Background: Bacillus cereus0–9, a Gram-positive, endospore-forming bacterium isolated from healthy wheat roots in our previous research, is considered to be an effective biocontrol strain against several soil-borne plant diseases. SpoVG, a regulator that is broadly conserved among many Gram-positive bacteria, may help this organism coordinate environmental growth and virulence to survive. This study aimed to explore the multiple functions of SpoVG inB. cereus0–9.Methods: The gene knockout strains were constructed by homologous recombination, and the sporulation process ofB. cereus0–9 and its mutants were observed by fluorescence staining method. We further determined the spore yields and biofilm formation abilities of test strains. Transcriptional fusion strains were constructed by overlapping PCR technique, and the promoter activity of the target gene was detected by measuring its fluorescence intensity.The biofilm production and colonial morphology ofB. cereus0–9 and its mutants were determined to study the functions of the target genes, and the transcription level of the target gene was determined by qRT-PCR.Results: According to observation of the sporulation process ofB. cereus0–9 in germination medium, SpoVG is crucial for regulating sporulation stage V ofB. cereus0–9, which is identical to that ofBacillus subtilisbut differs from that ofBacillus anthracis. In addition, SpoVG could influence biofilm formation ofB. cereus0–9. The transcription levels of two genes closely related to biofilm-formation,sipWandcalY, were downregulated in a ΔspoVGmutant. The role of SpoVG in regulating biofilm formation was further explored by deleting the genesabrBandsinRin the ΔspoVGmutant, respectively, generating the double mutant strains ΔspoVGΔabrBand ΔspoVGΔsinR. The phenotypes of these double mutants were congruent with those of the singleabrBandsinRdeletion strains, respectively, which showed increased biofilm formation. This indicated thatspoVGwas located upstream ofabrBandsinRin the regulatory pathway ofB. cereusbiofilm formation. Further, the results of qRT-PCR and the luminescence intensity of transcriptional fusion strains indicated thatspoVGgene deletion could inhibit the transcription of Spo0A.Conclusions: SpoVG, an important regulator in the sporulation ofB. cereus, is located upstream of Spo0A and participates in regulation of biofilm formation ofB. cereus0–9 through regulating the transcription level ofspo0A. Sporulation and biofilm formation are crucial mechanisms by which bacteria respond to adverse conditions. SpoVG is therefore an important regulator of Spo0A and is crucial for both sporulation and biofilm formation ofB. cereus0–9. This study provides a new insight into the regulatory mechanism of environmental adaptation in bacteria and a foundation for future studies on biofilm formation ofB. 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 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.

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.

Bacillus cereus 0-9, a Gram-positive endospore-forming bacterium isolated from healthy wheat roots, has biological control capacity against several soil-borne plant diseases of wheat such as sharp eyespot and take-all. The bacterium can produce various biofilms that differ in their architecture and formation mechanisms, possibly for adapting to different environments. The gapB gene, encoding a glyceraldehyde-3-phosphate dehydrogenase (GAPDH), plays a key role in B. cereus 0-9 biofilm formation. We studied the function of GapB and the mechanism of its involvement in regulating B. cereus 0-9 biofilm formation. GapB has GAPDH activities for both NAD⁺- and NADP⁺-dependent dehydrogenases and is a key enzyme in gluconeogenesis. Biofilm yield of the ΔgapB strain decreased by 78.5% compared with that of wild-type B. cereus 0-9 in lysogeny broth supplemented with some mineral salts (LBS), and the ΔgapB::gapB mutants were recovered with gapB gene supplementation. Interestingly, supplementing the LBS medium with 0.1–0.5% glycerol restored the biofilm formation capacity of the ΔgapB mutants. Therefore, GapB regulates biofilm formation relative to its function in gluconeogenesis. To illustrate how GapB is involved in regulating biofilm formation through gluconeogenesis, we carried out further research. The results indicate that the GapB regulated the B. cereus 0-9 biofilm formation independently of the exopolysaccharides and regulatory proteins in the typical SinI/R system, likely owing to the release of extracellular DNA in the matrix. Transcriptome analysis showed that the gapB deletion caused changes in the expression levels of only 18 genes, among which, lrgAB was the most significantly increased by 6.17-fold. We confirmed this hypothesis by counting the dead and living cells in the biofilms and found the number of living cells in the biofilm formed by the ΔgapB strain was nearly 7.5 times than that of wild-type B. cereus 0-9. Therefore, we concluded that the GapB is involved in the extracellular DNA release and biofilm formation by regulating the expression or activities of LrgAB. These results provide a new insight into the regulatory mechanism of bacterial biofilm formation and a new foundation for further studying the stress resistance of B. cereus.