mdRNA-Seq analysis of marine microbial communities from the northern Red Sea

Abstract

Metatranscriptomic differential RNA-Seq (mdRNA-Seq) identifies the suite of active transcriptional start sites at single-nucleotide resolution through enrichment of primary transcript 5′ ends. Here we analyzed the microbial community at 45 m depth at Station A in the northern Gulf of Aqaba, Red Sea, during 500 m deep mixing in February 2012 using mdRNA-Seq and a parallel classical RNA-Seq approach. We identified promoters active in situ for five different pico-planktonic genera (the SAR11 clade of Alphaproteobacteria, Synechococcus of Cyanobacteria, Euryarchaeota, Thaumarchaeota, and Micromonas as an example for picoeukaryotic algae), showing the applicability of this approach to highly diverse microbial communities. 16S rDNA quantification revealed that 24% of the analyzed community were group II marine Euryarchaeota in which we identified a highly abundant non-coding RNA, Tan1, and detected very high expression of genes encoding intrinsically disordered proteins, as well as enzymes for the synthesis of specific B vitamins, extracellular peptidases, carbohydrate-active enzymes, and transport systems. These results highlight previously unknown functions of Euryarchaeota with community-wide relevance. The complementation of metatranscriptomic studies with mdRNA-Seq provides substantial additional information regarding transcriptional start sites, promoter activities, and the identification of non-coding RNAs.

Winter mixing impacts gene expression in marine microbial populations in the Gulf of Aqaba

“…1) further supports this conclusion. The 45 m sample was obtained on the same day in the framework of a study that compared different RN A library preparation methods (Hou et al 2016). However, since this sample was collected several hours after the 2.5 and 440 m samples, we excluded it from the differential expression analysis performed here (detailed below).…”

“…The sample collected in the afternoon was obtained in the framework of a different project aiming to compare dRNA-seq (see below) with standard RNA-seq (Hou et al 2016). We also used the 45 m sample for the investigation of 16S-based community composition and taxonomic assignment of RNA reads (see Fig.…”

“…Following mapping to reference genomes, TSSs were inferred as previously described (Hou et al 2016) and outlined in Supplement 1. After detecting TSSs, averaged values of counts of forward and reverse reads assigned to each TSS were used as a measure for expression level of the associated gene.…”

“…Filters were subsequently placed in 1 ml of RN A resuspension buffer (10 mM N aOAc pH 5.2, 200 mM D (+)-sucrose, 100 mM NaCl, 5 mM EDTA), immediately frozen in liquid nitrogen, and kept at −80°C until further analysis. RN A extraction and library preparation was performed according to the mdRNA-seq approach (Sharma et al 2010, Hou et al 2016). The procedure is described below and detailed in Supplement 1 at www.int-res.com/articles/ suppl/a080p223_supp1.pdf.…”

“…Concurrently, we analyzed microbial transcriptional activity using metatranscriptomics. To capture genes that are expressed as a response to changes associated with convection, we assessed only nascent (recently transcribed) transcripts using meta-differential RN A-seq (mdRN Aseq, Hou et al 2016). We further analyzed the data examining the ecophysiological implications for representative species from abundant taxa, including photoautotrophic cyanobacteria, photoheterotrophic bacteria (SAR11), archaea, and picoeukaryotic algae.…”