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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Abstract
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.
Keywords
Scalindua Anammox Red Sea Genome binning Metagenomics Salt adaptationOver 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 [1]. 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 [2].
Maximum likelihood trees of anammox 16S rRNA and hzsA genes. a Maximum likelihood tree of 109 near full-length Brocadiales 16S rRNA genes matching >90% of the length the Ca. S. rubra sequence, originating from enrichment cultures, draft genomes, and clone libraries of marine environments. b Maximum likelihood tree of all available full-length hzsA gene sequences obtained from draft genomes. Sequences obtained in this study are indicated in bold. Trees were constructed using MEGA5 [36], bootstrapped with 1000 replicates, and visualized using the interactive tree of life (iTOL) v3 webserver [37]. Wedge height was scaled proportional to number of sequences. OMZ oxygen minimum zone, BSI brine-seawater interface, ETSP Eastern Tropical South Pacific
Metrics of the available Scalindua spp. draft genomes
The Ca. S. rubra draft genome encoded the genes required for hydrazine metabolism, hydrazine synthase [22] (SCARUB_01028–SCARUB_01030), and hydrazine dehydrogenase [23] (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 [24]. 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.
Protein isoelectric point distribution in eight genomes of anammox bacteria. Violin plots indicating the isoelectric point distribution of total protein set of all eight available anammox genomes, ordered from lowest to highest median value. Box plots (white bars) indicate 50% of the values around the median, indicated by a black circle. The three available genomes of Scalindua sp. are indicated by gray shading
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 [33]. 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 [34]. 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 [33]. 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.
Notes
Acknowledgements
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.
Accession Numbers
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.
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
Supplementary material
References
- 1.Bischoff JL (1969) Red Sea geothermal brine deposits: their mineralogy, chemistry, and genesis. In: Degens ET, Ross DA (eds) Hot brines and recent heavy metal deposits in the Red Sea. Springer, Berlin Heidelberg, Berlin, Heidelberg, pp 368–401CrossRefGoogle Scholar
- 2.Bougouffa S, Yang JK, Lee OO et al (2013) Distinctive microbial community structure in highly stratified deep-sea brine water columns. Appl Environ Microbiol 79:3425–3437. doi: 10.1128/AEM.00254-13 CrossRefPubMedPubMedCentralGoogle Scholar
- 3.Abdallah RZ, Adel M, Ouf A et al (2014) Aerobic methanotrophic communities at the Red Sea brine-seawater interface. Front Microbiol 5:487. doi: 10.3389/fmicb.2014.00487 CrossRefPubMedPubMedCentralGoogle Scholar
- 4.Guan Y, Hikmawan T, Antunes A et al (2015) Diversity of methanogens and sulfate-reducing bacteria in the interfaces of five deep-sea anoxic brines of the Red Sea. Res Microbiol. doi: 10.1016/j.resmic.2015.07.002 PubMedGoogle Scholar
- 5.Ngugi DK, Blom J, Alam I et al (2015) Comparative genomics reveals adaptations of a halotolerant thaumarchaeon in the interfaces of brine pools in the Red Sea. ISME J 9:396–411. doi: 10.1038/ismej.2014.137 CrossRefGoogle Scholar
- 6.Ngugi DK, Blom J, Stepanauskas R, Stingl U (2016) Diversification and niche adaptations of Nitrospina-like bacteria in the polyextreme interfaces of Red Sea brines. ISME J 10:1383–1399. doi: 10.1038/ismej.2015.214 CrossRefPubMedGoogle Scholar
- 7.Speth DR, In ‘t Zandt MH, Guerrero-Cruz S et al (2016) Genome-based microbial ecology of anammox granules in a full-scale wastewater treatment system. Nat Comms 7:11172. doi: 10.1038/ncomms11172 CrossRefGoogle Scholar
- 8.Lüke C, Speth DR, Kox MAR et al (2016) Metagenomic analysis of nitrogen and methane cycling in the Arabian Sea oxygen minimum zone. Peer J 4:e1924. doi: 10.7717/peerj.1924 CrossRefPubMedPubMedCentralGoogle Scholar
- 9.Speth DR, Russ L, Kartal B et al (2015) Draft genome sequence of anammox bacterium “Candidatus Scalindua brodae,” obtained using differential coverage binning of sequencing data from two reactor enrichments. Genome Announc. doi: 10.1128/genomeA.01415-14 PubMedPubMedCentralGoogle Scholar
- 10.van de Vossenberg J, Woebken D, Maalcke WJ et al (2013) The metagenome of the marine anammox bacterium “Candidatus Scalindua profunda” illustrates the versatility of this globally important nitrogen cycle bacterium. Environ Microbiol 15:1275–1289. doi: 10.1111/j.1462-2920.2012.02774.x CrossRefPubMedPubMedCentralGoogle Scholar
- 11.Borin S, Mapelli F, Rolli E et al (2013) Anammox bacterial populations in deep marine hypersaline gradient systems. Extremophiles 17:289–299. doi: 10.1007/s00792-013-0516-x CrossRefPubMedGoogle Scholar
- 12.Ultsch A, Mörchen F (2005) ESOM-Maps: tools for clustering, visualization, and classification with Emergent SOM. https://www.uni-marburg.de/fb12/datenbionik/pdf/pubs/2005/ultsch05esom
