Comparative Genomics and Physiological Investigation Supported Safety, Cold Adaptation, E cient Hydrolytic and Plant Growth-promoting Potential of Psychrotrophic Glutamicibacter Arilaitensis LJH19, Isolated from Night-Soil Compost

BACKGROUND
Night-soil compost (NSC) has traditionally been conserving water and a source of organic manure in northwestern Himalaya. Lately, this traditional method is declining due to modernization, its unhygienic conditions, and social apprehensions. Reduction in the age-old traditional practice has led to excessive chemical fertilizers and water shortage in the eco-sensitive region. In the current study, a bacterium has been analyzed for its safety, cold-adaptation, efficient degradation, and plant growth-promoting (PGP) attributes for its possible application as a safe bioinoculant in psychrotrophic bacterial consortia for improved night-soil composting.


RESULTS
Glutamicibacter arilaitensis LJH19, a psychrotrophic bacterium, was isolated from the NSC of Lahaul valley in northwestern Himalaya. The strain exhibited amylase (186.76 ± 19.28 U/mg), cellulase (21.85 ± 0.7 U/mg), and xylanase (11.31 ± 0.51 U/mg) activities at 10 °C. Possessing efficient hydrolytic activities at low-temperature garners the capability of efficient composting to LJH19. Additionally, the strain possessed multiple PGP traits such as indole acetic acid production (166.11 ± 5.7 μg/ml), siderophore production (85.72 ± 1.06% psu), and phosphate solubilization (44.76 ± 1.5 μg/ml). Enhanced germination index and germination rate of pea seeds under the LJH19 inoculation further supported the bacterium's PGP potential. Whole-genome sequencing (3,602,821 bps) and genome mining endorsed the cold adaptation, degradation of polysaccharides, and PGP traits of LJH19. Biosynthetic gene clusters for type III polyketide synthase (PKS), terpene, and siderophore supplemented the endorsement of LJH19 as a potential PGP bacterium. Comparative genomics within the genus revealed 217 unique genes specific to hydrolytic and PGP activity.


CONCLUSION
The physiological and genomic evidence promotes LJH19 as a potentially safe bio-inoculant to formulate psychrotrophic bacterial consortia for accelerated degradation and improved night-soil compost.


Background
The highland agro system of northwestern Himalaya lacks productivity and soil fertility due to extreme weather conditions like heavy snowfall, avalanches, landslides, soil erosion, and scanty rainfall [1]. To meet the high demand for manure and shortage of water during winter, the traditional method of composting human excreta (night-soil) using dry-toilets is prevalent in this region ( Fig. 1) [2][3][4]. Lately, the night-soil composting practice is declining, promoting excessive chemical fertilizers use in ecologically vulnerable high altitude farmlands [4]. Promotion of the safe and hygienic winter dry-toilets aided with scienti c intervention is necessary to sustain the agro-ecosystem and conservation of water in such highland areas.
The foul odour of the winter toilet has been one of the main reasons for the decline of this practice. In a cold climate, the lower microbial load in the initial composting process delays the composting process due to slower biomass degradation, and psychrotrophic bacteria play a crucial role in low-temperature composting [5]. Plant growth-promoting (PGP) bacteria play an important role in maintaining soil fertility by increasing the availability of the nutrients, such as iron, nitrogen, phosphorous and by producing phytohormones (indole acetic acid-IAA), growth regulators (siderophores), and solubilizing phosphate to modulate plant growth and development [6,7]. In the course of isolating the psychrotrophic, e ciently degrading strains with PGP potential to formulate psychrotrophic bacterial consortium for application in night-soil composting, we obtained a bacterial strain LJH19 with remarkable PGP traits and e cient hydrolytic activity in in-vitro assays. Owing to its cold adaptation, e cient hydrolytic activity, PGP potential, and origin from faecal compost, whole-genome sequencing was performed to elucidate the genetic basis of the catabolic activities, PGP traits, and analysis for pathogenicity determinants. Further, to explore the habitat-speci c gene repertoire, we performed comparative genomics of LJH19 with all the available strains of the same genus. Based on a unique genome region across the strain LJH19, a comparison was withdrawn with the closely related strains. Biosynthetic gene clusters in the genome of LJH19 were also identi ed, and further to evaluate bacterial safety, the presence of antibiotic resistance gene cluster across all the strains was assessed. To formulate an e cient psychrotrophic bacterial consortium, it is important to study each potential bacteria individually. The current study aims to establish strain LJH19 as a potential bio-inoculant for application in consortia for night-soil composting.

