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genera were obtained based on their hydrolytic activities in different substrates and plant growth-promoting traits (unpublished data).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 efficient 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 efficient 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 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 specific activity of 186.76 ± 19.28 U/mg (Supplementary Fig. S2) using corn starch as substrate, cellulase enzyme with a specific activity of 21.85 ± 0.7 U/mg (Supplementary Fig. S2) using CMC as a substrate and xylanase enzyme with a specific 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 efficient 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 reclassification 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 efficient 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.
Table 1
Physiological characterization, hydrolytic, plant growth promoting, and pathogenic attributes of G. arilaitensis LJH19
Characteristic
|
G. arilaitensis LJH19
|
Source
|
Night-soil compost
|
Growth condition a
|
|
Temperature range
|
4-37°C (10 °C)
|
pH range
|
7-11 (7)
|
NaCl range
|
1- 9% (1%)
|
Hydrolysis on agar plates b
|
|
Corn starch
|
+ ve (15)
|
Carboxymethylcellulose (CMC)
|
+ ve (5.66)
|
Birchwood xylan
|
+ ve (2.8)
|
Tributyrin
|
+ ve (1.8)
|
Enzyme assays
|
|
Amylase
|
186.76 ± 19.28 U/mg
|
Cellulase
|
21.85 ± 0.7 U/mg
|
Xylanase
|
11.31± 0.51 U/mg
|
PGP trait
|
|
IAA production
|
166.11 ± 5.7 µg/ml
|
Siderophore productionc
|
85.72 ± 1.06 percent siderophore unit (1.5)
|
Phosphate solubilisationd
|
44.76 ± 1.5 µg/ml (2.3)
|
Ammonia production
|
0.20 ± 0.01 µmoles/ml
|
Germination Index (GI)
|
116.348 ± 38.02 %
|
Pathogenic potential
|
|
Haemolysis on blood agar
|
- ve
|
Protease production
|
0.17 ± 0.002 U/mg
|
Biofilm production
|
- ve at 37 °C, weak producer at 15 °C
|
Antibiotic susceptibility test
|
AZM-, AMP-, CIP-, CHL-, E-, G-, K-, P-, RIF-, S-, TE-, VA-
|
Values in parentheses indicate aOptimum growth condition, benzymatic index at 10 °C , c Siderophore producing index; d phosphate solubilization index; +: Resistant; - : Sensitive; AZM: 15 mcg, Azithromycin; AMP: 10 mcg, Ampicillin; CIP: 5 mcg, Ciprofloxacin; CHL: 30 mcg, Chloramphenicol; E: 15 mcg Erythromycin; G:10 mcg, Gentamycin; K: 30 mcg, Kanamycin; P: 10 Units, Penicillin-G; RIF: 5 mcg, Rifampicin; S: 10 mcg, Streptomycin; TE: 30 mcg, Tetracycline; VA: 30 mcg, Vancomycin
|
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 efficiency 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 fixed 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 benefit 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 significantly 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 influence. The LJH19 strain capabilities to produce auxin and siderophore may have positively affected pea seeds' seed germination. In agreement with our findings, other Glutamicibacter strains have also shown PGP traits where a strain G. halophytocola KLMP 1580 have significantly 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 efficient 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 biofilm 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, biofilm 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 insignificant 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 efficient hydrolytic activity at varying temperatures (best activity at 10 °C), non-pathogenicity, and PGP potential, strain LJH 19 qualifies 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-specific 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 (Supplementary Fig. S7)
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 Re117T 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 specific to the strain LJH19, Re117, and BJ182 was 1740. A total of 217 genes were specific 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-specific gene from LJH19 classified by eggNOG falls in several COG categories (Fig. 3B). A list of the unique gene, its function, and COG classification 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 confirm this susceptibility profile, 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 confirm these results, the LJH19 genome was assessed for its pathogenic potential by PathogenFinder [35]. This web-based tool identifies the genome and provides a probability measure for the test strain to be pathogenic for humans. The predicted results identified LJH19 as a non-human pathogen with an average probability of 0.228 (Supplementary output file S1). None of the putative virulence or pathogenic genes were identified 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 identified 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-disulfide 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.
