MutL significantly regulates the formation of biofilms in B. subtilis YT1

doi:10.21203/rs.3.rs-4156921/v1

As a crucial and integral adaptation for thriving in diverse habitats, whether for survival or disease prevention and control, biofilm plays a vital role for most biocontrol bacteria, such as B. subtilis , Bacillus amyloliquefaciens, and plant-growth-promoting rhizobacteria (PGPR). However, the process of biofilm formation is intricate, and its regulatory mechanism remains unclear. In this study, we discovered that the regulatory protein MutL significantly influenced biofilm formation and exhibited a diminished colonization effectiveness on rice leaves. The mutant, lacking protein MutL expression, was unable to form biofilm with normal morphology and yielded only a quarter of the biofilm weight observed in the wild type B.subtilis YT1. In a petri dish confrontation assay examining the inhibitory effects on Rhizoctonia solani, no significant differences were observed between the mutant strain and the wild type YT1. Furthermore, through GFP fluorescent labeling technology, we conducted additional colonization tests, which demonstrated that the mutant failed to colonize rice stems effectively in the presence of R. solani. We hypothesize that the negative impact on biofilm formation resulted in inadequate colonization of rice stems, this combination accounts for the poor biocontrol efficacy against rice sheath blight, but it does not affect the normal growth of the strain or other biological phenotypes.

Biological sciences/Cell biology

Biological sciences/Microbiology

B. subtilis

biofilm formation

colonization

biological control

Bacillus spp. have the ability to form intricate and cell-aggregated communities called biofilms (Haggag and Timmusk, 2008). The presence of biofilm morphology enables Bacillus spp. to effectively resist pathogen infections (Stewart and Franklin, 2008; Vlamakis et al., 2013). Additionally, successful biofilm formation enhances the colonization of Bacillus spp. on plant surfaces (Weng et al., 2013). For example, PGPR PaeniBacillus polymyxa can colonize the root tips of Arabidopsis thaliana, enhancing the biocontrol effect against pathogens (Goswami et al., 2016). In B. subtilis 6051, biofilm formation, colonization, and surfactin secretion collectively contribute to biocontrol effects against Pseudomonas syringae (Bais et al., 2004).

However, the regulatory mechanisms of biofilm formation are complex. Previous studies have shown that the protein TasA and an exopolysaccharide (EPS) are the main components of biofilms. The protein TasA forms amyloid fibers that serve as the basic framework of the biofilm, while the EPS fills this framework (Ostrowski et al., 2011; Pintoet al., 2020). The eps operon, which is responsible for EPS biosynthesis, plays a crucial role in the regulatory mechanisms of biofilms in B. subtilis (Guttenplan et al., 2010). Defects in the protein EpsB and the PtkA gene lead to complete loss of biofilm formation in B. subtilis (Marvasi et al., 2010; Gerwig et al., 2014). The tapA-sipW-tasA operon is directly involved in biofilm synthesis and formation (Romero et al., 2011; Vlamakis et al., 2013). The DNA-binding protein SinR acts as the master regulator of biofilm formation, suppressing the expression of both operons (Stowe et al., 2014; Milton et al., 2020). Conversely, the protein RemA activates the expression of these operons (Prabhawathi et al., 2014;Bremer et al., 2022). The abrB gene also functions as a negative regulator of biofilm formation, and the AbrB-regulated genes sipW and yoaW are essential for normal biofilm formation (Weng et al., 2013).

Here, we present findings on a novel biofilm regulatory gene, mutL. We observed that the mutant with a defective mutL gene exhibited poor biofilm formation compared to the WT B. subtilis YT1. Previously, we successfully constructed a random mutant library of B. subtilis YT1 using the TnYLB-1 transposon (Zhou et al., 2013). Through biofilm phenotype screening, we identified a MutL mutant carrying the TnYLB-1 transposon that displayed significant defects in biofilm formation. We further disrupted the mutL gene in B. subtilis YT1 through homologous recombination, which resulted in a severe loss of biofilm. The biofilm network of the MutL mutant was less than one-tenth of that observed in the WT B. subtilis YT1. However, when observing colonization of the MutL mutant and the wild type B. subtilis YT1 on rice stems, we noted a significant reduction in the number of bacteria in the MutL mutant. These results indicate that the mutL gene plays a crucial role in the biofilm formation of B. subtilis YT1. The poor biofilm formation in the MutL mutant likely resulted in a significant reduction in its ability to colonize rice stems.