Results And Discussion
Physico-chemical properties of night-soil compost (NSC) samples and bacterial characterization The compost samples were collected from the compost pile randomly in triplicate from the collection chamber of the traditional night-soil toilet "ghop" (Fig. 1C). The compost pile was dug, and the sample was obtained from the core of the pile; the temperature, pH, and electrical conductivity (EC) of the collected samples were 9.9 °C, 10, and 1674 µS, respectively. The available nitrogen, phosphorous, and potassium in the collected NSC samples were 2297.6 ± 99.4 ppm, 117.11 ± 0.34 ppm, and 22534.11 ± 73.08 ppm, respectively.
In an attempt to explore the bacterial diversity from NSC, 130 bacterial strains belonging to varied taxonomic genera were obtained based on their hydrolytic activities in different substrates and plant growth-promoting traits (unpublished data). One such e cient hydrolytic bacterial colony was an opaque, yellow-pigmented bacterium LJH19 that showed multiple hydrolytic activities. The bacterium could survive at varying temperatures (4-37 °C) and showed optimum growth at 10°C, pH 7 ( Table 1). The bacterium showed hydrolytic activity against substrates like corn starch, CMC, and birchwood xylan at a varied temperature of 4-37°C, and the most e cient activity was obtained at 10 °C (Table 1, Supplementary Fig. S1). Gene sequence similarity based on partial 16S rRNA gene (NCBI accession no. MT349443) related the bacteria to G. arilaitensis Re117 with 100% identity and coverage of 96.5% in EzTaxon Biocloud (https://www.ezbiocloud.net/identify). Quantitatively, LJH19 showed enzyme activity Page 4/31 at varying temperatures (5, 10, 15, 20, 28, and 37 °C), and the best production was obtained at 10 °C. At 10°C, the strain LJH19 exhibited production of amylase enzyme with a speci c activity of 186.76 ± 19.28 U/mg ( Supplementary Fig. S2) using corn starch as substrate, cellulase enzyme with a speci c activity of 21.85 ± 0.7 U/mg ( Supplementary Fig. S2) using CMC as a substrate and xylanase enzyme with a speci c activity of 11.31± 0.51 U/mg ( Supplementary Fig. S2) using birchwood xylan as a substrate. It has been hypothesized that psychrotrophic bacteria play a crucial role in low-temperature composting, and it is critical for a bacterium to possess enzymatic activities to ensure e cient composting [5,8,9]. Like LJH19, other strains of genus Glutamicibacter have also been reported to possess hydrolytic enzymes such as amylase and cellulase [10,11]. Glutamicibacter strains have been reported from varied niche areas [12] and its reclassi cation originates from a much diverse genera Arthrobacter [13]. Genus Arthrobacter has also been reported from a harsh cold environment with potential hydrolytic enzymes [14,15]. With survival at a temperature as low as 4°C and e cient hydrolytic activity against complex polysaccharides (starch, cellulose, and xylan), the strain LJH19 was chosen as a potential candidate for psychrotrophic consortia for accelerated degradation of NSC at low ambient temperature. The nutrients released after the decomposition of polysaccharides have the tendency to leave the agricultural systems due to leaching, surface runoff, and eutrophication [16]. As a result, availability for plant uptake is always questionable; however, the inhabitant PGP bacteria improves nutrient uptake and produces phytohormones aiding the e ciency of applied compost [17]. Hence, LJH 19 was explored further to investigate its PGP potential for additional properties to become a suitable bioinoculant candidate for enhancement of soil nutrients at high altitude agro-ecosystems. In the qualitative assay for siderophore production, the LJH19 strain showed orange halo zones at varying temperatures and the best results of the siderophore index of 1.5 at 10°C (Table 1) (Supplementary Fig. S3D). Quantitatively, LJH19 exhibited considerable siderophore production of 85.72 ±1.06 % siderophore unit (psu) at 10°C. In the absorption spectra, we observed a peak at 292 nm supporting the presence of 2,3-dihydroxybenzoic acid (DHB) in the supernatant ( Supplementary Fig. S4). A previous study reported that in acidic medium DHB, a phenolic compound consisting of a catechol group absorbs below 330 nm showing two absorption bands with maxima at 254 nm and 292 nm, respectively [18]. DHB is an intermediate involved in the synthesis of catecholate type siderophore [19]. This evidence supports the presence of DHB in the supernatant, indicating the production of catecholate type siderophore by strain LJH19. Siderophore production by PGPB is vital for plant defense. Iron chelation by siderophores suppresses fungal pathogens in the rhizosphere [20]. LJH19 also demonstrated the ability to produce 166.11 ± 5.7 µg/ml of IAA after 72 Hrs of incubation with 200 µg/ml concentration of L-Trytophan at 10°C (Supplementary Fig.  