scholarly journals Construction of Aerobic/Anaerobic-Substrate-Induced Gene Expression Procedure for Exploration of Metagenomes From Subseafloor Sediments

2022 ◽  
Vol 12 ◽  
Author(s):  
Taisuke Wakamatsu ◽  
Saki Mizobuchi ◽  
Fumiaki Mori ◽  
Taiki Futagami ◽  
Takeshi Terada ◽  
...  
Keyword(s):  
Gene Expression ◽  
Aromatic Compounds ◽  
Fine Tuning ◽  
Anaerobic Conditions ◽  
Metagenomic Dna ◽  
Library Construction ◽  
Dna Library ◽  
Dna Libraries ◽  
Aerobic And Anaerobic ◽  
Aerobic And Anaerobic Conditions

Substrate-induced gene expression (SIGEX) is a high-throughput promoter-trap method. It is a function-based metagenomic screening tool that relies on transcriptional activation of a reporter gene green fluorescence protein (gfp) by a metagenomic DNA library upon induction with a substrate. However, its use is limited because of the relatively small size of metagenomic DNA libraries and incompatibility with screening metagenomes from anaerobic environments. In this study, these limitations of SIGEX were addressed by fine-tuning metagenome DNA library construction protocol and by using Evoglow, a green fluorescent protein that forms a chromophore even under anaerobic conditions. Two metagenomic libraries were constructed for subseafloor sediments offshore Shimokita Peninsula (Pacific Ocean) and offshore Joetsu (Japan Sea). The library construction protocol was improved by (a) eliminating short DNA fragments, (b) applying topoisomerase-based high-efficiency ligation, (c) optimizing insert DNA concentration, and (d) column-based DNA enrichment. This led to a successful construction of metagenome DNA libraries of approximately 6 Gbp for both samples. SIGEX screening using five aromatic compounds (benzoate, 3-chlorobenzoate, 3-hydroxybenzoate, phenol, and 2,4-dichlorophenol) under aerobic and anaerobic conditions revealed significant differences in the inducible clone ratios under these conditions. 3-Chlorobenzoate and 2,4-dichlorophenol led to a higher induction ratio than that for the other non-chlorinated aromatic compounds under both aerobic and anaerobic conditions. After the further screening of induced clones, a clone induced by 3-chlorobenzoate only under anaerobic conditions was isolated and characterized. The clone harbors a DNA insert that encodes putative open reading frames of unknown function. Previous aerobic SIGEX attempts succeeded in the isolation of gene fragments from anaerobes. This study demonstrated that some gene fragments require a strict in vivo reducing environment to function and may be potentially missed when screened by aerobic induction. The newly developed anaerobic SIGEX scheme will facilitate functional exploration of metagenomes from the anaerobic biosphere.

scholarly journals Modeling Gene Networks inSaccharomyces cerevisiaeBased on Gene Expression Profiles

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Yulin Zhang ◽  
Kebo Lv ◽  
Shudong Wang ◽  
Jionglong Su ◽  
Dazhi Meng
Keyword(s):  
Gene Expression ◽  
Gene Regulatory Network ◽  
Regulatory Network ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Clustering Coefficient ◽  
Anaerobic Conditions ◽  
Gene Regulatory ◽  
Aerobic And Anaerobic ◽  
Aerobic And Anaerobic Conditions

Detailed and innovative analysis of gene regulatory network structures may reveal novel insights to biological mechanisms. Here we study how gene regulatory network inSaccharomyces cerevisiaecan differ under aerobic and anaerobic conditions. To achieve this, we discretized the gene expression profiles and calculated the self-entropy of down- and upregulation of gene expression as well as joint entropy. Based on these quantities the uncertainty coefficient was calculated for each gene triplet, following which, separate gene logic networks were constructed for the aerobic and anaerobic conditions. Four structural parameters such as average degree, average clustering coefficient, average shortest path, and average betweenness were used to compare the structure of the corresponding aerobic and anaerobic logic networks. Five genes were identified to be putative key components of the two energy metabolisms. Furthermore, community analysis using the Newman fast algorithm revealed two significant communities for the aerobic but only one for the anaerobic network. David Gene Functional Classification suggests that, under aerobic conditions, one such community reflects the cell cycle and cell replication, while the other one is linked to the mitochondrial respiratory chain function.

scholarly journals Glycerol Is Metabolized in a Complex and Strain-Dependent Manner in Enterococcus faecalis