Strains and culture conditions

The strains and plasmids utilized in this study are outlined in Table 1(Zhou et al., 2016). The WT B. subtilis YT1 and the MutL mutant were cultured in Luria-Bertani (LB) broth medium (Kesel et al., 2017), which consisted of 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl. The cultures were incubated at 37℃ with shaking at 200 rpm. Escherichia coli DH5α was cultured in LB broth medium and used for vector preparation to disrupt the mutL gene in the wild type B. subtilis YT1. Rhizoctonia solani was cultured at 28℃ on potato dextrose agar (PDA) medium containing 200 g/L potato infusion, 20 g/L glucose, and 20 g/L agar at pH 7.0.

Table 1

Bacterial strains and plasmids used in this study
Strain/plasmid	Description	Source or reference
B. subtilis YT1	Wild type strain	CGMCC NO.29889
ΔmutL	ΔmutL::Spec^r, YT1 derivative	This study
ΔYT1-gfp	ΔYT1-gfp::Cm^r, YT1 tagged with green fluorescent protein	This study
ΔmutL-gfp	ΔmutL:: Spec^r, ΔmutL tagged with green fluorescent protein	This study
Plasmids
pUC19	Cloning vector, Ap^r	TakaRa U07650
pDG1728	Cloning vector, Amp^r Spec^r	BGSC No. ECE114
pUCSpec	pUC19 carrying spectinomycin cassette from pDG1728	This study
pUCSpec-MutL	pUCSpec carrying 741 bp fragment MutL	This study
pRp22-gfp		This study
^*Amp^r: ampicillin resistance, Spec^r: spectinomycin resistance, CGMCC: China General Microbiological Culture Collection Centre, Beijing, China

For biofilm formation assays, B. subtilis YT1 and the MutL mutant were grown in MSgg medium (Liu et al., 2022) consisting of 5 mM potassium phosphate (pH 7), 100 mM Mops (pH 7), 2 mM MgCl₂, 700 µM CaCl₂, 50 µM MnCl₂, 50 µM FeCl₃, 1 µM ZnCl₂, 2 µM thiamine, 0.5% glycerol, 0.5% glutamate, 50 µg/mL tryptophan, and 50 µg/mL phenylalanine. Antibiotics were added when necessary at the following concentrations: 100 µg/mL ampicillin, 100 µg/mL spectinomycin, 20 µg/mL neomycin, and 5 µg/mL chloramphenicol (Kantiwal and Pandey, 2022).

Construction of the WT B. subtilis YT1 mutant libraries and the mutL gene obtained

This study was conducted and completed previously in our laboratory, and the research methods have been published in Scientia Agricultura Sinica (Zhou et al., 2013). We constructed a random mutant library of B. subtilis YT1 using the plasmid pMarA carrying the transposon TnYLB-1, which demonstrated good randomness in the insertion of chromosomal loci. Several single colonies from this mutant library were inoculated into 4 mL of LB medium with spectinomycin for the mutants and incubated at 37℃ with shaking for 12 h. A control strain of B. subtilis YT1 single colony was also cultured without spectinomycin. Next, 200 µL of each culture was inoculated into 4 mL of MSgg medium and incubated statically at 37℃ for 24 h (Zhou et al., 2016). By visually inspecting the biofilm formation, we were able to observe any significant changes in the mutants compared to the WT strain B. subtilis YT1. Each sample was repeated three times to ensure accuracy. After screening thousands of mutants, we identified the MutL mutant as having significant deficiencies, and we confirmed that it had only one insertion site through Southern Blot analysis.

Construction of the ΔmutL mutant and tagging strains with GFP.