S3A). This infers the ability of LJH19 to produce the IAA in the presence of L-tryptophan, signifying that auxin production occurs through the tryptophan dependent pathway. Production of phytohormone IAA is essential for plant growth to proliferate lateral roots and root hairs [21]. Qualitative estimation of phosphate solubilization by LJH19 showed positive results at varying temperatures, and the best activity of 2.3 solubilization index was displayed at 10°C (Table 1). Quantitatively, LJH19 solubilized 44.76 ± 1.5 µg/ml of tri-calcium phosphate at 10°C after the 5th day of incubation in NBRIP broth ( Supplementary   Fig. S3C). The activity of bacteria decreased pH from 7 to 4.5, indicating the elevation of phosphate solubilization levels. This suggested that the presence of LJH19 in the compost can deliver available phosphorous to the plants. Since plants cannot uptake inorganic phosphate present in a xed or precipitated form in the soil, bacteria aids in increasing the availability of soluble P for plant acquisition through solubilization [22]. While performing in-vitro assays for ammonia production, strain LJH19 produced a low level of ammonia (0.20± 0.01 µmoles/ml) ( Supplementary Fig. S3B) after 10 days of incubation in peptone water. Ammonia production by bacteria is yet another feature of PGP to increase the availability of nitrogen [23]. However, these values are relatively low in the case of PGP attributes. In the composting case, ammonia gas released by bacteria is primarily responsible for the pungent smell and loss of organic nitrogen from the compost [24]. This may suggest that it doesn't directly bene t the plants but may maintain stable organic nitrogen content in the compost by not converting rich nitrogenous sources into ammonia gas.
The seed germination rate was signi cantly higher in the treated pea Pisum sativum var. Arkel seeds (83.33 ± 15.27 %) from that of the control (66.66 ± 15.27 %) ( Supplementary Fig. S5). The relative seed germination, relative root growth, and relative shoot growth were noticeably increased to 135.55 ± 33.55 %, 103.70 ± 33.60%, and 112.78 ± 12.14 %, respectively, subjected to the treatment of pea seeds with bacterial inoculation. The germination index was further recorded as 116.348 ± 38.02 % under the bacterial in uence. The LJH19 strain capabilities to produce auxin and siderophore may have positively affected pea seeds' seed germination. In agreement with our ndings, other Glutamicibacter strains have also shown PGP traits where a strain G. halophytocola KLMP 1580 have signi cantly promoted the growth of Limonium sinense under high salinity stress [25]. In another study, G. halophytocola KLMP 1580 was also reported to enhance tomato seedlings' growth [26]. Another Glutamicibacter species, G. creatinolyticus was reported as an e cient PGPR with IAA production [27]. Similarly, the closest related genus Arthrobacter has also been reported to exhibit excellent PGP attributes, having a potential role in recovering burned soils of holm-oak forests [15,28,29].
Owing to the source of LJH19 strain isolation from night-soil, it was mandatory to ensure its safety for humans before declaring it as a suitable candidate as a bioinoculant. Hence, the strain LJH19 was tested for its pathogenicity. In general, any pathogenic bacteria rely on various virulence factors to induce pathogenesis, including adhesion proteins, toxins like hemolysins, and proteases [30]. The initial screening of virulence of LJH19 performed on blood agar showed no hemolytic activity compared to the other tested hemolytic strains MTCC 96, MTCC121, MTCC 43, MTCC 2470 ( Supplementary Fig. S6A).
LJH19 was tested positive for protease activity with an enzymatic index of 12.5 ( Supplementary Fig.  S6B), but, quantitatively LJH19 showed very low protease activity (Table 1).
Furthermore, strain LJH19 was not observed to form bio lm on polystyrene at 37°C ( Supplementary Fig.   S6C). The adherence of bacteria to the host tissue cells is the initial step to induce the pathogenesis [31]. Therefore, bio lm formation is a notable virulence factor of pathogenic potential and is directly related to the strain's safety. In the antibiotic susceptibility test, the LJH19 strain exhibited susceptibility to all the twelve antibiotics tested ( Supplementary Fig. S6D), (Table 1).
