Insights into the physiological and genomic characterization of three bacterial isolates from a highly alkaline, terrestrial serpentinizing system

The terrestrial serpentinite-hosted ecosystem known as “The Cedars” is home to a diverse microbial community persisting under highly alkaline (pH ~ 12) and reducing (Eh < −550 mV) conditions. This extreme environment presents particular difficulties for microbial life, and efforts to isolate microorganisms from The Cedars over the past decade have remained challenging. Herein, we report the initial physiological assessment and/or full genomic characterization of three isolates: Paenibacillus sp. Cedars (‘Paeni-Cedars’), Alishewanella sp. BS5-314 (‘Ali-BS5-314’), and Anaerobacillus sp. CMMVII (‘Anaero-CMMVII’). Paeni-Cedars is a Gram-positive, rod-shaped, mesophilic facultative anaerobe that grows between pH 7–10 (minimum pH tested was 7), temperatures 20–40°C, and 0–3% NaCl concentration. The addition of 10–20 mM CaCl2 enhanced growth, and iron reduction was observed in the following order, 2-line ferrihydrite > magnetite > serpentinite ~ chromite ~ hematite. Genome analysis identified genes for flavin-mediated iron reduction and synthesis of a bacillibactin-like, catechol-type siderophore. Ali-BS5-314 is a Gram-negative, rod-shaped, mesophilic, facultative anaerobic alkaliphile that grows between pH 10–12 and temperatures 10–40°C, with limited growth observed 1–5% NaCl. Nitrate is used as a terminal electron acceptor under anaerobic conditions, which was corroborated by genome analysis. The Ali-BS5-314 genome also includes genes for benzoate-like compound metabolism. Anaero-CMMVII remained difficult to cultivate for physiological studies; however, growth was observed between pH 9–12, with the addition of 0.01–1% yeast extract. Anaero-CMMVII is a probable oxygen-tolerant anaerobic alkaliphile with hydrogenotrophic respiration coupled with nitrate reduction, as determined by genome analysis. Based on single-copy genes, ANI, AAI and dDDH analyses, Paeni-Cedars and Ali-BS5-314 are related to other species (P. glucanolyticus and A. aestuarii, respectively), and Anaero-CMMVII represents a new species. The characterization of these three isolates demonstrate the range of ecophysiological adaptations and metabolisms present in serpentinite-hosted ecosystems, including mineral reduction, alkaliphily, and siderophore production.

The terrestrial serpentinite-hosted ecosystem known as "The Cedars" is home to a diverse microbial community persisting under highly alkaline (pH 12) and reducing (Eh < −550 mV) conditions. This extreme environment presents particular difficulties for microbial life, and efforts to isolate microorganisms from The Cedars over the past decade have remained challenging. Herein, we report the initial physiological assessment and/or full genomic characterization of three isolates: Paenibacillus sp. Cedars ('Paeni-Cedars'), Alishewanella sp. , and Anaerobacillus sp. CMMVII ('Anaero-CMMVII'). Paeni-Cedars is a Gram-positive, rod-shaped, mesophilic facultative anaerobe that grows between pH 7-10 (minimum pH tested was 7), temperatures 20-40°C, and 0-3% NaCl concentration. The addition of 10-20 mM CaCl 2 enhanced growth, and iron reduction was observed in the following order, 2-line ferrihydrite > magnetite > serpentinite ~ chromite ~ hematite. Genome analysis identified genes for flavin-mediated iron reduction and synthesis of a bacillibactin-like, catecholtype siderophore. Ali-BS5-314 is a Gram-negative, rod-shaped, mesophilic, facultative anaerobic alkaliphile that grows between pH 10-12 and temperatures 10-40°C, with limited growth observed 1-5% NaCl. Nitrate is used as a terminal electron acceptor under anaerobic conditions, which was corroborated by genome analysis. The Ali-BS5-314 genome also includes genes for benzoatelike compound metabolism. Anaero-CMMVII remained difficult to cultivate for physiological studies; however, growth was observed between pH 9-12, with the addition of 0.01-1% yeast extract. Anaero-CMMVII is a probable oxygen-tolerant anaerobic alkaliphile with hydrogenotrophic respiration coupled with nitrate reduction, as determined by genome analysis. Based on single-copy genes, ANI,

