Heat stress reduces the contribution of diazotrophs to coral holobiont nitrogen cycling

Efficient nutrient cycling in the coral-algal symbiosis requires constant but limited nitrogen availability. Coral-associated diazotrophs, i.e., prokaryotes capable of fixing dinitrogen, may thus support productivity in a stable coral-algal symbiosis but could contribute to its breakdown when overstimulated. However, the effects of environmental conditions on diazotroph communities and their interaction with other members of the coral holobiont remain poorly understood. Here we assessed the effects of heat stress on diazotroph diversity and their contribution to holobiont nutrient cycling in the reef-building coral Stylophora pistillata from the central Red Sea. In a stable symbiotic state, we found that nitrogen fixation by coral-associated diazotrophs constitutes a source of nitrogen to the algal symbionts. Heat stress caused an increase in nitrogen fixation concomitant with a change in diazotroph communities. Yet, this additional fixed nitrogen was not assimilated by the coral tissue or the algal symbionts. We conclude that although diazotrophs may support coral holobiont functioning under low nitrogen availability, altered nutrient cycling during heat stress abates the dependence of the coral host and its algal symbionts on diazotroph-derived nitrogen. Consequently, the role of nitrogen fixation in the coral holobiont is strongly dependent on its nutritional status and varies dynamically with environmental conditions.Subject terms: Microbial ecology, Climate-change ecology     

Endophytic fungus Serendipita indica increased nutrition absorption and biomass accumulation in Cunninghamia lanceolata seedlings under low phosphate

Cunninghamia lanceolata is important forest tree species in southern China, and its successive plantations resulted in degradation of soil fertility in pure stands, causing decline in forest productivity. How to improve productivity in C. lanceolata pure stands is a tough task. Usage of mycorrhizal fungi might be a plausible access to the task. The objective is to study the possibility of the endophytic fungus Serendipita indica (named formerly as Piriformospora indica) in culture of C. lanceolata. Seeds were sowed in plastic pots with river sand. When seedlings had two true leaves, hyphae suspension solution of S. indica was added to near the roots of seedlings in each plastic pot. Such pots with seedlings were placed in a greenhouse and normal management was carried out for the seedlings. Symbiosis effects on root development, nutrition uptake and allocation, and biomass accumulation of C. lanceolata seedlings under low phosphate were investigated. The results showed that S. indica could symbiose with C. lanceolata. The symbiosis did not result in significant changes in root system architecture under low phosphate, but significantly increased nitrogen and phosphorus levels in leaves under low phosphate. Although the symbiosis did not significantly increased nitrogen allocation in leaves under low phosphate, it significantly increased phosphorus allocation in leaves. The interaction between S. indica and C. lanceolata resulted in increase in total biomass under low phosphate and changes in biomass allocation between shoots and roots. The results suggested that S. indica helps host plants to absorb more nutrients under low phosphate and to allocate more nitrogen and phosphate to leaves, promoting plant growth; the fungus might be used in pure stands of C. lanceolata because of its large-scaled axenic culture.     

Effects of Inoculum Level and Time of Microcoleus vaginatus on Control of Meloidogyne incognita on Tomato

Application of Microcoleus vaginatus, a blue-green alga (Cyanobacterium) at different levels along with Meloidogyne incognita, second stage larvae, in the rhizosphere of tomato plants; showed that the plant growth as well as yield of tomato were increased and gall formations and nematode populations decreased with the increase in inoculum level of M. vaginatus. An inoculum level of 20 ml endospores suspension of M. vaginatus (2.4 × 10⁶ endospores per ml) per plant was optimum to reduce nematode attack with a population density of 1000 larvae per kg soil. Plant growth and yield of fruits were greatly suppressed and gall formations on roots, and nematode populations in soil were increased when M. incognita larvae added five days prior to M. vaginatus inoculation. On the other hand, when M. vaginatus inoculated ten days before nematode inoculation, suppressive effect of M. incognta on plants was reduced and their population density as well as gall formations were also decreased significantly. The efficacy of simultaneous inoculation of both nematode and M. vaginatus was lied in between two treatments discussed above.     

