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. 2023 Sep 20;24(12):1510–1521. doi: 10.1111/mpp.13389

degQ associated with the degS/degU two‐component system regulates biofilm formation, antimicrobial metabolite production, and biocontrol activity in Bacillus velezensis DMW1

Chenjie Yu 1, Junqing Qiao 2, Qurban Ali 1, Qifan Jiang 1, Yan Song 1, Linli Zhu 1, Qin Gu 1, Rainer Borriss 3, Suomeng Dong 1, Xuewen Gao 1, Huijun Wu 1,✉
PMCID: PMC10632791  PMID: 37731193

Abstract

The gram‐positive bacterium Bacillus velezensis strain DMW1 produces a high level of antimicrobial metabolites that can suppress the growth of phytopathogens. We investigated the mechanism used by degQ and the degS/degU two‐component system to regulate the biocontrol characteristics of DMW1. When degQ and degU were deleted, the biofilm formation, cell motility, colonization activities, and antifungal abilities of ΔdegQ and ΔdegU were significantly reduced compared to wild‐type DMW1. The expression levels of biofilm‐related genes (epsA, epsB, epsC, and tasA) and swarming‐related genes (swrA and swrB) were all down‐regulated. We also evaluated the impact on secondary metabolites of these two genes. The degQ and degU genes reduced surfactin and macrolactin production and up‐regulated the production of fengycin, iturin, bacillaene, and difficidin metabolites. The reverse transcription‐quantitative PCR results were consistent with these observations. Electrophoretic mobility shift assay and microscale thermophoresis revealed that DegU can bind to the promoter regions of these six antimicrobial metabolite genes and regulate their synthesis. In conclusion, we provided systematic evidence to demonstrate that the degQ and degU genes are important regulators of multicellular behaviour and antimicrobial metabolic processes in B. velezensis DMW1 and suggested novel amenable strains to be used for the industrial production of antimicrobial metabolites.

Keywords: antimicrobial metabolites, Bacillus velezensis, biocontrol activity, biofilm formation, DNA–protein interaction

degQ and degU are important for the biocontrol activity of Bacillus velezensis DMW1, and DegU can regulate the production of secondary metabolites by targeting their promoter regions.

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1. INTRODUCTION

Plant growth‐promoting bacteria such as Bacillus subtilis, Bacillus velezensis, and Bacillus thuringiensis can be applied as biocontrol agents due to their broad‐spectrum effect against plant pathogens (such as fungi, bacteria, and nematodes; Liang et al., 2022; Ngalimat et al., 2021). The antagonistic activity of Bacillus spp. is associated with the production of antibacterial, nematicidal, and antifungal antibiotics (Ali et al., 2023; Gu et al., 2017). Among these antibiotics, the surfactin, iturin, and fengycin families of lipopeptides and the macrolactin, bacillaene, and difficidin types of polyketides are typical representatives (Chen et al., 2006; Yu et al., 2023). The iturin and fengycin lipopeptides exhibit activity against filamentous fungi (Cawoy et al., 2015). Surfactin has high surface activity as well as haemolytic, antibacterial, and antiviral properties (Meena et al., 2020). Surfactin is also associated with biofilm formation and root colonization of a Bacillus strain (Bais et al., 2004). Surfactin and fengycin can act as elicitors that induce disease resistance in plants (Li et al., 2019).

In strains of Bacillus spp. with biocontrol activity, lipopeptides are synthesized by nonribosomal peptide synthetases (NRPSs) or hybrid polyketide synthases (PKSs) and NRPSs through a multimodular template mechanism (Raaijmakers et al., 2010). PKSs synthesize polyketides via stepwise decarboxylative Claisen condensation reactions between the extender unit and the growing polyketide chain, resulting in enzyme‐bound ketoacyl intermediates (Caulier et al., 2019). To form an active holoenzyme, both NRPSs and PKSs must be posttranslationally modified by 4′‐phosphopantetheine transferase (encoded by the sfp gene), respectively in the peptidyl carrier protein (PCP) domain and the acyl carrier protein (ACP) domain (Mofid et al., 2004). Despite having complete srfA and pps operons for the synthesis of surfactin and fengycin, the model strain B. subtilis 168 cannot produce these two compounds due to a mutation in the sfp gene (Tsuge et al., 1999).

Two‐component signal transduction systems are the main systems through which Bacillus regulates multicellular behaviours. These include a sensor kinase that is autophosphorylated in response to a signal followed by the subsequent transfer of the phosphate group to a two‐domain response regulator (Murray et al., 2009). The response regulators bind target DNA promoters and activate or repress transcription. For example, the synthesis of the surfactin lipopeptide is regulated by the ComP/ComA system (Ogura et al., 2001). The transcription of the srfA operon is directly activated by the phosphorylated form of the factor ComA (ComA‐P), whose phosphorylation is controlled by ComP (Rahman et al., 2021). The DegS/DegU system is involved in the regulation of the biosynthesis of the lipopeptide bacillomycin D, dipeptide bacilysin, and poly‐γ‐dl‐glutamic acid (γ‐PGA) (Koumoutsi et al., 2007; Mariappan et al., 2012; Stanley & Lazazzera, 2005). In addition, Bacillus strains use the Rap‐Phr system, composed of the response regulator aspartate phosphatase (Rap) and its inhibitory oligopeptide (Phr), to regulate two‐component signal transduction (Auchtung et al., 2006). Rap proteins are transcriptional anti‐activator proteins that inhibit the binding of the response regulator ComA to DNA promoters, while the corresponding Phr peptides bind to Rap proteins and inhibit their activity (Bongiorni et al., 2005; Core & Perego, 2003).