Introduction
Serpentinization is a geologic process involving the aqueous alteration of ultramafic rock, leading to the production of energy (e.g., hydrogen gas) and carbon (e.g., small organic molecules) sources for microbial life. However, ecosystems that host these reactions are highly alkaline and reducing, often with limited availability of terminal electron acceptors. These extreme geochemical conditions are challenging for microorganisms, but unique microbial communities have been discovered within them (Suzuki et al., 2013;Quéméneur et al., 2015;Twing et al., 2017;Trutschel et al., 2022). From an astrobiology perspective, serpentinite-hosted ecosystems are proposed to have supported the emergence and evolution of life, and could provide insight into potential life on other planetary bodies, including ocean worlds and Mars (NASEM, 2019). Moreover, terrestrial serpentinizing environments (i.e., ophiolites) provide more readily available access to subsurface fluids and their microbial communities, as compared to marine counterparts (e.g., Lost City). This access enables investigations into the biogeochemical processes and the microbial ecophysiology of these systems to further understand the limits of life on Earth and in a broader planetary context (Jones et al., 2018;Merino et al., 2019).
Although the geochemical underpinnings of the serpentinization reaction have been understood since the mid 1960's with Dr. Ivan Barnes' landmark publication (Barnes et al., 1967), the unique chemical and energetic constraints of the fluids within ophiolites present particular challenges in the cultivation and isolation of pure culture strains for formal laboratory investigations. Over the past decades, much knowledge has been gained through analysis of the geochemical composition and microbial diversity of these systems in situ; however, the limited number of isolated representatives available to study in vitro has stymied our understanding of the ecophysiological role of these organisms and the multifaceted adaptations required by life to survive and thrive in these hyperalkaline and extremely reducing environments (e.g., metabolic capabilities, ATP production, and iron and trace metal acquisition).
In this study, we describe the preliminary physiological findings and in-depth genomic characterization of three isolates from The Cedars to commemorate the 55 th anniversary of one of the first publications detailing serpentinization, published by Barnes et al. (1967). The Cedars is an active serpentinite-hosted ecosystem located in Northern California, which was first described by Drs. Barnes and James O'Neil (Barnes and O'Neil, 1969). Modern biogeochemical analysis and modeling of The Cedars spring system began in 2005, with initial biomass determinations (via filtration) for the various springs ranging from 10 2 to 10 3 cells/mL in the mixed fluid source springs and as low as 10 cells/mL in the deep serpentinization fluid system. Despite the extremely low biomass inherent to The Cedars' fluids, the results of the first in situ enrichment experiments yielded encouraging results (10 6 -10 7 cells/cm 2 ) when glass slides were incubated in the springs for 3 weeks time (Morrill et al., 2013). That said, early attempts at cultivation and isolation of organisms for study in the laboratory proved unsuccessful, so focus turned towards identifying and describing the microbial community via 16S and 18S rRNA amplicon sequencing. This work (Suzuki et al., 2013) provided the first understanding of the diversity and composition of The Cedars microbial community, identified novel and uncultivated microorganisms, and provided evidence of both seasonal and spatial effects on the communities of the spring system.
Since then, hundreds-to-thousands of different cultivation conditions have been tested by several early career microbiologists, including undergraduate and graduate students and postdoctoral researchers in an effort to bring these elusive organisms into the laboratory. This initially led to the isolation and discovery of three species belonging to the novel genus Serpentinomonas, the dominant taxon in the mixed fluid springs (Suzuki et al., 2014). Members of the Serpentinomonas are hydrogen-utilizing obligate alkaliphiles, further characterized by Bird et al. (2021). Notably, S. maccroryi strain B1 holds the bacterial record for tolerating and growing at the highest pH (pH 12.5) (Merino et al., 2019).
Following the isolation of Serpentinomonas sp., a unique in-situ electrochemical enrichment approach led to the isolation of an alkalitolerant microbe, Paenibacillus sp. Cedars, capable of extracellular electron transfer (EET) and reduction of magnetite (Rowe et al., 2017). However, as discussed by Rowe et al. (2017), other isolates were comparatively less robust than Paenibacillus sp. or exhibited loss of activity after several transfers, including one closely related to the Alishewanella lineage of the Gammaproteobacteria. Subsequent Frontiers in Microbiology 03 frontiersin.org isolation attempts led to the successful cultivation of Alishewanella sp. BS5-314 under alkaliphilic conditions, further described for the first time in this study. Multiple attempts have also targeted the cultivation of anaerobic alkaliphiles, and thus far, only one anaerobic alkaliphile from The Cedars is cultivable: Cellulomonas sp. strain FA1 (Cohen et al., 2015;Kamennaya et al., 2020). Another anaerobe remains difficult to grow but cultivable, Anaerobacillus sp. CMMVII, with the first in-depth genomic characterization and initial physiological description described in this study.
In celebration of the anniversary of Dr. Ivan Barnes' monumental work, we present our current progress, understanding and hypotheses into the nature and ecophysiology of three diverse isolates from The Cedars described above: Paenibacillus sp. Cedars ('Paeni-Cedars'), Alishewanella sp. BS5-314 (' Ali-BS5-314′), and Anaerobacillus sp. CMMVII (' Anaero-CMMVII'). Herein, we report on the growth conditions for Paeni-Cedars (ATCC BAA-3230) and Ali-BS5-314 (ATCC TSD-356). We then discuss the putative metabolic and energetic pathways present in the genomes of all three microbes. The isolation, description, and characterization of these isolates provides new insights into our understanding of the diversity of life in serpentinizing fluids, the adaptations of organisms to survive in high pH/extremely reducing environments, and ultimately, the possibilities of life beyond Earth on other planetary bodies where these geochemical reactions are thought to exist.

