CK2 activity is crucial for proper glucagon expression.

AIMS/HYPOTHESIS: Protein kinase CK2 acts as a negative regulator of insulin expression in pancreatic beta cells. This action is mainly mediated by phosphorylation of the transcription factor pancreatic and duodenal homeobox protein 1 (PDX1). In pancreatic alpha cells, PDX1 acts in a reciprocal fashion on glucagon (GCG) expression. Therefore, we hypothesised that CK2 might positively regulate GCG expression in pancreatic alpha cells. METHODS: We suppressed CK2 kinase activity in αTC1 cells by two pharmacological inhibitors and by the CRISPR/Cas9 technique. Subsequently, we analysed GCG expression and secretion by real-time quantitative RT-PCR, western blot, luciferase assay, ELISA and DNA pull-down assays. We additionally studied paracrine effects on GCG secretion in pseudoislets, isolated murine islets and human islets. In vivo, we examined the effect of CK2 inhibition on blood glucose levels by systemic and alpha cell-specific CK2 inhibition. RESULTS: We found that CK2 downregulation reduces GCG secretion in the murine alpha cell line αTC1 (e.g. from 1094±124 ng/l to 459±110 ng/l) by the use of the CK2-inhibitor SGC-CK2-1. This was due to a marked decrease in Gcg gene expression through alteration of the binding of paired box protein 6 (PAX6) and transcription factor MafB to the Gcg promoter. The analysis of the underlying mechanisms revealed that both transcription factors are displaced by PDX1. Ex vivo experiments in isolated murine islets and pseudoislets further demonstrated that CK2-mediated reduction in GCG secretion was only slightly affected by the higher insulin secretion after CK2 inhibition. The kidney capsule transplantation model showed the significance of CK2 for GCG expression and secretion in vivo. Finally, CK2 downregulation also reduced the GCG secretion in islets isolated from humans. CONCLUSIONS/INTERPRETATION: These novel findings not only indicate an important function of protein kinase CK2 for proper GCG expression but also demonstrate that CK2 may be a promising target for the development of novel glucose-lowering drugs.

The microbial-driven nitrogen cycle and its relevance for plant nutrition.

Nitrogen (N) is a vital nutrient and an essential component of biological macromolecules, such as nucleic acids and proteins. Microorganisms represent major drivers of N-cycling processes in all ecosystems, including the soil and plant environment. The availability of N is a major growth limiting factor for plants and it is significantly affected by the plant microbiome. Plants and microorganisms form complex interaction networks resulting in molecular signaling, nutrient exchange and other distinct metabolic responses. In these networks, microbial partners influence growth and N use efficiency of plants either positively or negatively. Harnessing the beneficial effects of specific players within crop microbiomes is a promising strategy to counteract the emerging threats for human and planetary health due to the overuse of industrial N fertilizers. However, in addition to N-providing activities (e.g. the well-known symbiosis of legumes and Rhizobium bacteria), other plant-microorganism interactions must be considered to obtain a complete picture of how microbial driven N-transformations might affect plant nutrition. For this, we review recent insights into the tight interplay between plants and N-cycling microorganisms focusing on microbial N-transformation processes representing N sources and sinks that ultimately shape the plant N acquisition.

Some like it cold: the cellular organization and physiological limits of cold-tolerant nitrite-oxidizing Nitrotoga.

Chemolithoautotrophic production of nitrate is accomplished by the polyphyletic functional group of nitrite-oxidizing bacteria (NOB). A widely distributed and important NOB clade in nitrogen removal processes at low temperatures is Nitrotoga, which however remains understudied due to the scarcity of cultivated representatives. Here, we present physiological, ultrastructural and genomic features of Nitrotoga strains from various habitats, including the first marine species enriched from an aquaculture system. Immunocytochemical analyses localized the nitrite-oxidizing enzyme machinery in the wide irregularly shaped periplasm, apparently without contact to the cytoplasmic membrane, confirming previous genomic data suggesting a soluble nature. Interestingly, in two strains we also observed multicellular complexes with a shared periplasmic space, which seem to form through incomplete cell division and might enhance fitness or survival. Physiological tests revealed differing tolerance limits towards dissolved inorganic nitrogen concentrations and confirmed the generally psychrotolerant nature of the genus. Moreover, comparative analysis of 15 Nitrotoga genomes showed, e.g. a unique gene repertoire of the marine strain that could be advantageous in its natural habitat and confirmed the lack of genes for assimilatory nitrite reduction in a strain found to require ammonium for growth. Overall, these novel insights largely broaden our knowledge of Nitrotoga and elucidate the metabolic variability, physiological limits and thus potential ecological roles of this group of nitrite oxidizers.

