Thiolated nucleotides in yeast transfer RNA

By culturing Saccharomyces cerevisiae in growth medium containing Mg³⁵SO₄, we have determined the extent and variation of tRNA thiolation in this yeast. We find that 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U)¹ is the major, if not only, thiolated derivative in S. cerevisiae tRNA. In addition, a comparison of the chromatographic mobility of mcm⁵s²Up on cellulose thin layers with those reported for unknown uridine derivatives found in purified yeast tRNA digests, leads to the conclusion that at least two of these tRNAs contain this modification.     

Chemical synthesis of the 5-taurinomethyl(-2-thio)uridine modified anticodon arm of the human mitochondrial tRNALeu(UUR) and tRNALys

5-Taurinomethyluridine (τm⁵U) and 5-taurinomethyl-2-thiouridine (τm⁵s²U) are located at the wobble position of human mitochondrial (hmt) tRNA^Leu(UUR) and tRNA^Lys, respectively. Both hypermodified units restrict decoding of the third codon letter to A and G. Pathogenic mutations in the genes encoding hmt-tRNA^Leu(UUR) and hmt-tRNA^Lys are responsible for the loss of the discussed modifications and, as a consequence, for the occurrence of severe mitochondrial dysfunctions (MELAS, MERRF). Synthetic oligoribonucleotides bearing modified nucleosides are a versatile tool for studying mechanisms of genetic message translation and accompanying pathologies at nucleoside resolution. In this paper, we present site-specific chemical incorporation of τm⁵U and τm⁵s²U into 17-mers related to the sequence of the anticodon arms hmt-tRNA^Leu(UUR) and hmt-tRNA^Lys, respectively employing phosphoramidite chemistry on CPG support. Selected protecting groups for the sulfonic acid (4-(tert-butyldiphenylsilanyloxy)-2,2-dimethylbutyl) and the exoamine function (-C(O)CF₃) are compatible with the blockage of the canonical monomeric units. The synthesis of τm⁵s²U-modified RNA fragment was performed under conditions eliminating the formation of side products of 2-thiocarbonyl group oxidation and/or oxidative desulphurization. The structure of the final oligomers was confirmed by mass spectroscopy and enzymatic cleavage data.     

A circular dichroism study of modified nucleosides

The conformation of 5-methoxycarbonylmethyluridine and 5-methoxycarbonylmethyl-2-thiouridine was studied by means of circular dichroism in various solvents. In order to calculate the accurate spectral parameters of the Cotton effects, the circular dichroism spectra were resolved into component Gaussian functions which simultaneously fit the adsorption spectra. On the basis of circular dichroism and proton magnetic resonance spectra, these nucleosides were found to occur in the β-configuration with the ³E-gg-anti conformation preferred. Due to the fact that the long-wavelength Cotton effect of mcm⁵s²U is not masked by the Cotton effects of the other nucleic acid monomers, the molecular parameters of this band may be useful for the conformational analysis of tRNA segments.     

Familial dysautonomia (FD) patients have reduced levels of the modified wobble nucleoside mcmsU in tRNA

Familial dysautonomia (FD) is a recessive neurodegenerative genetic disease. FD is caused by a mutation in the IKBKAP gene resulting in a splicing defect and reduced levels of full length IKAP protein. IKAP homologues can be found in all eukaryotes and are part of a conserved six subunit protein complex, Elongator complex. Inactivation of any Elongator subunit gene in multicellular organisms cause a wide range of phenotypes, suggesting that Elongator has a pivotal role in several cellular processes. In yeast, there is convincing evidence that the main role of Elongator complex is in formation of modified wobble uridine nucleosides in tRNA and that their absence will influence translational efficiency. To date, no study has explored the possibility that FD patients display defects in formation of modified wobble uridine nucleosides as a consequence of reduced IKAP levels. In this study, we show that brain tissue and fibroblast cell lines from FD patients have reduced levels of the wobble uridine nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U). Our findings indicate that FD could be caused by inefficient translation due to lower levels of wobble uridine nucleosides.     

