scholarly journals Seasonal Change in Methanotrophic Diversity and Populations in a Rice Field Soil Assessed by DNA-Stable Isotope Probing and Quantitative Real-Time PCR

2010 ◽  
Vol 25 (3) ◽  
pp. 156-163 ◽  
Author(s):  
Daisuke Mayumi ◽  
Takuya Yoshimoto ◽  
Hiroo Uchiyama ◽  
Nobuhiko Nomura ◽  
Toshiaki Nakajima-Kambe
Keyword(s):  
Stable Isotope ◽  
Real Time ◽  
Seasonal Change ◽  
Real Time Pcr ◽  
Rice Field ◽  
Stable Isotope Probing ◽  
Field Soil ◽  
Quantitative Real Time Pcr ◽  
Rice Field Soil

scholarly journals Incorporation of plant residue-derived carbon into the microeukaryotic community in a rice field soil revealed by DNA stable-isotope probing

2011 ◽  
Vol 79 (2) ◽  
pp. 371-379 ◽  
Author(s):  
Jun Murase ◽  
Manami Shibata ◽  
Chol Gyu Lee ◽  
Takeshi Watanabe ◽  
Susumu Asakawa ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Rice Field ◽  
Stable Isotope Probing ◽  
Plant Residue ◽  
Field Soil ◽  
Rice Field Soil

scholarly journals Identification of Acetate-Assimilating Microorganisms under Methanogenic Conditions in Anoxic Rice Field Soil by Comparative Stable Isotope Probing of RNA

2006 ◽  
Vol 73 (1) ◽  
pp. 101-109 ◽  
Author(s):  
Tomoyuki Hori ◽  
Matthias Noll ◽  
Yasuo Igarashi ◽  
Michael W. Friedrich ◽  
Ralf Conrad
Keyword(s):  
Stable Isotope ◽  
Rice Field ◽  
Ferric Iron ◽  
Stable Isotope Probing ◽  
Polymorphism Analysis ◽  
Field Soil ◽  
Bacterial Populations ◽  
Density Fractions ◽  
Reverse Transcription Pcr ◽  
Rice Field Soil

ABSTRACT Acetate is the most abundant intermediate of organic matter degradation in anoxic rice field soil and is converted to CH4 and/or CO2. Aceticlastic methanogens are the primary microorganisms dissimilating acetate in the absence of sulfate and reducible ferric iron. In contrast, very little is known about bacteria capable of assimilating acetate under methanogenic conditions. Here, we identified active acetate-assimilating microorganisms by using a combined approach of frequent label application at a low concentration and comparative RNA-stable isotope probing with 13C-labeled and unlabeled acetate. Rice field soil was incubated anaerobically at 25°C for 12 days, during which 13C-labeled acetate was added at a concentration of 500 μM every 3 days. 13C-labeled CH4 and CO2 were produced from the beginning of the incubation and accounted for about 60% of the supplied acetate 13C. RNA was extracted from the cells in each sample taken and separated by isopycnic centrifugation according to molecular weight. Bacterial and archaeal populations in each density fraction were screened by reverse transcription-PCR-mediated terminal restriction fragment polymorphism analysis. No differences in the bacterial populations were observed throughout the density fractions of the unlabeled treatment. However, in the heavy fractions of the 13C treatment, terminal restriction fragments (T-RFs) of 161 bp and 129 bp in length predominated. These T-RFs were identified by cloning and sequencing of 16S rRNA as from a Geobacter sp. and an Anaeromyxobacter sp., respectively. Apparently these bacteria, which are known as dissimilatory iron reducers, were able to assimilate acetate under methanogenic conditions, i.e., when CO2 was the predominant electron acceptor. We hypothesize that ferric iron minerals with low bioavailability might have served as electron acceptors for Geobacter spp. and Anaeromyxobacter spp. under these conditions.

scholarly journals Syntrophic Oxidation of Propionate in Rice Field Soil at 15 and 30°C under Methanogenic Conditions

2012 ◽  
Vol 78 (14) ◽  
pp. 4923-4932 ◽  
Author(s):  
Yanlu Gan ◽  
Qiongfen Qiu ◽  
Pengfei Liu ◽  
Junpeng Rui ◽  
Yahai Lu
Keyword(s):  
Organic Matter ◽  
Rice Field ◽  
Stable Isotope Probing ◽  
Field Soil ◽  
Content Type ◽  
Rice Field Soil ◽  
Decomposition Of Organic Matter ◽  
Syntrophic Oxidation ◽  
Propionate Oxidation ◽  
Syntrophic Interaction

ABSTRACTPropionate is one of the major intermediary products in the anaerobic decomposition of organic matter in wetlands and paddy fields. Under methanogenic conditions, propionate is decomposed through syntrophic interaction between proton-reducing and propionate-oxidizing bacteria and H2-consuming methanogens. Temperature is an important environmental regulator; yet its effect on syntrophic propionate oxidation has been poorly understood. In the present study, we investigated the syntrophic oxidation of propionate in a rice field soil at 15°C and 30°C. [U-13C]propionate (99 atom%) was applied to anoxic soil slurries, and the bacteria and archaea assimilating13C were traced by DNA-based stable isotope probing.Syntrophobacterspp.,Pelotomaculumspp., andSmithellaspp. were found significantly incorporating13C into their nucleic acids after [13C]propionate incubation at 30°C. The activity ofSmithellaspp. increased in the later stage, and concurrently that ofSyntrophomonasspp. increased. AceticlasticMethanosaetaceaeand hydrogenotrophicMethanomicrobialesandMethanocellalesacted as methanogenic partners at 30°C. Syntrophic oxidation of propionate also occurred actively at 15°C.Syntrophobacterspp. were significantly labeled with13C, whereasPelotomaculumspp. were less active at this temperature. In addition,Methanomicrobiales,Methanocellales, andMethanosarcinaceaedominated the methanogenic community, whileMethanosaetaceaedecreased. Collectively, temperature markedly influenced the activity and community structure of syntrophic guilds degrading propionate in the rice field soil. Interestingly,Geobacterspp. and some other anaerobic organisms likeRhodocyclaceae,Acidobacteria,Actinobacteria, andThermomicrobiaprobably also assimilated propionate-derived13C. The mechanisms for the involvement of these organisms remain unclear.

