Genomic Insights of Wheat Root-Associated Lysinibacillus fusiformis Reveal Its Related Functional Traits for Bioremediation of Soil Contaminated with Petroleum Products

Abstract

The negative ecological impact of industrialization, which involves the use of petroleum products and dyes in the environment, has prompted research into effective, sustainable, and economically beneficial green technologies. For green remediation primarily based on active microbial metabolites, these microbes are typically from relevant sources. Active microbial metabolite production and genetic systems involved in xenobiotic degradation provide these microbes with the advantage of survival and proliferation in polluted ecological niches. In this study, we evaluated the ability of wheat root-associated L. fusiformis MGMM7 to degrade xenobiotic contaminants such as crude oil, phenol, and azo dyes. We sequenced the whole genome of MGMM7 and provided insights into the genomic structure of related strains isolated from contaminated sources. The results revealed that influenced by its isolation source, L. fusiformis MGMM7 demonstrated remediation and plant growth-promoting abilities in soil polluted with crude oil. Lysinibacillus fusiformis MGMM7 degraded up to 44.55 ± 5.47% crude oil and reduced its toxicity in contaminated soil experiments with garden cress (Lepidium sativum L.). Additionally, L. fusiformis MGMM7 demonstrated a significant ability to degrade Congo Red azo dye (200 mg/L), reducing its concentration by over 60% under both static and shaking cultivation conditions. However, the highest degradation efficiency was observed under shaking conditions. Genomic comparison among L. fusiformis strains revealed almost identical genomic profiles associated with xenobiotic assimilation. Genomic relatedness using Average Nucleotide Identity (ANI) and digital DNA-DNA hybridization (DDH) revealed that MGMM7 is distantly related to TZA38, Cu-15, and HJ.T1. Furthermore, subsystem distribution and pangenome analysis emphasized the distinctive features of MGMM7, including functional genes in its chromosome and plasmid, as well as the presence of unique genes involved in PAH assimilation, such as phnC/T/E, which is involved in phosphonate biodegradation, and nemA, which is involved in benzoate degradation and reductive degradation of N-ethylmaleimide. These findings highlight the potential properties of petroleum-degrading microorganisms isolated from non-contaminated rhizospheres and offer genomic insights into their functional diversity for xenobiotic remediation.

Keywords: azo dye; crude oil; pangenome; phenol; polycyclic aromatic hydrocarbons; xenobiotic assimilation.

Figure 1

Biosurfactant production by L. fusiformis MGMM7. Inoculation of 50 µL of distilled water (A), free-cell supernatant of MGMM7 (B), and 0.1% SDS solution (C) in the surface petroleum oil into Petri dishes amended with distilled water.

Figure 2

Qualitative analysis of crude oil degradation by L. fusiformis MGMM7. Control: MS medium amended with 1% crude petroleum (A); MS medium amended with 1% crude inoculated with L. fusiformis MGMM7 (B) after 21 days of incubation at 30 ± 1 °C.

Figure 3

Remediation of oil-contaminated soil by L. fusiformis MGMM7 in pot trial experiment. Plant growth ability under different soil conditions. Garden cress (Lepidium sativum L.) plants were grown in a climate chamber under controlled conditions: temperature, 28 °C; light intensity, 100%; and humidity, 70% for up to 21 days. The results are expressed as mean values ± standard deviation (SD). One-way ANOVA and Dunnett’s tests were used to estimate the significance levels at p < 0.05. Statistical difference among groups is labeled with different letters.

Figure 4

Growth and degradation ability of L. fusiformis MGMM7 in basal medium amended with or without phenol (2 mg/mL) at 30 ± 1 °C for 50 h (A) and UV-vis absorption spectra after 50 h of the incubation period (B).

Figure 5

Biotransformation of CR azo dye by L. fusiformis MGMM7 at 30 ± 1 °C for 24 h. Discoloration of CR azo dye (A) and UV-vis absorption spectra (B). Control sample (No. 1); CR azo dye degradation by L. fusiformis MGMM7 under shaking (No. 2) and static (No. 3) culture conditions.

Figure 6

Biotransformation of CR azo dye by L. fusiformis MGMM7 at 30 ± 1 °C for 72 h. Discoloration of CR azo dye (A) and UV-vis absorption spectra (B). Control sample (No. 1); CR azo dye biotransformation by L. fusiformis MGMM7 under shaking (No. 2), static + shaking (No. 3), and static (No. 4) culture conditions.

Figure 7

Average nucleotide identity based on BLAST (ANIb) (A) and digital DNA–DNA hybridization (dDDH) (B) estimation among L. fusiformis strains using JSpeciesWS and Genome-to-Genome Distance Calculator (GGDC) (formula 2 used was HSP length/total length). A distance metric (Euclidean distance) was used to determine the relatedness between strains. The resulting distances (%) were organized into an ANI and dDDH matrix, clustered based on distance patterns, and visualized as a color-coded heatmap. The heat map was generated using ClusVis.

Figure 8

Comparison of subsystem distribution among different categories based on RAST SEED. Heatmap cluster analysis of the subsystem distribution of L. fusiformis strains MGMM7, TZA38, Cu1-5, and HJ.T1 using RAST pipeline based on the relative abundances of the non-redundant protein dataset for each genomic strain. The Euclidean distance was used as a metric to evaluate the similarity/dissimilarity between each pair of abundance strain profiles.

Figure 9

Pangenome analysis of L. fusiformis strains. (A) Pie chart of the pangenome showing the core, soft core, shell, and cloud genes. (B) Heatmap showing the genes expressed in L. fusiformis strains TZA38, HJ.T1, and Cu1-5 and absent in L. fusiformis MGMM7. (C) Heatmap showing genes expressed in L. fusiformis MGMM7 but absent in TZA38, Cu1-5, and HJ.T1. L. fusiformis strains are listed following hierarchical clustering created using a Manhattan distance matrix based on the gene presence/absence gene content matrix.

Figure 10

A circular plot of genomic island distribution in L. fusiformis strain Cu1-5 (A), TZA38 (B), HJ.T1 (C), and MGMM7 (D) was predicted using genomic IslandViewer 4. Red, blue, and green bars represent the genetic elements predicted in each genome using the integrated, IslandPath-DIMOB, and IslandPick methods, respectively.