Sweet spheres: succession and CAZyme expression of marine bacterial communities colonizing a mix of alginate and pectin particles.

Polysaccharide particles are important substrates and microhabitats for marine bacteria. However, substrate-specific bacterial dynamics in mixtures of particle types with different polysaccharide composition, as likely occurring in natural habitats, are undescribed. Here, we studied the composition, functional diversity and gene expression of marine bacterial communities colonizing a mix of alginate and pectin particles. Amplicon, metagenome and metatranscriptome sequencing revealed that communities on alginate and pectin particles significantly differed from their free-living counterparts. Unexpectedly, microbial dynamics on alginate and pectin particles were similar, with predominance of amplicon sequence variants (ASVs) from Tenacibaculum, Colwellia, Psychrobium and Psychromonas. Corresponding metagenome-assembled genomes (MAGs) expressed diverse alginate lyases, several colocalized in polysaccharide utilization loci. Only a single, low-abundant MAG showed elevated transcript abundances of pectin-degrading enzymes. One specific Glaciecola ASV dominated the free-living fraction, possibly persisting on particle-derived oligomers through different glycoside hydrolases. Elevated ammonium uptake and metabolism signified nitrogen as an important factor for degrading carbon-rich particles, whereas elevated methylcitrate and glyoxylate cycles suggested nutrient limitation in surrounding waters. The bacterial preference for alginate, whereas pectin primarily served as colonization scaffold, illuminates substrate-driven dynamics within mixed polysaccharide pools. These insights expand our understanding of bacterial niche specialization and the biological carbon pump in macroalgae-rich habitats.

Cyanate as an energy source for nitrifiers.

Ammonia- and nitrite-oxidizing microorganisms are collectively responsible for the aerobic oxidation of ammonia via nitrite to nitrate and have essential roles in the global biogeochemical nitrogen cycle. The physiology of nitrifiers has been intensively studied, and urea and ammonia are the only recognized energy sources that promote the aerobic growth of ammonia-oxidizing bacteria and archaea. Here we report the aerobic growth of a pure culture of the ammonia-oxidizing thaumarchaeote Nitrososphaera gargensis using cyanate as the sole source of energy and reductant; to our knowledge, the first organism known to do so. Cyanate, a potentially important source of reduced nitrogen in aquatic and terrestrial ecosystems, is converted to ammonium and carbon dioxide in Nitrososphaera gargensis by a cyanase enzyme that is induced upon addition of this compound. Within the cyanase gene family, this cyanase is a member of a distinct clade also containing cyanases of nitrite-oxidizing bacteria of the genus Nitrospira. We demonstrate by co-culture experiments that these nitrite oxidizers supply cyanase-lacking ammonia oxidizers with ammonium from cyanate, which is fully nitrified by this microbial consortium through reciprocal feeding. By screening a comprehensive set of more than 3,000 publically available metagenomes from environmental samples, we reveal that cyanase-encoding genes clustering with the cyanases of these nitrifiers are widespread in the environment. Our results demonstrate an unexpected metabolic versatility of nitrifying microorganisms, and suggest a previously unrecognized importance of cyanate in cycling of nitrogen compounds in the environment.

Antagonistic species interaction drives selection for sex in a predator-prey system.

