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