Weak phylogenetic signal in physiological traits of methane‐oxidizing bacteria

The presence of phylogenetic signal is assumed to be ubiquitous. However, for microorganisms, this may not be true given that they display high physiological flexibility and have fast regeneration. This may result in fundamentally different patterns of resemblance, that is, in variable strength of phylogenetic signal. However, in microbiological inferences, trait similarities and therewith microbial interactions with its environment are mostly assumed to follow evolutionary relatedness. Here, we tested whether indeed a straightforward relationship between relatedness and physiological traits exists for aerobic methane‐oxidizing bacteria (MOB). We generated a comprehensive data set that included 30 MOB strains with quantitative physiological trait information. Phylogenetic trees were built from the 16S rRNA gene, a common phylogenetic marker, and the pmoA gene which encodes a subunit of the key enzyme involved in the first step of methane oxidation. We used a Blomberg's K from comparative biology to quantify the strength of phylogenetic signal of physiological traits. Phylogenetic signal was strongest for physiological traits associated with optimal growth pH and temperature indicating that adaptations to habitat are very strongly conserved in MOB. However, those physiological traits that are associated with kinetics of methane oxidation had only weak phylogenetic signals and were more pronounced with the pmoA than with the 16S rRNA gene phylogeny. In conclusion, our results give evidence that approaches based solely on taxonomical information will not yield further advancement on microbial eco‐evolutionary interactions with its environment. This is a novel insight on the connection between function and phylogeny within microbes and adds new understanding on the evolution of physiological traits across microbes, plants and animals.     

The potential of methane‐oxidizing bacteria for applications in environmental biotechnology

Methanotrophic bacteria possess a unique set of enzymes enabling them to oxidize, degrade and transform organic molecules and synthesize new compounds. Therefore, they have great potential in environmental biotechnology. The application of these unique properties was demonstrated in three case studies: (i) Methane escaping from leaky gas pipes may lead to massive mortality of trees in urban areas. Lack of oxygen within the soil surrounding tree roots caused by methanotrophic activity was identified as one of the reasons for this phenomenon. The similarity between metabolic reactions performed by the key enzymes of methanotrophs (methane monooxygenase) and ammonium oxidizers (ammonium monooxygenase) might offer a solution to this problem by applying commercially available nitrification and urease inhibitors. (ii) Methanotrophs are able to co‐metabolically degrade contaminants such as low‐molecular‐weight‐chlorinated hydrocarbons in soil and water in the presence of methane. Batch and continuous trichloroethylene degradation experiments in laboratory‐scale reactors using Methylocystis sp. GB 14 were performed, partly with cells entrapped in a polymer matrix. (iii) Using a short, two‐stage pilot‐scale process, the intracellular polymer accumulation of poly‐β‐hydroxybutyrate (PHB) in methanotrophs reached a maximum of 52%. Interestingly, an ultra‐high‐molecular‐weight PHB of 3.1 MDa was accumulated under potassium deficiency. Under strictly controlled conditions (temperature, pH and methane supply) this process can be nonsterile because of the establishment of a stable microbial community (dominant species Methylocystis sp. GB 25 ≥86% by biomass). The possibility to substitute methane with biogas from renewable sources facilitates the development of a methane‐based PHB production process that yields a high‐quality biopolymer at competitive costs.     

深部生物圈古菌的研究进展与展望

古菌作为深部生物圈中常见的原核生物,广泛分布于各类海洋沉积生境中,在沉积物生物地球化学循环中发挥着重要作用.由于不同的古菌类群对环境条件存在生理适应性差异,它们分别在近岸沿海和开阔大洋沉积物中构成了厌氧微生物生态系统和好氧微生物生态系统.本文通过对近岸与远洋、沉积物与上覆水体两个不同维度的古菌群落结构进行比较,以及对出...     

Responses of community structure of amoA‐encoding archaea and ammonia‐oxidizing bacteria in ammonia biofilter with rockwool mixtures to the gradual increases in ammonium and nitrate

Aims

To investigate community shifts of amoA‐encoding archaea (AEA) and ammonia‐oxidizing bacteria (AOB) in biofilter under nitrogen accumulation process.