Energetics of Growth of a Defined Mixed Culture of Desulfovibrio vulgaris and Methanosarcina barkeri: Interspecies Hydrogen Transfer in Batch and Continuous Cultures

Interspecies hydrogen transfer was studied in Desulfovibrio vulgaris-Methanosarcina barkeri mixed cultures. Experiments were performed under batch and continuous growth culture conditions. Lactate or pyruvate was used as an energy source. In batch culture and after 30 days of simultaneous incubation, these organisms were found to yield 1.5 mol of methane and 1.5 mol of carbon dioxide per mol of lactate fermented. When M. barkeri served as the hydrogen acceptor, growth yields of D. vulgaris were higher compared with those obtained on pyruvate without any electron acceptor other than protons. In continuous culture, all of the carbon derived from the oxidation of lactate was recovered as methane and carbon dioxide, provided the dilution rate was minimal. Increasing the dilution rate induced a gradual accumulation of acetate, causing acetate metabolism to cease at above μ = 0.05 h⁻¹. Under these conditions all of the methane produced originated from carbon dioxide. The growth yields of D. vulgaris were measured when sulfate or M. barkeri was the electron acceptor. Two key observations resulted from the present study. First, although sulfate was substituted by M. barkeri, metabolism of D. vulgaris was only slightly modified. The coculture-fermented lactate produced equimolar quantities of carbon dioxide and methane. Second, acetogenesis and methane formation from acetate were completely separable.     

Investigation of the microbial metabolism of carbon dioxide and hydrogen in the kangaroo foregut by stable isotope probing

Kangaroos ferment forage material in an enlarged forestomach analogous to the rumen, but in contrast to ruminants, they produce little or no methane. The objective of this study was to identify the dominant organisms and pathways involved in hydrogenotrophy in the kangaroo forestomach, with the broader aim of understanding how these processes are able to predominate over methanogenesis. Stable isotope analysis of fermentation end products and RNA stable isotope probing (RNA-SIP) were used to investigate the organisms and biochemical pathways involved in the metabolism of hydrogen and carbon dioxide in the kangaroo forestomach. Our results clearly demonstrate that the activity of bacterial reductive acetogens is a key factor in the reduced methane output of kangaroos. In in vitro fermentations, the microbial community of the kangaroo foregut produced very little methane, but produced a significantly greater proportion of acetate derived from carbon dioxide than the microbial community of the bovine rumen. A bacterial operational taxonomic unit closely related to the known reductive acetogen Blautia coccoides was found to be associated with carbon dioxide and hydrogen metabolism in the kangaroo foregut. Other bacterial taxa including members of the genera Prevotella, Oscillibacter and Streptococcus that have not previously been reported as containing hydrogenotrophic organisms were also significantly associated with metabolism of hydrogen and carbon dioxide in the kangaroo forestomach.     

Viable methanotrophic bacteria enriched from air and rain can oxidize methane at cloud-like conditions

Atmospheric methane is degraded by both photooxidation and, in topsoils, by methanotrophic bacteria, but this may not totally account for the global sink of this greenhouse gas. Topsoils are a prominent source of airborne bacteria, which can degrade some organic atmospheric compounds at rates similar to photooxidation. Although airborne methanotrophs would have direct access to atmospheric methane, their presence and activity in the atmosphere has not been investigated so far. We enriched airborne methanotrophs from air and rainwater and showed that they oxidized methane at atmospheric concentration. The majority of seven OTUs, detected using pmoA gene clone libraries, were affiliated to the type II methanotrophic genera Methylocystis and Methylosinus. Furthermore, 16S rRNA gene clone libraries revealed the presence of OTUs affiliated with the genera Hyphomicrobium and Variovorax, members of which can stimulate methane oxidation by yet unidentified mechanisms. Simulating cloud-like conditions revealed that although both low pH and the presence of common cloud-borne organics negatively affected methane oxidation, airborne methanotrophs were able to degrade atmospheric methane in most cases. We demonstrate here for the first time that viable methanotrophic bacteria are present in air and rain and thus expand our knowledge on the global distribution of methanotrophs to include the atmosphere. The fact that they can degrade methane to below atmospheric concentrations when inoculated into artificial cloud water leads to an important possible effect of these organisms: the atmosphere may not only function as a medium for microbial dissemination, but also as a site of active microbial methane turnover.     

Isolation and characterization of methane utilizing bacteria from wetland paddy ecosystem