The term “biofilm” describes microbial multicellular communities in which individual cells are bound together by an extracellular matrix that the community produces (Ali et al., 2022; Arnaouteli et al., 2021). The hydrophobin protein Bs1A, exopolysaccharides, and TasA protein fibres make up the biofilm matrix in B. subtilis (Hobley et al., 2013; Romero et al., 2010). The transcription repressors SinR and AbrB regulate the bs1A gene both directly and indirectly (Kobayashi & Iwano, 2012).

B. velezensis DMW1 was originally isolated from potato tubers and can produce antimicrobial lipopeptides and polyketides. DMW1 possesses broad‐spectrum antagonistic activity and exhibits strong biocontrol efficacy against Phytophthora sojae and Ralstonia solanacearum (Yu et al., 2023). However, the molecular mechanism accounting for DMW1 biocontrol activity is unknown. The small pleiotropic regulatory protein DegQ and the response regulator protein DegU are used by B. subtilis to regulate multicellular behaviour. In this study, we investigated the regulatory effects of the degQ and degU genes on the biocontrol ability of DMW1, including biofilm formation, motility ability, and antimicrobial metabolite production.

2. RESULTS

2.1. degQ and degU are required for biofilm formation in DMW1

To determine the roles of the degQ and degU genes in the biocontrol activity of B. velezensis DMW1, degQ and degU were successfully knocked out in DMW1 by a marker‐free CRISPR/Cas9 technique (Figure S1). We first analysed the colony morphology and biofilm formation of DMW1 and its mutants. Colonies of the wild‐type (WT) strain formed a solid surface and were highly structured. The ΔdegU mutant colonies had a relatively flat surface. The ΔdegQ mutant colonies had similar phenotypic characteristics, but the circular edge with undulating margins was different from those of WT DMW1 and the ΔdegU mutant (Figure 1a). The biofilm formation abilities were compared between WT DMW1, ΔdegQ, and ΔdegU in MSgg medium. WT DMW1 formed a robust biofilm at the liquid–solid interface. The ΔdegQ and ΔdegU mutants had a significantly decreased biofilm formation ability and the biofilm was smaller and thinner (Figure 1b). Crystal violet (CV) staining of the biofilm showed that biofilm formation was influenced in the ΔdegQ and ΔdegU mutants compared to the WT DMW1 strain (Figure 1c). The relative expression levels of the genes associated with biofilm formation were examined; the selected genes epsA, epsB, epsC, and tasA were significantly down‐regulated in ΔdegQ and ΔdegU mutants (Figure 1d). These results and the growth curve analysis (Figure S2) demonstrated that degQ and degU are critical for colony morphology and biofilm formation in B. velezensis DMW1.

FIGURE 1.

FIGURE 1

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Colony morphology and biofilm formation ability of Bacillus velezensis DMW1 and its mutants ΔdegQ and ΔdegU. The green fluorescent protein (GFP)‐labelled strains (DMW1‐gfp, ΔdegQ‐gfp, and ΔdegU‐gfp) were visualized using a fluorescent dissecting microscope. (a) Complex colony morphology of the wild‐type (WT) strain DMW1 and its mutants. (b) Biofilm formation ability of wild‐type strain DMW1 and its mutants. (c) Biofilm formation was quantitatively compared between different strains (not GFP‐labelled) by crystal violet staining. (d) The relative expression of genes involved in biofilm formation (epsA, epsB, epsC, and tasA) in wild‐type DMW1 and mutant strains (not GFP‐labelled) evaluated by reverse transcription‐quantitative PCR. Data are presented as the mean and standard deviation from three biological replicates. Different lowercase letters within the same row indicate statistically significant differences.

2.2. degQ and degU are required for swimming and swarming motility in DMW1

A plate assay with Luria–Bertani (LB) medium containing 0.3% and 0.7% (wt/vol) agar was used to test bacterial swimming and swarming motility (Figure 2a). After 5 h of incubation, the WT strain demonstrated significant swimming motility with swim diameters up to 7.4 cm. The ΔdegQ mutant exhibited moderate swimming motility with a swim diameter of 5.1 cm and the ΔdegU mutant had little swimming motility with a swim diameter of 1.3 cm. The swarming motility of WT DMW1, ΔdegQ, and ΔdegU was high, moderate, and low, with swarm diameters of 6.3, 4.3, and 1.6 cm, respectively (Figure 2b). We examined the relative expression of genes related to motility and found that the selected genes swrA and swrB were moderately down‐regulated in the ΔdegQ mutant and highly down‐regulated in the ΔdegU mutant. This was consistent with the phenotypic characteristics (Figure 2c).