Isolation of the three microorganisms
Paeni-Cedars was enriched on a two-electrode in-situ electrochemical system incubated in a highly alkaline pool (pH ~ 11) called Mortar Bed Springs (previously named Campsite Spring; coordinates 38.6191396, −123.1330681) (Rowe et al., 2017). Afterwards, Paeni-Cedars was isolated in nutrient-rich mLA medium at pH 9, and the mineral-and electrode-reducing capabilities were evaluated by Rowe et al. (2017). In this study, Paeni-Cedars was cultivated in nutrient-rich (mLA) or minimal (CSM-A, Cedars Medium-A) media. mLA medium was previously described in Rowe et al. (2017). CSM-A medium contained 1× salts solution (100× consisted of 5 mM Na 2 SO 4 , 37.8 mM NH 4 Cl, and 5 mM MgCl 2 •6H 2 O), 10 mM CAPS (pH 9), 0.1% yeast extract, and 2 mM CaCO 3 . After autoclaving, filter-sterilized solutions were added to a final concentration of 0.086 mM K 2 HPO 4 and 1× of vitamins and trace minerals previously described (Rowe et al., 2015). CSM-A was then dispensed to serum bottles to a liquidto-gas ratio of 35:65 (liquid:gas phase). Each bottle was crimp sealed with rubber stoppers (Cat. No.: 309018,Misumi USA), and then sparged with N 2 gas for 5 min before overpressurizing with H 2 gas (~10 s).

Physiological characterization
Physiological characterization of Ali-BS5-314 and Paeni-Cedars were conducted in 60 mL serum bottles (35:65 liquid:gas phase) crimped with rubber stoppers using CSM-N (Ali-BS5-314) and CSM-A (Paeni-Cedars) media. For physiological characterization, CSM-N medium gassing conditions were modified, similar to CSM-A medium, with the exception of overpressurizing with H 2 /CO 2 (80/20%) gas. For Ali-BS5-314, air was then added to a final concentration of 10% as filter-sterilized air in serum bottles crimped with rubber stoppers. Prior to inoculation for physiological studies, cultures of each isolate from a glycerol stock were incubated at 30°C in nutrient-rich media (R2A for Ali-BS5-314; mLA for Paeni-Cedars) for 24 h. Subsequently, 0.1% of the culture was transferred to fresh nutrient-rich media and incubated at 30°C for 24 h. The cells were then washed three times with the respective minimal media and inoculated at ~10 6 cells/ml. The range and optimum temperature and pH growth conditions were examined by incubating cultures from 10 to 50°C (10°C increments) and from pH 7-12 (pH 1 increments). At select timepoints, cell counts were performed by filtering fixed cells (1% formalin) stained with SYBR green through a 0.2 μm filter. Cells were lightly sonicated for 45 s to ensure even distribution due to cells that clump to carbonate precipitates. A fluorescence microscope was used for cell counting on a 10 × 10 grid and counts were conducted for 10 discrete fields of view.

DNA extraction and sequencing
DNA from Paeni-Cedars was extracted from a fresh cell pellet using Nucleobond AXG Column Kit (TakaraBio). The genome was sequenced by Takara Bio Inc. (Kusatsu, Japan) in a single-molecule real-time (SMRT) cell on a PacBio RSII sequencer (Pacific Bioscience, CA, USA) using a 15 kb insert library. This sequencing method consists of proprietary, long read, real-time detection sequencing based on terminal fluorophore attached nucleotides and zero-mode optical wave-guide detection system. A total of 90,234 inserts with a mean size of 16,260 bp (N50 22,314) were fully sequenced and assembled using the HGAP (Analysis Hierarchical Genome Assembly Process) protocol implemented in SMRT analysis (version 2.3, Pacific Bioscience). A total of three circular contigs were assembled: a 6,414,884 bp genomic contig with 181.03× mean coverage and two plasmids: 248,871 bp long with 208.87× mean coverage and 56,646 bp long with 113.26× mean coverage.
DNA from Anaero-CMMVII and Ali-BS5-314 were extracted from a fresh cell pellet using Qiagen AllPrep DNA/RNA Mini Kit. The genome was sequenced by MRDNA (TX, USA), using SMRT PacBio Sequel (Pacific Bioscience, CA, USA) with a 15 kb insert library. De Novo assembly of reads with mean size of 9.4 kb (Anaero-CMMVII) and 15.2 kb (Ali-BS5-314) was accomplished using HGAP implemented in SMRT analysis. For Anaero-CMMVII, a total of 24 polished reads were assembled (4,889,950 bp genome size) with 46× mean coverage using Falcon Assembler paired with Arrow polishing algorithm, and 80.15% of the bases were successfully realigned to the draft assembly with a mean concordance of 81.88%. For Ali-BS5-314, a total of 10 polished reads were assembled (3,747,989 bp genome size) with 2,234× mean coverage using Falcon Assembler paired with Arrow polishing algorithm, and 89.8% of the bases were successfully realigned to the draft assembly with a mean concordance of 89.7%.