Night-soil composting remains dormant during winters as the temperature goes to sub-zero conditions, and microbial degradation plays an insigni cant role in odour formation. However, in the summer months, where the temperature ranges from 5 to 25°C [1], slow microbial metabolism due to low microbial load produces a strong odour during composting. During this period, night-soil composting can be improved by supplementing it with a psychrotrophic bacterial consortium. Owing to the survivability at 4°C and e cient hydrolytic activity at varying temperatures (best activity at 10 °C), non-pathogenicity, and PGP potential, strain LJH 19 quali es as a potential bio-inoculant candidate for the preparation of a psychrotrophic consortium for accelerated degradation and quality improvement of NSC. Further, the whole genome sequencing, data mining, and comparative genomics of strain LJH19 bacterium were explored to obtain genetic bases on its potential to be a safe bio-inoculant for the consortia and to investigate the niche-speci c gene repertoire.

Genomic features of strain LJH19
RS hierarchical genome assembly was performed as described previously in Kumar et al. [32]. The assembly generated a draft genome (4 contigs) of 3,602,821 bp (N50 read length 2,610,692) with 59.60% GC content with average mean coverage of 153 X (Supplementary Table S1) (GenBank accession number: SPDS00000000). The NCBI Prokaryotic Genome Annotation Pipeline (URL: www.ncbi.nlm.nih.gov/genome/annotation_prok) prediction revealed a total of 3517 genes out of which 3396 were protein-coding genes (covering 96.56 % of the genome) and 99 RNA genes (30 rRNAs, 66 tRNAs, and 03 other RNA genes). There was no plasmid DNA in the genome of LJH19 as evident by no observation of bands in agarose gel electrophoresis after the plasmid isolation. Additionally, in silico analysis with PLSDB web-based tool supported no existence of plasmid in the genome of LJH19 Whole genome-based phylogenetic assessment and genome relatedness Phylogenetic tree based on extracted 16S rRNA gene sequence had an ambiguity. The strain LJH19 formed a cluster with another G. arilaitensis strain JB182, while the type strain G. arilaitensis Re117 T fell into a separate clade ( Fig. 2A). The true phylogeny of the isolate was obtained with the phylogenomic tree obtained from PhyloPhlAn, which uses around 400 most conserved gene sequences present across the isolates. Strain LJH19, type strain Re117, and JB182 were found to be in a single clade (Fig. 2B). To get the genome relatedness estimate, we have implemented the orthoANI estimation of the isolates from the genus. The orthoANI confers robustness in nucleotide identity analysis [33]. ANI matrix suggests the genome similarity of the strain LJH19 to subspecies level relatedness to the strain Re117 as its value was around 97% for the type strain Re117 and another strain JB182 (Fig. 2C).

Pan-genome analysis and Chromosomal map
Roary run for the group of the strains forming a clade with the type strains of G. arilaitensis and LJH19 resulted in a pan-genome of 9892 genes. A total of 634 genes were found to be core genes, whereas the gene clusters speci c to the strain LJH19, Re117, and BJ182 was 1740. A total of 217 genes were speci c to the strain LJH19. Chromosomal map showing the unique genomic regions across the strain LJH19 depicts the uniqueness of the strain LJH19 (Fig. 3A). All the strain-speci c gene from LJH19 classi ed by eggNOG falls in several COG categories (Fig. 3B). A list of the unique gene, its function, and COG classi cation is reported in Supplementary Table S2. Based on the annotation and unique genes data, an image illustrating a schematic representation of predicted genes associated with catabolic activities, transport, and plant growth promotion of the genome of LJH19 was generated (Fig. 4).

Genomic insights into the safety of LJH19
Virulence is a characteristic of pathogenicity which confers the ability to initiate and sustain infection for the organism. The occurrence of such determinants at the genetic level makes the organism potentially pathogenic with the ability to circulate such genes in the bacterial population [34]. LJH19 displayed a negative resistance phenotype to all the 12 antibiotics tested in the in vitro assays (Table 1; Fig. S6D). To con rm this susceptibility pro le, we performed an in silico investigation of the LJH19 genome and its phylogenetic relative. But, the RGI module of CARD 2020 with strict mode resulted in the detection of no antibiotic resistance gene cluster in LJH19 and its relatives. To further con rm these results, the LJH19 genome was assessed for its pathogenic potential by PathogenFinder [35]. This web-based tool identi es the genome and provides a probability measure for the test strain to be pathogenic for humans. The predicted results identi ed LJH19 as a non-human pathogen with an average probability of 0.228 (Supplementary output le S1). None of the putative virulence or pathogenic genes were identi ed in the tested genome. These results suggested the safety of strain LJH19.

