Microbial gene expression in Guaymas Basin subsurface sediments responds to hydrothermal stress and energy limitation

Abstract

Analyses of gene expression of subsurface bacteria and archaea provide insights into their physiological adaptations to in situ subsurface conditions. We examined patterns of expressed genes in hydrothermally heated subseafloor sediments with distinct geochemical and thermal regimes in Guaymas Basin, Gulf of California, Mexico. RNA recovery and cell counts declined with sediment depth, however, we obtained metatranscriptomes from eight sites at depths spanning between 0.8 and 101.9 m below seafloor. We describe the metabolic potential of sediment microorganisms, and discuss expressed genes involved in tRNA, mRNA, and rRNA modifications that enable physiological flexibility of bacteria and archaea in the hydrothermal subsurface. Microbial taxa in hydrothermally influenced settings like Guaymas Basin may particularly depend on these catalytic RNA functions since they modulate the activity of cells under elevated temperatures and steep geochemical gradients. Expressed genes for DNA repair, protein maintenance and circadian rhythm were also identified. The concerted interaction of many of these genes may be crucial for microorganisms to survive and to thrive in the Guaymas Basin subsurface biosphere.

Fig. 1. Guaymas Basin overview.

A Bathymetry of Guaymas Basin with IODP Expedition 385 drilling sites U1545 to U1552. Light green to deep blue contour lines indicate the increasing water depth in meters. The inner figure shows the overall sampling location in Guaymas Basin; red lines depict the transformation faults, and green lines show the oceanic spreading centers along the transformation faults in Guaymas Basin. Drilling sites included two adjacent sedimented sites in the northwestern flanking regions, with (U1546) and without (U1545) a deep sill intrusion at ~350-430 mbsf; two sites at the hydrothermally active Ringvent formation, where a shallow, recently emplaced hot sill creates steep temperature gradients and drives hydrothermal circulation (sites U1547 and U1548); one site 3.2 km offset from a cold seep on the northwestern flanking regions (U1549), and a cold seep site with a shallow hydrate area on the Sonora Margin (U1552). IODP 385 also drilled the northern axial valley (U1550) and the southwestern franking region known to receive terrestrial sediment input (U1551). B Temperature gradients in the Guaymas Basin subsurface (Source Data).

Fig. 2. Cell abundances for eight IODP385 drilling sites sampled for metatranscriptomic analysis.

A Cell abundance data vs. depth. B Cell abundance data vs. temperature.

Fig. 3. Heatmap showing expression levels (log₂(TPM + 1)) of selected metabolic and cellular processes in Guaymas Basin subsurface sediment samples.

Metabolic and cellular processes identified at the examined sites and depths using the KEGG database, and the BLASTx results generated with DIAMOND against NCBI-NR database (indicated with an asterisk). Processes involve pathways associated with energy-related metabolisms, genetic maintenance, survival strategies, and vitamin/co-factor biosynthesis (Supplementary Tables 2, 3, Source Data and “Methods”). Sites and depths are along the Y axis of the heatmap, and depths are given in meters below sea floor (mbsf). Expression levels are normalized as log₂ transformation of TPM + 1 (a value of 1 was added to TPM values to avoid zeros). TPM Transcripts Per Million, Gly/Gluc glycolysis/gluconeogenesis, TCA Krebs cycle, Fuc/Man/Gal Fructose/Mannose/Galactose, TCS two-component system, QS quorum sensing, As arsenic, Cu Copper, Cd cadmium, CoA Coenzyme A, CoQ complex Q, SM secondary metabolites, Tun-AFO Tungsten-containing aldehyde ferredoxin oxidoreductases.

Fig. 4. Heatmap of expressed genes (log₂(TPM + 1)) associated with methanogenesis, folate and glycine biosynthesis, sulfur and nitrogen cycling, and chemoautotrophy in Guaymas Basin subsurface sediments as identified in KEGG modules.

Sites and depths are along the Y axis, and depths are in meters below seafloor (mbsf). Expression levels are normalized as log₂ transformation of TPM + 1 (a value of 1 was added to TPM values to avoid zeros) (Supplementary Table 3, Source Data and “Methods”). TPM Transcripts Per Million, THF tetrahydrofolate, rTCA reductive Krebs cycle, Pta/Ack phosphate acetyltransferase/Acetate kinase, napA/B periplasmic nitrate reductases A/B, narG/H/I nitrate reductase subunits G/H/I, norB nitric oxide reductase, nosZ nitrous oxide reductase, dsrA dissimilatory sulfite reductase subunit A, apr adenylylsulfate reductase, sat sulfate adenylyltransferase. (*) indicates that these expressed genes were identified after manual curation of the decontaminated and non-redundant BLASTx results generated with DIAMOND against NCBI-NR database (Supplementary Table 2, Source Data and “Methods”).

Fig. 5. Overview of expressed genes for DNA maintenance and repair, RNA editing, and protein homeostasis and degradation used by Guaymas Basin subsurface bacteria and archaea.

Red, yellow and brown circles on the DNA molecule indicate expressed genes encoding DNA proteins (e.g., excinucleases, exonucleases, mismatch proteins) involved in the repair of single and double strand breaks, and mismatches (indicated with gaps, and as orange lines on the DNA molecule, respectively). Red areas on the DNA helix highlight roles of expressed genes for recombination, DNA competence and CRISPR/Cas9 editing. The red mark on the large ribosomal subunit (50 S) indicates the ribosome silencing factor that binds to the 50 S subunit and dissociates the two ribosomal subunits. The red dots on the tRNA molecule indicate positions that can be potentially modified according to the expressed genes detected in the metatranscriptomes. These modifications include thiolations (introduction of thiols in specific tRNA positions) and wobbling of tRNAs, that enables decoding of more than one synonymous mRNA codons (Supplementary Table 2).

Fig. 6. Heatmap of expressed genes (log₂(TPM + 1)) in Guaymas Basin subsurface sediment samples associated with prokaryotic mRNA modifications, DNA maintenance and repair, proteostasis, proteolysis and electron-transfer proteins as identified with BLASTx against NR.

Manual curation was performed on the decontaminated and non-redundant BLASTx results generated with DIAMOND against NCBI-NR database (Supplementary Table 2, Source Data and “Methods”). Sites and depths are along Y axis, and depths are given in meters below seafloor (mbsf). Expression levels are normalized as log₂ transformation of TPM + 1 (a value of 1 added to TPM values to avoid zeros). TPM Transcripts Per Million. vWA von Willebrand factor type A, srp signal recognition particles.