Methylotrophic bacteria which are known to utilize C1 compounds including methane. Research during past few decades increased the interest in finding out novel genera of methane degrading bacteria to efficiently utilize methane to decrease global warming effect. Moreover, evaluation of certain known plant growth promoting strains for their methane degrading potential may open up a new direction for multiple utility of such cultures. In this study, efficient methylotrophic cultures were isolated from wetland paddy fields of Gujarat. From the overall morphological, biochemical and molecular characterization studies, the isolates were identified and designated as Bacillus aerius AAU M 8; Rhizobium sp. AAU M 10; B. subtilis AAU M 14; Paenibacillus illinoisensis AAU M 17 and B. megaterium AAU M 29. Gene specific PCR analysis of the isolates, P. illinoisensis, B. aerius, Rhizobium sp. and B. subtilis showed presence of pmoA gene encoding α subunit particulate methane monooxygenase cluster. B. megaterium, P. illinoisensis, Rhizobium sp. and Methylobacterium extrorquens showed presence of mmoX gene encoding α subunit of the hydroxylase component of the soluble methane monooxygenase cluster. P. illinoisensis and Rhizobium sp. showed presence mxaF gene encoding α subunit region of methanol dehydrogenase gene cluster showing that both isolates are efficient utilizers of methane. To the best of our knowledge, this is the first time report showing presence of methane degradation enzymes and genes within the known PGPB group of organisms from wet land paddy agro-ecosystem, which is considered as one of the leading methane producer.     

Microbial communities of the discharge zone of oil- and gas-bearing fluids in low-mineral Lake Baikal

At the site of natural ingress of oil, microbial diversity in the Central Baikal bottom sediments differing in the chemical composition of pore waters was studied by molecular biological techniques. The sediments saturated with oil and methane were found to contain members of 10 bacterial and 2 archaeal phyla. The oxidized sediment layer contained methanotrophic bacteria belonging to the Alphaproteobacteria, which had a specific structure of the pmoA gene and clustered together with uncultured methanotrophs from cold ecosystems. The upper sediment layer also contained oil-oxidizing bacteria and the alkB genes most closely related to those of Rhodococcus. The microbial community of reduced sediments exhibited lower diversity and was represented mostly by the organisms involved in hydrocarbon biodegradation.     

闽江口芦苇沼泽湿地土壤产甲烷菌群落结构的垂直分布

应用PCR-RFLP技术及测序分析对闽江口芦苇湿地土壤产甲烷菌群落结构的垂直分布特征进行了研究。在构建的6个克隆文库中,每个克隆文库随机挑选100个克隆进行菌落PCR验证,共得到591个阳性克隆。PCR产物经限制性内切酶MspⅠ进行RFLP分析后得到37个不同的分类操作单元(OTUs)。对37个克隆子进行了序列测定,与GenBank数据库中的序列进行比对,最近相似性在91%—99%之间。RFLP分析和系统发育分析表明,闽江口芦苇湿地土壤中产甲烷菌群落包括3大类群:甲烷杆菌目(Methanobacteriales)、甲烷微菌目(Methanomirobiales)和甲烷八叠球菌目(Methanosarcinales)。不同土壤深度中产甲烷菌群落的分布呈现出不同的特征。土壤表层(0—10 cm)优势产甲烷菌类群为Methanoregula,约占76%;10—20 cm土层主要的产甲烷菌类群为Methanolinea和Methanoregula,分别约占23%和29%;20—30cm土层优势的产甲烷菌类群为Methanolinea,约占66%。Shannon指数(H’)和Simpson多样性指教(D)表明,10—20cm土层产甲烷菌多样性高于土壤表层(0—10 cm)和20—30 cm土层。37个测序OTUs中有26个OTUs属于不可培养的产甲烷菌序列,表明闽江口芦苇湿地土壤中存在大量不可培养的产甲烷菌。     

Microbial production of biogas form organic wastes

Biogas production through bacterial digestion of water hyacinth, rice straw and sawdust inoculated with cow dung was investigated in a batch loading of laboratory digestors. It was found that the overall gas yield was 368.00 l from water hyacinth having 77.13% methane, 310.00 l from rice straw with 77.80% methane, and 144.80 l from sandust containing 62.28% methane. Along with the production of gas, the change in pH and microbial population were also monitored. The pH of the digestion mixture was initially low and found to be increasing as the digestion progressed. The total microbial population of bacteria, fungi, actinomycetes, cellulolytic organisms, and coliform bacteria were found to decrease with time.     