Genomic insights into the cold adaptation of LJH19
Psychrotrophic bacteria isolated from high altitude ecosystems have unique adaptations to survive in a cold environment maintaining their growth and metabolism [32]. LJH19 was isolated from a night-soil compost of the high altitude ecosystem of Lahaul valley in northwestern Himalaya that experiences extreme temperature variations [1]. Psychrotrophic bacteria sustain these extreme factors with unique cold-adapted proteins active at low temperatures. There are reports on such cold-associated genes in the genome of cold-adapted bacteria [9,32,36]. LJH19 genome also predicted several cold associated genes encoding for proteins responsible for cold-active chaperons, general stress, osmotic stress, oxidative stress, membrane/cell wall alteration, carbon storage/ starvation, DNA repair, Toxin/Antitoxin modules were identi ed across the genome ( Table 2). This genomic evidence supports the versatility of the LJH19 strain to survive in a broad temperature range of 4 to 37 °C. Bacteria inhabiting high altitude regions are also prone to accumulation of Reactive oxygen species (ROS) such as hydrogen peroxide, superoxides, and hydroxyl radicals, and to prevent the damage caused by these radical bacteria synthesize antioxidative enzymes [9,32,37]. Similarly, the LJH19 genome predicted multiple genes encoding antioxidant enzymes such as catalase, superoxide dismutase, thioredoxin, and Thioredoxin-disul de reductase (Table 2). Additionally, the genome of the strain LJH19 also predicted genes encoding proteins involved in DNA repairs such as Recombinase, DNA repair protein RadA, DNA integrity scanning protein DisA, and DNA repair protein RecN ( Table 2) that may aid in the robust feature of strain LJH19 in surviving the extreme conditions. Genomic insights on nutritional versatility and adaptation to environmental stresses have been documented previously for strains of the Glutamicibacter genus [12]. Similar to genomic evidence on cold adaptation of LJH19, adaptation towards salt tolerance, oxidative and osmotic stress tolerance from varied ecological habitats such as cheese, coastal halophyte, rhizospheric soil, and coral Favia veroni have been reported previously (Supplementary Table S3) [26,[38][39][40]. Likewise, multiple reports on genomic evidence to support physiological adaptation for varied stress adaptations in the nearest genus Arthrobacter are also available [14,15,29,41] (Supplementary Table S3). The current study and other genomic insights supported the niche-speci c adaptational strategies of the genus Glutamicibacter in the varied ecological habitats.
Genomic insights into the hydrolytic potential of LJH19 The biodegradation of complex polysaccharide molecules by bacteria requires a cocktail of enzymes to depolymerize it to oligosaccharides and monomer sugars [42]. The genome of LJH19 showed the occurrence of multi copies of genes encoding for proteins responsible for the metabolism of a wide variety of complex polysaccharides like cellulose, starch, and xylan. Similar to the nding in LJH19, the genome of G. arilaitensis Re117 T strain also has been reported encoding genes involved in protein and lipid degradation [38] (Supplementary Table S3). The key enzymes encoded in the LJH19 genome are beta-glucosidase, alpha-amylase, beta-xylosidase, pullulanase, oligo-1,6-glucosidase, and glycosidases associated with the degradation of polysaccharides (Table 3, Fig. 4, Supplementary Table S3). These ndings endorse the experimental evidence of LJH19 showing enzymatic activities against complex polysaccharides that aids in the improved composting process. For the utilization of cellulosic substrates, psychrotrophic bacteria requires the ABC transporters speci c for the hydrolytic product, such as cellobiose, cellodextrin, β-D-Glucose. Cellulases such as beta-glucosidase cleave the β-(1,4)-glycosidic linkages within the cellulose polymer releasing cellobiose, glucose, and cellodextrin, which are then transported inside the cell via speci c transporters [43]. LJH19 genome also predicted genes encoding proteins that are components of transporter complexes engaged in the recognition and transport of monosaccharides and oligosaccharides such as maltose/maltodextrin, maltooligosaccharide, and cellobiose and transporters for hydrolyzed proteins (Table 3, Supplementary Table S3). Furthermore, genes such as triacylglycerol lipase were also predicted associated with fatty acid degradation ( Fig. 4; Supplementary Table S3). Within the cells, enzymes (like beta-glucosidase, oligo-1,6glucosidase, alpha-amylase) attack the polysaccharides producing smaller oligosaccharides and monomeric sugars, and nally, the monomeric sugars like glucose go into the glycolysis pathway and ultimately to the TCA cycle generating energy for cellular growth [44]. For better understanding, an overview of a similar mechanism has been represented in the LJH19 cell based on the prediction of genes encoding critical proteins for polysaccharides metabolism and transporters from the genome (Fig.4).