FIGURE 2.

FIGURE 2

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Swimming and swarming motility of Bacillus velezensis DMW1 and its mutants ΔdegQ and ΔdegU. (a) Swimming and swarming motility of wild‐type (WT) DMW1 and mutant strains. (b) The colony diameter was measured after 5 h (swimming) and 30 h (swarming). (c) The relative expression of the genes involved in swarming ability (swrA and swrB) in wild‐type DMW1 and mutant strains evaluated by reverse transription‐quantitative PCR. Data are presented as the mean and standard deviation from three biological replicates. Different lowercase letters within the same row indicate statistically significant differences.

2.3. ΔdegQ and ΔdegU showed decreased colonization ability

Biofilm formation and motility of biocontrol agents are important in bacterial colonization of plant surfaces (Gao et al., 2016; Weng et al., 2013). Given that ΔdegQ and ΔdegU mutants exhibited reduced biofilm formation and motility, the degree of colonization of DMW1 and its derivative strains on wheat seedling roots was monitored. DMW1‐gfp heavily colonized the surface of the primary roots' outer epidermal cells (Figure 3a). However, the ΔdegQ‐gfp and ΔdegU‐gfp mutant cells colonized fewer roots compared to the WT DMW1 strain (Figure 3a). Using isolation, serial dilution, plating, and fluorescence microscopy, B. velezensis (DMW1‐gfp, ΔdegQ‐gfp, and ΔdegU‐gfp) on wheat roots in a hydroponic system were counted 2, 4, 6, and 8 days after inoculation. The populations of the ΔdegQ and ΔdegU mutants were smaller than the WT DMW1 population at all time points, indicating the reduced colonization capacity of mutant strains (Figure 3b).

FIGURE 3.

FIGURE 3

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Colonization abilities of Bacillus velezensis DMW1 (WT) and its mutants ΔdegQ and ΔdegU. (a) Confocal laser scanning microscopy images of wheat roots colonized by DMW1‐gfp, ΔdegQ‐gfp, and ΔdegU‐gfp. Luria–Bertani medium was used as the control. Bar = 200 μm. (b) The number of bacterial colonies in wheat seedling roots was measured. Data are expressed in terms of log10(cfu per gram of fresh wheat root).

2.4. ΔdegQ and ΔdegU showed decreased control efficacy toward fungal disease

We conducted an antifungal activity experiment against the pathogen Fusarium graminearum on plates using vegetative cells and crude extract of WT DMW1 and its mutants ΔdegQ and ΔdegU. The WT strain showed a strong inhibitory effect against F. graminearum, with either the cells or crude extract, whereas the mutants had poor inhibition of mycelial growth (Figure 4a,b). In pot experiments, the lesions on wheat leaves were reduced after treatment with DMW1. After treatment with ΔdegQ and ΔdegU, the leaf lesions were not reduced compared to the control (Figure 4c,d). These data indicate that degQ and degU are involved in the regulation of antifungal activity and biocontrol efficacy in DMW1. This may be due to changes in the levels of antimicrobial metabolites.

FIGURE 4.

FIGURE 4

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Antagonistic activity of Bacillus velezensis DMW1 (wild type, WT) and its mutants ΔdegQ and ΔdegU against Fusarium graminearum. (a) In vitro inhibition of F. graminearum by Bacillus strains and crude extracts of their antimicrobial metabolites. (b) Control efficacy of DMW1 and its mutants ΔdegQ and ΔdegU against F. graminearum on detached wheat leaves. Luria–Bertani medium was used as the control (CK). (c) The lesion lengths on wheat leaves were recorded 4 days after inoculation. Different lowercase letters indicate statistically significant differences.

2.5. degQ and degU regulate the production of antimicrobial metabolites in DMW1

To investigate the specific regulation of antimicrobial metabolites by degQ and degU, the levels of major antimicrobial compounds (surfactin, macrolactin, iturin, fengycin, bacillaene, and difficidin) produced by DMW1 and its derivative strains were determined by high‐performance liquid chromatography (HPLC) analysis. Deletion of the degQ and degU genes led to increased yields of surfactin (approximately 3.1‐fold) and macrolactin (approximately 1.8‐fold) compared to WT DMW1 (Figure 5a,b). However, in comparison to the WT strain, the levels of other metabolites (iturin, fengycin, bacillaene, and difficidin) were reduced in ΔdegQ and ΔdegU (2.9‐, 4.0‐, 1.8‐, and 2.9‐fold, respectively; Figure 5c–f). In our previous research, iturin and fengycin were proved to be the antifungal factors of DMW1 (Yu et al., 2023). Hence, the loss of antifungal activity of ΔdegQ and ΔdegU was most likely due to the decreased content of iturin and fengycin. Surfactin and the three polyketides (macrolactin, bacillaene, and difficidin) possess antibacterial ability. The antagonistic effect against Xanthomonas oryzae pv. oryzae was slightly decreased by the knockout of degQ and degU. This may be due to the reduced production of difficidin, which has the best antibacterial ability among six major antimicrobial compounds (data not shown). These data indicate that degQ and degU are important in regulating the production of antimicrobial metabolites. This includes negatively regulating surfactin and macrolactin and positively regulating iturin, fengycin, bacillaene, and difficidin.