Characterization of htrB and msbB Mutants of the Light Organ Symbiont Vibrio fischeri

Bacterial lipid A is an important mediator of bacterium-host interactions, and secondary acylations added by HtrB and MsbB can be critical for colonization and virulence in pathogenic infections. In contrast, Vibrio fischeri lipid A stimulates normal developmental processes in this bacterium's mutualistic host, Euprymna scolopes, although the importance of lipid A structure in this symbiosis is unknown. To further examine V. fischeri lipid A and its symbiotic function, we identified two paralogs of htrB (designated htrB1 and htrB2) and an msbB gene in V. fischeri ES114 and demonstrated that these genes encode lipid A secondary acyltransferases. htrB2 and msbB are found on the Vibrio “housekeeping” chromosome 1 and are conserved in other Vibrio species. Mutations in htrB2 and msbB did not impair symbiotic colonization but resulted in phenotypic alterations in culture, including reduced motility and increased luminescence. These mutations also affected sensitivity to sodium dodecyl sulfate, kanamycin, and polymyxin, consistent with changes in membrane permeability. Conversely, htrB1 is located on the smaller, more variable vibrio chromosome 2, and an htrB1 mutant was wild-type-like in culture but appeared attenuated in initiating the symbiosis and was outcompeted 2.7-fold during colonization when mixed with the parent. These data suggest that htrB2 and msbB play conserved general roles in vibrio biology, whereas htrB1 plays a more symbiosis-specific role in V. fischeri.     

A Brazilian Population of the Asexual Fungus-Growing Ant Mycocepurus smithii (Formicidae,Myrmicinae, Attini) Cultivates Fungal Symbionts with Gongylidia-Like Structures

Attine ants cultivate fungi as their most important food source and in turn the fungus is nourished, protected against harmful microorganisms, and dispersed by the ants. This symbiosis evolved approximately 50–60 million years ago in the late Paleocene or early Eocene, and since its origin attine ants have acquired a variety of fungal mutualists in the Leucocoprineae and the distantly related Pterulaceae. The most specialized symbiotic interaction is referred to as “higher agriculture” and includes leafcutter ant agriculture in which the ants cultivate the single species Leucoagaricus gongylophorus. Higher agriculture fungal cultivars are characterized by specialized hyphal tip swellings, so-called gongylidia, which are considered a unique, derived morphological adaptation of higher attine fungi thought to be absent in lower attine fungi. Rare reports of gongylidia-like structures in fungus gardens of lower attines exist, but it was never tested whether these represent rare switches of lower attines to L. gonglyphorus cultivars or whether lower attine cultivars occasionally produce gongylidia. Here we describe the occurrence of gongylidia-like structures in fungus gardens of the asexual lower attine ant Mycocepurus smithii. To test whether M. smithii cultivates leafcutter ant fungi or whether lower attine cultivars produce gongylidia, we identified the M. smithii fungus utilizing molecular and morphological methods. Results shows that the gongylidia-like structures of M. smithii gardens are morphologically similar to gongylidia of higher attine fungus gardens and can only be distinguished by their slightly smaller size. A molecular phylogenetic analysis of the fungal ITS sequence indicates that the gongylidia-bearing M. smithii cultivar belongs to the so-called “Clade 1”of lower Attini cultivars. Given that M. smithii is capable of cultivating a morphologically and genetically diverse array of fungal symbionts, we discuss whether asexuality of the ant host maybe correlated with low partner fidelity and active symbiont choice between fungus and ant mutualists.     

Simultaneous Nitrification and Denitrification in Aerobic Chemostat Cultures of Thiosphaera pantotropha