The evolutionary maintenance of sexual reproduction has long challenged biologists as the majority of species reproduce sexually despite inherent costs. Providing a general explanation for the evolutionary success of sex has thus proven difficult and resulted in numerous hypotheses. A leading hypothesis suggests that antagonistic species interaction can generate conditions selecting for increased sex due to the production of rare or novel genotypes that are beneficial for rapid adaptation to recurrent environmental change brought on by antagonism. To test this ecology-based hypothesis, we conducted experimental evolution in a predator (rotifer)-prey (algal) system by using continuous cultures to track predator-prey dynamics and in situ rates of sex in the prey over time and within replicated experimental populations. Overall, we found that predator-mediated fluctuating selection for competitive versus defended prey resulted in higher rates of genetic mixing in the prey. More specifically, our results showed that fluctuating population sizes of predator and prey, coupled with a trade-off in the prey, drove the sort of recurrent environmental change that could provide a benefit to sex in the prey, despite inherent costs. We end with a discussion of potential population genetic mechanisms underlying increased selection for sex in this system, based on our application of a general theoretical framework for measuring the effects of sex over time, and interpreting how these effects can lead to inferences about the conditions selecting for or against sexual reproduction in a system with antagonistic species interaction.

Metagenomic recovery of two distinct comammox Nitrospira from the terrestrial subsurface.

The recently discovered comammox process encompasses both nitrification steps, the aerobic oxidation of ammonia and nitrite, in a single organism. All known comammox bacteria are affiliated with Nitrospira sublineage II and can be grouped into two distinct clades, referred to as A and B, based on ammonia monooxygenase phylogeny. In this study, we report high-quality draft genomes of two novel comammox Nitrospira from the terrestrial subsurface, representing one clade A and one clade B comammox organism. The two metagenome-assembled genomes were compared with other representatives of Nitrospira sublineage II, including both canonical and comammox Nitrospira. Phylogenomic analyses confirmed the affiliation of the two novel Nitrospira with comammox clades A and B respectively. Based on phylogenetic distance and pairwise average nucleotide identity values, both comammox Nitrospira were classified as novel species. Genomic comparison revealed high conservation of key metabolic features in sublineage II Nitrospira, including respiratory complexes I-V and the machineries for nitrite oxidation and carbon fixation via the reductive tricarboxylic acid cycle. In addition, the presence of the enzymatic repertoire for formate and hydrogen oxidation in the Rifle clades A and B comammox genomes, respectively, suggest a broader distribution of these metabolic features than previously anticipated.

High-speed, inline measurement of protein activity and inactivation processes by supercontinuum attenuation spectroscopy.

Some proteins such as catalase and glutamate dehydrogenase (GDH) are very sensitive to external factors such as irradiation or heat, which may cause inactivation. Since proteins are used in a wide field of applications, the entire activity has to be ensured during the whole process. By default, activity is measured by invasive and offline activity assays. To avoid the need for a time-consuming offline analysis, we developed an optical high-speed measurement technique, which may form the basis for the non-invasive inline control of enzyme processes e.g. in the textile or food industry. The technique is based on attenuation spectroscopy using a supercontinuum laser source in combination with multivariate data analysis, in particular partial least squares regression (PLSR). For verification of the approach, samples treated by various stresses were analyzed in parallel by activity assays and our new method. Applying this technique, we were able to determine the activity in the turbid catalase samples after heat treatment, addition of guanidine-HCl or irradiation with UV light by applying partial least squares regression. Furthermore, we demonstrate that the combination of broadband attenuation spectroscopy and PLSR enables us to determine also the activity of GDH in clear solutions after heat treatment.

Complete nitrification: insights into the ecophysiology of comammox Nitrospira.

Nitrification, the oxidation of ammonia via nitrite to nitrate, has been considered to be a stepwise process mediated by two distinct functional groups of microorganisms. The identification of complete nitrifying Nitrospira challenged not only the paradigm of labor division in nitrification, it also raises fundamental questions regarding the environmental distribution, diversity, and ecological significance of complete nitrifiers compared to canonical nitrifying microorganisms. Recent genomic and physiological surveys identified factors controlling their ecology and niche specialization, which thus potentially regulate abundances and population dynamics of the different nitrifying guilds. This review summarizes the recently obtained insights into metabolic differences of the known nitrifiers and discusses these in light of potential functional adaptation and niche differentiation between canonical and complete nitrifiers.

