Isoptericola sp. AK164 is Gram-positive, cocci-shaped, non-motile with 300–400 nm diameter, with characteristic circular and bright yellow colonies observed when grown on Zobell marine Agar at 30°C (Fig. 1C). AK164 tolerates heat and salinity stress and grows in temperatures up to 45°C and in/on up to 5 M NaCl-supplemented LB, which displays salt and high-temperature tolerance (Supplement Table S1). The ability of AK164 to tolerate salt stress contributes to the survival of AK164 in the coastal intertidal zones of mangroves (A. marina) and marine environments. The qualitative evaluation of PGP traits showed that AK164 can produce siderophores and IAA but is incapable of solubilizing phosphate (Supplement Table S1), giving AK164 a potential application as PGPR. In planta, AK164 caused enhancement of A. thaliana growth under both normal (½ MS and salt stress conditions of 100mM NaCl; beneficial increase > 110% compared with the non-colonized control plants). After 12 days of growth, A. thaliana seedlings treated with AK164 showed bigger shoot and root systems (Fig. 1A) and an increment of 50% in fresh weight (Fig. 1B).
Sequencing of the AK164 strain using PacBio technology resulted in a total of 14,559 reads with a mean read length of 73,030 bp and estimated genome coverage of 297X. The assembled genome consists of one contig of 35,767,03bp with a GC content of 73%. In total, 3211 coding genes have been annotated in the AK164 genome, covering 91.44% of the genome. RNA-noncoding genes, including 25 tRNAs and 9 rRNAs were predicted in the genome (Fig. 2, Table 1). Special emphasis was made on capturing the repeat regions in the genome as they play important roles in genome evolution and adaptation to environments. We identified mainly two kind of repeats in the genome, Tandem (22) and Interspersed (1094) (Fig. 2, Table 1).
Table 1
Summary of Isoptericola sp. AK164 genome features
Feature
|
Chromosome
|
Genome Size (bp)
|
3,576,703
|
GC content (%)
|
73.54
|
Gene Number
|
3211
|
% of Genome (Genes)
|
91.44
|
CDS
|
3211
|
16S-23S-5S operons
|
3-3-3
|
tRNAs
|
49
|
long terminal repeat LTR
|
13
|
DNA
|
5
|
Tandem repeats TRs
|
566
|
Minisatellite DNA
|
521
|
Microsatellite DNA
|
7
|
To determine the accurate taxonomic position of AK164, 16S and whole-genome-based taxonomic analysis was undertaken with the Type Strain Genome Server (TYGS) platform [24] as shown in Fig. 3A,B. The TYGS results show that AK164 is most closely related to Isoptericola sediminis JC619 with dDDH (d0) of 61.8% corresponding to high average branch support for the generated tree and high phylogenetic accuracy. However, to check the reliability of evolutionary distance assessment between bacterial species based on digital whole genome comparison, average nucleotide identity (ANI) was also measured. Complete genomes of Isopetricola species closely related to AK164 were retrieved from the NCBI GenBank database and subjected to Orthologous Average Nucleotide Identity calculation. As shown (Supplement Table S3 A, B) the highest OrthoANI value of 86.6% was obtained with Isoptericola sediminis JC619. However, the ANIo and dDDH values do not fit the species cut-off of 95% and 70%, respectively. Based on these results, AK164 may present a new species.
Function analysis on AK164 was conducted using different platforms (see Materials & Methods); by using BlastKOALA, we identified 1702 genes (53%) with assigned functions (Supplement Figure S1), while using KEGG gave 3042 genes (95%), COG 2570 genes (80%), GO 2290 genes (71%) (Supplement Figure S2, S3) and Pfam 2290 genes (71%). The AK164 chromosome contained nine 16S-23S-5S rRNA operons and 49 tRNAs (Supplement Table S5). Genome mining revealed the presence of genes involved in environmental stress tolerance and responses, e.g., trehalose, proline, choline, and betaine (glycine-betaine), important osmoprotectants produced under saline stress. Complete glutamate and proline biosynthesis pathways were identified and might be responsible for conferring salt tolerance. The genome of AK164 has a wide variety of enzymes and regulators that help bacteria to cope with oxidative stress, including superoxide dismutase (sod, AK164_GM001878; AK164_GM003108), catalase (katE, AK164_GM001364), peroxidase (AK164_GM002005) and two osmotically inducible proteins (osmC, AK164_GM001522, AK164_GM002579). These genes could explain the survival phenotypes of AK164 under high salt, osmotic, and heat stress. A complete pathway for the degradation of plant cell walls and their constituents, e.g., xylan and cellulose degradation, was also found and could contribute to bacterial internalization to plant roots (endophytes) (Supplement Table S2). Interestingly, all the genes required for flagella assembly were absent, confirming the non-motile nature of AK164.
The genome of AK164 revealed several genes involved in PGPR activity, e.g., AK164_GM000253 encodes for 1-aminocyclopropane-1-carboxylate (ACC) deaminase. ACC deaminase is involved in the metabolism of the immediate precursor of ethylene in ethylene biosynthesis and is one of the well-known PGPB traits (Shen et al. 2013). Several siderophores were identified in AK164, e.g., Enterochelin. Bacteria produce siderophores to scavenge iron from the extracellular space and use specific transporters to recover the siderophore–iron complex, ensuring their iron supply. AK164 has a large inventory of genes (122 genes) encoding ABC transporters, including mineral inorganic and metal ion transporters, cobalamin/Fe3+-siderophore transport, Fe3+-hydroxamate, Fe3+-siderophore, Mn2+/Zn2+ transport, nitrate/sulfonate/bicarbonate transport, phosphate transport and Oligosaccharide and amino acid transport, e.g., cellulase, chitobiose, glucose (Supplement Table S2). Many ABC transporters could be linked to the capacity of AK164 to survive in different ecological niches (Andres-Barrao et al. 2021). Along with the uptake and exchange of nutrients, bacteria require various protein secretion systems for growth and interaction with plants. AK164 harbors a general secretion (Sec) and a twin-arginine translation (Tat) secretion pathway along with several genes encoding for a type 2-secretion system (T2SS). Those pathways are the most commonly used secretion systems to transport proteins across the plasma membrane (Natale et al. 2008).
AntiSMASH analysis revealed the presence of six clusters for secondary metabolite biosynthesis, two of which were identified as terpene with high similarity to carotenoid biosynthetic gene cluster BGC0000644 from Dietzia sp. CQ4 and BGC0000636 from Brevibacterium linens. NRPS-independent-siderophore with high similarity with FW0622 BGC0002690 biosynthetic gene cluster from Verrucosispora sp. FIM060022 (Zhao et al. 2020). And Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) with a 70% similarity of BGC0000853: ectoine biosynthetic gene cluster from Streptomyces sp. The genes encoding for ectoine biosynthesis AK164_GM002457_ectA, AK164_GM002457_ectB, AK164_GM002457_ectC and its conversion to hydroxyectoine (AK164_GM001449_ ectD) were identified in AK164, and their presence could contribute to the osmotic and salt stress tolerance of AK164. Ectoine is an osmoprotectant agent found in several microorganisms and has a wide practical application in industries, including for skin protection and a potential medicine (Hermann et al. 2020). In addition, cluster of Lasso peptide were found, Lasso peptide is a novel class of bacteria-derived ribosomal assembled and post-translationally modified peptides; they are found throughout the bacterial domain and exhibit a versatile array of biological functions (Hegemann et al. 2015).