Cd2+ Treatment Changed Biofilm Formation by B.subtilis 1JN2
We found that biofilm formation by B.subtilis 1JN2 varied with the Cd2+ concentration (Figure 1). The biofilm colonies gradually reduced, and their surface became denser with Cd2+ concentration. Increasing Cd2+ concentration from 0 to 2 mM moderately inhibited biofilm formation by 1JN2; however, Cd2+ concentration of 3 mM or higher significantly inhibited biofilm formation by 1JN2. Unlike biofilm formation, there was no significant difference in the growth of 1JN2 in Cd2+ concentration of 0 to 2 mM Cd2+ (Yang et al., 2018).
The biofilm forms were also affected by Cd2+ treatment. Biofilm surfaces of the Cd2+-treated cells were complanate compared to the convex surfaces of the group without Cd2+ Moreover, the viscosity of the biofilms also increased with the Cd2+ concentration.
Cd2+ Treatment Influenced Tomato Roots Colonization by B.subtilis 1JN2
Since biofilm formation is an important step in root colonization, it was also imperative to evaluate the effect of Cd2+ on the root colonization ability of B.subtilis 1JN2. A GFP-tagged 1JN2 was constructed for the root colonization experiment in the greenhouse. The population of B.subtilis 1JN2, which colonized the tomato roots, decreased with an increase in Cd2+ concentration (Figure 2), indicating that biofilm inhibition caused by Cd2+ affected the colonization ability of 1JN2. The colonized position was also changed since many 1JN2 cells were found in tomato root cells compared to the root surface (Figure 2-C). Conversely, many 1JN2 colonies could be seen on the root surface of the group without Cd2+, showing that root surface colonization is important in preventing soil-borne pathogen infections.
RNA Sequencing and Identification of Differentially Expressed Genes (DEGs)
Cell samples were collected at 4-time points (6 h, 12 h, 18 h, and 24 h) after Cd2+ treatment. The samples collected at 6 h, 12 h, 18 h, and 24 h were denoted S2, S3, S4, and S5, respectively, while the blank control without Cd2+ was designated S1. The sequence data summary is presented in Table 1. More than 93% of the clean reads from each sample mapped to the reference genome, indicating that the obtained transcriptome data were suitable for further analysis. The raw data were deposited to NCBI SRA database with accession number: PRJNA646606 (https://www.ncbi.nlm.nih.gov/search/all/?term=PRJNA646606).
Table 1 Summary of RNA-seq data and the reads mapped to B.subtilis 1JN2 genome
Samples
|
Read Number
|
Base Number
|
GC Content
|
%≥Q30
|
S1-1
|
7996483
|
2398944900
|
45.09
|
95.83
|
S1-2
|
9237500
|
2771250000
|
45.08
|
96.31
|
S1-3
|
10721600
|
3216480000
|
44.98
|
94.94
|
S2-1
|
10667725
|
3200317500
|
44.49
|
95.43
|
S2-2
|
9264843
|
2779452900
|
44.54
|
93.99
|
S2-3
|
9528611
|
2858583300
|
44.29
|
94.29
|
S3-1
|
10855623
|
3256686900
|
44.37
|
94.71
|
S3-2
|
8856524
|
2656957200
|
44.13
|
94.52
|
S3-3
|
9460244
|
2838073200
|
44.43
|
93.75
|
S4-1
|
11315033
|
3394509900
|
44.59
|
94.51
|
S4-2
|
10009680
|
3002904000
|
44.66
|
94.13
|
S4-3
|
9704248
|
2911274400
|
44.35
|
93.06
|
S5-1
|
8895735
|
2668720500
|
44.68
|
90.67
|
S5-2
|
9932927
|
2979878100
|
44.69
|
93.12
|
S5-3
|
9358331
|
2807499300
|
44.71
|
94.85
|
The number of DEGs between the various time points post Cd2+ treatment of B.subtilis 1JN2 were analyzed using a Venn diagram (Figure 3a). The number of DEGs between S1 and S2 decreased with the prolongation of treatment time. There were 659 DEGs between S1 and S2, among which 308 were up-regulated, and 351 were down-regulated. However, the number of DEGs between S2 and S3, S3 and S4, S4 and S5 were 178, 36 and 6, respectively (Figure 3b). This implies that the cells undergo an important adjustment phase during the initial period of exposure to Cd2+ and later acquire resistance to Cd2+ stress. The same trend was observed with the PCA (principal component analysis) results (Figure 3c) which showed shorter distances between S3, S4, and S5, indicating no significant difference in their gene expression.
Cell Mobility-Related Genes Changed Significantly after Cd2+ Treatment
Since Cd2+ treatment modified the morphological features of strain 1JN2 biofilm, we checked whether it could also affect the mobility of the strain. The COG (Cluster of Orthologous Groups of Proteins) database is constructed based on the phylogenetic relationship between bacteria, algae, and eukaryotes. Applying BLAST to the database allows for orthologous classification of gene products. The COG (Cluster of Orthologous Groups of Proteins) database BLAST results showed that the most significant changes in gene products of the strain after exposure to Cd2+ were related to cell mobility, followed by chromatin structure and dynamics, amino acid and nucleotide transport and metabolism (Figure 4). Moreover, a comparison between S1 and S2 indicated that the strain could perceive and respond to Cd2+ stress after some period of exposure via regulatory metabolism. These results are consistent with the observed biofilm morphological changes of the strain.