Effect of sludge age on methanogenic and glycogen accumulating organisms in an aerobic granular sludge process fed with methanol and acetate

The influence of sludge age on granular sludge formation and microbial population dynamics in a methanol- and acetate-fed aerobic granular sludge system operated at 35°C was investigated. During anaerobic feeding of the reactor, methanol was initially converted to methane by methylotrophic methanogens. These methanogens were able to withstand the relatively long aeration periods. Lowering the anaerobic solid retention time (SRT) from 17 to 8 days enabled selective removal of the methanogens and prevented unwanted methane formation. In absence of methanogens, methanol was converted aerobically, while granule formation remained stable. At high SRT values (51 days), γ-Proteobacteria were responsible for acetate removal through anaerobic uptake and subsequent aerobic growth on storage polymers formed [so called metabolism of glycogen-accumulating organisms (GAO)]. When lowering the SRT (24 days), Defluviicoccus-related organisms (cluster II) belonging to the α-Proteobacteria outcompeted acetate consuming γ-Proteobacteria at 35°C. DNA from the Defluviicoccus-related organisms in cluster II was not extracted by the standard DNA extraction method but with liquid nitrogen, which showed to be more effective. Remarkably, the two GAO types of organisms grew separately in two clearly different types of granules. This work further highlights the potential of aerobic granular sludge systems to effectively influence the microbial communities through sludge age control in order to optimize the wastewater treatment processes.     

Spatial change of reservoir nitrite-dependent methane-oxidizing microorganisms

Nitrite-dependent anaerobic methane oxidation (n-damo), catalyzed by microorganisms affiliated with bacterial phylum NC10, can have an important contribution to the reduction of the methane emission from anoxic freshwater sediment to the atmosphere. However, information on the variation of sediment n-damo organisms in reservoirs is still lacking. The present study monitored the spatial change of sediment n-damo organisms in the oligotrophic freshwater Xinfengjiang Reservoir (South China). Sediment samples were obtained from six different sampling locations and two sediment depths (0–5 cm, 5–10 cm). Sediment n-damo bacterial abundance was found to vary with sampling location and layer depth, which was likely influenced by pH and nitrogen level. The presence of the n-damo pmoA gene was found in all these samples. A remarkable shift occurred in the diversity and composition of sediment n-damo pmoA gene sequences. A variety of distinctively different n-damo pmoA clusters existed in reservoir sediments. The pmoA sequences affiliated with Candidatus Methylomirabilis oxyfera formed the largest group, while a significant proportion of the obtained n-damo pmoA gene sequences showed no close relationship to those from any known NC10 species. In addition, the present n-damo process was found in reservoir sediment, which could be enhanced by nitrite nitrogen amendment.     

Anaerobic methane oxidation potential and bacteria in freshwater lakes: Seasonal changes and the influence of trophic status

Nitrite-dependent anaerobic methane oxidation (n-damo), mainly carried out by n-damo bacteria, is an important pathway for mitigating methane emission from freshwater lakes. Although n-damo bacteria have been detected in a variety of freshwater lakes, their potential and distribution, and associated environmental factors, remain unclear. Therefore, the current study investigated the potential and distribution of anaerobic methanotrophs in sediments from Erhai Lake and Dianchi Lake, two adjacent freshwater lakes in the Yunnan Plateau with different trophic status. Both lakes showed active anaerobic methane oxidation potential and harbored a high density of n-damo bacteria. Based on the n-damo pmoA gene, sediment n-damo bacterial communities mainly consisted of Candidatus Methylomirabils oxyfera and Candidatus Methylomirabils sinica, as well as novel n-damo organisms. Sediment anaerobic methane oxidation potential and the n-damo bacterial community showed notable differences among seasons and between lakes. The environmental variables associated with lake trophic status (e.g. total nitrogen, ammonia nitrogen, nitrate nitrogen, and total organic carbon) might have significant impacts on the anaerobic methane oxidation potential, as well as the abundance and community structure of n-damo bacteria. Therefore, trophic status could determine the n-damo process in freshwater lake sediment.     

Role of methylotrophs in the degradation of hydrocarbons during the Deepwater Horizon oil spill

The role of methylotrophic bacteria in the fate of the oil and gas released into the Gulf of Mexico during the Deepwater Horizon oil spill has been controversial, particularly in relation to whether organisms such as Methylophaga had contributed to the consumption of methane. Whereas methanotrophy remains unqualified in these organisms, recent work by our group using DNA-based stable-isotope probing coupled with cultivation-based methods has uncovered hydrocarbon-degrading Methylophaga. Recent findings have also shown that methylotrophs, including Methylophaga, were in a heightened state of metabolic activity within oil plume waters during the active phase of the spill. Taken collectively, these findings suggest that members of this group may have participated in the degradation of high-molecular-weight hydrocarbons in plume waters. The discovery of hydrocarbon-degrading Methylophaga also highlights the importance of considering these organisms in playing a role to the fate of oil hydrocarbons at oil-impacted sites.     

