temperatures (7°C) and elevated P CO₂ (0.2 kPa CO₂) at different levels of physiological organisation.

Results

For an integrated picture, we analysed the acclimation capacities of N. rossii by measuring routine metabolic rate (RMR), mitochondrial capacities (state III respiration) as well as intra- and extracellular acid–base status during acute thermal challenges and after long-term acclimation to changing temperature and hypercapnia. RMR was partially compensated during warm- acclimation (decreased below the rate observed after acute warming), while elevated P CO₂ had no effect on cold or warm acclimated RMR. Mitochondrial state III respiration was unaffected by temperature acclimation but depressed in cold and warm hypercapnia-acclimated fish. In both cold- and warm-exposed N. rossii, hypercapnia acclimation resulted in a shift of extracellular pH (pH_e) towards more alkaline values. A similar overcompensation was visible in muscle intracellular pH (pH_i). pH_i in liver displayed a slight acidosis after warm normo- or hypercapnia acclimation, nevertheless, long-term exposure to higher P CO₂ was compensated for by intracellular bicarbonate accumulation.

Conclusion

The partial warm compensation in whole animal metabolic rate indicates beginning limitations in tissue oxygen supply after warm-acclimation of N. rossii. Compensatory mechanisms of the reduced mitochondrial capacities under chronic hypercapnia may include a new metabolic equilibrium to meet the elevated energy demand for acid–base regulation. New set points of acid–base regulation under hypercapnia, visible at the systemic and intracellular level, indicate that N. rossii can at least in part acclimate to ocean warming and acidification. It remains open whether the reduced capacities of mitochondrial energy metabolism are adaptive or would impair population fitness over longer timescales under chronically elevated temperature and P CO₂.

     

Ecological and genomic analyses of candidate phylum WPS-2 bacteria in an unvegetated soil

Members of the bacterial candidate phylum WPS-2 (or Eremiobacterota) are abundant in several dry, bare soil environments. In a bare soil deposited by an extinct iron–sulfur spring, we found that WPS-2 comprised up to 24% of the bacterial community and up to 10⁸ cells per g of soil based on 16S rRNA gene sequencing and quantification. A single genus-level cluster (Ca. Rubrimentiphilum) predominated in bare soils but was less abundant in adjacent forest. Nearly complete genomes of Ca. Rubrimentiphilum were recovered as single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs). Surprisingly, given the abundance of WPS-2 in bare soils, the genomes did not indicate any capacity for autotrophy, phototrophy, or trace gas metabolism. Instead, they suggest a predominantly aerobic organoheterotrophic lifestyle, perhaps based on scavenging amino acids, nucleotides, and complex oligopeptides, along with lithotrophic capacity on thiosulfate. Network analyses of the entire community showed that some species of Chloroflexi, Actinobacteria, and candidate phylum AD3 (or Dormibacterota) co-occurred with Ca. Rubrimentiphilum and may represent ecological or metabolic partners. We propose that Ca. Rubrimentiphilum act as efficient heterotrophic scavengers. Combined with previous studies, these data suggest that the phylum WPS-2 includes bacteria with diverse metabolic capabilities.     

Response of the Endophytic Diazotroph Gluconacetobacter diazotrophicus on Solid Media to Changes in Atmospheric Partial O2 Pressure

Gluconacetobacter diazotrophicus is an N₂-fixing endophyte isolated from sugarcane. G. diazotrophicus was grown on solid medium at atmospheric partial O₂ pressures (pO₂) of 10, 20, and 30 kPa for 5 to 6 days. Using a flowthrough gas exchange system, nitrogenase activity and respiration rate were then measured at a range of atmospheric pO₂ (5 to 60 kPa). Nitrogenase activity was measured by H₂ evolution in N₂-O₂ and in Ar-O₂, and respiration rate was measured by CO₂ evolution in N₂-O₂. To validate the use of H₂ production as an assay for nitrogenase activity, a non-N₂-fixing (Nif⁻) mutant of G. diazotrophicus was tested and found to have a low rate of uptake hydrogenase (Hup⁺) activity (0.016± 0.009 μmol of H₂ 10¹⁰ cells⁻¹ h⁻¹) when incubated in an atmosphere enriched in H₂. However, Hup⁺ activity was not detectable under the normal assay conditions used in our experiments. G. diazotrophicus fixed nitrogen at all atmospheric pO₂ tested. However, when the assay atmospheric pO₂ was below the level at which the colonies had been grown, nitrogenase activity was decreased. Optimal atmospheric pO₂ for nitrogenase activity was 0 to 20 kPa above the pO₂ at which the bacteria had been grown. As atmospheric pO₂ was increased in 10-kPa steps to the highest levels (40 to 60 kPa), nitrogenase activity decreased in a stepwise manner. Despite the decrease in nitrogenase activity as atmospheric pO₂ was increased, respiration rate increased marginally. A large single-step increase in atmospheric pO₂ from 20 to 60 kPa caused a rapid 84% decrease in nitrogenase activity. However, upon returning to 20 kPa of O₂, 80% of nitrogenase activity was recovered within 10 min, indicating a “switch-off/switch-on” O₂ protection mechanism of nitrogenase activity. Our study demonstrates that colonies of G. diazotrophicus can fix N₂ at a wide range of atmospheric pO₂ and can adapt to maintain nitrogenase activity in response to both long-term and short-term changes in atmospheric pO₂.