Results and discussion
Potential abundance and relevance of the isolates in The Cedars These three isolates are likely part of the rare biosphere within the microbial communities of The Cedars' fluids. Alignment of genomic reads from The Cedars metagenomes (Suzuki et al., 2017) against each isolate genome resulted in overall alignment rate percentages of: 0.07-0.11% for Paeni-Cedars, 0.08-0.11% for Anaero-CMMVII, and 0.04-0.22% for Ali-BS5-314. The rarity of these isolates may be an artifact of the sampling capabilities; for example, at The Cedars, it is not feasible to obtain sediment cores or to directly sample deeper fluids. Nevertheless, rare species can have a disproportionate impact on the biogeochemical cycles ongoing within various ecosystems, as reviewed by Jousset et al. (2017), and necessitates the need to enrich and isolate novel species for physiological and genomic characterization.
The isolation and characterization of microorganisms from terrestrial serpentinite-hosted ecosystems can reveal important ecophysiological functions and adaptations that are missed by metagenomic studies (e.g., Suzuki et al., 2017). For example, the isolation of Paeni-Cedars revealed that mineral reduction can occur in The Cedars' springs, potentially serving as an important terminal electron accepting process in the absence of oxygen (Rowe et al., 2017). EET-capable microorganisms, such as Paeni-Cedars, were initially not identified in metagenomic surveys of The Cedars (Rowe et al., 2017;Suzuki et al., 2017), likely because of low relative read abundances or unknown EET mechanisms. As discussed below, the putative EET mechanism used by Paeni-Cedars is actually related to that observed in Listeria monocytogenes 10403S (Light et al., 2018). There may be other potential unknown EET pathways within The Cedars and other serpentinite-hosted ecosystems; Paeni-Cedars is only one isolate among the EET-active community enriched by Rowe et al. (2017). The isolate Anaero-CMMVII may also be able to reduce minerals but further studies are needed to confirm this activity. Rowe et al. (2017) also attempted to isolate an Alishewanella strain that seemed to have manganese reduction capabilities until the fifth round of transfer. However, the loss of growth and mineral reduction activity suggests the original culture may have included other microbial members responsible for manganese reduction. The isolation of Ali-BS5-314 (this study) further confirms that the Alishewanella species present in the spring waters of The Cedars cannot reduce minerals; however, this does not preclude the potential presence of other related Alishewanella strains with mineral reduction activity. Instead, the novelty of Ali-BS5-314, as discussed below, is its ability to grow at hyperalkaline pH > 10 and the potential for degrading aromatic compounds. Similarly, Anaero-CMMVII was observed to grow at pH > 9. The notable features of Anaero-CMMVII is that the isolate is likely an oxygen-tolerating anaerobe with the putative capability for hydrogenotrophic respiration. Further description of these three isolates is described below.
Taxonomic classification of the isolates GTDB-Tk, ANI, AAI, and dDDH were used to confirm the taxonomic classification of the isolates. Alignment of the 16S rRNA gene sequences against related species resulted in inconclusive identification, with only the genus-or family-level association for Paeni-Cedars (>98% identity) and Ali-BS5-314 (>98% identity) (Supplementary Table S3). For Anaero-CMMVII, the 16S rRNA gene sequences are >95% related to A. isosaccharinicus; however, this is based on the GTDB database (Supplementary Table S3), and further analysis using GTDB-Tk identified that Anaero-CMMVII is a novel species, as described below.
Anaero-CMMVII is a new species according to all metrics used in this study. The closest placement reference for Anaero-CMMVII is A. isosaccharinicus, with GTDB-Tk ANI 80.82% and AF 0.42 (Supplementary Table S4). GTDB-Tk threshold for clustering is ANI >97% and AF 0.65. Furthermore, pairwise dDDH of Anaero-CMMVII against possible related species are <19.4% (Supplementary Table S5), and ANI ranges from about 76 to 81% and AAI ranges from 71 to 79% (Supplementary Table S6).
Paeni-Cedars was previously reported to reduce magnetite with glucose as the electron donor (Rowe et al., 2017). We further examined its capability to reduce other Fe(III) minerals, such as 2-line ferrihydrite and Fe(III) minerals found at The Cedars and other serpentinite-hosted systems (e.g., serpentinite, olivine, chromite, hematite, and goethite). Phylogenetic tree for the three isolates. (A) Ali-BS5-314 and (B) Anaero-CMMVII and Paeni-Cedars. Representative genomes used to construct the phylogenetic trees are listed in Supplementary Tables S1, S2 and were retrieved via GToTree (Lee, 2019). FastTree (Price et al., 2010) via GToTree was used to generate the phylogenetic trees and viewed using the Interactive Tree of Life (iToL) v6 (Letunic and Bork, 2007). Local bootstrap values are depicted as circles at each node. Brackets indicate the number of genomes within a clade.
The physiological differences for Ali-BS5-314 and Paeni-Cedars suggests these two isolates may persist in separate niches within The Cedars springs fluids. Ali-BS5-314 seems to favor growth at conditions similar to the chemical composition of the fluids (pH 11.5-11.9, Ca 2+ 0.94-1.3 mM, and Mg 2+ 0.004-0.036 mM) (Morrill et al., 2013). In contrast, Paeni-Cedars grows pH < 10 and at much higher CaCl 2 concentrations. Along with the capability for mineral reduction, Paeni-Cedars may favor growth on or nearby solid substrates, including iron oxides and calcite precipitates. Rowe et al. (2017) also observed that EET activity was correlated with a drop in pH and that the optimal pH for mineral reduction was pH 9. One hypothesis suggested by Rowe et al. (2017) is that calcite precipitate microenvironments may accumulate protons and have lower pH, enabling Paeni-Cedars to persist. In contrast to Ali-BS5-314 and Paeni-Cedars, the isolate Anaero-CMMVII is likely found in anaerobic niches or deeper spring fluids that experience periodic exposure to oxygen.