FIGURE 5.

FIGURE 5

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Production of antimicrobial metabolites surfactin (a), macrolactin (b), iturin (c), fengycin (d), bacillaene (e), and difficidin (f) in Bacillus velezensis DMW1 (wild type, WT) and its mutants ΔdegQ and ΔdegU. Data are presented as the mean and standard deviation from three biological replicates. Different lowercase letters within the same row indicate statistically significant differences.

2.6. degQ and degU regulate the expression of the biosynthetic genes for antimicrobial metabolites

To further study the regulation of antimicrobial metabolites by degQ and degU at the transcript level, reverse transcription‐quantitative PCR (RT‐qPCR) analysis was performed to determine the expression levels of biosynthetic genes for each metabolite (surfactin, srfAA; macrolactin, mlnA; iturin, ituA; fengycin, fenA; bacillaene, baeA; and difficidin, dfnA) in WT DMW1 and its derivative strains. Knockout of degQ and degU resulted in the 46‐ and 26‐fold up‐regulation of srfAA and mlnA, respectively, compared to WT DMW1 (Figure 6a,b). The transcript levels of metabolite genes ituA, fenA, baeA, and dfnA were reduced by 2–5 times in the ΔdegQ and ΔdegU mutants compared to levels in DMW1 (Figure 6c–f). These data are consistent with the antimicrobial metabolite production results and confirm that degQ and degU regulate the expression of biosynthetic genes in DMW1.

FIGURE 6.

FIGURE 6

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Expression levels of antimicrobial metabolite genes in mutants ΔdegQ and ΔdegU relative to those for the wild‐type (WT) DMW1 strain. (a) srfAA (surfactin). (b) mlnA (macrolactin). (c) ituA (iturin). (d) fenA (fengycin). (e) baeA (bacillaene). (f) dfnA (difficidin). Data are presented as the mean and standard deviation from three biological replicates. Different lowercase letters within the same row indicate statistically significant differences.

2.7. DegU can bind to the promoter region of the biosynthetic operon for each antimicrobial metabolite

Because both the ΔdegQ and ΔdegU mutants were involved in regulating the transcription of antimicrobial metabolite genes, we conducted further experiments to understand the interaction between the DegU protein and promoters of biosynthetic operons. We tested the ability of recombinant DegU to bind to the srfA and mln promoters (which are negatively regulated) and the itu, fen, bae, and dfn promoters (which are positively regulated) using an electrophoretic mobility shift assay (EMSA; Figure 7). Mixing the purified recombinant DegU protein with the 5‐carboxyfluorescein (FAM)‐labelled promoters caused a shift in the mobility of all six DNA fragments. With the addition of unlabelled DNA fragments, a concentration‐dependent abolishment of the DNA shift was observed. The EMSA revealed DegU binding with p srfA , p mln , p itu , p fen , p bae , and p dfn in a dose‐dependent manner (Figure 7). To quantify the binding affinity of DegU to the promoters of the six operons, a microscale thermophoresis (MST) assay was performed. MST results showed that DegU bound to p srfA , p mln , p itu , p fen , p bae , and p dfn with affinity K d values of 0.67, 0.13, 0.03, 1.17, 0.11, and 0.14 μM, respectively. These data were consistent with the EMSA results (Figure 7). Based on the EMSA and MST results, DegU was shown to negatively or positively regulate the biosynthesis of antimicrobial metabolites (surfactin, macrolactin, iturin, fengycin, bacillaene, and difficidin) by binding to their operon promoters.

FIGURE 7.

FIGURE 7

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DegU of Bacillus velezensis DMW1 directly bound to the promoters of srfA (a), mln (b), itu (c), fen (d), bae (e), and dfn (f). Upper panels, electrophoretic mobility shift assay. Different concentrations of DegU were added to mixtures containing 80 ng of FAM probe. Unlabelled probes were added as cold excess competitors. Lower panels represent microscale thermophoresis measurements of binding affinity. A constant concentration (20 μM) of FAM‐labelled DNA fragments was used to titrate against increasing concentrations of DegU.

The promoter of the srfA operon (p srfA ) was selected for further study. Two truncated fragments of p srfA , promoter P01 (−328 to −36; from the 328th base upstream of the transcription start site [TSS] to the 36th base upstream of the TSS) and promoter P02 (−35 to +263; from the 35th base upstream of the TSS to the 263rd base downstream of the TSS) were labelled with a FAM probe for EMSA (Figure 8a). Figure 8b shows that no bound probe was present in the mixture of DegU and promoter P01, whereas the addition of DegU led to a shift of promoter P02, which is similar to p srfA . These results demonstrate that DegU can directly bind to the P02 promoter containing the −35 and −10 regions, which is the binding site of RNA polymerase II.