Methods and Results

A laboratory‐scale rockwool biofilter with an irrigated water circulation system was operated for 436 days with ammonia loading rates of 49–63 NH₃ g m^?3 day^?1. The AEA and AOB communities were investigated by denaturing gradient gel electrophoresis, sequencing and real‐time PCR analysis based on amoA genes. The results indicated that changes in abundance and community compositions occurred in a different manner between archaeal and bacterial amoA during the operation. However, both microbial community structures mainly varied when free ammonia (FA) concentrations in circulation water were increasing, which caused a temporal decline in reactor performance. Dominant amoA sequences after this transition were related to Thaumarchaeotal Group I.1b, Nitrosomonas europaea lineages and one subcluster within Nitrosospira sp. cluster 3, for archaea and bacteria, respectively.

Conclusions

The specific FA in circulation water seems to be the important factor, which relates to the AOB and AEA community shifts in the biofilter besides ammonium and pH.

Significance and Impact of the Study

One of the key factors for regulating AEA and AOB communities was proposed that is useful for optimizing biofiltration technology.     

Microbial diversity and community structure of a highly active anaerobic methane‐oxidizing sulfate‐reducing enrichment

Anaerobic oxidation of methane (AOM) is an important methane sink in the ocean but the microbes responsible for AOM are as yet resilient to cultivation. Here we describe the microbial analysis of an enrichment obtained in a novel submerged‐membrane bioreactor system and capable of high‐rate AOM (286 μmol g_{dry weight}^?1 day^?1) coupled to sulfate reduction. By constructing a clone library with subsequent sequencing and fluorescent in situ hybridization, we showed that the responsible methanotrophs belong to the ANME‐2a subgroup of anaerobic methanotrophic archaea, and that sulfate reduction is most likely performed by sulfate‐reducing bacteria commonly found in association with other ANME‐related archaea in marine sediments. Another relevant portion of the bacterial sequences can be clustered within the order of Flavobacteriales but their role remains to be elucidated. Fluorescent in situ hybridization analyses showed that the ANME‐2a cells occur as single cells without close contact to the bacterial syntrophic partner. Incubation with ¹³C‐labelled methane showed substantial incorporation of ¹³C label in the bacterial C₁₆ fatty acids (bacterial; 20%, 44% and 49%) and in archaeal lipids, archaeol and hydroxyl‐archaeol (21% and 20% respectively). The obtained data confirm that both archaea and bacteria are responsible for the anaerobic methane oxidation in a bioreactor enrichment inoculated with Eckernförde bay sediment.     

Function of coenzyme F420-dependent NADP reductase in methanogenic archaea containing an NADP-dependent alcohol dehydrogenase

Methanogenic archaea growing on ethanol or isopropanol as the electron donor for CO₂ reduction to CH₄ contain either an NADP-dependent or a coenzyme F₄₂₀-dependent alcohol dehydrogenase. We report here that in both groups of methanogens, the N ⁵, N ¹⁰-methylenetetrahydromethanopterin dehydrogenase and the N ⁵, N ¹⁰-methylenetetrahydromethanopterin reductase, two enzymes involved in CO₂ reduction to CH₄, are specific for F₄₂₀. This raised the question how F₄₂₀H₂ is regenerated in the methanogens with an NADP-dependent alcohol dehydrogenase. We found that these organisms contain catabolic activities of an enzyme catalyzing the reduction of F₄₂₀ with NADPH. The F₄₂₀-dependent NADP reductase from Methanogenium organophilum was purified and characterized. The N-terminal amino acid sequence showed 42% sequence identity to a putative gene product in Methanococcus jannaschii, the total genome of which has recently been sequenced. Received: 12 May 1997 / Accepted: 1 July 1997     

Activities and distribution of methanogenic and methane‐oxidizing microbes in marine sediments from the Cascadia Margin