Expanded metabolic versatility of ubiquitous nitrite-oxidizing bacteria from the genus Nitrospira.

Nitrospira are a diverse group of nitrite-oxidizing bacteria and among the environmentally most widespread nitrifiers. However, they remain scarcely studied and mostly uncultured. Based on genomic and experimental data from Nitrospira moscoviensis representing the ubiquitous Nitrospira lineage II, we identified ecophysiological traits that contribute to the ecological success of Nitrospira. Unexpectedly, N. moscoviensis possesses genes coding for a urease and cleaves urea to ammonia and CO2. Ureolysis was not observed yet in nitrite oxidizers and enables N. moscoviensis to supply ammonia oxidizers lacking urease with ammonia from urea, which is fully nitrified by this consortium through reciprocal feeding. The presence of highly similar urease genes in Nitrospira lenta from activated sludge, in metagenomes from soils and freshwater habitats, and of other ureases in marine nitrite oxidizers, suggests a wide distribution of this extended interaction between ammonia and nitrite oxidizers, which enables nitrite-oxidizing bacteria to indirectly use urea as a source of energy. A soluble formate dehydrogenase lends additional ecophysiological flexibility and allows N. moscoviensis to use formate, with or without concomitant nitrite oxidation, using oxygen, nitrate, or both compounds as terminal electron acceptors. Compared with Nitrospira defluvii from lineage I, N. moscoviensis shares the Nitrospira core metabolism but shows substantial genomic dissimilarity including genes for adaptations to elevated oxygen concentrations. Reciprocal feeding and metabolic versatility, including the participation in different nitrogen cycling processes, likely are key factors for the niche partitioning, the ubiquity, and the high diversity of Nitrospira in natural and engineered ecosystems.

NxrB encoding the beta subunit of nitrite oxidoreductase as functional and phylogenetic marker for nitrite-oxidizing Nitrospira.

Nitrospira are the most widespread and diverse known nitrite-oxidizing bacteria and key nitrifiers in natural and engineered ecosystems. Nevertheless, their ecophysiology and environmental distribution are understudied because of the recalcitrance of Nitrospira to cultivation and the lack of a molecular functional marker, which would allow the detection of Nitrospira in the environment. Here we introduce nxrB, the gene encoding subunit beta of nitrite oxidoreductase, as a functional and phylogenetic marker for Nitrospira. Phylogenetic trees based on nxrB of Nitrospira were largely congruent to 16S ribosomal RNA-based phylogenies. By using new nxrB-selective polymerase chain reaction primers, we obtained almost full-length nxrB sequences from Nitrospira cultures, two activated sludge samples, and several geographically and climatically distinct soils. Amplicon pyrosequencing of nxrB fragments from 16 soils revealed a previously unrecognized diversity of terrestrial Nitrospira with 1801 detected species-level operational taxonomic units (OTUs) (using an inferred species threshold of 95% nxrB identity). Richness estimates ranged from 10 to 946 coexisting Nitrospira species per soil. Comparison with an archaeal amoA dataset obtained from the same soils [Environ. Microbiol. 14: 525-539 (2012)] uncovered that ammonia-oxidizing archaea and Nitrospira communities were highly correlated across the soil samples, possibly indicating shared habitat preferences or specific biological interactions among members of these nitrifier groups.

Metabolic and phylogenetic diversity in the phylum Nitrospinota revealed by comparative genome analyses.

The most abundant known nitrite-oxidizing bacteria in the marine water column belong to the phylum Nitrospinota. Despite their importance in marine nitrogen cycling and primary production, there are only few cultured representatives that all belong to the class Nitrospinia. Moreover, although Nitrospinota were traditionally thought to be restricted to marine environments, metagenome-assembled genomes have also been recovered from groundwater. Over the recent years, metagenomic sequencing has led to the discovery of several novel classes of Nitrospinota (UBA9942, UBA7883, 2-12-FULL-45-22, JACRGO01, JADGAW01), which remain uncultivated and have not been analyzed in detail. Here, we analyzed a nonredundant set of 98 Nitrospinota genomes with focus on these understudied Nitrospinota classes and compared their metabolic profiles to get insights into their potential role in biogeochemical element cycling. Based on phylogenomic analysis and average amino acid identities, the highly diverse phylum Nitrospinota could be divided into at least 33 different genera, partly with quite distinct metabolic capacities. Our analysis shows that not all Nitrospinota are nitrite oxidizers and that members of this phylum have the genomic potential to use sulfide and hydrogen for energy conservation. This study expands our knowledge of the phylogeny and potential ecophysiology of the phylum Nitrospinota and offers new avenues for the isolation and cultivation of these elusive bacteria.