FIGURE 8.

FIGURE 8

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Electrophoretic mobility shift assay of DegU and truncated promoters of srfA. (a) Scheme of two truncated promoters of P srfA , P01 and P02. (b, c) Different concentrations of DegU were added to mixtures containing 80 ng of FAM probe. Unlabelled probes were added as cold excess competitors.

3. DISCUSSION

Bacillus spp. include many biological control agents and promote plant growth and control plant diseases, in addition to being promising alternatives to commercial chemicals (Afzal et al., 2019; Backer et al., 2018). B. velezensis DMW1 is an endophytic and genetically amenable strain with strong biocontrol characteristics. Detailed research on the biocontrol mechanism of DMW1 should be conducted to further its use as a biocontrol agent.

The behaviour of B. subtilis is enabled by the transcription factor DegU, which is the response regulator in the DegS/DegU two‐component system (Murray et al., 2009). The pleiotropic regulatory protein DegQ regulates the expression of many secreted enzymes, intracellular proteases, and degradative enzymes through enhancement of the phosphorylated form of DegU (Kobayashi, 2007). In the present study, we created the mutant strains ΔdegQ and ΔdegU to investigate their roles in the biocontrol mechanism of B. velezensis DMW1.

We found that the deletion of degQ and degU in DMW1 adversely affected colony morphology and biofilm formation (Figure 1). These data were consistent with reports showing that degQ and degU affect the colony form and biofilm formation of Bacillus spp. (Verhamme et al., 2007; Wang et al., 2015). Many regulatory factors are associated with biofilm formation. For example, ResD/ResE can improve the biosynthesis of terminal oxidases to activate biofilm formation (Zhou et al., 2018). SinR and AbrB can suppress biofilm formation, while phosphorylated Spo0A can relieve this suppression (Molle et al., 2003). In addition to biofilm formation, knockout of degQ and degU was shown to inhibit the transcription of swrA and swrB and to decrease the motility of the DMW1 strain (Figure 2). The ΔdegU mutant had reduced swimming and swarming motility compared to ΔdegQ. The swarming motility of B. subtilis can be activated by very low levels of DegU‐P (Verhamme et al., 2007). Hence, the moderate motility of the ΔdegQ mutant may have been due to the low phosphorylation level of DegU. Also, the root colonization abilities of ΔdegQ and ΔdegU were considerably weaker than that of WT DMW1 (Figure 3). This demonstrates that the biofilm formation ability and motility in Bacillus are closely associated with its colonization ability.

Bacillus spp. produce many secondary metabolites with antimicrobial activity that inhibit phytopathogens and help protect plants. The antifungal activity assay showed that the mutants ΔdegQ and ΔdegU and their crude extracts had little antifungal activity compared to the WT strain (Figure 4). Therefore, the degQ and degU genes might regulate the production of antimicrobial metabolites in DMW1. The lipopeptides iturin and fengycin exhibit strong antagonistic effects against F. graminearum and are the major antifungal substances produced by Bacillus spp. (Hanif et al., 2019; Xie et al., 2022). Our HPLC results showed that the deletion of degQ and degU significantly reduced iturin and fengycin yields based on the calculation of peak areas, which corroborate our predictions (Figure 5c,d). Similar reports have shown that the introduction of degQ in B. subtilis 168 results in increased production of plipastatin (analogue of fengycin) and iturin A (Tsuge et al., 1999, 2005). Knockout of degU in Bacillus amyloliquefaciens fmbJ reduces the yield of bacillomycin D and fengycin (Sun et al., 2021), but the underlying mechanism is unknown. In addition to iturin and fengycin, degQ and degU negatively regulated the biosynthesis of surfactin and macrolactin while positively regulating bacillaene and difficidin production in DMW1 (Figure 5). The biosynthesis of antimicrobial metabolites in Bacillus spp. is regulated by a complex molecular network. CodY and Spo0A positively regulate bacillomycin D biosynthesis; RapA4 inhibits the production of surfactin and PhrA4 suppresses the activity of RapA4. AbrB can inhibit the production of bacitracin via direct binding to the promoter regions of the bacABC operon (Liang et al., 2020; Sun et al., 2021; Wang et al., 2017). The present study is the first to demonstrate the regulatory effect of degQ and degU on the polyketides of Bacillus spp.