Flagella Synthesis- and Assembly-related Genes Increased in Strain 1JN2 after Cd2+ Treatment
The flagellum is the most important motor organ which plays an important role in the mobility and chemotaxis of bacterial cells. Due to the observed significant effects of Cd2+ on the strain’s mobility, we assessed its effects on flagella encoding and assembly genes. KEGG (Kyoto Encyclopedia of Genes and Genomes) is a database for systematic gene function analysis and genomic information. The significant pathway enrichments can indicate the main biochemical, metabolic, or signal transduction pathways associated with differentially expressed genes. According to KEGG analysis, flagella encoding and assembly genes were significantly enhanced than the control group 12 hours after Cd2+ treatment (Figure 5). The differential genes at the later sampling time points were mainly involved in metabolic-related reactions, such as glycolysis/gluconeogenesis and biosynthesis of valine, leucine, isoleucine, pantothenate, and CoA. Figure 6 also illustrates similar results.
Effect of Cd2+ on the Extracellular Secretions of Strain 1JN2
We evaluated the effects of Cd2+ stress on the extracellular secretions (polysaccharides and proteins) of strain 1JN2 and found that the extracellular protein-related genes (tapA-sipW-tasA) were not expressed in the strain. However, the expression of EPS encoding genes (epsA-O) decreased significantly after Cd2+ treatment, indicating that Cd2+ reduces polysaccharides levels in B. subtilis 1JN2 biofilm (Figure 7). According to Yu et al. (2016), γ-PGA plays an important role in the polymorphism of B. Subtilis biofilm; therefore, we assessed the expression of the γ-PGA encoding genes in this study. Moreover, the genes encoding γ-PGA (pgsB-pgsC-pgsA) were highly expressed after Cd2+ treatment, but there was no significant difference in the expression at the last four sampling time points. This indicated that changes in the extracellular secretions mainly occurred within a certain period after 1JN2 exposure to Cd2+ treatment. Similar results were obtained by comparing the changes in EPS and γ-PGA levels at different time points after Cd2+ treatment. The EPS levels remained unchanged at very low concentrations, while γ-PGA content increased significantly compared to the control group (Figure 8).
Additionally, studies have reported that surfactants secretion by Bacillus increases after exposure to cations, thus, we evaluated the expression of surfactin-encoding genes. The expression levels of the genes related to the surfactin-related genes (srfAA-srfAB-srfAC) were also significantly increased following Cd2+ treatment (Figure 7).
Effect of Cd2+ on the Regulation Pathway of 1JN2 Biofilm Formation
According to related reports, multiple histidine kinases (KinA, KinB, KinC, KinD, and KinE) mainly sense exogenous environmental signals and collectively act on Spo0A either directly through protein phosphorylation or indirectly via a Phospho-relay (Mcloon et al., 2011). We examined the expression of the regulatory factors of the biofilm formation pathway of 1JN2 and found that the expression of kinC significantly increased after exposure to Cd2+, reaching its highest level at 24 h (Figure 7). This shows that KinC plays an important role in detecting the change in exogenous ion signals. It has been reported that KinC induces low levels of Spo0A-P in response to potassium cations evacuation caused by surfactin-generated pores in B.subtilis membrane (Lopez et al., 2009). But the main regulatory factor showed a downward trend. Therefore, increased kinC levels reduced the expression of Spo0A after exposure to Cd2+ (Figure 7). To explain this, we also examined the changing trends of Sda and DnaA encoding genes following Cd2+ treatment. Sda is a critical protein that controls the sporulation or biofilm states in bacterial cells. It can prevent the transfer of phosphate groups from histidine kinase to Spo0F, thereby blocking or delaying Spo0A activity (Whitten et al., 2007; Yan et al., 2016). The replication initiation protein, DnaA, activates sda, which effectively prevents phosphate group accumulation and activates Spo0A, thereby preventing cells from premature sporulation stage (Burkholder et al., 2001). Sda expression and activity are reduced when the cell enters the stationary phase, therefore, reversing the Spo0A and proteolytic activities of the existing Sda protein (Ruvolo et al., 2006). Thus, the increased expression levels of dnaA and sdaAB after exposure to Cd2+may have led to the changes in Spo0A. Meanwhile, the two-component regulatory system SinI/SinR limited the production of extracellular polysaccharides, similar to the effects of increased expression of AbrB encoding genes after Cd2+ treatment.
We also evaluated the changes in the γ-PGA regulatory factors and found that SigD significantly increased after Cd2+ treatment. This activated the expression of flagella encoding and assembly genes by regulating γ-PGA production through another two-component regulatory system called DegU/S. The components of this regulatory pathway (sigD, degS, and degU) increased consistently, resulting in γ-PGA production, which in turn enhanced the mobility of the strain. The changes in the expression levels of γ-PGA and EPS are consistent with the previously reported switch-like mechanism (Yu et al., 2016).
Real-time PCR Validation of the Selected DEGs
Five genes were selected for qPCR assay to confirm the reproducibility and accuracy of the transcriptome data. We found less variation between the two data sets from RNA-seq and RT-PCR analyses (Figure 9).