Table 2
Genes encoding known cold & stress response and DNA repair proteins as predicted in the genome of G. arilaitensis LJH19
Category and GenBank ID
|
Description
|
Category and GenBank ID
|
Description
|
Cold active chaperones
|
|
Osmotic Stress/ Oxidative stress
|
|
TFH54768, TFH56297, TFH57075
|
Cold-shock protein
|
TFH56153
|
Glycine betaine ABC transporter substrate-binding protein
|
TFH56633
|
Co-chaperone GroES
|
TFH56253
|
Sarcosine oxidase subunit beta family protein
|
TFH56634, TFH54762
|
Chaperonin GroEL
|
TFH56254
|
Sarcosine oxidase subunit delta family protein
|
TFH57159
|
Molecular chaperone DnaJ
|
TFH56255
|
Sarcosine oxidase subunit alpha family protein
|
TFH54768, TFH56297, TFH57075
|
Cold-shock protein
|
TFH56256
|
Sarcosine oxidase subunit gamma family protein
|
TFH56633
|
Co-chaperone GroES
|
TFH56949
|
Superoxide dismutase
|
TFH56416
|
Molecular chaperone DnaK
|
TFH57402, TFH54880
|
Catalase
|
TFH56424
|
ATP-dependent chaperone ClpB
|
TFH56696, TFH56988, TFH56162
|
OsmC family peroxiredoxin
|
TFH57532
|
Heat shock protein HslJ / META domain-containing protein
|
TFH55537
|
Organic hydroperoxide resistance protein
|
TFH56416
|
Molecular chaperone DnaK
|
TFH55668, TFH56321, TFH56523, TFH54754, TFH55543
|
Thioredoxin
|
TFH56424
|
ATP-dependent chaperone ClpB
|
TFH55669
|
Thioredoxin-disulfide reductase
|
Carbon storage/starvation
|
Carbon storage/starvation
|
TFH55955, TFH57498
|
Thioredoxin-dependent thiol peroxidase
|
TFH54400
|
Carbon starvation protein A
|
TFH56887
|
Thioredoxin family protein
|
TFH56944, TFH57627, TFH57801
|
1-acyl-sn-glycerol-3-phosphate acyltransferase
|
TFH54908
|
Thioredoxin domain-containing protein
|
Membrane/cell wall alteration
|
|
TFH56279
|
Sodium/proline symporter PutP
|
TFH55154
|
3-oxoacyl-ACP synthase III
|
TFH54956
|
Na+/H+ antiporter NhaA
|
TFH57491
|
Phytoene desaturase
|
TFH54753
|
Trehalose-6-phosphate synthase
|
TFH57492
|
Phytoene/squalene synthase
|
TFH54752
|
Trehalose-phosphatase
|
General Stress response
|
|
DNA repair
|
|
TFH55892, TFH56176, TFH56200, TFH56262, TFH57611, TFH55142, TFH54651, TFH55629
|
Universal stress protein
|
TFH54856, TFH54452
|
Recombinase family protein
|
TFH57700, TFH57339
|
GlsB/YeaQ/YmgE family stress response membrane protein
|
TFH55191
|
Recombinase RecA
|
TFH54980, TFH56483
|
Serine/threonine protein kinase
|
TFH57358
|
Tyrosine recombinase XerC
|
TFH57322
|
Peroxide stress protein YaaA
|
TFH57661
|
Site-specific tyrosine recombinase XerD
|
TFH54953
|
50S ribosomal protein L25
|
TFH57734
|
Recombinase
|
TFH57081
|
SOS response-associated peptidase
|
TFH54405
|
ATP-dependent DNA helicase RecQ
|
Toxin/Antitoxin modules
|
|
TFH55567
|
DNA repair protein RadA
|
TFH54837
|
Type II toxin-antitoxin system prevent-host-death family antitoxin
|
TFH57167
|
DNA repair protein RecO
|
TFH54532
|
Type II toxin-antitoxin system VapB family antitoxin
|
TFH55568
|
DNA integrity scanning protein DisA
|
TFH54610
|
Type II toxin-antitoxin system HipA family toxin
|
TFH57664
|
DNA repair protein RecN
|
TFH56658
|
Type II toxin-antitoxin system Phd/YefM family antitoxin
|
TFH57433
|
ATP-dependent DNA helicase RecG
|
TFH57154
|
Type II toxin-antitoxin system PemK/MazF family toxin
|
TFH56747
|
ATP-dependent DNA helicase UvrD2
|
TFH57340
|
Toxin component of a toxin/antitoxin system
|
TFH57103
|
Holliday junction branch migration protein RuvA
|
TFH55640
|
Serine/threonine-protein kinase
|
TFH57104
|
Holliday junction branch migration DNA helicase RuvB
|
|
|
TFH57121
|
Holliday junction resolvase RuvX
|
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, 38–40]. 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-specific 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 finding in LJH19, the genome of G. arilaitensis Re117T 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 findings 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 specific 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 specific 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).