Thiosphaera pantotropha is capable of simultaneous heterotrophic nitrification and aerobic denitrification. Consequently, its nitrification potential could not be judged from nitrite accumulation, but was estimated from complete nitrogen balances. The maximum rate of nitrification obtained during these experiments was 93.9 nmol min⁻¹ mg of protein⁻¹. The nitrification rate could be reduced by the provision of nitrate, nitrite, or thiosulfate to the culture medium. Both nitrification and denitrification increased as the dissolved oxygen concentration fell, until a critical level was reached at approximately 25% of air saturation. At this point, the rate of (aerobic) denitrification was equivalent to the anaerobic rate. At this dissolved oxygen concentration, the combined nitrification and denitrification was such that cultures receiving ammonium as their sole source of nitrogen appeared to become oxygen limited and the nitrification rate fell. It appeared that, under carbon-and energy-limited conditions, a high nitrification rate was correlated with a reduced biomass yield. To facilitate experimental design, a working hypothesis for the mechanism behind nitrification and denitrification by T. pantotropha was formulated. This involved the basic assumption that this species has a “bottleneck” in its cytochrome chain to oxygen and that denitrification and nitrification are used to overcome this. The nitrification potential of other heterotrophic nitrifiers has been reconsidered. Several species considered to be “poor” nitrifiers also simultaneously nitrify and denitrify, thus giving a falsely low nitrification potential.     

Simultaneous removal of inorganic nutrients and organic carbon by symbiotic co-culture of <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> and <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

The co-culture system of photosynthetic microalgae Chlorella vulgaris and aerobic heterotrophic bacteria Pseudomonas putida was investigated as a possible combination of symbiotic mixed culture for the simultaneous removal of nutrients (ammonium and phosphate) and organic contaminants. Using synthetic municipal wastewater, the co-culture system exhibited symbiotic enhancement in the removal of nutrients and organic carbon compared to each of axenic cultures. The co-culture system performed successfully in removing both of ammonium and chemical oxygen demand (COD), showing around 80% removal for 4 days. Strategies of nitrogen and phosphorous starvation in C. vulgaris for two days prior to main treatment did not increase the performance of nutrients removal, indicating that the nutrient starvation as a pretreatment is unnecessary. Without alkalinity (as bicarbonate), nutrients and COD were not removed significantly, implying that the existence of alkalinity is essential for symbiotic treatment of both nutrients and organics. Results demonstrated that coculture system composed of C. vulgaris and P. putida can be a potential candidate of mixed culture system for the simultaneous removal of nutrients and organic carbon in wastewater treatment using a single reactor.     

Development of a Quantitative Assay for Mycobacterial Endogenous Arabinase and Ensuing Studies of Arabinase Levels and Arabinan Metabolism in Mycobacterium smegmatis

Treatment of either Mycobacterium tuberculosis or M. smegmatis with ethambutol results both in inhibition of arabinan synthesis and in copious loss of previously formed arabinan from the cell wall. The loss of arabinan has been shown to be due to the action of an endogenous arabinase. To better understand this phenomenon, a quantitative assay for endogenous arabinase was developed. Using the assay it was determined that various subcellular fractions of M. smegmatis showed significant amounts of endogenous arabinase activity. Surprisingly, treatment with ethambutol yielded only minor changes in the amounts of endogenous arabinase activities. Endogenous arabinase was present in the cell wall, and consistently, incubation of the M. smegmatis cell wall in only buffer resulted in the release of arabinan, mimicking the effect of ethambutol on whole cells. To determine if cell wall arabinan is rapidly turned over, the arabinan was labeled in the early log phase of culture by feeding [¹⁴C]glucose, followed by a “chase” with nonradioactive glucose. Most of the labeled arabinan remained in the cell wall after the culture was grown to late log phase. Thus, there is active arabinase in the cell wall, but arabinan is not rapidly removed unless ethambutol is present. Purification of the endogenous arabinase, using the assay described, is ongoing to help further discern its biological function.     

Bone Marrow Mesenchymal Stem Cells Attenuate Mitochondria Damage Induced by Hypoxia in Mouse Trophoblasts

