Gang Wang

Copper serves as an indispensable trace element in living organisms, playing pivotal roles in fundamental physiological processes including enzyme assembly, antioxidant defense, and metabolic regulation. However, excess copper can induce oxidative damage. MerR-family regulator CueR acts as the core mediator of copper homeostasis in prokaryotes, but its mechanism of action in P. chlororaphis zm-1, a plant biocontrol bacterium that inhibits peanut stem rot, remains unclear. In this study, we investigated the regulatory function of CueR using molecular techniques including gene deletion, electrophoretic mobility shift assay, and quantitative real-time PCR. The results demonstrated that the transcriptional regulator gene cueR and the P _1B -type ATPase gene cueA (involved in copper efflux) form the cueRA operon in strain zm-1, with a 4-bp overlap and co-transcriptional feature. CueR directly binds to the promoter region and positively activates cueRA expression upon copper ion induction, thereby driving CueA-mediated efflux of excess copper ions. The deletion of cueR or cueA significantly reduced copper tolerance, increased intracellular copper content, and impaired peanut rhizosphere colonization ability of the strain, while these phenotypes were restored in the complementation strains. This study deciphers the regulatory mechanism of the cueRA operon in P. chlororaphis and establishes a correlation analysis between this mechanism and the rhizosphere colonization capacity of the strain. The findings provide a molecular basis for understanding the diversity of copper homeostasis regulation in Pseudomonas and optimizing the biocontrol application of this bacterium. IMPORTANCE: The CueR-regulated copper efflux system is essential for bacterial survival under copper overload conditions. While this system has been extensively characterized in human pathogens, its function in plant-beneficial bacteria remains poorly understood. Here, we investigate the agricultural biocontrol strain P. chlororaphis zm-1 and delineate the regulatory mechanism of CueR through molecular approaches, leading to the identification of the cueRA operon. Furthermore, we establish a functional link between CueR and rhizosphere colonization, thereby providing a framework for understanding CueR-mediated regulation in plant-associated bacteria.

Bacillus cereus 0-9 is a biocontrol strain with abundant antimicrobial peptide (AMP) biosynthesis gene clusters. This strain exhibits significant antibacterial activity against Gram-positive bacteria. Notably, the deletion of gene abrB, encoding global transcriptional regulator AbrB, resulted in a 62.74% increase in the antibacterial efficacy of strain 0-9. EMSA studies revealed that the gene cluster lanT, responsible for producing key components of antibacterial activity, is dual-regulated by AbrB. In the ΔabrB strain, five novel AMPs were identified, with MICs ranging from 125 to 250 μg/mL against Corynebacterium glutamicum, surpassing the antibacterial efficacy of Nisin Z. Furthermore, the presence of K⁺, Ni²⁺, Zn²⁺, and Ca²⁺ ions significantly enhances the antibacterial activity of the peptides TPV21, LLV7, and CPR6. The genes FRY47_03100 and FRY47_15105, which encode precursor proteins associated with LLV7 and CPR6, are also directly regulated by AbrB. This research expands our understanding of Bacillus-derived AMPs regulated by AbrB in B. cereus 0-9.

The biofilm formation of Bacillus cereus brings new challenges to food hygiene control. We investigated the multifunctional role of FtsH, an ATP-dependent metalloprotease, in regulating biofilm formation and environmental adaptation in B. cereus 0-9. Through homologous recombination, we generated ΔftsH and related mutants, subsequently evaluating their biofilm production using crystal violet staining, colonial morphology, and sporulation capacity, respectively. Transcriptional analysis using luminescent reporter strains revealed that biofilm-associated genes (sipW and calY) are downregulated in a FtsH-dependent manner in the mutants. Our findings demonstrate FtsH modulates Spo0E protein stability (confirmed by Western blot) and consequently influences Spo0A phosphorylation levels. Thus, FtsH as a crucial regulator of both sporulation and biofilm formation in B. cereus 0-9, mirroring its function in Bacillus subtilis while differing from Bacillus anthracis. It provides novel insights into bacterial stress response mechanisms and offers potential applications for developing biofilm eradication strategies, thereby addressing critical disinfection challenges in food processing environments.

