Transcriptome analysis of Agrobacterium sp. ATCC 31749 wild strain and ΔfnrN based on RNA-seq
ABSTRACT: Purpose:first,we want to find the genes revelant to curdlan synthesis and oxygen regulation, second, we want to research the function of fnrN gene in Agrobacterium sp. ATCC 31749. Method: samples of cell growth phase, curdlan-producing phase (normoxia) and curdlan-producing phase (micro-oxygen treated) in both Agrobacterium sp. ATCC 31749 wild strain and ΔfnrN strain were collectecd to extract mRNA. Each sample was treated in duplicate. The softwares we used include fastqc, trimmomatic, TopHat2 and Cufflinks. Illumina Hiseq4000 was used to complete the research. Overall design: 12 samples: samples of cell growth phase, curdlan-producing phase (normoxia) and curdlan-producing phase (micro-oxygen treated) in both Agrobacterium sp. ATCC 31749 wild strain and ΔfnrN strain were collectecd to extract mRNA. Each sample was treated in duplicate.
Project description:Gene Expression Analysis of Curdlan Production in Agrobacterium sp. ATCC 31749 Overall design: Two conditions are compared with four biological replicates of each condition, corresponding to eight total samples. The control condition was sampled at 22 hours during the exponential growth phase when no curdlan is produced. The second condition was sampled at 70 hours after the initiation of curdlan production, corresponding to approximately 100 hours.
Project description:Gene Expression Analysis of Curdlan Production in Agrobacterium sp. ATCC 31749 Two conditions are compared with four biological replicates of each condition, corresponding to eight total samples. The control condition was sampled at 22 hours during the exponential growth phase when no curdlan is produced. The second condition was sampled at 70 hours after the initiation of curdlan production, corresponding to approximately 100 hours.
Project description:Investigation of mRNA expression level changes in S. pombeOfd2 (SPAP8A3.02c) deletion strain compared to wild-type strain at both normal oxygen (normoxia) and low oxygen (hypoxia) growth conditions. Four samples in total consisting of two S. pombeyeast strains, wild type (WT) and a gene deleted strain for Ofd2 (SPAP8A3.02c), analysed for gene expression under two growth conditions, normal oxygen (normoxia) and low oxygen (hypoxia). Five micrograms of total RNA from two independent experiments were pooled and mRNA levels were quantified by Roche NimbleGen using the S. pombe 72K array service
Project description:Saccharomyces cerevisiae is currently widely used as a model to study chronological aging of metazoan cells. Chronological aging is typically studied in aerobic stationary phase (SP) cultures, i.e. the final stage of batch cultures in which growth is arrested due to exogenous carbon source exhaustion. Survival of yeast cells in SP defines their chronological lifespan (CLS). S. cerevisiae SP cultures have strongly contributed to the understanding of cellular mechanisms involved in aging and indicated a key role for oxygen. Oxygen is the natural starting point for reactive oxygen species (ROS) that may both have malignant and beneficial effects on aging. In addition, oxygen allows yeast to grow on ethanol and organic acids formed during the initial respiro-fermentative growth phase on glucose. This post-diauxic phase is hallmarked by reduced growth rates, increased expression of genes involved in SP survival, and increased stress resistance. To date, the role of oxygen and respiration in aging has mostly been studied using respiratory deficient mutants, and respiration repressing agents. However, genetic or chemical interventions may result in unwanted side effects that influence survival in SP. We therefore followed a different approach to evaluate the impact of oxygen availability on yeast robustness in SP, i.e. its CLS and stress resistance, by using the capability of S. cerevisiae to grow under anaerobic conditions. A thorough physiological comparison of strictly anaerobic and aerobic SP cultures revealed that the presence of oxygen during growth and aging of S. cerevisiae strongly affects its energetic status, longevity and stress tolerance in a positive way. Combining the physiological data with genome-wide expression analysis revealed that the oxygen-dependent diauxic growth phase enabled the full induction of robustness in S. cerevisiae, and points to appropriate pre-conditioning of cells as a crucial factor to survive starvation. These findings highlight the importance of exogenous energy availability in the conditions leading to growth arrest, and bring new insight on the role of oxygen in the aging of eukaryotes. The goal of the present study is to evaluate the impact of oxygen availability on yeast longevity. More specifically, the questions addressed are whether the presence of a ‘conditioning’ post-diauxic phase and the ability to utilize efficiently reserves, characteristics of aerobicity, affects yeast survival during stationary phase. To this end, S. cerevisiae was cultivated in well controlled bioreactors under the presence or absence of oxygen. S. cerevisiae’s response to oxygen availability was monitored by an in-depth physiological analysis combined with genome wide expression analysis.