We investigated methane production and oxidation and the depth distribution and phylogenetic affiliation of a functional gene for methanogenesis, methyl coenzyme M reductase subunit A (mcrA), at two sites of the Integrated Ocean Drilling Program Expedition 311. These sites, U1327 and U1329, are respectively inside and outside the area of gas hydrate distribution on the Cascadia Margin. Radiotracer experiments using ¹⁴C‐labelled substrates indicated high potential methane production rates in hydrate‐bearing sediments [128–223 m below seafloor (mbsf)] at U1327 and in sediments between 70 and 140 mbsf at U1329. Tracer‐free experiments indicated high cumulative methane production in sediments within and below the gas hydrate layer at U1327 and in sediments below 70 mbsf at U1329. Stable tracer experiments using ¹³C‐labelled methane showed high potential methane oxidation rates in near‐surface sediments and in sediments deeper than 100 mbsf at both sites. Results of polymerase chain reaction amplification of mcrA in DNA were mostly consistent with methane production: relatively strong mcrA amplification was detected in the gas hydrate‐bearing sediments at U1327, whereas at U1329, it was mainly detected in sediments from around the bottom‐simulating reflector (126 mbsf). Phylogenetic analysis of mcrA separated it into four phylotype clusters: two clusters of methanogens, Methanosarcinales and Methanobacteriales, and two clusters of anaerobic methanotrophic archaea, ANME‐I and ANME‐II groups, supporting the activity measurement results. These results reveal that in situ methanogenesis in deep sediments probably contributes to gas hydrate formation and are inconsistent with the geochemical model that microbial methane currently being generated in shallow sediments migrates downward and contributes to the hydrate formation. At Site U1327, gas hydrates occurred in turbidite sediments, which were absent at Site U1329, suggesting that a geological setting suitable for a gas hydrate reservoir is more important for the accumulation of gas hydrate than microbiological properties.     

艾比湖湿地芦苇根际土壤氨氧化古菌的多样性和群落结构

【目的】旨在揭示耐盐植物芦苇根际与非根际土壤AOA群落结构间的差异,为深入研究盐生植物根际土壤微生物与耐盐性之间的关系提供理论基础。【方法】应用高通量测序技术以氨单加氧酶基因(amoA)为分子标记,对新疆艾比湖湿地荒漠生态系统不同季节(春、夏、秋)芦苇根际与非根际土壤氨氧化古菌(AOA)的多样性和群落结构进行研究。【结果】结果表明,不同季节芦苇根际土壤AOA多样性和丰富度存在差异,相比非根际土壤,夏季和秋季芦苇根际土壤AOA多样性较低丰富度较高,春季多样性较高丰富度较低。芦苇根际土壤中AOA的多样性为春季夏季秋季。AOA群落组成分析表明,土壤样品中AOA群落主要集中在泉古菌门(Crenarchaeota)和奇古菌门(Thaumarchaeota),其中泉古菌门为主要优势菌门。RDA分析表明,含水量(SM)、有机质(SOM)、总氮(TN)和pH是影响芦苇根际土壤AOA群落多样性和丰富度的主要环境因子。【结论】不同季节芦苇根际土壤AOA多样性及丰富度存在差异,相比非根际土壤,芦苇根际土壤AOA更丰富。     

Community structures of ammonia‐oxidising archaea and bacteria in high‐altitude lakes on the Tibetan Plateau

1. Community structures of planktonic ammonia‐oxidising archaea (AOA) and bacteria (AOB) were investigated for five high‐altitude Tibetan lakes, which could be classified as freshwater, oligosaline or mesosaline, to develop a general view of the AOA and AOB in lakes on the Tibetan Plateau. 2. Based on PCR screening of the ammonia monooxygenase α‐subunit (amoA) gene, AOA were present in 14 out of 17 samples, whereas AOB were detected in only four samples. Phylogenetic analyses indicated that the AOB communities were dominated by a unique monophylogenetic lineage within Nitrosomonas, which may represent a novel cluster of AOB. AOA, on the other hand, were distinct among lakes with different salinities. 3. Multivariate statistical analyses indicated a heterogeneous distribution of the AOA communities among lakes largely caused by lake salinity, whereas the uniform chemical properties within lakes and their geographical isolation may favour relatively homogeneous AOA communities within lakes. 4. Our results suggest a wide occurrence of AOA in Tibetan lakes and provide the first evidence of salinity‐related differentiation of AOA community composition as well as potential geographical isolation of AOA in inland aquatic environments.     