Bacillus cereus 0-9 is a biocontrol strain isolated from a healthy wheat root, and studying cell separation after division in this strain will improve our understanding of its growth, environmental adaptation, and spread. In this work, we identified the deletion of dsfB resulted in a chaining phenotype. Four genes, lysM1, lysM2, lysM3, and lysM4, were associated with daughter cell separation in strain 0-9. Furthermore, DsfB bound to the promoter regions of lysM1 and lysM2 and induced their transcription. The peptidoglycan hydrolase activity of the lysM1 and lysM2 gene products was confirmed in vitro by site-directed mutagenesis and biochemical analyses. The addition of purified LysM1 or LysM2 proteins in vitro or the overexpression of their coding genes inhibited the chaining phenotype of ΔdsfB. Taken together, our data indicate that DsfB is involved in daughter cell separation via the positive regulation of lysM1 and lysM2 expression in B. cereus 0-9.

Bacillus cereus 0–9 is a biocontrol microorganism that antagonizes Gram-positive bacteria and pathogenic fungi, such as Staphylococcus aureus and Gaeumannomyces graminis, through the secretion of antimicrobial peptides. However, its low antibacterial activity limits its biocontrol application. In this study, a significant enhancement in antibacterial activity against S. aureus was achieved by overexpressing glucose dehydrogenase from Bacillus subtilis (BsGDH) in B. cereus 0–9, expanding the activity from 6.98 to 11.59 U/mL, representing a 66% improvement. To further improve its biocontrol capability, we aimed to improve the catalytic efficiency of BsGDH by screening 11 low-conserved residues in the protein’s second-shell via conservation analysis and molecular docking. Following three rounds of saturation mutagenesis, the specific enzyme activity and Kcat/Km value of the variant N97F/N192S/E198G reached to 289.74 U/mg and 4.95 µM⁻¹·min⁻¹, representing 5.66 and 11.38 times greater than that of the wild-type BsGDH, respectively. Molecular docking suggested that residues Gly94, Gly14, and Ile191 form a triangular region enhancing substrate affinity and enzymatic activity. Furthermore, the Root Mean Square Fluctuation analysis from molecular dynamics showed significant conformational changes in five regions of the mutants (α2 helix, α3 helix, α5 helix + β4 sheet, α8 helix + β5 sheet, and α13–14 helix), increasing the flexibility of the active pocket. Ultimately, the antibacterial activity of B. cereus 0–9 expressing N97F/N192S/E198G reached 22.79 U/mL, 2.26 times higher than that of B. cereus 0–9. This study offers a promising candidate for enhancing NAD(P)+ metabolic cycling and antimicrobial peptide synthesis in cells for industrial applications.

Background: Bacteria usually secrete a variety of extracellular enzymes to degrade extracellular macromolecules to meet their nutritional needs and enhance their environmental adaptability. Bacillus cereus 0–9, a biocontrol bacterial strain isolated from wheat roots, has three genes annotated as encoding amylases in the genome, but their functions are unknown, and whether they are involved in the colonization process of the bacterium remains to be further studied. Methods: Mutant gene strains and fluorescently tagged strains were constructed by homologous recombination, and amylase protein was expressed in the prokaryotic Escherichia coli BL21(DE3) expression system. The iodine staining method was used to measure the activity of amylase proteins. We further observed the colonization abilities of the test strains in wheat roots through frozen section technology. Results: The results showed that there were three amylase-encoding genes, amyC, amyP and amyS, in the B. cereus 0–9 genome. Among the three amylase encoding genes, only amyS produced extracellular amylase whose secretion was related to signal peptide at position 1–27. The AmyS protein encoded by the amyS gene is an α-amylase. The growth of Rhizoctonia cerealis was inhibited 84.7% by B. cereus 0–9, but the biocontrol ability of the ΔamyS strain decreased to 43.8% and that of ΔamyS/amyS was restored when the amyS gene was complemented. Furthermore, the biocontrol ability of the ΔamySec strain was decreased to 46.8%, almost the same as that of the ΔamyS mutant. Due to the deletion of the amyS gene, the colonization capacities of ΔamyS (RFP) and ΔamySec (RFP) in wheat roots decreased, while that of ΔamyS/amyS (RFP) was restored after the amyS gene was complemented, indicating that the amyS gene influences the colonization of B. cereus 0–9 in wheat roots. In addition, the colonization and biocontrol abilities of the mutant were restored after the addition of sugars, such as glucose and maltose. Conclusions: B. cereus 0–9 encodes three genes annotated as amylases, amyC, amyP and amyS. Only the deletion of the amyS gene with a signal peptide did not produce extracellular amylase. The AmyS protein encoded by the amyS gene is an α-amylase. Our results indicated that the amyS gene is closely related to the colonization abilities of B. cereus 0–9 in wheat roots and the biocontrol abilities of B. cereus 0–9 to fight against R. cerealis. The extracellular amylase produced by B. cereus 0–9 can hydrolyze starch and use glucose, maltose and other nutrients to meet the needs of bacterial growth. Therefore, it is very possible that the secretion and hydrolytic activities of extracellular amylase can promote the colonization of B. cereus 0–9 in wheat roots and play important roles in the prevention and control of plant diseases. Our results contribute to exploring the mechanisms of microbial colonization in plant roots.