Genome analyses: overview
The putative genome features for all three isolates are summarized in Table 4 and conceptual models are depicted in Figures 5-7. All gene annotations, including KEGG, InterProScan, and FeGenie, can be found in Supplementary Tables S8-S13.

Genome analyses: energy conservation and extracellular electron transport
Based on physiological observations and genome analyses, Ali-BS5-314 and Paeni-Cedars are facultative anaerobes, and Anaero-CMMVII is a probable oxygen-tolerant anaerobe. The respiratory electron transport chain (ETC) of all three isolates contains succinate dehydrogenase (sdhABC or sdhABCD), cytochrome bc 1 complex (cytbc 1 ), cytochrome c oxidase (ccO), and cytochrome bd (cydAB or cydABCD). All three microbes also use an H + -coupled F-type ATPase for oxidative phosphorylation. Alignment of the c-subunit of the ATPase against other H + or Na + coupled F-type ATPases confirmed that the genomes of all three isolates encode for an H + coupled F-type ATPase (Supplementary Figure S4; Hicks et al., 2010). Besides these pH range (optimum) <7 d -10 (7-9) 6.5-11 (7) 10-12 (11) 6.5-9.5 (6.5-9) N/A 9 d -12 (N/A) 8.5-11 (9.8-10) NaCl range (%) (optimum) 0-3 0-9 (0) 0-5 1-2 (1) 0-5 N/A 0-6 (2)  Alexander and Priest, 1989;Shida et al., 1997;Priest, 2009;Liu et al., 2015), (3)  Frontiers in Microbiology 08 frontiersin.org similarities, the three isolates have differences in their energy conservation mechanisms. The ETC of Ali-BS5-314 includes both nitrate reductase (NarGHJI) or fumarate reductase (FrdABCD). However, our physiological studies suggest that only nitrate can be used as a terminal electron acceptor during anaerobic respiration (Figure 4). This demonstrates that Ali-BS5-314 has flexibility for anaerobic or aerobic respiration, which may be required to persist in The Cedars. Dissolved oxygen is likely present in surface springs and shallow groundwater of The Cedars (Morrill et al., 2013), but there may be spatial and temporal changes resulting in anaerobic conditions or niches, requiring Ali-BS5-314 to rely on nitrate reduction in the environment. However, nitrate concentrations in The Cedars' springs fluids were low μM levels (Cook et al., 2021), potentially immediately utilized by the microbial community. The genome of Ali-BS5-314 lacks NADH dehydrogenase, indicating that other pathways are used for NADH oxidation, such as glutamate synthesis (van Heeswijk et al., 2013) or gluconeogenesis. NADH regeneration pathways include glycolysis and the action of the Rnf complex (Rhodobacter nitrogen fixation electron transport complex [rnfABCDEG]). The Rnf complex also results in the electrogenic pumping of Na + /H + ions extracellularly, inducing an ion gradient that can be used for Na + /H + -dependent symporters and by the F-type ATPase. This complex may be important for Ali-BS5-314 to grow under hyperalkaline conditions, further discussed in the section "Genome Analysis: adaptations to alkaliphily, osmotic stress, and radicals".
The ETC of Anaero-CMMVII has NADH dehydrogenase (nuoABCDHIJKLMN), but the gene operon lacks the subunits nuoEFG. These subunits are known to contain the domain involved in NADH binding and oxidation (Sazanov, 2007). Instead, NADH dehydrogenase likely utilizes alternative electron donors, such as reduced ferredoxin. It is also unlikely that the nuo operon encodes for other closely related complexes, such as MBX (archaeal respiratory complex), FPO (methanogenic respiratory complex), and MBH ([NiFe] hydrogenase), as demonstrated by a maximum likelihood phylogenetic tree (Supplementary Figure S5) of the catalytic subunits in NUO, MBX, FPO, and MBH (Schut et al., 2016). Notably, the genome of Anaero-CMMVII does contain the genes hyaABC, which encode for group 1d [NiFe] hydrogenase (Peters et al., 2015). This suggests that energy conservation in Anaero-CMMVII likely involves hydrogenotrophic respiration by the action of the group 1d [NiFe] hydrogenase, HyaABC (Peters et al., 2015). The gene neighborhood of hyaABC includes nitrate reductase (napGH-napABD-napG), suggesting H 2 oxidation is coupled with the reduction of nitrate. Oxygen may also be used as an electron acceptor (Peters et al., 2015).
The ETC of Paeni-Cedars includes an additional terminal menaquinol oxidase, encoded by qoxABCD. This likely functions with the other terminal oxidases in aerobic respiration (Götz and Mayer, 2013). As mentioned above, Paeni-Cedars is capable of EET and reduction of magnetite (Rowe et al., 2017) and other iron minerals (Table 3). We inspected the genome of Paeni-Cedars for the presence of genetic markers gleaned from hypothesized EET mechanisms for Gram-positive and Gram-negative bacteria. We confirmed the presence of genes involved in EET mechanisms for Gram-positive bacteria only. Paeni-Cedars encodes an operon implicated in flavin-mediated iron reduction in Listeria monocytogenes 10403S (Light et al., 2018). In L. monocytogenes, this operon consists of eight genes, including flavin transporters (fmnB, fmnA), cell surface electron transport proteins (eetAB, pplA), demethylmenaquinone synthases (dmkA, dmkB), and type II NADH dehydrogenases (ndh2).
Homologs (e-value<1E-15) to seven of these genes are present in a single operon in Paeni-Cedars, while the eighth, one of the demethylmenaquinone (DMK) synthases (dmkA), is present elsewhere in the genome. The Paeni-Cedars EET operon also contains the genes for FAD transport via the ECF (energy-coupling factor) system, with the genes ecfA1-ecfA2-ecfT (ecfT is identified as fmnA in the L. monocytogenes EET operon) and an unknown gene for riboflavin transport (ribU) (Gutiérrez-Preciado et al., 2015). FAD transport is needed for FmnB to catalyze FMNylation of PplA, enabling electron transfer from DMK to FMN groups on PplA or free flavin shuttles for iron reduction (Light et al., 2018).