Project description:ChIP-seq analysis was used to identify B. dermatitidis genes bound by the GATA transcription factor encoded by SREB during growth as yeast at 37oC SREB was engineered to contain an in-frame 3x-hemagglutinin (HA) epitope tag at the C-terminus. The SREB-3xHA construct was under control of its native promoter and contained the 3-untranslated region. Using Agrobacterium tumefaciens, B. dermatitidis ATCC 26199 was transformed with the SREB-3xHA construct (referred to a SREB-3xHA strain in this document). The SREB-3xHA construct was functional because retransformation of SREBΔ with the construct complented the null mutant. Chromatin was extracted and sheared from ATCC 26199 and SREB-3xHA yeast grown in liquid Histoplasma macrophage medium (HMM) containing 10 μM iron sulfate (FeSO4) at 37oC. ATCC 26199 was the untagged control strain.
Project description:(1) The identification of genes that are transcriptionally regulated in response to exposure to hypoxia (~0.2% oxygen) in H99 (WT) and strains CM092 and CM098. (2) The identification of genes that are differentially-regulated in the strain CM093 compared to H99 (WT) Keywords: hypoxia response, comparative transcriptional hybridization 8 cultures for each strain (4 grown in normoxia and 4 grown in hypoxia) are analyzed on 4 microarrays for each strain (normoxia hybridized against hypoxia). Two out of four microarrays contain dye-swaps.
Project description:The anaerobic gut microbe Akkermansia muciniphila ATCC BAA-835 lives in the mucus layer where it is exposed to oxygen. To investigate how it survives the changing oxygen concentrations, we exposed a exponentially growing culture to oxygen. The experiment was performed in parallel fermentor systems. To one system we added 0.2l/h oxygen after the culture reached an OD of 0.1, while the other remained anaerobic. Samples were taken just before addition of oxygen, about 1h after and when the cell reached stationary phase.
Project description:Welan gum is mainly produced by Sphingomonas sp. ATCC 31555 and has broad applications in industry such as that in cement production. Both carbon and nitrogen sources are essential for welan production. However, how nitrogen sources affect the metabolism and gene transcription of welan remains elusive. Here, we used next-generation sequencing RNA-seq to analyze the transcriptome of Sphingomonas sp. ATCC 31555 in the presence of inorganic or organic nitrogen sources. Enriched gene expression and pathway analysis suggest that organic nitrogen sources significantly enhanced the expression of genes in central metabolic pathways of Sphingomonas sp. ATCC 31555 and those critical for welan synthesis compared to that observed using inorganic nitrogen sources. The present study improves our understanding of the molecular mechanism underlying the use of nitrogen in welan synthesis in Sphingomonas sp., as well as provides an important transcriptome resource for Sphingomonas sp. in relation to nitrogen sources. Sphingomonas sp. ATCC 31555 strain (stored in our laboratory) was first seeded in an inoculum medium (20 g/L glucose, 3 g/L yeast extract, 3 g/L malt extract, and 5 g/L fish meal protein peptone, pH 7.0), and then cultured in a fermentation medium containing 40 g/L sucrose, 4.0 g/L nitrogen source, 0.6 g/L KH2PO4, and 0.2 g/L MgSO4.7H2O at 37°C. The nitrogen sources used in the present study were as follows: NaNO3 (4.0 g/L) as inorganic nitrogen (IN), beef extract (4.0 g/L) as organic nitrogen (ON), and NaNO3 (1.5 g/L) + beef extract (2.5 g/L) as complex nitrogen (CN). All cultivations were conducted in flasks with constant rotary shaking at 400–1,000 rpm and 37°C.