ObjectiveWe aimed to observe the change of mitochondrial function and structure as well as the cell function induced by hypoxia in mouse trophoblasts, and moreover, to validate the restoration of these changes after co-culture with bone marrow mesenchymal stem cells (hereinafter referred to as “MSCs”). Further, we explored the mechanism of MSCs attenuating the functional damage of trophoblasts caused by hypoxia.MethodsCells were divided into two groups, trophoblasts and MSCs+trophoblasts respectively, and the two groups of cells were incubated with normoxia or hypoxia. Chemiluminescence was used to assay the β-HCG and progesterone in cell culture supernatants quantitatively. Western blotting and PCR were applied to detect the expression of Mfn2, MMP-2, MMP-9 and integrin β1 in the two groups. The mitochondrial membrane potential of each group of cells was detected with JC-1 dye and the ATP content was measured by the phosphomolybdic acid colorimetric method. We utilized transmission electron microscopy for observing the ultrastructure of mitochondria in trophoblasts. Finally, we assessed the cell apoptosis with flow cytometry (FCM) and analyzed the expression of the apoptosis related genes—Bcl-2, Bax, Caspase3 and Caspase9 by western blotting.ResultsThe results showed that the Mfn2 expression was reduced after 4 h in hypoxia compared with that in normoxia, but increased in the co-culture group when compared with that in the separated-culture group (p<0.05). In addition, compared with the separated-culture group, theβ-HCG and progesterone levels in the co-culture group were significantly enhanced (p<0.05), and so were the expressions of MMP-2, MMP-9 and integrin β1 (p<0.05). Moreover, it exhibited significantly higher in ATP levels and intensified about the mitochondrial membrane potential in the co-culture group. TEM revealed disorders of the mitochondrial cristae and presented short rod-like structure and spheroids in hypoxia, however, in the co-culture group, the mitochondrial cristae had a relatively regular arrangement and the mitochondrial ultrastructure showed hyperfusion. The expression of Bax, Caspase3 and Caspase9 was decreased in the co-culture group when compared with that in trophoblast cells cultured alone (p<0.05), while the Bcl-2 levels and the Apoptosis Index (AI) were markedly increased in the co-culture group (p<0.05).ConclusionBone marrow mesenchymal stem cells can attenuate mitochondria damage and cell apoptosis induced by hypoxia; the mechanism could be upregulating the expression of Mfn2 in mouse trophoblasts and changing mitochondrial structure.     

Molecular Analysis of Diazotroph Diversity in the Rhizosphere of the Smooth Cordgrass, Spartina alterniflora

N₂ fixation by diazotrophic bacteria associated with the roots of the smooth cordgrass, Spartina alterniflora, is an important source of new nitrogen in many salt marsh ecosystems. However, the diversity and phylogenetic affiliations of these rhizosphere diazotrophs are unknown. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified nifH sequence segments was used in previous studies to examine the stability and dynamics of the Spartina rhizosphere diazotroph assemblages in the North Inlet salt marsh, near Georgetown, S.C. In this study, plugs were taken from gel bands from representative DGGE gels, the nifH amplimers were recovered and cloned, and their sequences were determined. A total of 59 sequences were recovered, and the amino acid sequences predicted from them were aligned with sequences from known and unknown diazotrophs in order to determine the types of organisms present in the Spartina rhizosphere. We recovered numerous sequences from diazotrophs in the γ subdivision of the division Proteobacteria (γ-Proteobacteria) and from various anaerobic diazotrophs. Diazotrophs in the α-Proteobacteria were poorly represented. None of the Spartina rhizosphere DGGE band sequences were identical to any known or previously recovered environmental nifH sequences. The Spartina rhizosphere diazotroph assemblage is very diverse and apparently consists mainly of unknown organisms.     

The Thioamides Methimazole and Thiourea Inhibit Growth of M. avium Subspecies paratuberculosis in Culture

Background

Thyrotoxicosis is conceptualized as an “autoimmune” disease with no accepted infectious etiology. There are increasingly compelling data that another “autoimmune” affliction, Crohn disease, may be caused by Mycobacterium avium subspecies paratuberculosis (MAP). Like M. tb, MAP is systemic. We hypothesized that some cases of thyrotoxicosis may be initiated by a MAP infection. Because other thioamides treat tuberculosis, leprosy and M. avium complex, we hypothesized that a mode of action of some thioamide anti-thyrotoxicosis medications may include MAP growth inhibition.

Methods

The effect of the thioamides, thiourea, methimazole and 6-propo-2-thiouracil (6-PTU) were studied in radiometric Bactec® culture, on ten strains of three mycobacterial species (six of MAP, two of M. avium and two of M. tb. complex). Data are presented as “cumulative growth index,” (cGI) or “percent decrease in cumulative GI” (%-ΔcGI).