Genome analyses: carbon metabolism
All three microbes are capable of glycolysis and gluconeogenesis; and converting CO 2 to HCO 3 (or the reversible reaction) by the action of carbonic anhydrase. Carbonic anhydrase may be a necessary enzyme for microorganisms in The Cedars' springs given the high pH conditions and the presence of calcite precipitates. For glycolysis and gluconeogenesis, the Anaero-CMMVII genome lacks glucokinase (glk), which is involved in the phosphorylation of glucose to glucose-6-phosphate. Instead, Anaero-CMMVII likely relies on the phospho-enolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) to catalyze the transport and phosphorylation of sugars (Deutscher et al., 2006).
In contrast to the other two isolates, the Ali-BS5-314 genome likely encodes only two sugar-associated ABC transporters: AfuABC for the uptake of hexose/heptose-phosphate metabolites (e.g., glucose-6-phosphate) (Sit et al., 2015) and BenEK for the uptake of benzoate-like compounds (Patrauchan et al., 2005). Ali-BS5-314 may also catabolize benzoate-like compounds. In our growth studies, protease peptone was provided as a nutrient source, and it is likely that similar aromatic compounds are in The Cedars BS5 spring fluids, based on the observation of various aromatics in other marine (Pasini et al., 2013;Plümper et al., 2017) and terrestrial (Seyler et al., 2022) serpentinite-hosted ecosystems. After uptake by the transporter BenEK, these benzoate-like compounds can be degraded to catechol-like metabolites by the action of dioxygenase (BenABC) and dihydrodiol dehydrogenase (BenD) (Patrauchan et al., 2005). The gene neighborhood of the benzoate degradation pathway also includes genes (catRBCA) for catechol degradation by the 3-oxoadipate pathway (Harwood and Parales, 1996;Nojiri et al., 2002) and genes (pcaIJFD) for further degradation to acetyl-CoA by the protocatechuic acid degradation pathway (Clarkson et al., 2017).

