Millimeter-scale vertical partitioning of nitrogen cycling in hypersaline mats reveals prominence of genes encoding multi-heme and prismane proteins

Abstract

Microbial mats are modern analogues of the first ecosystems on the Earth. As extant representatives of microbial communities where free oxygen may have first been available on a changing planet, they offer an ecosystem within which to study the evolution of biogeochemical cycles requiring and inhibited by oxygen. Here, we report the distribution of genes involved in nitrogen metabolism across a vertical oxygen gradient at 1 mm resolution in a microbial mat using quantitative PCR (qPCR), retro-transcribed qPCR (RT-qPCR) and metagenome sequencing. Vertical patterns in the presence and expression of nitrogen cycling genes, corresponding to oxygen requiring and non-oxygen requiring nitrogen metabolism, could be seen across gradients of dissolved oxygen and ammonium. Metagenome analysis revealed that genes annotated as hydroxylamine dehydrogenase (proper enzyme designation EC 1.7.2.6, hao) and hydroxylamine reductase (hcp) were the most abundant nitrogen metabolism genes in the mat. The recovered hao genes encode hydroxylamine dehydrogenase EC 1.7.2.6 (HAO) proteins lacking the tyrosine residue present in aerobic ammonia oxidizing bacteria (AOB). Phylogenetic analysis confirmed that those proteins were more closely related to ɛHao protein present in Campylobacterota lineages (previously known as Epsilonproteobacteria) rather than oxidative HAO of AOB. BLAST analysis of some transcribed proteins indicated that they likely functioned as a nitrate reductase. The presence of hao sequences related with ɛHao protein, as well as numerous hcp genes encoding a prismane protein, suggest the presence of a nitrogen cycling pathway previously described in Nautilia profundicola as ancestral to the most commonly studied present day nitrogen cycling pathways.

Community ecology and functional potential of bacteria, archaea, eukarya and viruses in Guerrero Negro microbial mat

“…The microbial mats were collected in June 2019 in a concentration area in salterns managed by Exportadora de Sal S.A. (ESSA), Guerrero Negro, Baja California Sur, México, with coordinates: A4; 27•41015.1" N 113•54052.1" W, as previously described [9]. At the time of mat collection, salinity of brine was 125 ppt, temperature 24.4 C, pH 8.3, ammonium concentration 0.12 µM, oxygen dissolved 7 mg/L and nitrate concentration was below the limit of detection (< 0.5 µM).…”

“…At the time of mat collection, salinity of brine was 125 ppt, temperature 24.4 C, pH 8.3, ammonium concentration 0.12 µM, oxygen dissolved 7 mg/L and nitrate concentration was below the limit of detection (< 0.5 µM). Samples from the microbial mats were collected with a stainless-steel corer of 1 cm diameter as previously described [9]. Three replicate cores were placed into sterile centrifuge tubes (Falcon®, Corning, Corning, NY, USA), capped, and immediately frozen in liquid nitrogen.…”

“…Mats located in salterns managed for the production of salt are permanently submerged by a hypersaline water column which serves to protect the vertical structure of the mats -relative to mats found in intertidal environments subjected to more profound environmental disturbance [8]. In a recent study using PCR and qPCR techniques, vertical patterns of the abundance and expression of oxygen requiring and non-oxygen requiring nitrogen cycling genes were found to correlate with measured oxygen concentrations [9]. Metagenome analyses of the same samples were used to investigate the vertical distribution of other genes involved in nitrogen transformations.…”

“…Metagenome analyses of the same samples were used to investigate the vertical distribution of other genes involved in nitrogen transformations. Among the ndings were that hydroxylamine dehydrogenase and hydroxylamine reductase (HAO and HCP proteins, respectively) were the most abundant (demonstrated the highest normalized coverage) nitrogen metabolism genes in the mat [9]. Recovered HAO proteins lacked a key tyrosine residue present in aerobic ammonia oxidizing bacteria (AOB) and was phylogenetically more closely related to Hao protein present in Campylobacterota lineages -providing new information with regard to the evolution of HAO proteins in mats and suggesting the presence of a primitive nitrogen cycling pathway in GN [9].…”

“…Among the ndings were that hydroxylamine dehydrogenase and hydroxylamine reductase (HAO and HCP proteins, respectively) were the most abundant (demonstrated the highest normalized coverage) nitrogen metabolism genes in the mat [9]. Recovered HAO proteins lacked a key tyrosine residue present in aerobic ammonia oxidizing bacteria (AOB) and was phylogenetically more closely related to Hao protein present in Campylobacterota lineages -providing new information with regard to the evolution of HAO proteins in mats and suggesting the presence of a primitive nitrogen cycling pathway in GN [9]. GN microbial mats are characterized by extreme vertical chemical gradients at micrometer to millimeter spatial scales, and these vertical gradients have been shown to correspond to changes in microbial community composition [6].…”