Novel Alphaproteobacteria transcribe genes for nitric oxide transformation at high levels in a marine oxygen-deficient zone

Abstract

Marine oxygen-deficient zones (ODZs) are portions of the ocean where intense nitrogen loss occurs primarily via denitrification and anammox. Despite many decades of study, the identity of the microbes that catalyze nitrogen loss in ODZs is still being elucidated. Intriguingly, high transcription of genes in the same family as the nitric oxide dismutase (nod) gene from Methylomirabilota has been reported in the anoxic core of ODZs. Here, we show that the most abundantly transcribed nod genes in the Eastern Tropical North Pacific ODZ belong to a new order (UBA11136) of Alphaproteobacteria, rather than Methylomirabilota as previously assumed. Gammaproteobacteria and Planctomycetia also transcribe nod, but at lower relative abundance than UBA11136 in the upper ODZ. The nod-transcribing Alphaproteobacteria likely use formaldehyde and formate as a source of electrons for aerobic respiration, with additional electrons possibly from sulfide oxidation. They also transcribe multiheme cytochrome (here named ptd) genes for a putative porin-cytochrome protein complex of unknown function, potentially involved in extracellular electron transfer. Molecular oxygen for aerobic respiration may originate from nitric oxide dismutation via cryptic oxygen cycling. Our results implicate Alphaproteobacteria order UBA11136 as a significant player in marine nitrogen loss and highlight their potential in one-carbon, nitrogen, and sulfur metabolism in ODZs.IMPORTANCEIn marine oxygen-deficient zones (ODZs), microbes transform bioavailable nitrogen to gaseous nitrogen, with nitric oxide as a key intermediate. The Eastern Tropical North Pacific contains the world's largest ODZ, but the identity of the microbes transforming nitric oxide remains unknown. Here, we show that highly transcribed nitric oxide dismutase (nod) genes belong to Alphaproteobacteria of the novel order UBA11136, which lacks cultivated isolates. These Alphaproteobacteria show evidence for aerobic respiration, using oxygen potentially sourced from nitric oxide dismutase, and possess a novel porin-cytochrome protein complex with unknown function. Gammaproteobacteria and Planctomycetia transcribe nod at lower levels. Our results pinpoint the microbes mediating a key step in marine nitrogen loss and reveal an unexpected predicted metabolism for marine Alphaproteobacteria.

Keywords: Alphaproteobacteria; denitrification; marine; nitric oxide; nitrogen; oxygen; oxygen-deficient zone.

Fig 1

Marine Nod clades, gene neighborhoods, and depth profiles of transcription. (A) A maximum likelihood phylogeny of nitric oxide dismutase (Nod) amino acid sequences in marine (blue) and select terrestrial (brown) taxa, primarily from marine MAGs (20–22) and ETNP ODZ metagenomes (18). Branch support was evaluated using 1,000 rapid bootstrap replicates, with bootstrap values shown for deep branches. The tree is drawn to scale, with branch lengths in number of substitutions per site. Bold sequences represent those present in multiple ETNP ODZ metagenomes (see Table S3 for duplicate accession numbers). “PF” indicates genes from the particle fraction (>1.6 μm fraction) of filters. “FL” indicates genes from the free-living fraction (0.2–1.6 μm) collected on Sterivex filters. The most highly transcribed ETNP ODZ sequence is indicated with an asterisk. The qNor sequence Geobacillus stearothermophilus was used as the outgroup. (B) Gene neighborhoods surrounding nod genes in select taxa. GenBank contigs: Cecembia calidifontis SGXG01000001, Scalindua japonica BAOS01000045, Gammaproteobacteria NP964 PBRC01000062, Gammaproteobacterium HdN1 FP929140, Deltaproteobacteria NZCL01000067, Candidatus Methylomirabilis oxyfera FP565575, and Rhodospirillaceae NP1106 PCBZ01000014. Unlabeled gray genes are hypothetical. (C, D) Oxygen concentrations (gray lines), nitrite concentrations (black circles), and nod transcripts (squares, as reads per kilobase per million mapped reads [RPKM]) with depth in ETNP ODZ P1 (onshore) and P2 (offshore) sites (25).

Fig 2

Alphaproteobacteria phylogeny with order UBA11136 expanded and nod-containing MAGs bolded. The phylogeny was constructed using the alphaproteobacterial phylogenetic marker NADH ubiquinone oxidoreductase subunit L as in Cevallos and Degli Esposti (28). Taxonomic names are from Cevallos and Degli Esposti (28) and GTDB Release 08-RS214. The scale bar represents amino acid substitutions per site. The full phylogeny is shown in Fig. S1.

Fig 3

Schematic of the electron transport chain in nod-containing Alphaproteobacteria. Increasing transcriptional activity is indicated from lighter to darker blue (Table S5). Red circles with black lines indicate hemes. Hypothetical Ptd proteins are labeled A, B, C, D, E, F, and G (Table S8). Proposed electron transfer from formate to Complex I is shown. Highly transcribed Nod protein and predicted O₂ generation is shown as feeding into A1-type CCO Complex IV. Additional electrons for CytC and the electron transport chain are proposed to come from sulfur oxidation carried out by the flavocytochrome c sulfide dehydrogenase (FccAB, FCC), and sulfane-sulfur dehydrogenase (SoxCD) with the multi-enzyme carrier complex (SoxYZ).

Fig 4

Gene neighborhoods of pentaheme–tetraheme–decaheme genes from select organisms. Depicted heme spacing is approximate. All organisms are from saline environments (seawater, marine sediment, or saline spring).