Dataset: Thalassiosira pseudonana CCMP1335 endometabolite uptake by Ruegeria pomeroyi DSS-3

Name: Thalassiosira pseudonana CCMP1335 endometabolite uptake by Ruegeria pomeroyi DSS-3
Published: 2024-07-15
Keywords: marine microbial cocultures, transcriptomes, temperature acclimated strains, Ruegeria pomeroyi, Emiliania huxleyi, Synechococcus sp. WHOI8102, oceans

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Cite This Dataset

University of Georgia, Athens GA 30602

Project:

Effects of Climate Change Variables on Microbial Autotroph-Heterotroph Carbon Flux (CC_Auto_Hetero_Fluxes)

Principal Investigator:

Mary Ann Moran (University of Georgia, UGA)

Scientist:

Malin Olofsson (University of Georgia, UGA)

Technician:

Christa Smith (University of Georgia, UGA)

BCO-DMO Data Manager:

Karen Soenen (Woods Hole Oceanographic Institution, WHOI BCO-DMO)

Version:

Version Date:

2024-07-15

Restricted:

Validated:

Yes

Current State:

Final no updates expected

Thalassiosira pseudonana CCMP1335 endometabolite uptake by Ruegeria pomeroyi DSS-3

Abstract:

The ocean’s temperature increase has fundamental implications for physiological rates and processes of marine microbes. In this study, a marine diatom Thalassiosira pseudonana CCMP1335 was acclimated for three months at temperatures below (14°C), equal to (20°C), and above (28°C). Heterotrophic bacterium Ruegeria pomeroyi DSS-3 was inoculated into cultures, and transporter expression was compared between temperatures. R. pomeroyi transporter expression leveraged as a biosensor of available diatom exometabolites indicated temperature-related substitution of diatom osmolytes dimethylsulfoniopropionate (DMSP), dihydroxypropanesulfonate (DHPS), and homarine (dominating carbon transfer at lower temperatures) with glycine betaine and choline (dominating at higher temperatures). T. pseudonana endometabolome pools and biosynthetic pathway expression indicated increased availability of amino acids and glycerol-3-phosphate at higher temperatures. Overall trends across datasets supported a greater importance of organic sulfur compounds in diatom-bacterial metabolite transfer at lower temperatures and greater importance of organic nitrogen compounds at higher temperatures.

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Description

All the raw spectra, NMRPipe scripts for spectrum processing, and MATLAB scripts for data analysis are deposited in Metabolomics Workbench under Project ID PR001837 (see related datasets).

Methods & Sampling

Temperature experiment: Axenic cultures of the diatom Thalassiosira pseudonana CCMP1335 (National Center for Marine Algae) were acclimated to three temperature conditions (below optimal: 14°C, optimal: 20°C, and above optimal: 28°C) for three months with weekly transfers (approximately 120 generations) in L1 medium with 35 ppt artificial seawater under 120 µmol photons m-2 s-1 (ULM-500 Light Meter, Walz) and a 16:8 h light:dark cycle. Diatom cultures were stepwise limited by B12 through three transfers. Ruegeria pomeroyi DSS-3 was grown overnight in ½ YTSS medium, harvested in exponential growth phase, washed five times in sterile artificial seawater at 6,000 RCF, and inoculated into diatom flasks with 4 replicates per acclimation temperature. Flasks were harvested at late exponential growth phase on days 3 (28°C), 4 (20°C), and 6 (14°C), with sampling occurring 7 h into the light cycle.

From axenic and inoculated flasks, cultures were sequentially filtered through 2.0 µm-pore-size Isopore filters (Millipore, Burlington, MA) and 0.2 µm-pore-size Supor filters (PALL, Port Washington, NY) to collect 600 ml for diatom endometabolits analysis, bacterial cells for RNA extraction. RNA filters were immediately flash-frozen in liquid nitrogen and stored at -80°C until processing. RNA was subsequently extracted from filters using the ZymoBIOMICS RNA Miniprep Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s protocol.

Stranded RNASeq libraries were prepared by the Joint Genome Institute (JGI) and sequenced on an Illumina NovaSeq.

Metabolites were analyzed by NMR spectroscopy using a 600 MHz AVANCE III HD instrument (Bruker) equipped with a 5 mm TCI cryoprobe and pulse programs of 1H-13C heteronuclear single quantum correlation (HSQC, hsqcetgpprsisp2.2 by Bruker nomenclature) and 1H-13C HSQC-total correlation spectroscopy (HSQC-TOCSY, hsqcdietgpsisp.2). TopSpin (Bruker) version 3.5 was used for NMR operation. Data were processed by NMRPipe.

Peak intensity was extracted by rNMR version 1.11 and data were analyzed by MATLAB (MathWorks) version R2023b. Peak intensity was normalized by biovolume and auto-scaled. Metabolites were annotated based on chemical shift (HSQC) and correlation information (HSQC-TOCSY). Chemical shift values for candidate peaks were obtained from the Biological Magnetic Resonance Data Bank and the Human Metabolome Database, and raw reference spectra from BMRB were used for validation.

Draw down experiment: Thalassiosira pseudonana CCMP1335 cells harvested from axenic flasks were rinsed from thawed filters into 10 ml sterile L1 medium, sonicated to lyse cells, and passed through a pre-combusted GF/F filter. Ruegeria pomeroyi DSS-3 was inoculated into a 2.7 ml aliquot of concentrated endometabolomes and grown for 10 h in 96-well plates with shaking at 30°C in dark conditions. Subsamples of 1 ml were collected at inoculation and at the end of the experiment for metabolite quantification. From these subsamples, 540 µl were mixed with 60 µL of the phosphate buffer and transferred to NMR tubes. Metabolites were analyzed by NMR spectroscopy using a 600 MHz AVANCE III HD instrument (Bruker) equipped with a 5 mm TCI cryoprobe and pulse programs of 1H-13C heteronuclear single quantum correlation (HSQC, hsqcetgpprsisp2.2 by Bruker nomenclature) and 1H-13C HSQC-total correlation spectroscopy (HSQC-TOCSY, hsqcdietgpsisp.2). TopSpin (Bruker) version 3.6.4 was used for NMR operation.

Data were processed by NMRPipe. Peak intensity was extracted by rNMR version 1.11 and data were analyzed by MATLAB (MathWorks) version R2023b. Peak intensity was normalized by biovolume and auto-scaled. Metabolites were annotated based on chemical shift (HSQC) and correlation information (HSQC-TOCSY). Chemical shift values for candidate peaks were obtained from the Biological Magnetic Resonance Data Bank and the Human Metabolome Database, and raw reference spectra from BMRB were used for validation.

Data Processing Description

Reads were filtered and trimmed using the JGI QC pipeline, followed by evaluation of artifact sequences by kmer matching (kmer=25) using BBDuk, allowing one mismatch; detected artifacts were trimmed from the 3´end of the reads. Quality trimming was performed using the phred trimming method set at Q6 and reads under the minimum length of 25 bases or 1/3 of the original read length were removed. Filtered reads from each library were aligned to the reference genome using HISAT2 version 2.2.0 and strand-specific coverage bigwig files were generated using deepTools v3.1. FeatureCounts was used to generate the raw gene counts, and DESeq2 (version 1.28.1) was used to determine relative expression differences between pairs of conditions. T. pseudonana gene annotations and biosynthesis pathways were based on Biocyc and PhyloDB. R. pomeroyi annotations were based on Roseobase (http://roseobase.org)