Metal-ion-induced expression of gene fragments from subseafloor micro-organisms in the Kumano forearc basin, Nankai Trough

Wakamatsu, Taisuke; Morono, Yuki; Futagami, Taiki; Terada, T.; Nishikawa, Shuh-ichi; Morisawa, Takashi; Ohshita, Koki; Inagaki, Fumio; Ashiuchi, Makoto

doi:10.1111/jam.14061

Cited by 2 publications

(9 citation statements)

References 59 publications

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“…The insert length (0.8–8 kbp) was confirmed by restriction enzyme digestion ( Eco RI/ Hin dIII) of plasmids from randomly selected 56 clones (28 clones from each library). The sum length of DNA inserts (approximately 6 Gbp for both libraries) exceeded that of our previous attempts (0.05 Gbp) ( Futagami et al, 2013 ; Wakamatsu et al, 2018 ), and those reported by Uchiyama et al (2005) , Uchiyama and Watanabe (2008) , Uchiyama and Miyazaki (2013) , (0.1 Gbp) and by Meier et al (2016) (1 Gbp).…”

Section: Resultscontrasting

confidence: 61%

“…The next optimization step concerned the DNA size selection, which is the most important step in plasmid library preparation. In previous reports, inserted DNA was size-selected by electrophoresis and gel excision ( Futagami et al, 2013 ; Wakamatsu et al, 2018 ). Although a TOPO-vector with a dT-overhang at its 3′ end was used in the above-mentioned studies, clones with green fluorescence, which indicated that no DNA insert was introduced into the plasmid, were frequently observed.…”

Section: Resultsmentioning

confidence: 99%

“…We next optimized the electroporation transformation procedure. In the previous studies ( Futagami et al, 2013 ; Wakamatsu et al, 2018 ), ethanol precipitation was used to concentrate the ligation products. However, there were problems of arching or high current-induced reduction of transformation efficiency happened upon the electroporation step.…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, to date, induction analyses have been performed only under aerobic conditions. Although approximately a quarter of inducible genes identified by aerobic SIGEX screening shared similarity with anaerobic microbial genes ( Futagami et al, 2013 ; Wakamatsu et al, 2018 ), transcription factors that do not function under oxidative conditions have been reported ( Unden and Schirawski, 1997 ; Pop et al, 2004 ; Mesa et al, 2009 ). The potential of using gfp under anaerobic conditions was shown for a facultative anaerobic bacterium Enterobacter aerogenes under anaerobic culture conditions, by transferring the culture to aerobic conditions ( Zhang et al, 2005 ).…”

Section: Discussionmentioning

confidence: 99%

“…For example, Uchiyama et al (2005) , Uchiyama and Watanabe (2008) , Uchiyama and Miyazaki (2013) , and Meier et al (2016) analyzed groundwater and soil samples, respectively, and obtained genes induced by aromatic compounds. Futagami et al (2013) and Wakamatsu et al (2018) retrieved gene fragments from deep subseafloor microbes recovered from the Kumano forearc basin (Nankai Trough, Japan). The authors identified fragments responsive to organohalides, 2,4,6-tribromophenol (2,4,6-TBP) or 2,4,6-trichlorophenol (2,4,6-TCP), individually or by a mixture of 2,4,6-TBP, 2,4,6-TCP, and 2,4,6-triiodophenol, and metal ions (Ni 2+ , Ga 3+ , or Fe 3+ ).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2022

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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.

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