Biogeochemistry of methane and methanogenic archaea in permafrost

This study summarizes the findings of our research on the genesis of methane, its content and distribution in permafrost horizons of different age and origin. Supported by reliable data from a broad geographical sweep, these findings confirm the presence of methane in permanently frozen fine-grained sediments. In contrast to the omnipresence of carbon dioxide in permafrost, methane-containing horizons (up to 40.0 mL kg(-1)) alternate with strata free of methane. Discrete methane-containing horizons representing over tens of thousands of years are indicative of the absence of methane diffusion through the frozen layers. Along with the isotopic composition of CH(4) carbon (delta(13)C -64 per thousand to -99 per thousand), this confirms its biological origin and points to in situ formation of this biogenic gas. Using (14)C-labeled substrates, the possibility of methane formation within permafrost was experimentally shown, as confirmed by delta(13)C values. Extremely low values (near -99 per thousand) indicate that the process of CH(4) formation is accompanied by the substantial fractionation of carbon isotopes. For the first time, cultures of methane-forming archaea, Methanosarcina mazei strain JL01 VKM B-2370, Methanobacterium sp. strain M2 VKM B-2371 and Methanobacterium sp. strain MK4 VKM B-2440 from permafrost, were isolated and described.     

High diversity of ammonia‐oxidizing archaea in permanent and seasonal oxygen‐deficient waters of the eastern South Pacific

The community structure of putative aerobic ammonia‐oxidizing archaea (AOA) was explored in two oxygen‐deficient ecosystems of the eastern South Pacific: the oxygen minimum zone off Peru and northern Chile (11°S–20°S), where permanent suboxic and low‐ammonium conditions are found at intermediate depths, and the continental shelf off central Chile (36°S), where seasonal oxygen‐deficient and relatively high‐ammonium conditions develop in the water column, particularly during the upwelling season. The AOA community composition based on the ammonia monooxygenase subunit A (amoA) genes changed according to the oxygen concentration in the water column and the ecosystem studied, showing a higher diversity in the seasonal low‐oxygen waters. The majority of the archaeal amoA genotypes was affiliated to the uncultured clusters A (64%) and B (35%), with Cluster A AOA being mainly associated with higher oxygen and ammonium concentrations and Cluster B AOA with permanent oxygen‐ and ammonium‐poor waters. Q‐PCR assays revealed that AOA are an abundant community (up to 10⁵amoA copies ml^?1), while bacterial amoA genes from β proteobacteria were undetected. Our results thus suggest that a diverse uncultured AOA community, for which, therefore, we do not have any physiological information, to date, is an important component of the nitrifying community in oxygen‐deficient marine ecosystems, and particularly in rich coastal upwelling ones.     

Thermodynamic characterization of proton‐ionizable functional groups on the cell surfaces of ammonia‐oxidizing bacteria and archaea

The ammonia‐oxidizing archaeon Nitrosopumilus maritimus strain SCM1 (strain SCM1), a representative of the Thaumarchaeota archaeal phylum, can sustain high specific rates of ammonia oxidation at ammonia concentrations too low to sustain metabolism by ammonia‐oxidizing bacteria (AOB). One structural and biochemical difference between N. maritimus and AOB that might be related to the oligotrophic adaptation of strain SCM1 is the cell surface. A proteinaceous surface layer (S‐layer) comprises the outermost boundary of the strain SCM1 cell envelope, as opposed to the lipopolysaccharide coat of Gram‐negative AOB. In this work, we compared the surface reactivities of two archaea having an S‐layer (strain SCM1 and Sulfolobus acidocaldarius) with those of four representative AOB (Nitrosospira briensis, Nitrosomonas europaea, Nitrosolobus multiformis, and Nitrosococcus oceani) using potentiometric and calorimetric titrations to evaluate differences in proton‐ionizable surface sites. Strain SCM1 and S. acidocaldarius have a wider range of proton buffering (approximately pH 10–3.5) than the AOB (approximately pH 10–4), under the conditions investigated. Thermodynamic parameters describing proton‐ionizable sites (acidity constants, enthalpies, and entropies of protonation) are consistent with these archaea having proton‐ionizable amino acid side chains containing carboxyl, imidazole, thiol, hydroxyl, and amine functional groups. Phosphorous‐bearing acidic functional groups, which might also be present, could be masked by imidazole and thiol functional groups. Parameters for the AOB are consistent with surface structures containing anionic oxygen ligands (carboxyl‐ and phosphorous‐bearing acidic functional groups), thiols, and amines. In addition, our results showed that strain SCM1 has more reactive surface sites than the AOB and a high concentration of sites consistent with aspartic and/or glutamic acid. Because these alternative boundary layers mediate interaction with the local external environment, these data provide the basis for further comparisons of the thermodynamic behavior of surface reactivity toward essential nutrients.     