- 13.Dick GJ, Andersson AF, Baker BJ et al (2009) Community-wide analysis of microbial genome sequence signatures. Genome Biol 10:R85. doi: 10.1186/gb-2009-10-8-r85 CrossRefPubMedPubMedCentralGoogle Scholar
- 14.Tyson GW, Chapman J, Hugenholtz P et al (2004) Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature 428:37–43. doi: 10.1038/nature02340 CrossRefPubMedGoogle Scholar
- 15.Bankevich A, Nurk S, Antipov D et al (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. doi: 10.1089/cmb.2012.0021 CrossRefPubMedPubMedCentralGoogle Scholar
- 16.Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359. doi: 10.1038/nmeth.1923 CrossRefPubMedPubMedCentralGoogle Scholar
- 17.Seemann T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–2069. doi: 10.1093/bioinformatics/btu153 CrossRefPubMedGoogle Scholar
- 18.Strous M, Pelletier E, Mangenot S et al (2006) Deciphering the evolution and metabolism of an anammox bacterium from a community genome. Nature 440:790–794. doi: 10.1038/nature04647 CrossRefPubMedGoogle Scholar
- 19.Hira D, Toh H, Migita CT et al (2012) Anammox organism KSU-1 expresses a NirK-type copper-containing nitrite reductase instead of a NirS-type with cytochrome cd1. FEBS Lett 586:1658–1663. doi: 10.1016/j.febslet.2012.04.041 CrossRefPubMedGoogle Scholar
- 20.Ferousi C, Speth DR, Reimann J et al (2013) Identification of the type II cytochrome c maturation pathway in anammox bacteria by comparative genomics. BMC Microbiol 13:265. doi: 10.1016/j.sbi.2008.02.002 CrossRefPubMedPubMedCentralGoogle Scholar
- 21.Oshiki M, Shinyako-Hata K, Satoh H, Okabe S (2015) Draft genome sequence of an anaerobic ammonium-oxidizing bacterium, “Candidatus Brocadia sinica”. Genome Announc. doi: 10.1128/genomeA.00267-15 PubMedPubMedCentralGoogle Scholar
- 22.Dietl A, Ferousi C, Maalcke WJ et al (2015) The inner workings of the hydrazine synthase multiprotein complex. Nature 527:394–397. doi: 10.1038/nature15517 CrossRefPubMedGoogle Scholar
- 23.Maalcke WJ, Reimann J, de Vries S et al (2016) Characterization of anammox hydrazine dehydrogenase, a key N 2-producing enzyme in the global nitrogen cycle. J Biol Chem jbc.M116.735530. doi: 10.1074/jbc.M116.735530
- 24.Englert C, Horne M, Pfeifer F (1990) Expression of the major gas vesicle protein gene in the halophilic archaebacterium Haloferax mediterranei is modulated by salt. Mol Gen Genet 222:225–232. doi: 10.1007/BF00633822 CrossRefPubMedGoogle Scholar
- 25.Sleator RD, Hill C (2002) Bacterial osmoadaptation: the role of osmolytes in bacterial stress and virulence. FEMS Microbiol Rev 26:49–71. doi: 10.1111/j.1574-6976.2002.tb00598.x CrossRefPubMedGoogle Scholar
- 26.Rice P, Longden I, Bleasby A (2000) EMBOSS: the European molecular biology open software suite. Trends Genet 16:276–277. doi: 10.1016/S0168-9525(00)02024-2 CrossRefPubMedGoogle Scholar
- 27.Zaccai G, Cendrin F, Haik Y, Borochov N, Eisenberg H (1989) Stabilization of halophilic malate dehydrogenase. J Mol Biol 208:491–500. doi: 10.1016/0022-2836(89)90512-3 CrossRefPubMedGoogle Scholar
- 28.van de Vossenberg J, Rattray JE, Geerts W et al (2008) Enrichment and characterization of marine anammox bacteria associated with global nitrogen gas production. Environ Microbiol 10:3120–3129. doi: 10.1111/j.1462-2920.2008.01643.x CrossRefPubMedGoogle Scholar
- 29.Rattray JE, van de Vossenberg J, Hopmans EC et al (2008) Ladderane lipid distribution in four genera of anammox bacteria. Arch Microbiol 190:51–66. doi: 10.1007/s00203-008-0364-8 CrossRefPubMedGoogle Scholar
- 30.Oren A (1999) Bioenergetic aspects of halophilism. Microbiol Mol Biol Rev 63:334–348PubMedPubMedCentralGoogle Scholar
- 31.Bowers KJ, Mesbah NM, Wiegel J (2009) Biodiversity of poly-extremophilic bacteria: does combining the extremes of high salt, alkaline pH and elevated temperature approach a physico-chemical boundary for life? Saline Syst 5:9. doi: 10.1186/1746-1448-5-9 CrossRefPubMedPubMedCentralGoogle Scholar
- 32.Bowers KJ, Wiegel J (2011) Temperature and pH optima of extremely halophilic archaea: a mini-review. Extremophiles 15:119–128. doi: 10.1007/s00792-010-0347-y CrossRefPubMedGoogle Scholar
- 33.Empadinhas N, da Costa M (2008) Osmoadaptation mechanisms in prokaryotes: distribution of compatible solutes. Int Microbiol 11:151–161. doi: 10.2436/im.v11i3.9665 PubMedGoogle Scholar
- 34.Kartal B, Koleva M, Arsov R et al (2006) Adaptation of a freshwater anammox population to high salinity wastewater. J Biotechnol 126:546–553. doi: 10.1016/j.jbiotec.2006.05.012 CrossRefPubMedGoogle Scholar
- 35.Parks DH, Imelfort M, Skennerton CT et al (2015) CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res 25:1043–1055. doi: 10.1101/gr.186072.114 CrossRefPubMedPubMedCentralGoogle Scholar
- 36.Tamura K, Peterson D, Peterson N et al (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739. doi: 10.1093/molbev/msr121 CrossRefPubMedPubMedCentralGoogle Scholar
- 37.Letunic I, Bork P (2016) Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res 44:W242–W245. doi: 10.1093/nar/gkw290 CrossRefPubMedPubMedCentralGoogle Scholar
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