Unveiling unique microbial nitrogen cycling and nitrification driver in coastal Antarctica.

Largely removed from anthropogenic delivery of nitrogen (N), Antarctica has notably low levels of nitrogen. Though our understanding of biological sources of ammonia have been elucidated, the microbial drivers of nitrate (NO3-) cycling in coastal Antarctica remains poorly understood. Here, we explore microbial N cycling in coastal Antarctica, unraveling the biological origin of NO3- via oxygen isotopes in soil and lake sediment, and through the reconstruction of 1968 metagenome-assembled genomes from 29 microbial phyla. Our analysis reveals the metabolic potential for microbial N2 fixation, nitrification, and denitrification, but not for anaerobic ammonium oxidation, signifying a unique microbial N-cycling dynamic. We identify the predominance of complete ammonia oxidizing (comammox) Nitrospira, capable of performing the entire nitrification process. Their adaptive strategies to the Antarctic environment likely include synthesis of trehalose for cold stress, high substrate affinity for resource utilization, and alternate metabolic pathways for nutrient-scarce conditions. We confirm the significant role of comammox Nitrospira in the autotrophic, nitrification process via 13C-DNA-based stable isotope probing. This research highlights the crucial contribution of nitrification to the N budget in coastal Antarctica, identifying comammox Nitrospira clade B as a nitrification driver.

Functional Investigation of IGF1R Mutations in Multiple Myeloma.

High expression of the receptor tyrosine kinase (RTK) insulin-like growth factor-1 receptor (IGF1R) and RTK mutations are associated with high-risk/worse prognosis in multiple myeloma (MM). Combining the pIGF1R/pINSR inhibitor linsitinib with the proteasome inhibitor (PI) bortezomib seemed promising in a clinical trial, but IGF1R expression was not associated with therapy response. Because the oncogenic impact of IGF1R mutations is so far unknown, we investigated the functional impact of IGF1R mutations on survival signaling, viability/proliferation and survival response to therapy. We transfected four human myeloma cell lines (HMCLs) with IGF1RWT, IGF1RD1146N and IGF1RN1129S (Sleeping Beauty), generated CRISPR-Cas9 IGF1R knockouts in the HMCLs U-266 (IGF1RWT) and L-363 (IGF1RD1146N) and tested the anti-MM activity of linsitinib alone and in combination with the second-generation PI carfilzomib in seven HMCLs. IGF1R knockout entailed reduced proliferation. Upon IGF1R overexpression, survival signaling was moderately increased in all HCMLs and slightly affected by IGF1RN1129S in one HMCL, whereby the viability remained unaffected. Expression of IGF1RD1146N reduced pIGF1R-Y1135, especially under serum reduction, but did not impact downstream signaling. Linsitinib and carfilzomib showed enhanced anti-myeloma activity in six out of seven HMCL irrespective of the IGF1R mutation status. In conclusion, IGF1R mutations can impact IGF1R activation and/or downstream signaling, and a combination of linsitinib with carfilzomib might be a suitable therapeutic approach for MM patients potentially responsive to IGF1R blockade.

A Nitrospira metagenome illuminates the physiology and evolution of globally important nitrite-oxidizing bacteria.

