Metaproteogenomic Profile of a Mesopelagic Adenylylsulfate Reductase: Course-Based Discovery Using the Ocean Protein Portal

Abstract

Adenylylsulfate reductase (Apr) is a flavoprotein with a dissimilatory sulfate reductase function. Its ability to catalyze the reverse reaction in sulfur oxidizers has propelled a complex phylogenetic history of transfers with sulfate reducers and made this enzyme an important protein in ocean sulfur cycling. As part of a graduate course, we analyzed metaproteomic data from the Ocean Protein Portal and observed evidence of Apr alpha (AprA) and beta (AprB) subunits in the Central Pacific Ocean. The protein was originally taxonomically attributed toChlorobium tepidum TLS, a green sulfur bacterium. However, our phylogenomic and oceanographic contextual analysis contradicted this label, instead showing that this protein is consistent with the genomic material from the newly discovered Candidatus Lambdaproteobacteriaclass, implying that the ecological role of this lineage in oxygen minimum twilight zones is underappreciated. This study illustrates how metaproteogenomic analysis can contribute to more accurate metagenomic/proteomic annotations and comprehensive ocean biogeochemical processes conducive to course-based research experiences.

Keywords: biogeochemistry; course-based research; metaproteogenomics; ocean metaproteomics.

Figure 1

Visualization of Apr abundance in an oxygen minimum zone of the Central Pacific. Section heatmap of (A) AprA and (B) AprB vertical distributions along the ocean transect, color gridded by abundance. (C) Oxygen concentrations along stations, color gridded by abundance. (D) Vertical profiles of the total 211 AprA spectral counts along expedition FK160115 sites.

Figure 2

Abridged (1–420 amino acids) visualization of AprA multiple sequence alignment between the homologous OPP sequence, Candidatus Lambdaproteobacteria bacterium (Can. Lambda), and Chlorobium tepidum TLS (C. tepidum). The colored line denotes the FAD-conserved domain. Individual amino acids are colored by the CLUSTALX general color scheme.

Figure 3

Maximum likelihood tree of AprA homologs. The OPP query position is marked in red, and Candidatus Lambdaproteobacteria taxa are colored yellow. (A) Radial unrooted tree with 100 collapsed lineages. (B) Midpoint-rooted tree with collapsed clades of 242 taxa. Support values indicate the approximate likelihood ratio test (aLRT)/bootstrap values less than 100. Collapsed groups are labeled and colored by taxonomy.

Figure 4

Structures of AprA and AprB from modeling and PDB. (A) AprA and AprB from the OPP query produced by AlphaFold (v2.3.1.). (B) 3D structure of Apr inD. gigas from Protein Data Bank ID 3GYX, including a FAD cofactor and iron–sulfur clusters. Coloring based on conserved protein domains and structures. Further information about modeling confidence is found in Supporting Information Figures 1, 2.

Figure 5

Metagenomic reads across the Ocean Gene Atla Tara expedition. (A) Krona pie chart abundance weighted taxonomic distribution of homologs by percentage with colored legends. (B) Bubble plots of homolog abundances plotted by O₂ and depth against sampling depth at the upper layer zone (SRF), DCM, mesopelagic layer (MES), and marine epipelagic mixed layer (MIX). Sample circles are proportional to homolog abundance.