Genomic insights into plant growth-promoting potential of LJH 19
A series of genes encoding enzymes related to PGP traits predicted in the LJH19 genome were amidase, isochorismate synthase, isochorismatase family protein YecD, nitrite reductase, nitrate reductase, and alkaline phosphatase (Table 3). Quantitatively LJH19 strain showed auxin production by utilizing Ltryptophan, and it got endorsed by the genomic evidence that predicted tryptophan dependent pathway utilizing L-tryptophan (Fig 4, Table 3). Genes encoding amidase, N-acetyltransferase, and acetaldehyde dehydrogenase for auxin synthesis were predicted in the LJH19 genome (Table 3, Fig 4). Auxin plays a vital role in the development of lateral plant roots and stem elongation [21]. The experimental studies also have shown remarkable siderophore production by LJH19 strain that is an important plant defense, suppressing fungal pathogens in the rhizosphere [20]. Upon genome mining, genes involved in the synthesis of polyamines (PAs), putrescine (Put), and spermidine (Spd) were also identi ed in the LJH19 genome (Table 3 and Supplementary Table S4). In bacteria, these active molecules are involved in the biosynthesis of siderophores, improve the survival rate in freezing conditions, and stabilize spheroplasts and protoplasts from osmotic shock [45].
As discussed earlier, experimental evidence suggested that LJH19 is involved in the catecholate type siderophore production. The genomic insights further strengthened these ndings by predicting the genes involved in the biosynthesis of enterobactin and petrobactin (Table 3). These results indicate that LJH19 has the potential to produce a wide variety of siderophores. Most of the enzymes involved in enterobactin biosynthesis were predicted except the genes involved in the conversion of 2,3-Dihydro-2,3dihydroxybenzoate to enterobactin marked as a red arrow in Fig. 4 (Supplementary Table S4). LJH19 genome also predicted the genes encoding the transporters required for the import and export of synthesized enterobactin. In respect to petrobactin's biosynthesis, spermidine molecules are used for synthesis using citrate backbone [46]. Further, genes encoding the transporters required to import and export both synthesized enterobactin and petrobactin and transporters for hydroxamate type siderophores were predicted in the genome (Supplementary Table S2, S4). In addition to auxin and siderophore, the LJH19 genome also predicted few genes encoding phosphatases, inositol-phosphatases, and gluconate permease ( LJH19 strain has also been noted to carry genes involved in nitrate/nitrite transport pathways, including the genes associated with denitri cation and nitrate reduction like nitrite reductase and nitrate reductase (Fig 4, Table 3, Supplementary Table S4). Nitrite reductase encoded by the NirD gene converts nitrite to ammonium and further converted to glutamate by glutamate synthetase for amino acid metabolism (Fig.  4). Thus, LJH19 may deliver plants with available nitrogen sources via enzymatic conversion.
The cold-tolerant LJH19 has shown potential PGP properties physiologically, and genomic evidence has supported the function. Similar genomic insights for saline tolerant strain G. halophytocola KLBMP 5180 has also been reported to carry the genes related to PGP, such as siderophores and spermidine biosynthesis [26]. Like LJH19, KLBMP 5180 also harbored genes such as agmatinase, spermidine synthase, siderophore ABC transport system ATP-binding protein, siderophore ABC transporter substratebinding protein (Supplementary Table S3). Similarly, G. halophytocola DR408 genome also carried PGP genes involved in siderophore production and phosphate solubilization [39] (Supplementary Table S3).
Although few reports of genomic evidence of PGP potential of Glutamicibacter species are available in the literature, the closest related genus Arthrobacter has multiple reports on genetic evidence of PGP traits [15,29,47,48]. Among the Arthrobacter species, A. agilis L77 [15] and A. alpinus R3.8 [29] possessing PGP traits such as phosphate solubilization, IAA, and ammonia production are also reported for cold adaptation.

Genomic insights into secondary metabolic gene cluster of LJH19
Phylum actinobacteria are very well known for their ability to produce a variety of secondary metabolites [49]. Secondary metabolites gene clusters search using antiSMASH v5.0 resulted in the identi cation of three biosynthetic gene clusters, namely type III polyketide synthase (PKS), terpene, and siderophore (Fig.  5). Type III PKS are involved in the synthesis of numerous metabolites and have a variety of biological and physiological roles, such as antimicrobials and defense systems in bacteria [50]. Such a gene cluster that has a probable biological function in the production of antimicrobial metabolites goes in favor of LJH19 as a PGP bacterium for being a biological control agent against phytopathogens [51]. The presence of a carotenoid gene cluster supports the indicative yellow color of the LJH19 colonies. Besides pigmentation, carotenoid's major function in bacteria is to protect the cell from UV radiations, oxidative damage and modify membrane uidity [52]. Siderophore production is another attribute that has several ecological applications in plant growth promotion and acts in plant defense against various pathogens [53]. Prediction of the siderophore gene cluster in the genome of LJH19 endorses the experimental evidence of catecholate type siderophore production by LJH19. It supports the presence of several siderophores associated genes in the genome of LJH19.