Principal Findings

Methimazole was the most effective thioamide at inhibiting MAP growth. At 128µg/ml: MAP UCF-4; 65%-ΔcGI & MAP ATCC 19698; 90%-ΔcGI. Thiourea inhibited MAP “Ben” maximally; 70%-ΔcGI. Neither methimazole nor thiourea inhibited M. avium or M. tb. at the doses tested. 6-PTU has no inhibition on any strain studied, although a structurally analogous control, 5-PTU, was the most inhibitory thioamide tested.

Significance

We show inhibition of MAP growth by the thioamides, thiourea and methimazole in culture. These data are compatible with the hypothesis that these thioamides may have anti-prokaryotic in addition to their well-established eukaryotic actions in thyrotoxic individuals.     

Diazotrophs are overlooked contributors to carbon and nitrogen export to the deep ocean

Diazotrophs are widespread microorganisms that alleviate nitrogen limitation in 60% of our oceans, thereby regulating marine productivity. Yet, the group-specific contribution of diazotrophs to organic matter export has not been quantified, which so far has impeded an accurate assessment of their impact on the biological carbon pump. Here, we examine the fate of five groups of globally-distributed diazotrophs by using an original combination of mesopelagic particle sampling devices across the subtropical South Pacific Ocean. We demonstrate that cyanobacterial and non-cyanobacterial diazotrophs are exported down to 1000 m depth. Surprisingly, group-specific export turnover rates point to a more efficient export of small unicellular cyanobacterial diazotrophs (UCYN) relative to the larger and filamentous Trichodesmium. Phycoerythrin-containing UCYN-B and UCYN-C-like cells were recurrently found embedded in large (>50 µm) organic aggregates or organized into clusters of tens to hundreds of cells linked by an extracellular matrix, presumably facilitating their export. Beyond the South Pacific, our data are supported by analysis of the Tara Oceans metagenomes collected in other ocean basins, extending the scope of our results globally. We show that, when diazotrophs are found in the euphotic zone, they are also systematically present in mesopelagic waters, suggesting their transport to the deep ocean. We thus conclude that diazotrophs are a significant part of the carbon sequestered in the deep ocean and, therefore, they need to be accounted in regional and global estimates of export.Subject terms: Biogeochemistry, Microbiology     

Isolation of a Multiheme Protein with Features of a Hydrazine-Oxidizing Enzyme from an Anaerobic Ammonium-Oxidizing Enrichment Culture

A multiheme protein having hydrazine-oxidizing activity was purified from enriched culture from a reactor in which an anammox bacterium, strain KSU-1, was dominant. The enzyme has oxidizing activity toward hydrazine but not hydroxylamine and is a 130-kDa homodimer composed of a 62-kDa polypeptide containing eight hemes. It was therefore named hydrazine-oxidizing enzyme (HZO). With cytochrome c as an electron acceptor, the V_max and K_m for hydrazine are 6.2 ± 0.3 μmol/min · mg and 5.5 ± 0.6 μM, respectively. Hydrazine (25 μM) induced an increase in the proportion of reduced form in the spectrum, whereas hydroxylamine (500 μM) did not. Two genes coding for HZO, hzoA and hzoB, were identified within the metagenomic DNA from the culture. The genes encode the same amino acid sequence except for two residues. The sequences deduced from these genes showed low-level identities (<30%) to those of all of the hydroxylamine oxidoreductases reported but are highly homologous to two hao genes found by sequencing the genome of “Candidatus Kuenenia stuttgartiensis” (88% and 89% identities). The purified enzyme might therefore be a novel hydrazine-oxidizing enzyme having a critical role in anaerobic ammonium oxidation.     