The response regulator DegU can bind target DNA promoters and activate or repress transcription. For example, DegU binds to p bmy , the promoter of the bacillomycin D biosynthetic operon, and directly triggers the expression of the metabolite (Koumoutsi et al., 2007). Unphosphorylated DegU binds to the inverted repeat on the comK promoter, which is crucial for the competence development of Bacillus (Shimane & Ogura, 2004). Unlike DegU, the regulatory effect of DegQ is indirect and mediated by DegU. This is because DegQ has no resemblance to normal transcriptional regulators, such as DNA‐binding proteins, and DegQ overexpression cannot compensate for the loss of DegU in terms of bacillomycin D production (Xu et al., 2014). The RT‐qPCR results showed that the knockout of degQ and degU resulted in the up‐regulation of srfAA and mlnA. In contrast, the ituA, fenA, baeA, and dfnA genes were significantly down‐regulated compared to WT DMW1. These data imply that DegU might alter gene expression by binding to the promoter region of the operon (Figure 6). Our EMSA and MST results revealed that the recombinant DegU could directly target the promoters of srfA, mln, itu, fen, bae, and dfn (Figure 7). Consistent with the EMSA, MST, RT‐qPCR, and antimicrobial metabolite production results, DegU was demonstrated to regulate the biosynthesis of antimicrobial metabolites (iturin, fengycin, surfactin, macrolactin, difficidin, and bacillaene) by binding to their operon promoters and thus regulating their transcript levels. ComA can activate the transcription of srfA by binding to the srfA promoter (Nakano et al., 1991). To investigate the suppression mechanism of DegU on srfA, two truncated promoters (P01 and P02) were examined using EMSA. DegU could not bind to P01, which contained the ComA binding site, but it was able to bind to P02, a fragment from the 35th base upstream of the TSS to the 263rd base downstream of the TSS (Figure 8). Given that −35 and −10 hexameric sequences can be recognized by RNA polymerase II, we speculated that DegU might inhibit transcription by forming steric hindrance to block the binding of RNA polymerase II to the promoter.

In conclusion, the results indicate that the degQ and degU genes influence the biocontrol activity of DMW1 by altering multicellular behaviour and the production of antimicrobial metabolites. The ΔdegQ and ΔdegU mutants had decreased biofilm formation, cell motility, root colonization, and antifungal efficacy. The degQ and degU genes could negatively regulate the production of surfactin and macrolactin while positively regulating iturin, fengycin, bacillaene, and difficidin production. The response regulator DegU regulates the expression levels of antimicrobial metabolite operons by targeting their promoter regions. This provides further control of the dynamic balance of secondary metabolism in DMW1. However, the determination of the specific mechanism used by DegU, with different phosphorylation levels, to regulate different antimicrobial metabolites will require additional research.

4. EXPERIMENTAL PROCEDURES

4.1. Microbial strains and growth conditions

The bacterial strains used in this study are listed in Table S1. B. velezensis DMW1 was stored at the Laboratory of Biocontrol and Bacterial Molecular Biology, Nanjing Agricultural University. DMW1 and its derivative strains were grown in LB medium at 37°C and, where appropriate, MSgg medium was used. The fungal strain F. graminearum PH‐1 was routinely grown in potato dextrose agar (PDA) at 25°C.

4.2. Mutant construction

The knockout mutants ∆degQ and ∆degU were constructed using the markerless CRISPR/Cas9 system (Altenbuchner, 2016; Yu et al., 2023). Briefly, the knockout vectors were constructed by integrating single‐guide RNA (sgRNA) and homologous fragments into the original plasmid pJOE8999. The knockout vectors were transformed into the DMW1 strain according to a previous report (Anagnostopoulos & Spizizen, 1961). Afterwards, the positive colonies were verified for the loss of whole open reading frames of degQ and degU by colony PCR and sequencing, followed by culturing at 42°C and 50°C for 24 h to eliminate the temperature‐sensitive knockout vectors. The primers used in this study are listed in Table S2.

4.3. Biofilm and colony morphology assays

The CV staining method was used to estimate biofilm formation (Morikawa et al., 2006). B. velezensis DMW1 and its mutant strains ΔdegQ and ΔdegU were cultured overnight and transferred into LB medium at 37°C with shaking at 200 rpm until OD600 = 1.0. Subsequently, 2 μL of the bacterial suspensions was transferred to a 24‐well plate containing 2 mL of MSgg liquid medium and incubated at 37°C without shaking (Kearns et al., 2005). After 48 h, the culture was discarded, and 5 mL of 1% (wt/vol) CV was added to stain the biofilm for 15 min. Then, the CV was removed and the tubes were washed three times with sterile water. After the tubes were dried, 4 mL of ethanol was added to dissolve the CV, and the attached cells were quantified by measuring their optical density at 575 nm. The experiment was repeated three times each with three replications.

For colony morphology analysis, 5 μL of overnight suspensions of DMW1 and mutant strains were added to the centre of an MSgg solid plate and incubated at 37°C for 24 h. The biofilm and colony images were made using an SMZ25 stereomicroscope (Nikon).

4.4. Growth curve assay

B. velezensis DMW1 and its mutant strains ΔdegQ and ΔdegU were cultured overnight at 37°C in LB medium with shaking (200 rpm) and subsequently diluted 1:100 in the same medium. The OD600 of each strain was measured at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 36, and 48 h, and the experiments were repeated three times.