In this study, eight rap-related genes were found in the Bacillus cereus 0–9 genome; five rap genes were located on chromosomes and three on large plasmids. A total of five Rap proteins in B. cereus 0–9 were annotated as ‘tetratricopeptide repeat proteins’. SMART Server analysis showed that the eight Rap proteins had typical tetrapeptide repeat sequence (TPR) domains. Biofilm assays and crystal violet staining showed that overexpression of the rapp1 and rap5 genes affected the biofilm formation of B. cereus 0–9, and the activities of Rapp1 and Rap5 proteins were inhibited by their corresponding cognate Phr, suggesting that the Rap–Phr quorum sensing (QS) system might also exist in the B. cereus 0–9 strain. In addition, overexpression of rap1 genes inhibited in the extracellular amylase decomposition capacity of B. cereus 0–9. The results of the sporulation assay indicated that overexpression of the eight rap genes inhibited the spore formation of B. cereus 0–9 to varying degrees. These results provide a reference for research on the regulation of the Rap–Phr QS system in B. cereus.

Background: Bacteria usually secrete a variety of extracellular enzymes to degrade extracellular macromolecules to meet their nutritional needs and enhance their environmental adaptability. Bacillus cereus 0-9, a biocontrol bacterial strain isolated from the wheat root, has three genes annotated as amylase in the genome, but its function is unknown, and whether it is involved in its colonization process remains to be further studied. Methods: The gene mutant strains and fluorescently tagged strains were constructed by homologous recombination, and the amylase protein obtained by the prokaryotic expression system Escherichia coli BL21(DE3). Iodine staining method was used to measure the activities of amylase proteins. We further observed the colonization abilities of test strains in wheat roots through frozen section technology.Results: The results showed that there were three amylase-encoding genes of amyC, amyP and amyS in the B. cereus 0-9 genome. Among the three amylases, only the ΔamyS mutant did not produce extracellular amylase, and the secretion ability was related to the signal peptide at positions 1-27. AmyS protein encoded by amyS gene is an α-amylase. The growth of R. cerealis was inhibited 84.7% by B. cereus 0-9, but the inhibited rate of ΔamyS strain decreased to 43.8%, and that of ΔamyS/amyS was restored when the amyS gene complementation. Further, the inhibited rate of ΔamySec strain was decreased to 46.8%, almost the same as ΔamyS mutant. Due to the deletion of amyS gene, the colonization capacity of ΔamyS (RFP) and ΔamySec (RFP) in wheat roots declined, while that of ΔamyS/amyS (RFP) was restored after the amyS gene was complemented, indicating that amyS gene influences the colonization of B. cereus 0-9 in wheat roots.For another, the colonization and biocontrol ability of the mutant was also restored after the addition of sugars such as glucose and maltose.Conclusions: B. cereus 0-9 has three genes of amyC, amyP and amyS annotated as amylase. Among them, only the deletion of amyS gene with signal peptide did not produce extracellular amylase. AmyS protein encoded by amyS gene is an α-amylase. Our results indicated the amyS gene is closely related to the colonization abilities of B. cereus 0-9 in wheat root, and the biocontrol abilities of B. cereus 0-9 to fight against R. cerealis. The extracellular amylase produced by B. cereus 0-9 can hydrolyze starch, and use the glucose, maltose and other nutrients to meet the needs of bacterial growth and multiply. Therefore, it is very possible that the secretion and hydrolytic activity of extracellular amylase can promote the colonization of B. cereus 0-9 in wheat roots, and play an important role in the prevention and control of plant diseases. Our results contribute to explore the mechanisms of microbial colonization in plant roots.