Assessment of Changes in Microbial Community Structure during Operation of an Ammonia Biofilter with Molecular Tools

Biofiltration has been used for two decades to remove odors and various volatile organic and inorganic compounds in contaminated off-gas streams. Although biofiltration is widely practiced, there have been few studies of the bacteria responsible for the removal of air contaminants in biofilters. In this study, molecular techniques were used to identify bacteria in a laboratory-scale ammonia biofilter. Both 16S rRNA and ammonia monooxygenase (amoA) genes were used to characterize the heterotrophic and ammonia-oxidizing bacteria collected from the biofilter during a 102-day experiment. The overall diversity of the heterotrophic microbial population appeared to decrease by 38% at the end of the experiment. The community structure of the heterotrophic population also shifted from predominantly members of two subdivisions of the Proteobacteria (the beta and gamma subdivisions) to members of one subdivision (the gamma subdivision). An overall decrease in the diversity of ammonia monooxygenase genes was not observed. However, a shift from groups dominated by organisms containing Nitrosomonas-like and Nitrosospira-like amoA genes to groups dominated by organisms containing only Nitrosospira-like amoA genes was observed. In addition, a new amoA gene was discovered. This new gene is the first freshwater amoA gene that is closely affiliated with Nitrosococcus oceanus and the particulate methane monooxygenase gene from the methane oxidizers belonging to the gamma subdivision of the Proteobacteria.     

Enhancement of bioenergy production from organic wastes by two-stage anaerobic hydrogen and methane production process

The present study investigated a two-stage anaerobic hydrogen and methane process for increasing bioenergy production from organic wastes. A two-stage process with hydraulic retention time (HRT) 3 d for hydrogen reactor and 12 d for methane reactor, obtained 11% higher energy compared to a single-stage methanogenic process (HRT 15 d) under organic loading rate (OLR) 3 gVS/(L d). The two-stage process was still stable when the OLR was increased to 4.5 gVS/(L d), while the single-stage process failed. The study further revealed that by changing the HRT_hydrogen:HRT_methane ratio of the two-stage process from 3:12 to 1:14, 6.7%, more energy could be obtained. Microbial community analysis indicated that the dominant bacterial species were different in the hydrogen reactors (Thermoanaerobacterium thermosaccharolyticum-like species) and methane reactors (Clostridiumthermocellum-like species). The changes of substrates and HRT did not change the dominant species. The archaeal community structures in methane reactors were similar both in single- and two- stage reactors, with acetoclastic methanogens Methanosarcina acetivorans-like organisms as the dominant species.     

Characterization of C1-Metabolizing Prokaryotic Communities in Methane Seep Habitats at the Kuroshima Knoll, Southern Ryukyu Arc, by Analyzing pmoA, mmoX, mxaF, mcrA, and 16S rRNA Genes

Samples from three submerged sites (MC, a core obtained in the methane seep area; MR, a reference core obtained at a distance from the methane seep; and HC, a gas-bubbling carbonate sample) at the Kuroshima Knoll in the southern Ryuku arc were analyzed to gain insight into the organisms present and the processes involved in this oxic-anoxic methane seep environment. 16S rRNA gene analyses by quantitative real-time PCR and clone library sequencing revealed that the MC core sediments contained abundant archaea (~34% of the total prokaryotes), including both mesophilic methanogens related to the genus Methanolobus and ANME-2 members of the Methanosarcinales, as well as members of the δ-Proteobacteria, suggesting that both anaerobic methane oxidation and methanogenesis occurred at this site. In addition, several functional genes connected with methane metabolism were analyzed by quantitative competitive-PCR, including the genes encoding particulate methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), methanol dehydrogenese (mxaF), and methyl coenzyme M reductase (mcrA). In the MC core sediments, the most abundant gene was mcrA (2.5 × 10⁶ copies/g [wet weight]), while the pmoA gene of the type I methanotrophs (5.9 × 10⁶ copies/g [wet weight]) was most abundant at the surface of the MC core. These results indicate that there is a very complex environment in which methane production, anaerobic methane oxidation, and aerobic methane oxidation all occur in close proximity. The HC carbonate site was rich in γ-Proteobacteria and had a high copy number of mxaF (7.1 × 10⁶ copies/g [wet weight]) and a much lower copy number of the pmoA gene (3.2 × 10² copies/g [wet weight]). The mmoX gene was never detected. In contrast, the reference core contained familiar sequences of marine sedimentary archaeal and bacterial groups but not groups specific to C₁ metabolism. Geochemical characterization of the amounts and isotopic composition of pore water methane and sulfate strongly supported the notion that in this zone both aerobic methane oxidation and anaerobic methane oxidation, as well as methanogenesis, occur.