Lunar-Rhythmic Molting in Laboratory Populations of the Noble Crayfish Astacus astacus (Crustacea,Astacidea): An Experimental Analysis

Juvenile noble crayfish, Astacusastacus (Crustacea, Astacidea) in the second year of age were kept in the laboratory for a twelve-month period under continuing “summer conditions” (LD 16:8, 19°C). Molting processes in this population could be synchronized by artificial moonlight cycles. Peaks of exuviations occurred at “new moons”. Males showed a slightly higher degree of synchronization than females. A phase-shift of the artificial lunar cycle in relation to the natural cycle resulted in a corresponding shift of the molting cycle. This clearly demonstrates that changes in the nocturnal light regime provide the primary external information for the lunar-monthly molting rhythm. There is a first indication that lunar photic stimuli do not act directly but as a zeitgeber which entrains an endogenous molting rhythm to the lunar cycle. Moreover, the results of the long-term experiments suggest that the hibernal resting period of A . astacus in the field (no molts between October and April) may also involve some endogenous programming. Continuing artificial summer conditions can delay but not completely suppress this resting period. The adaptive significance of the phenomena and how the findings may be applied to improve the management of crowded crayfish stocks are discussed.     

High‐resolution imaging sheds new light on a multi‐tier symbiotic partnership between a “walking” solitary coral,a sipunculan,and a bivalve from East Africa

Marine symbioses are integral to the persistence of ecosystem functioning in coral reefs. Solitary corals of the species Heteropsammia cochlea and Heterocyathus aequicostatus have been observed to live in symbiosis with the sipunculan worm Aspidosiphon muelleri muelleri, which inhabits a cavity within the coral, in Zanzibar (Tanzania). The symbiosis of these photosymbiotic corals enables the coral holobiont to move, in fine to coarse unconsolidated substrata, a process termed as “walking.” This allows the coral to escape sediment cover in turbid conditions which is crucial for these light‐dependent species. An additional commensalistic symbiosis of this coral‐worm holobiont is found between the Aspidosiphon worm and the cryptoendolithic bivalve Jousseaumiella sp., which resides within the cavity of the coral skeleton. To understand the morphological alterations caused by these symbioses, interspecific relationships, with respect to the carbonate structures between these three organisms, are documented using high‐resolution imaging techniques (scanning electron microscopy and µCT scanning). Documenting multi‐layered symbioses can shed light on how morphological plasticity interacts with environmental conditions to contribute to species persistence.     

Oxygen Uptake and Hydrogen-Stimulated Nitrogenase Activity from Azorhizobium caulinodans ORS571 Grown in a Succinate-Limited Chemostat

Succinate-limited continuous cultures of an Azorhizobium caulinodans strain were grown on ammonia or nitrogen gas as a nitrogen source. Ammonia-grown cells became oxygen limited at 1.7 μM dissolved oxygen, whereas nitrogen-fixing cells remained succinate limited even at dissolved oxygen concentrations as low as 0.9 μM. Nitrogen-fixing cells tolerated dissolved oxygen concentrations as high as 41 μM. Succinate-dependent oxygen uptake rates of cells from the different steady states ranged from 178 to 236 nmol min⁻¹ mg of protein⁻¹ and were not affected by varying chemostat-dissolved oxygen concentration or nitrogen source. When equimolar concentrations of succinate and β-hydroxybutyrate were combined, oxygen uptake rates were greater than when either substrate was used alone. Azide could also used alone as a respiratory substrate regardless of nitrogen source; however, when azide was added following succinate additions, oxygen uptake was inhibited in ammonia-grown cells and stimulated in nitrogen-fixing cells. Use of 25 mM succinate in the chemostat resevoir at a dilution rate of 0.1 h⁻¹ resulted in high levels of background respiration and nitrogenase activity, indicating that the cells were not energy limited. Lowering the reservoir succinate to 5 mM imposed energy limitation. Maximum succinate-dependent nitrogenase activity was 1,741 nmol of C₂H₄h⁻¹ mg (dry weight)⁻¹, and maximum hydrogen-dependent nitrogenase activity was 949 nmol of C₂H₄ h⁻¹ mg (dry weight)⁻¹. However, when concentration of 5% (vol/vol) hydrogen or greater were combined with succinate, nitrogenase activity decreased by 35% in comparison to when succinate was used alone. Substitution of argon for nitrogen in the chemostat inflow gas resulted in “washout,” proving that ORS571 can grow on N₂ and that there was not a nitrogen source in the medium that could substitute.     