Unique coenzyme binding mode of hyperthermophilic archaeal sn‐glycerol‐1‐phosphate dehydrogenase from Pyrobaculum calidifontis

A gene encoding an sn‐glycerol‐1‐phosphate dehydrogenase (G1PDH) was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. In contrast to conventional G1PDHs, the expressed enzyme showed strong preference for NADH: the reaction rate (V_max) with NADPH was only 2.4% of that with NADH. The crystal structure of the enzyme was determined at a resolution of 2.45 Å. The asymmetric unit consisted of one homohexamer. Refinement of the structure and HPLC analysis showed the presence of the bound cofactor NADPH in subunits D, E, and F, even though it was not added in the crystallization procedure. The phosphate group at C2’ of the adenine ribose of NADPH is tightly held through the five biased hydrogen bonds with Ser40 and Thr42. In comparison with the known G1PDH structure, the NADPH molecule was observed to be pushed away from the normal coenzyme binding site. Interestingly, the S40A/T42A double mutant enzyme acquired much higher reactivity than the wild‐type enzyme with NADPH, which suggests that the biased interactions around the C2’‐phosphate group make NADPH binding insufficient for catalysis. Our results provide a unique structural basis for coenzyme preference in NAD(P)‐dependent dehydrogenases. Proteins 2016; 84:1786–1796. © 2016 Wiley Periodicals, Inc.     

施氮肥对华北平原土壤氨氧化细菌和古菌数量及群落结构的影响

利用荧光定量PCR、末端限制性片段长度多样性(T-RFLP)和基因克隆文库技术,比较了4种施氮水平(不施氮肥,0 kg N/hm~2,CK;施低水平氮肥,75 kg N/hm~2,N1;施中水平氮肥,150 kg N/hm~2,N2;施高水平氮肥,225 kg N/hm~2,N3)下华北平原地区小麦季表层(0—20 cm)土壤总细菌、氨氧化细菌(AOB)和氨氧化古菌(AOA)的丰度和群落结构。结果表明,土壤总细菌、AOB和AOA数量分别在每克干土5.74×10~9—7.50×10~9、8.89×10~6—2.66×10~7和3.83×10~8—7.78×10~8之间。不同施氮量土壤AOA数量均高于AOB数量,AOA/AOB值在81.72—14.38之间。增施氮肥显著显著提高AOB数量(P0.05),对总细菌和AOA数量的影响不显著(P0.05)。与CK相比,处理N1、N2和N3中AOB数量分别提高了0.64、1.50和1.99倍。增施氮肥显著改变了AOB和AOA的群落结构,且不同施氮量处理中AOB群落结构差异更大。系统进化分析显示,施氮肥小麦土壤AOB主要为Nitrosospira属类群,分布在Cluster 3的两个分支中;AOA分布在Cluster S的4个分支中。相关性分析显示,AOB数量与全氮和铵态氮含量呈显著正相关关系,与土壤pH和碳氮比呈显著负相关关系(P0.05);AOA数量与硝态氮含量和土壤pH呈显著正相关关系,与铵态氮含量呈显著负相关关系(P0.05)。研究结果表明:增施氮肥可显著改变华北平原地区碱性土壤AOB数量与群落结构,该地区小麦土壤中AOB比AOA对氮肥响应更敏感。     