Conclusion
Night-soil compost is a rich nutrient source and, when supplemented to the soil, increases its fertility. In this study, G. arilaitensis LJH19 isolated from NSC demonstrated the ability to hydrolyze complex polysaccharides richly found in night-soil and agricultural residues like starch, cellulose, and xylan. The bacterium survived extreme cold conditions and exhibited several PGP traits such as auxin production, siderophore production, and phosphate solubilization at low ambient temperature. The strain displayed its capabilities as a safe bacterium by demonstrating negative hemolysis and bio lm formation.
Genomic search reinforced the bacterium's safety with the absence of any virulence and antibiotic resistance genes. A comprehensive genomic analysis predicted and excavated key genes related to cold adaptation, polysaccharide metabolism, and plant growth promotion. These results indicated that G. arilaitensis LJH19 may serve as a safe bioinoculant and may contribute to e cient psychrotrophic bacterial formulations for improved night-soil degradation and enrichment of the soil with PGP attributes.
To the best of our knowledge, the current study is the pioneering scienti c intervention addressing NSC's issue in the high Himalaya.

Materials And Method
Sampling source, strain isolation, and hydrolytic potential NSC samples were collected from the collection chamber of the night-soil composting toilet, locally termed as "Ghop" of Jundah village (32.64°N 76.84°E) of Lahaul valley (Fig. 1C). The samples were collected from the core of the compost pile in sterile plastic bottles using stainless steel spatula in triplicates and stored in icebox containing ice packs. The samples were then immediately transported to the laboratory and processed. The temperature was noted at the time of sampling itself by inserting the handheld digital thermometer (MEXTECH, India) into the compost pile's core. Air-dried solid sample was mixed with Milli-Q water at a ratio of 1:10 vortexed and kept overnight to check pH and electrical conductivity (EC) using digital pH and EC meter (Eutech, India). The samples were dried at room temperature, nely grounded, and sieved to analyze total available nitrogen, phosphorus, and potassium.
All the chemical analysis was performed as per the standard methods for testing compost materials [54].
The bacterial strain LJH19 was isolated from NSC samples while screening for potential psychrotrophic hydrolytic bacteria. The isolation was carried out using serial dilution and spread plate methods on nutrient agar (NA) medium (HiMedia) at 10°C. The bacterial isolation was performed in Class II, Type A2 Biological Safety Cabinet (Thermo Scienti c, US). The optimum growth conditions of the LJH19 strain were determined by incubating the culture at various temperatures (4-50°C), NaCl concentration (1-10%), and pH (2 to 10) range. The production of hydrolytic enzymes by the LJH19 strain was initially screened using a plate assay method. An exponentially grown culture of LJH19 were spot inoculated on Carboxymethylcellulose (CMC) agar [55], Starch agar (Hi-Media), Xylan agar [56], and Tributyrin agar (Hi-Media) plates and incubated at 10°C for 48 hrs. The clear halo zones around the colony indicated positive results. The enzymatic index (EI=Diameter of the halo of hydrolysis/Diameter of the colony) was calculated as described previously by Vermelho [57]. For quantitative estimation of polysaccharide degrading enzymes viz. cellulase, xylanase, and amylase, the microplate-based 3, 5-dinitrosalicylic acid colorimetry method was followed using 1% (w/v) carboxymethylcellulose (CMC), 1% birchwood xylan (HiMedia) and 1% soluble starch (HiMedia) as the substrate [58].
Haemolysin and protease assay, bio lm formation and antibiotic susceptibility pro le For assessment of pathogenic potential, we assayed LJH19 for protease and hemolysin activity using a plate assay method [34]. The strains Staphylococcus aureus subsp. aureus (MTCC 96), Bacillus subtilis (MTCC121), Escherichia coli (MTCC 43), Micrococcus luteus (MTCC 2470) were used as a positive control for hemolytic activity. Hemolytic activity was interpreted according to Buxton [59].
Bio lm formation was evaluated according to Basson and Igbinosa [34,60] with slight modi cations. The adhered cells were stained with 200 μL of 0.5% crystal violet for 10 min. The optical density (OD) readings from respective wells were obtained at 595 nm. The cutoff OD (ODc) for the test was set using the formula (Mean OD of negative control + 3x Standard deviation), and results were interpreted as previously described by Basson et al. [60].