Background: Peanut stem rot is a serious plant disease that causes great economic losses. At present, there are no effective measures to prevent or control the occurrence of this plant disease. Biological control is one of the most promising plant disease control measures. In this study, Pseudomonas chlororaphis subsp. aurantiaca strain zm-1, a bacterial strain with potential biocontrol properties isolated by our team from the rhizosphere soil of Anemarrhena asphodeloides, was studied to control this plant disease. Methods: We prepared extracts of Pseudomonas chloroaphis zm-1 extracellular antibacterial compounds (PECEs), determined their antifungal activities by confrontation assay, and identified their components by UPLC-MS/MS. The gene knockout strains were constructed by homologous recombination, and the biocontrol efficacy of P. chlororaphis zm-1 and its mutant strains were evaluated by pot experiments under greenhouse conditions and plot experiments, respectively. Results: P. chlororaphis zm-1 could produce extracellular antifungal substances and inhibit the growth of Sclerotium rolfsii, the main pathogenic fungus causing peanut stem rot. The components of PECEs identified by UPLC-MS/MS showed that three kinds of phenazine compounds, i.e., 1-hydroxyphenazine, phenazine-1-carboxylic acid (PCA), and the core phenazine, were the principal components. In particular, 1-hydroxyphenazine produced by P. chlororaphis zm-1 showed antifungal activities against S. rolfsii, but 2-hydroxyphenazine did not. This is quite different with the previously reported. The extracellular compounds of two mutant strains, ΔphzH and ΔphzE, was analysed and showed that ΔphzE did not produce any phenazine compounds, and ΔphzH no longer produced 1-hydroxyphenazine but could still produce PCA and phenazine. Furthermore, the antagonistic ability of ΔphzH declined, and that of ΔphzE was almost completely abolished. According to the results of pot experiments under greenhouse conditions, the biocontrol efficacy of ΔphzH dramatically declined to 47.21% compared with that of wild-type P. chlororaphis zm-1 (75.63%). Moreover, ΔphzE almost completely lost its ability to inhibit S. rolfsii (its biocontrol efficacy was reduced to 6.19%). The results of the larger plot experiments were also consistent with these results. Conclusions: P. chlororaphis zm-1 has the potential to prevent and control peanut stem rot disease. Phenazines produced and secreted by P. chlororaphis zm-1 play a key role in the control of peanut stem rot caused by S. rolfsii. These findings provide a new idea for the effective prevention and treatment of peanut stem rot.

Isocitrate dehydrogenase (IDH), a key enzyme in the TCA cycle, participates in the formation of biofilms in Staphylococcus aureus, but it remains to be clarified whether it is involved in the formation of Bacillus cereus biofilms. In this study, we scanned the genome of B. cereus 0-9 and found a gene encoding isocitrate dehydrogenase (FRY47_22620) named icdH. The IcdH protein was expressed and purified. The enzyme activity assay showed that the protein had IDH activity dependent on NADP⁺, indicating that this gene encoded an IDH. The ΔicdH mutant and its complemented strains were obtained by a homologous recombination strategy, and crystal violet data and CLSM were measured. The results showed that the biofilm yield of the mutant ΔicdH decreased, and the biofilm morphology also changed, while the growth of ΔicdH was not affected. The extracellular pH and citric acid content results showed that the ΔicdH mutant exhibited citric acid accumulation and acidification of the extracellular matrix. In addition, the addition of excess Fe³⁺ restored the biofilm formation of the ΔicdH mutant. It is speculated that IDH in B. cereus may regulate biofilm formation by modulating intracellular redox homeostasis. In addition, we found that the icdH deletion of B. cereus 0-9 could result in a reduced sporulation rate, which was significantly different from sporulation in B. subtilis caused by interruption of the stage I sporulation process due to icdH loss. All the above results provide us with new insights for further research on IDH.