scholarly journals Relative and Absolute Quantitative Real-Time PCR-Based Quantifications ofhcnCandphlDGene Transcripts in Natural Soil Spiked withPseudomonassp. Strain LBUM300

2010 ◽  
Vol 77 (1) ◽  
pp. 41-47 ◽  
Author(s):  
Nadine J. DeCoste ◽  
Vijay J. Gadkar ◽  
Martin Filion
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Transcriptional Analysis ◽  
Relative Quantification ◽  
Field Soil ◽  
Quantitative Real Time Pcr ◽  
Qrt Pcr ◽  
The Absolute ◽  
Bacterial Concentrations

ABSTRACTTranscriptional analysis of microbial gene expression using relative quantitative real-time PCR (qRT-PCR) has been hampered by various technical problems. One such problem is the unavailability of an exogenous standard robust enough for use in a complex matrix like soil. To circumvent this technical issue, we made use of a recently developed artificial RNA (myIC) as an exogenous “spike-in” control. Nonsterile field soil was inoculated with various concentrations of the test bacteriumPseudomonassp. strain LBUM300, ranging from 4.3- to 8.3-log bacterial cells per gram of soil. Total soil RNA was extracted at days 0, 7, and 14 postinoculation, and using two-step TaqMan assays,phlD(encoding the production of 2,4-diacetylphloroglucinol) andhcnC(encoding the production of hydrogen cyanide) gene expression was monitored. For relative quantification, a defined quantity ofin vitro-synthesized myIC RNA was spiked during the RNA extraction procedure. Absolute qRT-PCR was also performed in parallel. Both the absolute and relative quantifications showed similar transcriptional trends. Overall, the transcriptional activity ofphlDandhcnCchanged over time and with respect to the bacterial concentrations used. Transcripts of thephlDandhcnCgenes were detected for all five bacterial concentrations, but thephlDtranscript copy numbers detected were lower than those detected forhcnC, regardless of the initial bacterial concentration or sampling date. For quantifying a low number of transcripts, the relative method was more reliable than the absolute method. This study demonstrates for the first time the use of a relative quantification approach to quantifying microbial gene transcripts from field soil using an exogenous spike-in control.

scholarly journals KLK11 promotes the activation of mTOR and protein synthesis to facilitate cardiac hypertrophy

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yi Wang ◽  
Hongjuan Liao ◽  
Yueheng Wang ◽  
Jinlin Zhou ◽  
Feng Wang ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Angiotensin Ii ◽  
Cardiac Hypertrophy ◽  
Western Blot ◽  
Real Time ◽  
Real Time Pcr ◽  
Mtor Signaling ◽  
Heart Weight ◽  
Cardiomyocyte Hypertrophy ◽  
Quantitative Real Time Pcr

Abstract Background Cardiovascular diseases have become the leading cause of death worldwide, and cardiac hypertrophy is the core mechanism underlying cardiac defect and heart failure. However, the underlying mechanisms of cardiac hypertrophy are not fully understood. Here we investigated the roles of Kallikrein 11 (KLK11) in cardiac hypertrophy. Methods Human and mouse hypertrophic heart tissues were used to determine the expression of KLK11 with quantitative real-time PCR and western blot. Mouse cardiac hypertrophy was induced by transverse aortic constriction (TAC), and cardiomyocyte hypertrophy was induced by angiotensin II. Cardiac function was analyzed by echocardiography. The signaling pathway was analyzed by western blot. Protein synthesis was monitored by the incorporation of [3H]-leucine. Gene expression was analyzed by quantitative real-time PCR. Results The mRNA and protein levels of KLK11 were upregulated in human hypertrophic hearts. We also induced cardiac hypertrophy in mice and observed the upregulation of KLK11 in hypertrophic hearts. Our in vitro experiments demonstrated that KLK11 overexpression promoted whereas KLK11 knockdown repressed cardiomyocytes hypertrophy induced by angiotensin II, as evidenced by cardiomyocyte size and the expression of hypertrophy-related fetal genes. Besides, we knocked down KLK11 expression in mouse hearts with adeno-associated virus 9. Knockdown of KLK11 in mouse hearts inhibited TAC-induced decline in fraction shortening and ejection fraction, reduced the increase in heart weight, cardiomyocyte size, and expression of hypertrophic fetal genes. We also observed that KLK11 promoted protein synthesis, the key feature of cardiomyocyte hypertrophy, by regulating the pivotal machines S6K1 and 4EBP1. Mechanism study demonstrated that KLK11 promoted the activation of AKT-mTOR signaling to promote S6K1 and 4EBP1 pathway and protein synthesis. Repression of mTOR with rapamycin blocked the effects of KLK11 on S6K1 and 4EBP1 as well as protein synthesis. Besides, rapamycin treatment blocked the roles of KLK11 in the regulation of cardiomyocyte hypertrophy. Conclusions Our findings demonstrated that KLK11 promoted cardiomyocyte hypertrophy by activating AKT-mTOR signaling to promote protein synthesis.

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