To obtain a spectinomycin resistance gene and its promoter, we used the primers SpecF (5'-TTTGGATCCCTGCAGCCCTGGCGAATG-3') and SpecR (5'-TTTGAATTCAGATCCCCCTATGCAAGG-3') (Zhou et al., 2016) (BamHI and EcoRI restriction sites are underlined) from plasmid pDG1728. The complete spectinomycin cassette, digested with the BamHI and EcoRI restriction enzymes, was cloned into the plasmid pUC19, resulting in the construction of pUCSpec. We then obtained a PCR product of mutL (741 bp) from the genomic DNA of the WT B. subtilis YT1 using the primers MutLF (5'-TTTAAGCTTATTTACGGGACGGCGGTTGC-3') and MutLR (5'-TTTGGATCCGCTGGGCGCTCGGAATAAGC-3') (HindIII and BamHI restriction sites are underlined). The plasmid pUCSpec-MutL was created by inserting this digestion product into pUCSpec. The ΔmutL mutant was generated by transforming the pUCSpec-MutL plasmid into B. subtilis YT1 through the process of genetic transformation. Additionally, we successfully constructed the positive emission green fluorescent protein (GFP) strains ΔYT1-gfp and ΔmutL-gfp by transforming the plasmid pRp22-gfp into the WT B. subtilis YT1 and the ΔmutL mutant, respectively.

Antifungal activity of WT B. subtilis YT1 and ΔmutL mutant against R. solani

The mycelial plugs of R. solani, measuring 7 mm in diameter, were cultivated in PDA medium for three days and then centered on LB plates. Two microliters of the B. subtilis BsYT1 WT strain and the ser mutant culture were carefully placed onto a 6 mm circular filter paper disk, uniformly distributed around the mycelial plugs. The plates were subsequently sealed with parafilm and incubated at 28℃. The antib zone of inhibition was determined after 24 to 72 h of incubation (Zhou et al., 2016). This experiment was conducted three times to ensure reproducibility.

Colonization detection in rice plants using confocal microscopy and assessment of biocontrol efficacy against rice sheath blight

The R. solani bacterial cakes, with a diameter of 6 mm, were cultured for 3 days in solid PDA medium. These cakes were then inoculated into 100 mL of liquid PDA medium with 200 matchsticks without Commelina. The cakes were incubated in a 500 mL flask without shaking at 28℃ for one week. Thirty seeds of a rice cultivar were soaked in water for 24 h and then sown in a nursery containing sterile organic soil. Five days before rice heading, 10 matchsticks of R. solani were inoculated into 2–3 cm rice leaves of each rice plant. Approximately 20 mL of broth, cultured for 48 h, from the WT B. subtilis YT1-gfp and ΔmutL-gfp mutant strains were evenly sprayed onto the rice plants marked by the matchsticks. From 0 to 12 days after the initial inoculation, the rice leaves marked by the matchsticks were observed and captured using a confocal microscope equipped with a 40x objective lens (Carl Zeiss LSM710, Germany). The lesion area length of the WT B. subtilis YT1-gfp and ΔmutL-gfp mutant strains was measured as the primary criterion for assessing the biocontrol effects against rice sheath blight.Each treatment group consisted of eight individual pots (Zhou et al., 2016).

Genomic transcript levels analyzed between WT B. subtilis YT1 and the ΔmutL mutant using the Illumina HiSeqTM2500 platform.

Total biofilm RNA from both the WT B. subtilis YT1 and the ΔmutL mutant, cultured in a static MSgg medium (4 mL in 12-well microtiter plates) at 37℃ for 24 h, was extracted using an RNAprep pure Cell/Bacteria Kit. The extracted RNA was qualified using the 2100 Bioanalyzer test. Subsequently, 10 µg of total RNA was treated with 5U DNaseI (Takara, Japan) for 30 minutes at 37℃, followed by purification using an RNeasy MinElute Cleanup Kit (Qiagen, Germany) and elution with 15 µL of RNase-free water. Ribo-Zero™Magnetic Kit (Gram-negative or Gram-positive bacteria; Epicentre, USA) was utilized to eliminate all rRNA by conducting a reaction at 68℃ for 10 minutes and room temperature for 5 minutes. Next, a cDNA library was constructed using a NEB Next ® Ultra TM Directional RNA Library Prep Kit for Illumina (NEB, USA), followed by cluster generation and sequencing on an Illumina HiseqTM2500 platform.