Genome analyses: nitrogen metabolism
All three isolates have different nitrogen metabolisms. Ali-BS5-314 reduces nitrate and assimilates ammonia; Anaero-CMMVII is capable of dissimilatory nitrate reduction; and Paeni-Cedars uses the glycine cleavage and urea degradation pathways. However, in the natural setting of The Cedars, the isolates would likely experience limited nitrate and ammonia availability, with spring concentrations between 1 and 2 μM (Cook et al., 2021). Frontiers in Microbiology 11 frontiersin.org Genome analysis of Ali-BS5-314 identified that this microbe is capable of nitrate reduction by the action of nitrate reductase (NarGHJI). This is supported by the physiological studies, discussed above. Nitrate reduction occurs intracellularly, based on the localization of the catalytic subunit (narG) to the cytoplasm (Supplementary Figure S6; PSORTb localization prediction Supplementary Table S9) and the identification of nitrate/nitrite transporters (NarK1 and NarK2) (Sharma et al., 2006). Subsequent reduction to nitrogen or ammonia is likely not feasible. Instead, ammonia is transported intracellularly by the bidirectional ammonium transporter Amt and assimilated. For example, Ali-BS5-314 may use ammonia in the conversion of glutamate to glutamine (GlnA) and vice versa (GltB) (van Heeswijk et al., 2013). In Ali-BS5-314, ammonia could freely diffuse through the outer membrane into the periplasm for uptake by the transporter Amt (Andrade and Einsle, 2007). Potential buildup of ammonium intracellularly is prevented by regulation of Amt (Soupene et al., 2002;Andrade and Einsle, 2007).
For Anaero-CMMVII, dissimilatory nitrate reduction is feasible. The major facilitator superfamily proteins NarK1 and NarK2 are used to transport nitrate/nitrite (Sharma et al., 2006). Nitrate reduction is conducted by the action of nitrate reductase (NapABD). The gene operon for napABD does not contain napC, which encodes for a cytochrome needed for quinone cycling. Nitrite can then be converted to nitric oxide (NirK) or ammonia (NrfAH). Nitric oxide is a diffusible free radical gas that may be involved in oxidative stress response, cell signaling, and defense (Orsini et al., 2020). The genome also contains the gene nmo, which encodes for nitronate monooxygenase that acts to oxidize alkyl nitronates (e.g., nitroalkane) to nitrite or other compounds using molecular oxygen (Gadda and Francis, 2010).
Nitrogen metabolism in Paeni-Cedars may occur via the glycine cleavage system (GCS) and urea degradation. These two pathways result in the production of ammonia, which is needed for other metabolic pathways (e.g., glutamate synthesis). GCS is a multienzyme system which acts to oxidize glycine, resulting in CO 2 , NH 3 , 5,10-methylenetetrahydrofolate (5,10-CH 2 -THF), and a reduced pyridine nucleotide (Okamura-Ikeda et al., 2010). In Paeni-Cedars, the GCS is feasible with the genes gcvHT (GCS proteins) and gcvPAB (glycine dehydrogenase). The genome also encodes the urea cycle via the arginine biosynthesis pathway, yielding urea. Urea can then breakdown into CO 2 and NH 3 by the action of urease. Paeni-Cedars may acquire arginine (or polyamines) and urea by ABC transporters. In particular, transported polyamines could be oxidized by the action of copper amine oxidase, releasing ammonia and hydrogen peroxide; other genes, discussed below, will then convert H 2 O 2 to H 2 O and O 2 . The Paeni-Cedars genome also encodes for a nitrite or monovalent anion bi-directional transporter (NirC), which has been linked to the transport nitrite, formate, hydrosulfide, lactate, acetate, and bicarbonate (Atkovska and Hub, 2017). The reason for nitrite transport remains unknown, but could prevent nitrite build-up intracellularly as a result of nitroalkane breakdown by the action of Nmo.

Genome analyses: sulfur metabolism
All three microbes are capable of assimilatory sulfate reduction. Both Ali-BS5-314 and Paeni-Cedars transport sulfate into the cell by an ABC transporter, while Anaero-CMMVII uses sulfate permease (CysP). All three microbes convert sulfate to adenosyl phosphosulfate (APS) by the action of sulfate adenylyltransferase (CysND for Ali-BS5-314; Sat for Paeni-Cedars and Anaero-CMMVII). Subsequent conversion of APS to sulfide through multiple steps involves the genes cysC (adenylylsulfate kinase), cysH (phosphoadenosine phosphosulfate reductase), and cysJI (sulfite reductase). Sulfide can then be transformed to L-cysteine by the action of CysK (cysteine synthase A).
Paeni-Cedars may also acquire sulfur from alkanesulfonates or organosulfur compounds (Kahnert et al., 2000). Intracellular uptake  (Eren et al., 2015). a Read length from PacBio. b Reads from The Cedars metagenomes (Suzuki et al., 2017) were mapped to the isolate genomes. The metagenomes are under the DDBJ BioProject accession number PRJDB2971. The Bowtie2 (Langmead and Salzberg, 2012) overall alignment rate percentage is reported.
Frontiers in Microbiology 12 frontiersin.org of alkanesulfonates occurs by the action of an ATP transporter, and the genome of Paeni-Cedars contains the genes ssuBACC. Subsequently, the alkanesulfonates can then be converted to sulfite by a gene encoding for a monooxygenase, which may function similarly as SsuD. The genome of Paeni-Cedars also contains several copies of ssuE (FMN reductase) and other genes for FMN reductase, although these genes are not in the same gene neighborhood as other ssu genes. This suggests that there is potential for Paeni-Cedars to have two sulfur metabolic pathways.

