Draft Genome of Scalindua rubra, Obtained from the Interface Above the Discovery Deep Brine in the Red Sea, Sheds Light on Potential Salt Adaptation Strategies in Anammox Bacteria
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Several recent studies have indicated that members of the phylum Planctomycetes are abundantly present at the brine-seawater interface (BSI) above multiple brine pools in the Red Sea. Planctomycetes include bacteria capable of anaerobic ammonium oxidation (anammox). Here, we investigated the possibility of anammox at BSI sites using metagenomic shotgun sequencing of DNA obtained from the BSI above the Discovery Deep brine pool. Analysis of sequencing reads matching the 16S rRNA and hzsA genes confirmed presence of anammox bacteria of the genus Scalindua. Phylogenetic analysis of the 16S rRNA gene indicated that this Scalindua sp. belongs to a distinct group, separate from the anammox bacteria in the seawater column, that contains mostly sequences retrieved from high-salt environments. Using coverage- and composition-based binning, we extracted and assembled the draft genome of the dominant anammox bacterium. Comparative genomic analysis indicated that this Scalindua species uses compatible solutes for osmoadaptation, in contrast to other marine anammox bacteria that likely use a salt-in strategy. We propose the name Candidatus Scalindua rubra for this novel species, alluding to its discovery in the Red Sea.
KeywordsScalindua Anammox Red Sea Genome binning Metagenomics Salt adaptation
Over 25 brine pools have been discovered along the rift through the middle of the Red Sea. These brine pools are characterized by anoxic, salty water, and in some cases geothermal activity . The high salinity of the brine pools prevents mixing with the overlying seawater creating a brine-seawater interface (BSI) featuring steep salt and, in the case of hot brines, temperature gradients. Several studies using 16S rRNA gene amplicon community profiling and shotgun metagenomics have recently revealed the abundant presence of Planctomycetes (5–35%) in the BSI above the Discovery Deep, Atlantis II Deep, and Kebrit Deep brine pools [2, 3, 4]. As these are low-oxygen environments, detection of Planctomycetes likely indicates the presence of anammox bacteria. Furthermore, recent studies have shown the presence of ammonia-oxidizing Archaea and nitrite-oxidizing Bacteria in the Atlantis II Deep BSI, indicating an active nitrogen cycle in these systems [5, 6]. To further investigate the presence and nature of anammox bacteria in the Red Sea BSI, we employed genome-resolved shotgun metagenomics of the BSI above the Discovery Deep, where 16S rRNA gene amplicon community profiling indicated that Planctomycetes were more abundant than in other brine pools .
Metrics of the available Scalindua spp. draft genomes
The Ca. S. rubra draft genome encoded the genes required for hydrazine metabolism, hydrazine synthase  (SCARUB_01028–SCARUB_01030), and hydrazine dehydrogenase  (SCARUB_00654). The genes encoding hydrazine synthase subunits B and C are not fused in Ca. S. rubra, suggesting that the fusion of these genes in Ca. S. profunda and Ca. S. brodae is a recent event. Like the other Scalindua species, Ca. S. rubra encodes a heme-cd 1 type nitrite reductase (nirS) (SCARUB_03231). In contrast to Ca. S. profunda, neither Ca. S. brodae nor Ca. S. rubra encode a cyanase. Another interesting feature in the Ca. S. rubra genome is the apparent capability to synthesize gas vesicles, as 11 gas vesicle synthesis proteins are present. Although gas vesicles are often regulated by light intensity, gas vesicle formation is induced by high salinity in halophilic Archaeon Haloferax mediterranei . It is possible that Ca. S. rubra uses gas vesicles to stabilize its position within the BSI and prevent osmotic and/or heat shock as a result of the steep gradients in the BSI. The cellular location of gas vesicles in the already complicated cell architecture of an anammox bacterium is an interesting topic for further investigation.
We searched the Ca. S. rubra draft genome for proteins required for biosynthesis and transport of common compatible solutes. Many organisms use the amino acids glutamate, glutamine, or proline as compatible solutes . All anammox bacteria can synthesize these amino acids, and thus, it is possible that Ca. S. rubra utilizes any or all three of these amino acids. This could also provide an explanation for the adaptation of freshwater anammox species Ca. K. stuttgartiensis to marine salt concentrations . None of the Scalindua species is capable of synthesizing amino acid-derived compatible solutes glycine-betaine or (hydroxy)ectoine, but all three encode a glycine-betaine transporter. Furthermore, none of the Scalindua genomes encode the potential for biosynthesis of glycerate-derived compatible solutes or mannitol or sorbitol . Conclusive evidence on the presence, and nature, of compatible solutes in Ca. S. rubra will require biomass for experimental verification of the amino acid content.
In conclusion, we have presented the draft genome of a moderately halophilic anammox bacterium, Ca. S. rubra. Our analysis of the adaptations to salt stress in this genome has shed new light on previous results of salt adaptation in anammox bacteria.
Daan R. Speth was supported by BE-Basic FP 07.002.01. Bas E. Dutilh was supported by the Netherlands Organization for Scientific Research (NWO) Vidi grant 864.14.004. Mike S. M. Jetten was supported by the European Research Council advanced grants 232937 and 339880 and the NWO gravitation SIAM 024002002.
The raw sequencing reads described in this paper has been deposited to Genbank/EBI/DDBJ under SRA accession number SRX1894129. The assembled, annotated draft genome has been deposited at DDBJ/ENA/GenBank under the accession MAYW00000000. The version described in this paper is version MAYW01000000.
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Conflict of Interest
The authors declare that they have no conflict of interest.
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