Comparative transcriptomics reveal different genetic adaptations of biofilm formation in Bacillus subtilis isolate 1JN2 in response to Cd2+ treatment

Abstract

Biofilm plays important roles in the life cycle of Bacillus species, such as promoting host and object surface colonization and resisting heavy metal stress. This study utilized transcriptomics to evaluate the impacts of cadmium on the components, morphology, and function of biofilms of Bacillus subtilis strain 1JN2. Under cadmium ion stress, the morphology of the B. subtilis 1JN2 biofilm was flattened, and its mobility increased. Moreover, differential gene expression analysis showed that the main regulator of biofilm formation, Spo0A, decreased in expression under cadmium ion stress, thereby inhibiting extracellular polysaccharide synthesis through the SinI/SinR two-component regulatory system and the AbrB pathway. Cadmium ion treatment also increased the SigD content significantly, thereby increasing the expression of the flagella encoding and assembly genes in the strain. This promoted poly-γ-glutamic acid production via the DegS/DegU two-component regulatory system and the conversion of biofilm extracellular polysaccharide to poly-γ-glutamic acid. This conferred cadmium stress tolerance in the strain. Additionally, the cadmium ion-mediated changes in the biofilm composition affected the colonization of the strain on the host plant root surface. Cadmium ions also induced surfactin synthesis. These findings illustrate the potential of Bacillus species as biocontrol strains that can mitigate plant pathogenic infections and heavy metal stress. The results also provide a basis for the screening of multifunctional biocontrol strains.

Keywords: Bacillus subtilis; adaptation; biofilm; cadmium; stress response; transcriptomics analysis.

Figure 1

Impacts of Cd2⁺ on the biofilm colonies of B. subtilis 1JN2. 5 ul of 1JN2 suspension (10⁶ CFU/ml) was spotted on the surface of LBGM agar plate with a Cd2⁺ gradient of 0, 1, 2, 3, 4 and 5 mM, the picture was taken at 72 h post incubation at 30°C.

Figure 2

Confocal microscope detection of B. subtilis 1JN2 on the root of tomato with (B) or without (A) Cd2⁺ treatment. Fluorescent analysis was carried out using ZEISS LSM 700. GFP was selected in Smart Setup, the excitation wavelength is 488 nm, and the emission wavelength is 493–550 nm.

Figure 3

Analysis of the differentially expressed genes of B. subtilis 1JN2 after Cd2⁺ treatment. (A) Venn diagram analysis, (B) numbers of DEGs between time points. Samples collected at 6 h, 12 h, 18 h, and 24 h were named as S2, S3, S4, S5 and the blank control that without Cd2⁺ was named as S1. Comparison between S1 and S2 means the DEGs between the two samples, and so on.

Figure 4

Cluster of Orthologous Groups of Proteins (COG) function classification of the DEGs of B. subtilis 1JN2 after Cd2⁺ treatment. (A–D) Means the comparison of DEGs according to COG function classification between S1 and S2, S2 and S3, S3 and S4, S4 and S5 separately.

Figure 5

Scatter plot of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of the DEGs of B. subtilis 1JN2 after Cd2⁺ treatment. (A–D) Means the comparison of DEGs according to KEGG pathway enrichment between S1 and S2, S2 and S3, S3 and S4, S4 and S5 separately.

Figure 6

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation of the genes involved in flagella encoding and assembly of B. subtilis 1JN2 after Cd2⁺ treatment. Genes markered with red indicate up-regulation.

Figure 7

Relative expression level of the genes involved in extracellular secretion and regulation of B. subtilis 1JN2 after Cd2⁺ treatment. The histogram was based on the expression level in FPKM (Fragments per Kilobase of Transcript per Million Fragments Mapped) value.

Figure 8

The content of EPS and γ-PGA of the strain 1JN2 after Cd²⁺ treatment. 3 mM Cd²⁺ was added in the broth and the supernatants at 6, 12, 18 and 24 h after inoculation were used to detect the content of EPS and γ-PGA. T means treated with Cd²⁺ and C means control without Cd²⁺.

Figure 9

Validation of the selected DEGs by real-time PCR. Samples collected at 6 h, 12 h, 18 h, and 24 h were named as S2, S3, S4, S5 and the blank control that without Cd2⁺ was named as S1, 1–1, 1–2 and 1–3 means the three replicates of each sample.