Metabolism of Benzoate, Cyclohex-1-ene Carboxylate, and Cyclohexane Carboxylate by “Syntrophus aciditrophicus” Strain SB in Syntrophic Association with H2-Using Microorganisms

The metabolism of benzoate, cyclohex-1-ene carboxylate, and cyclohexane carboxylate by “Syntrophus aciditrophicus” in cocultures with hydrogen-using microorganisms was studied. Cyclohexane carboxylate, cyclohex-1-ene carboxylate, pimelate, and glutarate (or their coenzyme A [CoA] derivatives) transiently accumulated during growth with benzoate. Identification was based on comparison of retention times and mass spectra of trimethylsilyl derivatives to the retention times and mass spectra of authentic chemical standards. ¹³C nuclear magnetic resonance spectroscopy confirmed that cyclohexane carboxylate and cyclohex-1-ene carboxylate were produced from [ring-¹³C₆]benzoate. None of the metabolites mentioned above was detected in non-substrate-amended or heat-killed controls. Cyclohexane carboxylic acid accumulated to a concentration of 260 μM, accounting for about 18% of the initial benzoate added. This compound was not detected in culture extracts of Rhodopseudomonas palustris grown phototrophically or Thauera aromatica grown under nitrate-reducing conditions. Cocultures of “S. aciditrophicus” and Methanospirillum hungatei readily metabolized cyclohexane carboxylate and cyclohex-1-ene carboxylate at a rate slightly faster than the rate of benzoate metabolism. In addition to cyclohexane carboxylate, pimelate, and glutarate, 2-hydroxycyclohexane carboxylate was detected in trace amounts in cocultures grown with cyclohex-1-ene carboxylate. Cyclohex-1-ene carboxylate, pimelate, and glutarate were detected in cocultures grown with cyclohexane carboxylate at levels similar to those found in benzoate-grown cocultures. Cell extracts of “S. aciditrophicus” grown in a coculture with Desulfovibrio sp. strain G11 with benzoate or in a pure culture with crotonate contained the following enzyme activities: an ATP-dependent benzoyl-CoA ligase, cyclohex-1-ene carboxyl-CoA hydratase, and 2-hydroxycyclohexane carboxyl-CoA dehydrogenase, as well as pimelyl-CoA dehydrogenase, glutaryl-CoA dehydrogenase, and the enzymes required for conversion of crotonyl-CoA to acetate. 2-Ketocyclohexane carboxyl-CoA hydrolase activity was detected in cell extracts of “S. aciditrophicus”-Desulfovibrio sp. strain G11 benzoate-grown cocultures but not in crotonate-grown pure cultures of “S. aciditrophicus”. These results are consistent with the hypothesis that ring reduction during syntrophic benzoate metabolism involves a four- or six-electron reduction step and that once cyclohex-1-ene carboxyl-CoA is made, it is metabolized in a manner similar to that in R. palustris.     

Enrichment and physiological characterization of a novel comammox Nitrospira indicates ammonium inhibition of complete nitrification

The recent discovery of bacteria within the genus Nitrospira capable of complete ammonia oxidation (comammox) demonstrated that the sequential oxidation of ammonia to nitrate via nitrite can also be performed within a single bacterial cell. Although comammox Nitrospira exhibit a wide distribution in natural and engineered ecosystems, information on their physiological properties is scarce due to the limited number of cultured representatives. Additionally, most available genomic information is derived from metagenomic sequencing and high-quality genomes of Nitrospira in general are limited. In this study, we obtained a high (90%) enrichment of a novel comammox species, tentatively named “Candidatus Nitrospira kreftii”, and performed a detailed genomic and physiological characterization. The complete genome of “Ca. N. kreftii” allowed reconstruction of its basic metabolic traits. Similar to Nitrospira inopinata, the enrichment culture exhibited a very high ammonia affinity (K_{m(app)_NH3} ≈ 0.040 ± 0.01 µM), but a higher nitrite affinity (K_{m(app)_NO2}- = 12.5 ± 4.0 µM), indicating an adaptation to highly oligotrophic environments. Furthermore, we observed partial inhibition of ammonia oxidation at ammonium concentrations as low as 25 µM. This inhibition of “Ca. N. kreftii” indicates that differences in ammonium tolerance rather than affinity could potentially be a niche determining factor for different comammox Nitrospira.Subject terms: Bacterial genomics, Environmental microbiology, Bacterial physiology