Linker sequences containing low-quality clean reads were removed using fastx_clipper. Unwanted, low-quality reads (> 20 bases) from 3’to 5’end were eliminated using a FASTQ Quality filter (FASTX-Toolkit, v0.0.13). Clean reads less than 50 bp were excluded. Quality assessment of the sequencing data was performed with Fastqc software (v0.10.0). Differential gene expression analysis was carried out by mapping clean reads (Bowtie 2, v2.1.0) and utilizing the MA-plot-based method with the random sampling model. Gene expression levels in both the WT YT1 and ΔmutL mutant strains were determined. Genes showing significant differential expression with fold change > 2, FDR(q value) < 0.001, and at least one sample with an RPKM > 20 were selected for further analysis.

The acquisition of the mutL gene

Through biofilm phenotype testing and bioinformatic analysis of a random insertion mutant library of WT B. subtilis YT1, the mutL gene, which is associated with decreased biofilm phenotype, was identified. By employing homologous recombination, a single-gene knockout mutant with a MutL deficiency was successfully generated and characterized.

This MutL mutant showed a significant reduction in biofilm formation, and its structure was barely visible, with only subtle projections distinguishing it from the well-defined three-dimensional architecture and intricate patterns of WT B. subtilis YT1 (Fig. 1). Analysis of the biofilm network width (NW) for the MutL mutant revealed that its density was merely one-tenth that of the wild type. In terms of colony morphology, the MutL mutant lacked a continuous edge and clear projections, presenting an incomplete and planar structure in contrast to the robust three-dimensional growth of WT B. subtilis YT1 (Fig. 2). However, the mutant strain can grow and reproduce normally, without significant differences compared to the WT YT1 strain (Fig. 2). These findings indicate that the mutL gene plays a significant role in B. subtilis YT1's biofilm development.

No significant difference in the antibacterial activity

On Petri dishes effectively inoculated with R. solani, both the WT YT1 and the mutant ΔmutL were able to consistently form normal and relatively fixed antibacterial zones after 24–72 h of confrontation cultivation. The fungal mycelium of the pathogen was unable to cross this antibacterial zone and continue its expansion, exhibiting an upward growth pattern of the mycelium. No significant difference in the size of the antibacterial zones was observed between the two strains (Fig. 3).

Significant decrease in colonization ability of the mutant ΔmutL

This study utilized GFP labeling to observe the colonization patterns of the ΔmutL mutant and WT B. subtilis YT1 on rice stems infected with R. solani over a period of 12 days. Both the number of bacteria and the apparent green fluorescence increased in both strains in the initial 8 days, with the maximum number of bacteria observed on the eighth day (Fig. 4). However, significant clustering was observed only in the WT B. subtilis YT1 as the amount of bacteria increased. Although the maximum number of bacteria was similar between the two strains on the eighth day, the bacterial quantity was noticeably higher in the WT B. subtilis YT1. The amount of bacteria decreased from day nine to day twelve, but clustering continued in the WT B. subtilis YT1, while the ΔmutL mutant did not exhibit any clustering until the twelfth day, indicating a substantial reduction in its colonization ability(Fig. 4). These results suggest that the colonization of the ΔmutL mutant was significantly reduced compared to the WT B. subtilis YT1, with no cluster movement observed.

Significant reduction in the biocontrol efficacy of the ΔmutL mutant against rice sheath blight

Between the first and twelfth day of observation, the B. subtilis YT1 strain exhibited peak biocontrol efficacy of 63.2% on the fourth day, after which it progressively decreased to 42.9% by the twelfth day (Table 2). This decline may be attributed to the initial onset of R. solani spread on the fourth day. In contrast, the mutL mutant strain demonstrated a biocontrol efficiency of 15.8%, significantly inferior to that of the WT B. subtilis YT1. As the disease progressed rapidly, the biocontrol efficacies of both the WT B. subtilis YT1 and the mutL mutant substantially declined. Nonetheless, at any given time point, the biocontrol effects of the B. subtilis YT1 were consistently superior to those of the ΔmutL mutant. Particularly on the twelfth day, the biocontrol efficiency of the ΔmutL mutant was only about 24.9% of the WT B. subtilis YT1 (Table 2). These findings indicate that the absence of regulation by the mutL gene led to a significant decrease in the biocontrol efficacy of the MutL mutant over time.