Nitrospira are barely studied and mostly uncultured nitrite-oxidizing bacteria, which are, according to molecular data, among the most diverse and widespread nitrifiers in natural ecosystems and biological wastewater treatment. Here, environmental genomics was used to reconstruct the complete genome of "Candidatus Nitrospira defluvii" from an activated sludge enrichment culture. On the basis of this first-deciphered Nitrospira genome and of experimental data, we show that Ca. N. defluvii differs dramatically from other known nitrite oxidizers in the key enzyme nitrite oxidoreductase (NXR), in the composition of the respiratory chain, and in the pathway used for autotrophic carbon fixation, suggesting multiple independent evolution of chemolithoautotrophic nitrite oxidation. Adaptations of Ca. N. defluvii to substrate-limited conditions include an unusual periplasmic NXR, which is constitutively expressed, and pathways for the transport, oxidation, and assimilation of simple organic compounds that allow a mixotrophic lifestyle. The reverse tricarboxylic acid cycle as the pathway for CO2 fixation and the lack of most classical defense mechanisms against oxidative stress suggest that Nitrospira evolved from microaerophilic or even anaerobic ancestors. Unexpectedly, comparative genomic analyses indicate functionally significant lateral gene-transfer events between the genus Nitrospira and anaerobic ammonium-oxidizing planctomycetes, which share highly similar forms of NXR and other proteins reflecting that two key processes of the nitrogen cycle are evolutionarily connected.

A novel Nitrospira lineage isolated from activated sludge using elevated temperatures.

The genus Nitrospira represents the dominant nitrite-oxidizing clade in most wastewater treatment plants (WWTPs) globally, and several Nitrospira strains have been isolated from activated sludge. Using a pre-enrichment strategy with alternating nitrifying and denitrifying conditions, followed by incubation at elevated temperatures, we isolated a novel Nitrospira species, named Nitrospira tepida. This moderately thermophilic species with optimal growth between 37 and 45°C is only distantly related to other Nitrospira and forms a novel lineage VII within the genus, together with few environmental 16S rRNA gene sequences predominantly detected in thermal wastewater or oxygen-limited systems. Genomic and physiological analyses revealed remarkable differences between N. tepida and two other isolates previously obtained from the same WWTP, suggesting niche differentiation between these nitrite oxidizers. N. tepida grows in aggregates, and tolerates nitrite and nitrate concentrations of up to 20 mM and 40 mM, respectively. The Km value for nitrite of N. tepida is 77 ± 26 µM. In summary, this novel Nitrospira lineage seems to be well-adapted for wastewater treatment processes at elevated temperatures and limited aeration, conditions that potentially reduce operational costs of such systems.

Complete Genome Sequence of Nitrospina watsonii 347, Isolated from the Black Sea.

Here, we present the complete genome sequence of Nitrospina watsonii 347, a nitrite-oxidizing bacterium isolated from the Black Sea at a depth of 100 m. The genome has a length of 3,011,914 bp with 2,895 predicted coding sequences. Its predicted metabolism is similar to that of Nitrospina gracilis with differences in defense against reactive oxygen species.

Towards a More Comprehensive Picture of the MicroRNA-23a/b-3p Impact on Impaired Male Fertility.

The expression levels of various genes involved in human spermatogenesis are influenced by microRNAs (miRNAs), specifically microRNA-23a/b-3p. While certain genes are essential for spermatogenesis and male germ cell function, the regulation of their expression remains unclear. This study aimed to investigate whether microRNA-23a/b-3p targets genes involved in spermatogenesis and the impact of this targeting on the expression levels of these genes in males with impaired fertility. In-silico prediction and dual-luciferase assays were used to determine the potential connections between microRNA-23a/b-3p overexpression and reduced expression levels of 16 target genes. Reverse transcription-quantitative PCR (RT-qPCR) was conducted on 41 oligoasthenozoospermic men receiving infertility treatment and 41 age-matched normozoospermic individuals to verify the lower expression level of target genes. By employing dual-luciferase assays, microRNA-23a-3p was found to directly target eight genes, namely NOL4, SOX6, GOLGA6C, PCDHA9, G2E3, ZNF695, CEP41, and RGPD1, while microRNA-23b-3p directly targeted three genes, namely SOX6, GOLGA6C, and ZNF695. The intentional alteration of the microRNA-23a/b binding site within the 3' untranslated regions (3'UTRs) of the eight genes resulted in the loss of responsiveness to microRNA-23a/b-3p. This confirmed that NOL4, SOX6, GOLGA6C, PCDHA9, and CEP41 are direct targets for microRNA-23a-3p, while NOL4, SOX6, and PCDHA9 are direct targets for microRNA-23b-3p. The sperm samples of oligoasthenozoospermic men had lower expression levels of target genes than age-matched normozoospermic men. Correlation analysis indicated a positive correlation between basic semen parameters and lower expression levels of target genes. The study suggests that microRNA-23a/b-3p plays a significant role in spermatogenesis by controlling the expression of target genes linked to males with impaired fertility and has an impact on basic semen parameters.