Table 3
Genes encoding proteins involved in catabolic activity, plant growth promoting activity, transport and cold adaptation predicted in the genome of G. arilaitensis LJH19
Category and GenBank ID
|
Description
|
Category and GenBank ID
|
Description
|
Catabolic activity
|
Plant Growth Promoting activity
|
TFH56992, TFH56993
|
ATP-dependent Clp protease proteolytic subunit
|
TFH55608, TFH57060
|
amidase
|
TFH56994
|
ATP-dependent Clp protease ATP-binding subunit ClpX
|
TFH56909
|
Anthranilate synthase component I
|
TFH54429
|
Putative esterase
|
TFH56082
|
Nitrite reductase [NAD(P)H]
|
TFH56413
|
pullulanase-type alpha-1,6-glucosidase
|
TFH56083
|
nitrite reductase (NAD(P)H) small subunit
|
TFH57366
|
trypsin-like serine protease
|
TFH56086
|
nitrite reductase
|
TFH56438
|
MarP family serine protease
|
TFH57000
|
nitrite/sulfite reductase
|
TFH57809
|
Alpha-amylase
|
TFH55619
|
nitrate reductase
|
TFH54465
|
alpha/beta fold hydrolase
|
TFH56511
|
Isochorismate synthase
|
TFH54414
|
Beta-glucosidase
|
TFH57125
|
chorismate synthase
|
TFH54614
|
Xylose isomerase
|
TFH57182
|
Anthranilate phosphoribosyltransferase
|
Transporters
|
|
TFH57559, TFH55965
|
Isochorismatase family protein YecD
|
TFH56366
|
spermidine/putrescine ABC transporter substrate-binding protein
|
TFH55761
|
Acetylornithine aminotransferase
|
TFH54902, TFH55612, TFH56015, TFH56106, TFH56479, TFH56632, TFH56864, TFH57051, TFH57718
|
amino acid permease
|
TFH54544
|
alkaline phosphatase
|
TFH56389
|
amino acid ABC transporter ATP-binding protein
|
TFH54792
|
Inositol-1-monophosphatase
|
TFH55562
|
phosphate ABC transporter ATP-binding protein
|
TFH56913
|
tryptophan synthase subunit beta
|
TFH55563
|
phosphate ABC transporter permease PstA
|
TFH56914
|
tryptophan synthase subunit alpha
|
TFH55564
|
phosphate ABC transporter permease subunit PstC
|
TFH56912
|
indole-3-glycerol phosphate synthase TrpC
|
TFH55565, TFH57701
|
phosphate ABC transporter substrate-binding protein PstS
|
TFH55232
|
Ornithine carbamoyltransferase
|
TFH55134
|
phosphate/phosphite/phosphonate ABC transporter substrate-binding protein
|
TFH56276
|
ornithine decarboxylase
|
TFH55132
|
phosphonate ABC transporter, permease protein PhnE
|
TFH55236
|
Argininosuccinate lyase
|
TFH55133
|
phosphonate ABC transporter ATP-binding protein
|
TFH55205
|
phosphoribosylanthranilate isomerase PriA
|
TFH56773
|
peptide ABC transporter substrate-binding protein
|
TFH56212
|
Formimidoylglutamase* (Arginase)
|
TFH54587
|
aliphatic sulfonate ABC transporter substrate-binding protein
|
TFH54789
|
agmatinase
|
TFH54588
|
ABC transporter permease
|
TFH55596
|
inorganic diphosphatase
|
TFH55268
|
short-chain fatty acid transporter
|
TFH56916
|
glutamate synthase subunit beta
|
TFH56023
|
D-serine/D-alanine/glycine transporter
|
TFH57767
|
glutamate synthase large subunit
|
TFH55620, TFH56087
|
MFS transporter (nitrate)
|
TFH56292
|
FMN-binding glutamate synthase family protein
|
TFH56177
|
gluconate permease
|
TFH56820
|
Glutamine synthetase
|
TFH55044
|
iron-enterobactin ABC transporter permease
|
|
|
TFH55045
|
Fe(3+)-siderophore ABC transporter permease
|
|
|
TFH54561, TFH54802
|
siderophore-interacting protein
|
|
|
TFH54562
|
Fe2+-enterobactin ABC transporter substrate-binding protein
|
|
|
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-1,6-glucosidase, alpha-amylase) attack the polysaccharides producing smaller oligosaccharides and monomeric sugars, and finally, 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 L-tryptophan, 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 identified 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 findings 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-2,3-dihydroxybenzoate 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 (Table 2, Fig 4 and Supplementary Table S4) involved in phosphate metabolism. LJH19 strain has also been noted to carry genes involved in nitrate/nitrite transport pathways, including the genes associated with denitrification 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 substrate-binding 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 identification 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 fluidity [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.