Genome analyses: iron and other trace metal acquisition
The genomes of all three microbes have genes for ABC transporters to uptake trace metals for growth and enzyme function. In the natural environment, The Cedars spring fluids contain low nM levels of iron, copper, nickel and cobalt (Supplementary Table S14), indicating that microorganisms require adaptations to transport metals from the surrounding minerals or fluids. Ali-BS5-314 is capable of iron (as Fe 3+ ) and molybdate uptake. In comparison, Paeni-Cedars and Anaero-CMMVII can uptake various metals, including iron (as Fe 2+ or Fe 3+ ), molybdate, nickel, or zinc. Notably, the genome of Anaero-CMMVII also encodes an ABC transporter specifically for tungstate transport. Enzymes containing tungstate are often observed in prokaryotic obligate anaerobes (Hille, 2002), although Anaero-CMMVII is a likely oxygen-tolerant anaerobe.
Frontiers in Microbiology 14 frontiersin.org well-known that alkaliphilic and alkali-tolerant microbes maintain an intracellular pH lower than the external pH through ion transport and/or synthesis of acidic compounds (Krulwich et al., 2014). All three genomes contain genes (mrpABCDEFG and nhaC) for Na + /H + antiporters (Ito, 2017), in addition to proton pumping within the electron transport chain, oxidative phosphorylation, and flagellar rotation. Paeni-Cedars and Ali-BS5-314 also have genes (mscM, mscS, and/or mscL) for encoding mechanosensitive ion channels, which allow for osmotic adjustment (Cox et al., 2018). Osmotic stress adaptations are used by all three microbes. These adaptations are needed in the natural environment of The Cedars, where the conductivity of spring fluids ranges from 740 and 3,010 μS/ cm because of the high Ca 2+ , Na + and Cl − content (Morrill et al., 2013). Notably, all three microbes could synthesize glutamate as a primary organic osmolyte, as demonstrated in the halophile Halobacillus halophilus (Saum and Müller, 2008). In contrast, only Anaero-CMMVII and Paeni-Cedars may uptake osmoprotectants (e.g., glycine betaine and proline) by an ABC transporter. The ectoine biosynthesis pathway [ectABC(D)] is also present in the genomes of Anaero-CMMVII and Paeni-Cedars; ectoine is an osmolyte and is produced by microbes in response to osmotic stress (Czech et al., 2018). However, the genome of Anaero-CMMVII lacks ectD, indicating that ectoine cannot be subsequently transformed to the osmolyte 5-hydroxyectoine. The putative uptake and synthesis of osmolytes by Paeni-Cedars may enable growth under high CaCl 2 conditions (5-20 mM) ( Figure 4).
All three microbes have adaptations against radicals and hydrogen peroxide, including genes encoding for superoxide dismutase, catalases, and peroxidases. In addition, the cytochrome bd complex (CydAB or CydABCD) is present in the three isolates, as described in the section "Genome analyses: Energy conservation and extracellular electron transport" and is also known to protect against oxidative and nitrosative stress conditions, potentially responding to high pH conditions (Giuffrè et al., 2014). Paeni-Cedars may also use transported polyamines, discussed above, to act as radical scavengers (Chattopadhyay et al., 2003;Shah et al., 2008), in addition to other biological functions (Khazaal et al., 2021).
Paeni-Cedars and Anaero-CMMVII may form endospores in response to adverse environmental conditions. The genomes of both isolates contain the sporulation master regulator gene, spo0A, and other genes involved in sporulation, including sspA (encodes for α/β-type small acid-soluble sporulation protein) and dipicolinate synthase (dpaA [spoVFA] and dpaB [spoVFB]) (Galperin et al., 2022). Spore formation may enable both isolates to survive in the Conceptual cell model of Anaero-CMMVII based on genome analysis. Annotations for Anaero-CMMVII can be found in Supplementary Tables S8, S11, S13. Abbreviations also found in Figures 5, 6 description. napABD, nitrate reductase; napGH, genes for ferredoxin-type protein; nrfAH, nitrite reductase; nirK, nitrite reductase, NO forming; nuo, NADH dehydrogenase. Pink, phosphate transporter; gray, function related to metal transport or storage; blue, function related to the uptake, assimilation, or breakdown of sugars, amino acids, vitamin, fatty acids; orange, function related to the nitrogen cycle; dark red, flagellar complex; black, ROS/stress response; dark teal, oxidative phosphorylation; white/black, function related to ion homeostasis; green, function related to sulfur cycle; *putative. Microbiology  15 frontiersin.org nutrient-limited and alkaliphilic conditions of fluids in The Cedars springs.

Conclusion
Serpentinite-hosted ecosystems are important locations to understand the origins and limits of life in a planetary context. The highly alkaline and reducing fluids are host to microbial communities that often persist under extreme conditions and nutrient limitations. However, the ecophysiology of these microbes remains elusive because of the lack of pure culture representatives available for investigation in the laboratory environment. In this study, we characterized three isolates (Ali-BS5-314, Paeni-Cedars, and Anaero-CMMVII) from The Cedars through physiological and genomic studies. These three isolates are each unique in their metabolic capacities and optimal growth conditions. Moreover, Ali-BS5-314 and Anaero-CMMVII are alkaliphiles, while Paeni-Cedars is alkali-tolerant. Further studies are needed to understand the mechanisms for alkaliphily and stress response. These isolates also have biotechnological potential for processes done at alkaline pH. Based on genome analyses, Ali-BS5-314 can putatively metabolize benzoate-like compounds, and Anaero-CMMVII can degrade complex carbohydrates. Notably, Paeni-Cedars has been demonstrated to perform EET, and is believed to be capable of synthesizing siderophores or metabolizing complex carbohydrates and alkanesulfonates. Overall, this work demonstrates how the enrichment, isolation, and investigation of novel microbes from serpentinizing systems in the laboratory setting is paramount to furthering our understanding of the nature of life in these unique systems and are necessary for studies in both astrobiology and biotechnology.

Author contributions
JT and CB contributed equally to the experimental design and physiological characterization of The Cedars microorganisms. NM, MT, ML, and CT supported the physiological characterization studies. NM isolated Ali-BS5-314 and analyzed the genomes of all isolates. LB isolated Anaero-CMMVII. ELC worked with Paeni-Cedars and sequenced the genome. SS, AO, LB, and KN advised the physiological and genomic characterization studies. YH advised the genomic analyses. LB-A and DR analyzed the siderophores from The Cedars. All authors contributed to the article and approved the submitted version.