4.5. Swimming and swarming motility assay

LB solid medium was prepared by the addition of 0.3% and 0.7% agar for swimming and swarming assays, respectively, according to Ansari and Ahmad (2019) with some modifications. WT DMW1 and its mutants ΔdegQ and ΔdegU were cultivated overnight at 37°C and 200 rpm and were then transferred into new LB medium and cultivated until they reached an OD600 of 1.0. Then, 5 μL of the bacterial cultures was dropped on the centre of an agar plate and incubated at 37°C. The colony diameter was measured after 5 h (swimming) and 30 h (swarming). The swimming and swarming experiments were repeated three times.

4.6. Determination of Bacillus colonization ability in the roots of wheat seedlings

The Bacillus strains were labelled with green fluorescent protein (GFP) using the CRISPR/Cas9 system to study root colonization. The wheat seeds (cv. Jimai 22) were surface sterilized with 70% ethanol and 5% NaOCl and then washed three times with sterile water. Seeds were placed on sterile filter paper with sterilized water and incubated in a dark environment at 25°C for germination. After 2 days, equally sized wheat seedlings were selected and soaked in the culture of Bacillus strains (DMW1‐gfp, ΔdegQ‐gfp, and ΔdegU‐gfp, OD600 = 1.0) at 30°C for 1 h. Then, the seedlings were transplanted into tubes with 20 mL of 0.5× Murashige & Skoog medium and incubated in a greenhouse chamber at 28°C with a 16:8 h (light:dark) photoperiod. After 4 days, the seedling roots were cut into 0.5‐ to 1‐cm pieces and observed under a confocal laser scanning microscope (LSM710; Zeiss). Root samples were taken at 0, 2, 4, 6, and 8 days after inoculation, and 0.1 g of roots was homogenized in 2 mL phosphate‐buffered saline using an automatic grinder (Tissuelyser‐64; Jinxin Ltd). The homogenate was serially diluted and placed on LB plates for growth. After 24 h, the bacterial colonies were counted.

4.7. In vitro and in vivo antifungal assay

The in vitro antifungal activity of Bacillus strains was studied on PDA plates. A 6‐mm‐diameter plug with fungal mycelia was placed in the centre of a PDA plate. After incubation for 24 h, 5 μL of bacterial suspension or crude extract was added 3 cm away from the plate centre. Images were taken 2 days later, and the diameter of inhibitory zones was measured. The experiment was repeated three times independently, with three replicates for each treatment. For the in vivo antifungal assay, 7‐day‐old leaves of wheat seedlings were used. The cultures of Bacillus strains (OD600 = 1.0) were sprayed evenly on the detached leaves, followed by placing a mycelial plug on the centre of the leaves after 1 day. The leaves were kept at 100% humidity at 25°C. Images were taken and lesion length was recorded after 4 days of incubation. The experiment was repeated three times, with 18 leaves for each treatment.

4.8. The detection of antimicrobial metabolite production

The extraction of crude metabolites from DMW1 and its derivative mutants was performed using the method described by Yu et al (2021). WT DMW1 and its derivative mutants were grown in LB medium for 48 h at 37°C with shaking at 200 rpm. Sterilized resin XAD16N (Sigma) was added to the culture during fermentation to absorb compounds. Afterwards, resin XAD16N was redissolved with methanol and incubated at 37°C for 4 h. The extracts were concentrated using a rotary evaporator, and the crude extract of metabolites was obtained by suspension in methanol and filtration (0.22 μm nylon). Then, an HPLC system was used to detect the production of antimicrobial metabolites synthesized by DMW1 and mutant strains. The mobile phase used in the study included A (HPLC‐grade acetonitrile containing 0.1% trifluoroacetic acid) and B (Milli‐Q water containing 0.1% trifluoroacetic acid). The detection procedure was a gradient elution from 5% A to 95% A, and then holding 95% A for 10 min. UV absorption at 210 nm and 280 nm was used to detect multiple lipopeptides and polyketides, respectively. The injection volume was 5 μL and the flow rate was 1 mL/min. The corresponding peak for each metabolite was identified by the combined genetic and chemical approach used in a previous report (Figure S3; Yu et al., 2023). The peak area was calculated by Waters Empower 3 software.

4.9. RT‐qPCR

DMW1 and its mutant strains were cultured for 48 h, and total RNA was isolated using RNAiSO reagent (TaKaRa) following the manufacturer's guidelines. Afterwards, RNA was reverse‐transcribed into cDNA using HiScript III RT SuperMix (Vazyme) according to the manufacturer's instructions. The expression levels of swarming‐related genes (swrA, swrB), biofilm‐related genes (epsA, epsB, epsC, tasA), and metabolite biosynthetic genes (ituA, fenA, srfAA, mlnA, baeA, dfnA) of WT DMW1, ∆degQ, and ∆degU were detected in a 7500 fast real‐time PCR detector with the 2−∆∆Ct method. The 16S rRNA gene was used for normalization. The experiment was repeated three times independently, with three replicates for each treatment.

