Three highly alkaliphilic bacterial strains designated as A1^T, H1^T and B1^T were isolated from two highly alkaline springs at The Cedars, a terrestrial serpentinizing site. Cells from all strains were motile, Gram-negative and rod-shaped. Strains A1^T, H1^T and B1^T were mesophilic (optimum, 30 °C), highly alkaliphilic (optimum, pH 11) and facultatively autotrophic. Major cellular fatty acids were saturated and monounsaturated hexadecenoic and octadecanoic acids. The genome size of strains A1^T, H1^T and B1^T was 2574013, 2475906 and 2623236 bp, and the G+C content was 66.0, 66.2 and 66.1mol%, respectively. Analysis of the 16S rRNA genes showed the highest similarity to the genera Malikia (95.1–96.4%), Macromonas (93.0–93.6%) and Hydrogenophaga (93.0–96.6%) in the family Comamonadaceae. Phylogenetic analysis based on 16S rRNA gene and phylogenomic analysis based on core gene sequences revealed that the isolated strains diverged from the related species, forming a distinct branch. Average amino acid identity values of strains A1^T, H1^T and B1^T against the genomes of related members in this family were below 67%, which is below the suggested threshold for genera boundaries. Average nucleotide identity by blast values and digital DNA– DNA hybridization among the three strains were below 92.0 and 46.6% respectively, which are below the suggested thresholds for species boundaries. Based on phylogenetic, genomic and phenotypic characterization, we propose Serpentinimonas gen. nov., Serpentinimonas raichei sp. nov. (type strain A1^T=NBRC 111848^T=DSM 103917^T), Serpentinimonas barnesii sp. nov. (type strain H1^T= NBRC 111849^T=DSM 103920^T) and Serpentinimonas maccroryi sp. nov. (type strain B1^T=NBRC 111850^T=DSM 103919^T) belonging to the family Comamonadaceae. We have designated Serpentinimonas raichei the type species for the genus because it is the dominant species in The Cedars springs.

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Water from The Cedars springs that discharge from serpentinized ultramafic rocks feature highly basic (pH=∼12), highly reducing (E h <'550 mV) conditions with low ionic concentrations. These conditions make the springs exceptionally challenging for life. Here, we report the metagenomic data and recovered draft genomes from two different springs, GPS1 and BS5. GPS1, which was fed solely by a deep groundwater source within the serpentinizing system, was dominated by several bacterial taxa from the phyla OD1 ('Parcubacteria') and Chloroflexi. Members of the GPS1 community had, for the most part, the smallest genomes reported for their respective taxa, and encoded only archaeal (A-type) ATP synthases or no ATP synthases at all. Furthermore, none of the members encoded respiration-related genes and some of the members also did not encode key biosynthesis-related genes. In contrast, BS5, fed by shallow water, appears to have a community driven by hydrogen metabolism and was dominated by a diverse group of Proteobacteria similar to those seen in many terrestrial serpentinization sites. Our findings indicated that the harsh ultrabasic geological setting supported unexpectedly diverse microbial metabolic strategies and that the deep-water-fed springs supported a community that was remarkable in its unusual metagenomic and genomic constitution.

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Water from The Cedars springs that discharge from serpentinized ultramafic rocks feature highly basic (pH=∼12), highly reducing (E h <'550 mV) conditions with low ionic concentrations. These conditions make the springs exceptionally challenging for life. Here, we report the metagenomic data and recovered draft genomes from two different springs, GPS1 and BS5. GPS1, which was fed solely by a deep groundwater source within the serpentinizing system, was dominated by several bacterial taxa from the phyla OD1 ('Parcubacteria') and Chloroflexi. Members of the GPS1 community had, for the most part, the smallest genomes reported for their respective taxa, and encoded only archaeal (A-type) ATP synthases or no ATP synthases at all. Furthermore, none of the members encoded respiration-related genes and some of the members also did not encode key biosynthesis-related genes. In contrast, BS5, fed by shallow water, appears to have a community driven by hydrogen metabolism and was dominated by a diverse group of Proteobacteria similar to those seen in many terrestrial serpentinization sites. Our findings indicated that the harsh ultrabasic geological setting supported unexpectedly diverse microbial metabolic strategies and that the deep-water-fed springs supported a community that was remarkable in its unusual metagenomic and genomic constitution.@en

The spring waters trom The Cedars ultra-mafic rocks have extremely high pH (-12) and relatively high concentrations of Ca2+, H2 and CH4 due to the active serpentinization. In addition, the waters contain low concentrations of dissolved carbon and sodium, and undetectable levels of ammonium, phosphate and electron acceptors. This combination of water chemistry is supposed to be very challenging tor life; however, certain Betaproteobacteria have been trequently observed in serpentinization sites globally. Three closely-related, highly-alkaliphilic Betaprotebacterial strains were isolated trom The Cedars springs and we have proposed these as a new genus "Serpentinomonas'. All three strains grow autotrophically with H2 as the electron donor and CaCO3 as the carbon source at high pH (optimum pH 11 ), and the Serpentinomonas strains are numerically dominant in The Cedars and other terrestrial serpentinizing ecosystems. These features indicate that Serpentinomonas strains are highly adapted to the geochemistry of serpentinizing environment. Having the strains in culture has allowed both physiological and genomic studies of the metabolic capabilities of Serpentinomonas strains (Suzuki. et al., 2014}. However it remains unknown how they manage to grow in these harsh environments where low levels of protons and sodium make it extremely difficult to establish a cation motive force, and where high levels of calcium reduce the available phosphate and inorganic carbon significantly. A model alkaliphilic organism, Bacillus pseudofirmus OF4, is known to have a unique W-ATP synthase that is capable of translocating protons into the cytosol at high pH effectively (Preiss Let al., 2013}. However, the proteins in our Serpentinomonas strains lack the specific motif in the c-rings that is proposed to be critica! tor the success of the alkaliphilic Bacilli; therefore, Serpentinomonas strains must have acquired different, as yet unknown, adaptations. Genomic analyses revealed that genome size of Serpentinomonas is significantly smaller (-2.5 Mbp) than those of the most closely related genus Hydrogenophaga species (- 5Mb). To identify proteins involved in alkaliphilic properties of Serpentinomonas strains, we employed a comparative genomics approach that subtracted proteins encoded in the genomes of Serpentinomonas strains trom the shared proteins encoded in the genomes of closely-related neutrophilic Betaproteobacteria. The approach led to the identification of 380 genes that are present exclusively in Serpentinomonas strains. The identified genes mostly encoded proteins tor cation transporters and alkaline phosphatases, and those genes were further shown to be of significance by direct evidence of their transcription in The Cedars springs. Meanwhile Serpentinomonas lost 2386 genes compared with a type genus Hydrogenophaga flava1. The deleted genes include amino acids and sugar transporters, RNA polymerase sigma-70 factor, and fatty acids metabolisms. While studies of alkaliphiles trom calcium-rich highly-alkaline environment are limited, the alkaliphilic mechanisms are likely different from those of alkaliphiles from high salt environment. The genomic features of Serpentinomonas indicate that extremely low availability of proton and phosphate are the major selective forces that drive the genomic evolution of this genus in a serpentinizing ecosystem. @en