Table 2

Biocontrol of rice sheath blight in pot cultures of the WT *B. subtilis* YT1 and the ΔmutL
Treatment	4 d		8 d		12 d
Treatment	Lesion size (cm²)	Control efficiency (%)	Lesion size (cm²)	Control efficiency (%)	Lesion size (cm²)	Control efficiency (%)
YT1	1.4(0.1)^b	63.2^a	7.8(0.3)^c	50.6^a	16.5(0.7)^c	42.9^a
ΔmutL	3.2(0.2)^a	15.8^b	13.7(0.4)^ab	13.3^b	25.8(0.9)^ab	10.7^b
CK	3.8(0.2)^a		15.8(0.4)^a		28.9(0.9)^a

*Data are the average of eight pots ± the standard deviation of three independent experiments with eight pots.

Differential genes expression in B. subtilis YT1 and the ΔmutL mutant

The identification of differentially expressed genes was achieved through the analysis of gene transcription (Table 3). This table lists the key regulatory genes that have been reported in recent years to directly and significantly control the formation of biofilms. In this study, compared to the control YT1, some reported and confirmed signaling pathway regulatory factors in the mutant ΔmutL strain showed significant abnormal expression. For example, there was a notable decrease in the expression of genes senN, capB, and the appA family, as well as abnormal increases in the expression of genes spo0A, sinR, and abrB. The transcriptional expression levels of tasA, a protein involved in biofilm matrix formation, and the eps family proteins, which are also components of the biofilm skeleton, were also found to be decreased, although not significantly.

Table 3

Analysis of differentially expressed genes in wild type B. subtilis YT1 and the ΔmutL mutant
Gene	Relative expression level		Function
	B. subtilis YT1	ΔmutL	Function
senN	1	0.41	SenN transcriptional regulator
capB	1	0.44	γ-PGA synthesis
appA -D	1	0.44	Periplasmic oligopeptide-binding protein
nprE	1	0.52	Extracellular neutral metalloprotease
epsA-B	1	0.6	Substrate for biofilm formation
tasA	1	0.87	Substrate for biofilm formation
sipW-yqxM	1	1.37	Type I signal peptidase protein for biofilm
kinA-D	1	1.58	Two-component sensor histidine kinase
spo0A	1	1.8	Response regulator
degQ	1	1.96	Pleiotropic regulator
sinR	1	2.09	Master regulator of biofilm formation
abrB	1	2.5	Transcriptional regulator for transition state

B. subtilis , as a model organism strain, is extensively studied. Due to varying environmental conditions, B. subtilis exists in multiple forms in nature, including free-living, motile, and dormant cell (Steinberg and Kolodkin-Gal, 2018; Kantiwal and Pandey, 2023). As an outstanding biocontrol agent, B. subtilis is commonly employed to manage fungal diseases in crops such as rice and wheat. Diseases like wheat take-all, wheat stripe rust, and wheat powdery mildew can be efficiently inhibited by B. subtilis E1R-J (Liu et al. 2009; Li et al. 2013; Gao et al. 2015). Additionally, rice sheath blight, a prevalent fungal disease in rice crops that causes over 15% yield loss, can be effectively controlled by B. subtilis MBI 600 (Kumar et al. 2012, 2013).

Biofilms are highly organized communities of microorganisms composed of amyloid fibers (TasA) and exopolysaccharides (EPS), which adhere to the surfaces of cells (Branda et al. 2006; Romero D et al. 2010; López et al. 2010). As an effective adaptation strategy for survival in diverse environments, biofilms are crucial for the prevention and treatment of diseases (Stewart and Franklin, 2008; López et al. 2010; Vlamakis et al. 2013). In Bacillus amyloliquefac SQR9, the formation of biofilms can significantly enhance the colonization capacity and biocontrol efficacy against cucumber and watermelon wilt diseases (Weng et al. 2013).