4.10. EMSA

DNA fragments, including promoter regions of antimicrobial metabolite operons (ituA, fenA, srfAA, mlnA, baeA, and dfnA), were amplified using primers labelled with FAM. The purified PCR product (80 ng) was mixed with different concentrations of DegU protein in a 20‐μL reaction mixture containing 10 mM Tris (pH 7.5), 50 mM KCl, 1 mM dithiothreitol, and 0.4% glycerol. Subsequently, the reaction mixture was incubated at room temperature for 30 min, followed by electrophoresis on a 6% native‐PAGE gel at 4°C in 0.5× TBE buffer. The DNA fragments were visualized using a ChemiDoc MP system (Bio‐Rad) at an excitation wavelength of 492 nm. In this assay,  different amounts of unlabelled DNA were added to the reaction as cold excess competitors.

4.11. MST assay

The binding of recombinant DegU to the promoters of antimicrobial metabolite operons was detected using Monolith NT.115 (NanoTemper Technologies) as described by Xu et al. (2018). A constant concentration (20 μM) of the FAM‐labelled DNA fragments used in the EMSA was titrated against increasing concentrations of DegU in MST buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.05% Tween 20). The mixtures were loaded into the MST instrument using premium‐coated capillaries at 25°C. Data were analysed using NanoTemper analysis software v. 1.2.101 (NanoTemper Technologies). All experiments were performed in triplicate.

4.12. Statistical analysis

Data were statistically analysed using one‐way analysis of variance followed by Duncan's multiple range test (p < 0.05) using SPSS software (SPSS Inc.).

Supporting information

Figure S1. Verification of positive colonies of ∆degQ and ∆degU. Lines 1–4 were amplified using the primers KOdegQ‐VF/KOdegQ‐VR, while lines 5–7 were amplified using the primers KOdegU‐VF/KOdegU‐VR. Lines 1, 2, and 5 represent the corrected colonies of mutant strains. Lines 3 and 6 represent the sterile water negative controls, and lines 4 and 7 represent the positive controls of genomic DNA of wild‐type DMW1.

Click here for additional data file. (1.5MB, tif)

Figure S2. Growth curves of wild‐type DMW1 and its mutants ∆degQ and ∆degU.

Click here for additional data file. (155.6KB, tif)

Figure S3. High‐performance liquid chromatography analysis of the metabolites in DMW1 and its mutants ∆degQ and ∆degU. The fermentation time was 48 h. Lipopeptides (a) and polyketides (b) were detected by UV absorption at 210 and 280 nm, respectively. WT, wild type DMW1; itu, iturin; fen, fengycin; srf, surfactin; bae, bacillaene; mln, marcolactin; dfn, difficidin.

Click here for additional data file. (204.8KB, tif)

Table S1. Bacterial strains and plasmids used in this study.

Click here for additional data file. (23KB, docx)

Table S2. Primers used in this study.

Click here for additional data file. (28.4KB, docx)

ACKNOWLEDGEMENTS

This work was supported by the Korea‐China Cooperation Research Project (2022YFE0198100), the Natural Science Foundation of Jiangsu Province (BK 20201239), the National Natural Science Foundation of China (31972325, 32172490), the Natural Science Foundation for Excellent Youth Scholars of Jiangsu Province, China (BK 20200078), and the Guidance Foundation of Sanya Institute of Nanjing Agricultural University (NAUSY‐M18).

Yu, C. , Qiao, J. , Ali, Q. , Jiang, Q. , Song, Y. , Zhu, L. et al. (2023) degQ associated with the degS/degU two‐component system regulates biofilm formation, antimicrobial metabolite production, and biocontrol activity in Bacillus velezensis DMW1 . Molecular Plant Pathology, 24, 1510–1521. Available from: 10.1111/mpp.13389

Chenjie Yu and Junqing Qiao contributed equally to this work.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. Verification of positive colonies of ∆degQ and ∆degU. Lines 1–4 were amplified using the primers KOdegQ‐VF/KOdegQ‐VR, while lines 5–7 were amplified using the primers KOdegU‐VF/KOdegU‐VR. Lines 1, 2, and 5 represent the corrected colonies of mutant strains. Lines 3 and 6 represent the sterile water negative controls, and lines 4 and 7 represent the positive controls of genomic DNA of wild‐type DMW1.

Click here for additional data file. (1.5MB, tif)

Figure S2. Growth curves of wild‐type DMW1 and its mutants ∆degQ and ∆degU.

Click here for additional data file. (155.6KB, tif)

Figure S3. High‐performance liquid chromatography analysis of the metabolites in DMW1 and its mutants ∆degQ and ∆degU. The fermentation time was 48 h. Lipopeptides (a) and polyketides (b) were detected by UV absorption at 210 and 280 nm, respectively. WT, wild type DMW1; itu, iturin; fen, fengycin; srf, surfactin; bae, bacillaene; mln, marcolactin; dfn, difficidin.

Click here for additional data file. (204.8KB, tif)

Table S1. Bacterial strains and plasmids used in this study.

Click here for additional data file. (23KB, docx)

Table S2. Primers used in this study.

Click here for additional data file. (28.4KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon request.

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