TY - JOUR
T1 - Activity and interactions of methane seep microorganisms assessed by parallel transcription and FISH-NanoSIMS analyses
AU - Dekas, Anne E.
AU - Connon, Stephanie A.
AU - Chadwick, Grayson L.
AU - Trembath-Reichert, Elizabeth
AU - Orphan, Victoria J.
N1 - Funding Information:
We thank the pilots, crew and science party of AT15-11 and AT15-59, especially Lisa Levin, Jake Bailey and Shana Goffredi; Abigail Green-Saxena and Joshua Steele for valuable discussions; Bethany Jenkins for consultation with RNA extraction protocol development; John Eiler and Yunbin Guan for assistance with NanoSIMS measurements. We additionally thank three anonymous reviewers for their careful critique of the manuscript. Funding was provided by the US Department of Energy, Office of Science, Office of Biological and Environmental Research (DE-SC0003940 and DE-SC0004949 to VJO), the National Science Foundation (MCB-0348492 to VJO and a Graduate
Funding Information:
Research Fellowship to AED) and the Gordon and Betty Moore Foundation (GBMF no. 3780 to VJO, and via support for The Caltech Center for Microanalysis housing the CAMECA NanoSIMS 50 L). The writing of this manuscript by AED was partially performed while funded by a Lawrence Postdoctoral Fellowship under the auspices of the US Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.
PY - 2016/3/1
Y1 - 2016/3/1
N2 - To characterize the activity and interactions of methanotrophic archaea (ANME) and Deltaproteobacteria at a methane-seeping mud volcano, we used two complimentary measures of microbial activity: a community-level analysis of the transcription of four genes (16S rRNA, methyl coenzyme M reductase A (mcrA), adenosine-5′-phosphosulfate reductase α-subunit (aprA), dinitrogenase reductase (nifH)), and a single-cell-level analysis of anabolic activity using fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS). Transcript analysis revealed that members of the deltaproteobacterial groups Desulfosarcina/Desulfococcus (DSS) and Desulfobulbaceae (DSB) exhibit increased rRNA expression in incubations with methane, suggestive of ANME-coupled activity. Direct analysis of anabolic activity in DSS cells in consortia with ANME by FISH-NanoSIMS confirmed their dependence on methanotrophy, with no 15 NH 4 + assimilation detected without methane. In contrast, DSS and DSB cells found physically independent of ANME (i.e., single cells) were anabolically active in incubations both with and without methane. These single cells therefore comprise an active 'free-living' population, and are not dependent on methane or ANME activity. We investigated the possibility of N 2 fixation by seep Deltaproteobacteria and detected nifH transcripts closely related to those of cultured diazotrophic Deltaproteobacteria. However, nifH expression was methane-dependent. 15 N 2 incorporation was not observed in single DSS cells, but was detected in single DSB cells. Interestingly, 15 N 2 incorporation in single DSB cells was methane-dependent, raising the possibility that DSB cells acquired reduced 15 N products from diazotrophic ANME while spatially coupled, and then subsequently dissociated. With this combined data set we address several outstanding questions in methane seep microbial ecosystems and highlight the benefit of measuring microbial activity in the context of spatial associations.
AB - To characterize the activity and interactions of methanotrophic archaea (ANME) and Deltaproteobacteria at a methane-seeping mud volcano, we used two complimentary measures of microbial activity: a community-level analysis of the transcription of four genes (16S rRNA, methyl coenzyme M reductase A (mcrA), adenosine-5′-phosphosulfate reductase α-subunit (aprA), dinitrogenase reductase (nifH)), and a single-cell-level analysis of anabolic activity using fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS). Transcript analysis revealed that members of the deltaproteobacterial groups Desulfosarcina/Desulfococcus (DSS) and Desulfobulbaceae (DSB) exhibit increased rRNA expression in incubations with methane, suggestive of ANME-coupled activity. Direct analysis of anabolic activity in DSS cells in consortia with ANME by FISH-NanoSIMS confirmed their dependence on methanotrophy, with no 15 NH 4 + assimilation detected without methane. In contrast, DSS and DSB cells found physically independent of ANME (i.e., single cells) were anabolically active in incubations both with and without methane. These single cells therefore comprise an active 'free-living' population, and are not dependent on methane or ANME activity. We investigated the possibility of N 2 fixation by seep Deltaproteobacteria and detected nifH transcripts closely related to those of cultured diazotrophic Deltaproteobacteria. However, nifH expression was methane-dependent. 15 N 2 incorporation was not observed in single DSS cells, but was detected in single DSB cells. Interestingly, 15 N 2 incorporation in single DSB cells was methane-dependent, raising the possibility that DSB cells acquired reduced 15 N products from diazotrophic ANME while spatially coupled, and then subsequently dissociated. With this combined data set we address several outstanding questions in methane seep microbial ecosystems and highlight the benefit of measuring microbial activity in the context of spatial associations.
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U2 - 10.1038/ismej.2015.145
DO - 10.1038/ismej.2015.145
M3 - Article
C2 - 26394007
AN - SCOPUS:84942155451
VL - 10
SP - 678
EP - 692
JO - ISME Journal
JF - ISME Journal
SN - 1751-7362
IS - 3
ER -