新疆艾比湖和伊吾湖可培养嗜盐古菌多样性

新疆地区盐湖密布,蕴藏着丰富的微生物资源。为保护和利用微生物物种与基因资源,作者从新疆准噶尔盆地的艾比湖和天山山间盆地的伊吾湖分离纯化嗜盐微生物。采用PCR方法扩增其中65株嗜盐古菌16SrRNA基因序列。序列分析表明,分离的嗜盐古菌分属6个属,艾比湖以Haloterrigena和Natrinema属的菌株为主,伊吾湖由Haloarcula和Halorubrum两个属的菌株构成。通过多样性指数、丰富度指数、均匀度指数和物种相对多度模型对分离的菌株进行多样性分析和比较,结果表明,盐湖嗜盐古菌的多样性指数、丰富度指数和均匀度指数具有一定相关性,艾比湖可培养嗜盐古菌的多样性高于伊吾湖。研究发现了一些新的物种资源,表明新疆盐湖中孕育的特色微生物资源亟待保护与利用。     

Crystal structure of archaeal ketopantoate reductase complexed with coenzyme a and 2‐oxopantoate provides structural insights into feedback regulation

Coenzyme A (CoA) plays essential roles in a variety of metabolic pathways in all three domains of life. The biosynthesis pathway of CoA is strictly regulated by feedback inhibition. In bacteria and eukaryotes, pantothenate kinase is the target of feedback inhibition by CoA. Recent biochemical studies have identified ketopantoate reductase (KPR), which catalyzes the NAD(P)H‐dependent reduction of 2‐oxopantoate to pantoate, as a target of the feedback inhibition by CoA in archaea. However, the mechanism for recognition of CoA by KPR is still unknown. Here we report the crystal structure of KPR from Thermococcus kodakarensis in complex with CoA and 2‐oxopantoate. CoA occupies the binding site of NAD(P)H, explaining the competitive inhibition by CoA. Our structure reveals a disulfide bond between CoA and Cys84 that indicates an irreversible inhibition upon binding of CoA. The structure also suggests the cooperative binding of CoA and 2‐oxopantoate that triggers a conformational closure and seems to facilitate the disulfide bond formation. Our findings provide novel insights into the mechanism that regulates biosynthesis of CoA in archaea. Proteins 2016; 84:374–382. © 2016 Wiley Periodicals, Inc.     

几种农田土壤中古菌、泉古菌和细菌的数量分布特征

真核生物、细菌和古菌三者共同构成了生命的三域系统.古菌作为第3种生命形式,不仅能在高温、强酸和高盐等极端环境下生存,而且在海洋、湖泊和土壤等生境中也广泛分布,预示其在整个生态系统中有着不可估量的作用.本文以2个农田剖面土壤和2个长期施肥试验站祁阳(QY)和封丘(FQ)的土壤为对象,以实时定量PCR方法为主要研究手段,对土壤中古菌(包括泉古菌)和细菌的16S rRNA基因拷贝数丰度变化进行了研究.结果表明:土壤泉古菌16S rRNA基因拷贝数要低于古菌l～2个数量级,两者与细菌相比,16S rRNA基因拷贝数大小顺序为土壤泉古菌＜古茵＜细菌,而古菌和泉古菌16S rRNA基因拷贝数与细菌的比值均随土壤深度加深而增大.不同施肥处理对土壤古菌和泉古茵的数量有显著影响.QY试验站土壤古菌和细菌的数量与土壤pH值显著相关(分别为r=0.850,P＜0.01和r=0.676,P＜0.05).FQ古菌、泉古菌和细菌与土壤pH值相关性不显著,与土壤有机质含量相关性均达显著水平(分别为r=0.783,P＜0.05;r=0.827,P＜0.05;r=0.767,P＜0.05).了解古菌包括泉古菌在农田土壤中的分布,可为评价其在生态系统和物质循环中的作用提供重要的理论依据.