2009 ◽  
Vol 192 (3) ◽  
pp. 779-785 ◽  
Author(s):  
Alain Bizzini ◽  
Chen Zhao ◽  
Aurélie Budin-Verneuil ◽  
Nicolas Sauvageot ◽  
Jean-Christophe Giard ◽  
...  
Keyword(s):  
Gene Expression ◽  
Enterococcus Faecalis ◽  
Glycerol Dehydrogenase ◽  
Dihydroxyacetone Phosphate ◽  
Dependent Manner ◽  
Anaerobic Conditions ◽  
Dihydroxyacetone Kinase ◽  
Aerobic And Anaerobic ◽  
Strain Dependent ◽  
Aerobic And Anaerobic Conditions

ABSTRACT Enterococcus faecalis is equipped with two pathways of glycerol dissimilation. Glycerol can either first be phosphorylated by glycerol kinase and then oxidized by glycerol-3-phosphate oxidase (the glpK pathway) or first be oxidized by glycerol dehydrogenase and then phosphorylated by dihydroxyacetone kinase (the dhaK pathway). Both pathways lead to the formation of dihydroxyacetone phosphate, an intermediate of glycolysis. It was assumed that the glpK pathway operates during aerobiosis and that the dhaK pathway operates under anaerobic conditions. Because this had not been analyzed by a genetic study, we constructed mutants of strain JH2-2 affected in both pathways. The growth of these mutants on glycerol under aerobic and anaerobic conditions was monitored. In contrast to the former model, results strongly suggest that glycerol is catabolized simultaneously by both pathways in the E. faecalis JH2-2 strain in the presence of oxygen. In accordance with the former model, glycerol is metabolized by the dhaK pathway under anaerobic conditions. Comparison of different E. faecalis isolates revealed an impressive diversity of growth behaviors on glycerol. Analysis by BLAST searching and real-time reverse transcriptase PCR revealed that this diversity is based not on different gene contents but rather on differences in gene expression. Some strains used preferentially the glpK pathway whereas others probably exclusively the dhaK pathway under aerobic conditions. Our results demonstrate that the species E. faecalis cannot be represented by only one model of aerobic glycerol catabolism.

scholarly journals Impact of Anaerobiosis on Expression of the Iron-Responsive Fur and RyhB Regulons

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Nicole A. Beauchene ◽  
Kevin S. Myers ◽  
Dongjun Chung ◽  
Dan M. Park ◽  
Allison M. Weisnicht ◽  
...  
Keyword(s):  
Gene Expression ◽  
Small Rna ◽  
Iron Homeostasis ◽  
Target Genes ◽  
Arms Race ◽  
Anaerobic Conditions ◽  
Content Type ◽  
Expression Of Genes ◽  
Aerobic And Anaerobic ◽  
Aerobic And Anaerobic Conditions

ABSTRACTIron, a major protein cofactor, is essential for most organisms. Despite the well-known effects of O2on the oxidation state and solubility of iron, the impact of O2on cellular iron homeostasis is not well understood. Here we report that inEscherichia coliK-12, the lack of O2dramatically changes expression of genes controlled by the global regulators of iron homeostasis, the transcription factor Fur and the small RNA RyhB. Using chromatin immunoprecipitation sequencing (ChIP-seq), we found anaerobic conditions promote Fur binding to more locations across the genome. However, by expression profiling, we discovered that the major effect of anaerobiosis was to increase the magnitude of Fur regulation, leading to increased expression of iron storage proteins and decreased expression of most iron uptake pathways and several Mn-binding proteins. This change in the pattern of gene expression also correlated with an unanticipated decrease in Mn in anaerobic cells. Changes in the genes posttranscriptionally regulated by RyhB under aerobic and anaerobic conditions could be attributed to O2-dependent changes in transcription of the target genes: aerobic RyhB targets were enriched in iron-containing proteins associated with aerobic energy metabolism, whereas anaerobic RyhB targets were enriched in iron-containing anaerobic respiratory functions. Overall, these studies showed that anaerobiosis has a larger impact on iron homeostasis than previously anticipated, both by expanding the number of direct Fur target genes and the magnitude of their regulation and by altering the expression of genes predicted to be posttranscriptionally regulated by the small RNA RyhB under iron-limiting conditions.IMPORTANCEMicrobes and host cells engage in an “arms race” for iron, an essential nutrient that is often scarce in the environment. Studies of iron homeostasis have been key to understanding the control of iron acquisition and the downstream pathways that enable microbes to compete for this valuable resource. Here we report that O2availability affects the gene expression programs of twoEscherichia colimaster regulators that function in iron homeostasis: the transcription factor Fur and the small RNA regulator RyhB. Fur appeared to be more active under anaerobic conditions, suggesting a change in the set point for iron homeostasis. RyhB preferentially targeted iron-containing proteins of respiration-linked pathways, which are differentially expressed under aerobic and anaerobic conditions. Such findings may be relevant to the success of bacteria within their hosts since zones of reduced O2may actually reduce bacterial iron demands, making it easier to win the arms race for iron.

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