Plant growth-promoting (PGP) attributes
Indole acetic acid (IAA) production by LJH19 was studied according to Goswami et al. [62] by supplementing the nutrient broth (100 ml) with L-Trytophan (200 μg ml−1). The IAA test medium inoculated with strain LJH19 was incubated at 10˚C, and the colorimetric assay for detection of IAA was performed at room temperature. Siderophore production and phosphate solubilization by LJH19 has initially screened on Chromeazurol S (CAS) agar [63] and Pikovskya's agar (HI Media) at 10°C.
Siderophore producing index (SI) and phosphate solubilization index (PSI) was calculated by dividing zone size by colony diameter.
Quantitative estimation of siderophore was done using CAS-shuttle assay [62] by growing LJH19 in ironfree CAS-broth (pH 6.8) at 10°C at 150 rpm. Further, to determine the chemical nature of siderophore, we examined the absorption maxima ( max) of cell-free supernatant in UV-3092 UV/Visible spectrophotometer. Ammonia production was quanti ed spectrophotometrically [64]. LJH19 was grown in peptone water at 10°C for 10 days at 150 rpm. Inorganic phosphate solubilization was estimated by the vanado-molybdate method [65] using NBRIP broth containing 0.5% tricalcium phosphate (TCP).
Seed germination activity of strain LJH19 was carried out using pea seeds (Pisum sativum var. Arkel). The bacterium was grown in nutrient broth for 48 h at 10˚C and centrifuged at 10000 rpm to obtain a cell pellet. The pellet was then resuspended in sterile distilled water. Pea seeds were surface sterilized using 5% sodium hypochlorite for 10 mins, followed by several washes with sterilized distilled water. The surface-sterilized seeds were treated with bacterial suspension for 10 mins and allowed to dry under aseptic conditions. Five seeds per pot were then sown in moist sterile vermiculite. The seeds treated with sterile distilled water with no bacterial inoculant served as the control. The pots were then incubated in a controlled growth chamber with three replicates under mixed incandescent and uorescent illumination of 550 µmol photon/m2/s with a 16/8-h light/dark cycle at 25 ± 2˚C and 40% to 50% relative humidity for 7 days. The number of bacterial cells per seed was approx. 10 8 CFU determined using serial dilution method [66]. The germination index was then calculated using the equation as described by Mondal et al. [67].
Strain identi cation, genome sequencing, annotation, phylotaxono-genomics, and gene content analysis The genomic DNA was extracted using the conventional CTAB method [68], and for identi cation, partial 16S rRNA gene sequencing was performed using 27F and 1492R primers as described previously [69]. Plasmid DNA isolation was performed using PureLink® Quick Plasmid Miniprep Kit (Invitrogen, US).
To provide a genetic basis to the experimental evidence, we performed whole-genome sequencing using PacBio RS-II (Paci c Biosciences, US) as previously [37,70] [76]. Additional annotation and the manual review was performed using prokka v1.14.6 [77] and JGI Prokaryotic Automatic Annotation Pipeline [78]. Data mining across the genome of LJH19 was carried out to identify potential genes for endorsement of its potential safe psychrotrophic bio-inoculant candidate as described earlier [32,36]. The orthoANI v1.2 [79] was performed to infer the taxonomic relatedness of the strain LJH19. ANI value matrix obtained was used for generating heatmap using the webserver of Morpheus (https://software.broadinstitute.org/morpheus). Further, 10 strains forming a clade were considered for pan-genome analysis with a 95% cutoff using Roary v3.6.0 [80]. The unique gene present in the strain LJH19 was fetched and annotated with eggNOG mapper v1 [81] (http://eggnogdb.embl.de/app/home#/app/home). Chromosomal maps [82]for comparison of the closely related strains and visualization of the unique genomic region across the strain LJH19 to the type strain RE117 and JB182 are marked in the gure (Fig. 3).
To identify the biosynthetic gene clusters (BGCs) in the genome of LJH19, we used a web-based server of antiSMASH v5.0 (https://antismash.secondarymetabolites.org/#!/start) [83], and a cluster image of the identi ed biosynthetic gene was prepared with EASY g v2.2.2 [84]. To assess the presence of the antibiotic gene cluster across the strains, a web-based server of Resistance gene identi er (RGI) v5.     Schematic representation of the predicted genes encoding catabolic activities, transport and plant growth promotion in G. arilaitensis LJH19. The selected key genes involved in the pathway indicated by blue