MutL, as a highly conserved protein, is widely present in bacterial genomes and its main reported function is an important part of DNA mismatch repair (MMR)(Guarné, 2012). MMR is a conservative pathway that protects genome integrity by correcting replication errors to achieve repair. This process requires the coordinated action of two proteins (MutS and MutL) to initiate the mismatch repair response, and the basic steps include recognizing mismatches, distinguishing newly synthesized strands from parental strands, removing and re-synthesizing erroneous strands(Liu, et al. 2016).

B. subtilis YT1, being an effective biocontrol bacteria, has been utilized in rice fields for a long time, and has shown significant effectiveness between 63.9% to 84.6% against rice sheath blight and rice false smut (unpublished data). The random mutant library of B. subtilis YT1 was effectively created using TnYLB-1 transposon in prior studies (Zhou et al. 2013). The ΔmutL mutant, identified through transposon mutagenesis and confirmed via Southern Blot, exhibited significant deficiencies in biofilm formation. Following this, we successfully generated a single knockout mutant, ΔmutL, through homologous recombination in B. subtilis YT1. However, the biofilm formed by this mutant was minimal and lacked distinct structures. Biofilm dry weight analysis revealed that the ΔmutL mutant only weighed one-tenth of the WT B. subtilis YT1. The agar plate confrontation test showed that the antibacterial activity was not significantly affected, but the colonization capacity exhibited a significant decrease, with the loss of the community aggregation effect. This ultimately led to a significant decrease in the control effectiveness against rice sheath blight disease, indicating that mutL is a key node gene that directly affects biofilm formation as a single factor. Transcriptome data has shown that these different genes reported are all expressed to varying degrees, with one important gene, capB, being significantly down-regulated. The main mechanism of action of capB is to regulate the assembly and utilization of glutamate in the process of biofilm formation. The resulting polyglutamic acid γ-PGA is an important filler in the structure of biofilms and is indispensable. In this study, the transcription of this gene was significantly down-regulated. Therefore, our research may achieve global regulation of biofilm formation through the glutamate-γ-PGA signaling pathway.

we confirmed the significant positive regulatory role of the MutL protein in the biofilm formation process of strain YT1 ultimately. After multiple confirmations, the experimental results were stable and reliable, which fully demonstrates that the MutL protein plays a key role in the biofilm formation process and even the growth and development process of Bacillus species. This provides a new research area for the study of biofilm mechanisms. Furthermore, subsequent work in this study will aim to reveal the detailed mechanism of its action, contributing to the study of biofilm regulatory pathways and laying a solid theoretical foundation for the improvement of microbial pesticides by Bacillus species.

Conflict of Interest

All authors report no conflict of interest.

Author Contribution

Authorship contribution statement：1. Huafei and Baoyou，Conceived and designed the experiments,Performed the experiments, Implemented methods, Analyzed the data, Wrote the paper.2. Baoyan, Min and Haining, Performed the experiments, Implemented methods.3. Hongtao, Shaoli and Binghui, Analyzed the data.

Acknowledgments

This work was supported by the Project of Shandong Natural Science Foundation (ZR2020KC026); the Shandong Province Fruit Industry Technology System (SDAIT-06-11); the Shandong Province Key R&D Program (2021CXGC010602, 2021CXGC010802); the Monitoring and Control Project of Crop Diseases, Pests and Rodents Epidemic in the Ministry of Agriculture and Rural Affairs (152307026); the Science and Technology Planning Project of Yantai (2021NYNC015, 2022XCZX094); the Shandong Province Science and Technology Small and Medium Enterprises Innovation Ability Enhancement Project (2023TSGC0829, 2023TSGC0894).

Data Availability

Data Availability Statements：The data that support the findings of this study are available from the corresponding author upon reasonable request.

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No competing interests reported.

MutL significantly regulates the formation of biofilms in B. subtilis YT1

Status:

Version 1

Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Strains and culture conditions

RESULTS

No significant difference in the antibacterial activity

Significant decrease in colonization ability of the mutant ΔmutL

Significant reduction in the biocontrol efficacy of the ΔmutL mutant against rice sheath blight

DISCUSSION

Declarations