Exploring the diversity and genomics of cultivable Bacillus-related endophytic bacteria from the medicinal plant Galium aparine L

Abstract

Introduction: Endophytes are crucial partners that contribute to the plants' health and overall wellbeing. Apart from the elucidation of the relationship between plants and their microbiota, the metabolic potential of endophytes is also of a special interest. Therefore, it is crucial to isolate and taxonomically identify endophytes, as well as to investigate their genomic potential to determine their significance in plant health and potential as bioactive metabolite producers for industrial application.

Methods: In this study, we isolated ten endophytic bacterial strains from different tissues of medicinal plant Galium aparine L. and performed de novo assembly of their genomes using short and long reads. Comparative genomic analysis was conducted to assess the accurate taxonomic identification of the strains. The investigation also focused on the presence of mobile genetic elements and their significance concerning endophytic lifestyles. We performed functional annotation of coding sequences, particularly targeted genes that encode carbohydrate enzymes and secondary metabolites within gene clusters.

Results: Through sequencing using two complementary methods, we obtained 10 bacterial genomes, ranging in size, coding density and number of mobile genetic elements. Our findings provide a first insight into the cultivable bacterial community of the medicinal plant Galium aparine L., their genome biology, and potential for producing valuable bioactive metabolites. Obtained whole genome sequences allowed for complete phylogenetic analysis, which revealed crucial insights into the taxonomic status of bacteria and resulted in the discovery of two putatively novel bacterial species from the Bacillus and Priestia genera, suggesting that plants are hiding a reservoir of novel species with potentially useful properties and unknown mechanisms related to their relationship with plant host.

Keywords: Galium aparine L.; bacterial endophytes; genome mining; mobile genetic elements; phylogenetics; plant microbiome; secondary metabolites.

FIGURE 1

Phylogenetic tree of Galium aparine L’s endophytes and their closest type-strain matches based on whole genome alignments. Single nucleotide polymorphisms (SNPs) and indels within the multiple sequence alignments were constructed by the reference sequence Alignment based Phylogeny builder (REALPHY) and extracted for subsequent phylogeny reconstruction using MEGA v11.0.9 by the Neighbor-joining method, with a bootstrap of 1,000 replications (Bertels et al., 2014; Tamura et al., 2021). Bootstrap confidence levels are indicated at the internodes.

FIGURE 2

Average Nucleotide Identity (ANI) and digital DNA:DNA hybridization (dDDH) values between isolated bacterial endophytes’ genomes. ANI (%; ANIm results) values are indicated in the upper triangle and dDDH (%; formula d4 results) in the lower triangle.

FIGURE 3

Presence of genes associated with plant growth promotion according to SEED functions from The Rapid Annotation using Subsystem Technology (RAST).

FIGURE 4

Comparison of the distribution of carbohydrate-active enzyme (CAZyme) classes identified in Galium aparine L.’s bacterial endophytes. AA, auxiliary activity; CBM, carbohydrate-binding module; CE, carbohydrate esterase; GH, glycoside hydrolase; GT, glycosyltransferase; PL, polysaccharide lyase.

FIGURE 5

Genes associated with polysaccharide degradation.

FIGURE 6

Comparison of the distribution of biosynthetic gene clusters (BGCs) types. NRPS, non-ribosomal peptide synthetase; PKS, polyketide synthase; RiPP, ribosomally synthesized and post-translationally modified peptides.