Trichlorobacter ammonificans, a dedicated acetate-dependent ammonifier with a novel module for dissimilatory nitrate reduction to ammonia.

Dissimilatory nitrate reduction to ammonia (DNRA) is a common biochemical process in the nitrogen cycle in natural and man-made habitats, but its significance in wastewater treatment plants is not well understood. Several ammonifying Trichlorobacter strains (former Geobacter) were previously enriched from activated sludge in nitrate-limited chemostats with acetate as electron (e) donor, demonstrating their presence in these systems. Here, we isolated and characterized the new species Trichlorobacter ammonificans strain G1 using a combination of low redox potential and copper-depleted conditions. This allowed purification of this DNRA organism from competing denitrifiers. T. ammonificans is an extremely specialized ammonifier, actively growing only with acetate as e-donor and carbon source and nitrate as e-acceptor, but H2 can be used as an additional e-donor. The genome of G1 does not encode the classical ammonifying modules NrfAH/NrfABCD. Instead, we identified a locus encoding a periplasmic nitrate reductase immediately followed by an octaheme cytochrome c that is conserved in many Geobacteraceae species. We purified this octaheme cytochrome c protein (TaNiR), which is a highly active dissimilatory ammonifying nitrite reductase loosely associated with the cytoplasmic membrane. It presumably interacts with two ferredoxin subunits (NapGH) that donate electrons from the menaquinol pool to the periplasmic nitrate reductase (NapAB) and TaNiR. Thus, the Nap-TaNiR complex represents a novel type of highly functional DNRA module. Our results indicate that DNRA catalyzed by octaheme nitrite reductases is a metabolic feature of many Geobacteraceae, representing important community members in various anaerobic systems, such as rice paddy soil and wastewater treatment facilities.

The coral symbiont Candidatus Aquarickettsia is variably abundant in threatened Caribbean acroporids and transmitted horizontally.

The symbiont "Candidatus Aquarickettsia rohweri" infects a diversity of aquatic hosts. In the threatened Caribbean coral, Acropora cervicornis, Aquarickettsia proliferates in response to increased nutrient exposure, resulting in suppressed growth and increased disease susceptibility and mortality of coral. This study evaluated the extent, as well as the ecology and evolution of Aquarickettsia infecting threatened corals, Ac. cervicornis, and Ac. palmata and their hybrid ("Ac. prolifera"). Aquarickettsia was found in all acroporids, with coral host and geographic location impacting the infection magnitude. Phylogenomic and genome-wide single-nucleotide variant analysis of Aquarickettsia found phylogenetic clustering by geographic region, not by coral taxon. Analysis of Aquarickettsia fixation indices suggests multiple sequential infections of the same coral colony are unlikely. Furthermore, relative to other Rickettsiales species, Aquarickettsia is undergoing positive selection, with Florida populations experiencing greater positive selection relative to other Caribbean locations. This may be due in part to Aquarickettsia proliferating in response to greater nutrient stress in Florida, as indicated by greater in situ replication rates in these corals. Aquarickettsia was not found to significantly codiversify with either the coral animal or the coral's algal symbiont (Symbiodinium "fitti"). Quantitative PCR analysis showed that gametes, larvae, recruits, and juveniles from susceptible, captive-reared coral genets were not infected with Aquarickettsia. Thus, horizontal transmission of Aquarickettsia via coral mucocytes or an unidentified host is more likely. The prevalence of Aquarickettsia in Ac. cervicornis and its high abundance in the Florida coral population suggests that coral disease mitigation efforts focus on preventing early infection via horizontal transmission.