Biological control against bacterial wilt and colonization of mulberry by an endophytic Bacillus subtilis strain

Forty-five bacterial isolates were collected from surface-sterilized leaves of mulberry ( Morus alba L.). By screening their antagonistic activities against Ralstonia solanacearum in vitro , four isolates showed a remarkable inhibitory effect. The evaluation of the antagonistic strains against bacterial wilt of mulberry indicated that the strain Lu144 effectively reduced disease incidence. In the greenhouse, Lu144 displayed effective biological control against bacterial wilt of mulberry when it was applied to sterile or nonsterile soil before the infection by the pathogen. Based on bacteriological properties and 16S rRNA gene sequencing, Lu144 was identified as a strain of Bacillus subtilis . The endophytic population and infection process of Lu144 in mulberry seedlings was explored following recovery of the green fluorescent protein (GFP)-labeled Lu144 and examination of the labeled strain by confocal laser scanning microscopy. Interestingly, the infection of GFP-labeled Lu144 cells into the mulberry seedlings occurred through the cracks formed at the lateral root junctions and the zone of differentiation and elongation, and the cells were able to develop and transfer in mulberry and mainly in the intercellular spaces of different tissues. The population of the GFP-labeled Lu144 inoculant was larger and more stable in leaves than that in roots and stems.     

Macrolactin N, a new peptide deformylase inhibitor produced by Bacillus subtilis

A new 24-membered ring lactone, macrolactin N, was isolated from a culture broth of Bacillus subtilis and its structure was established by various spectral analysis. Macrolactin N inhibited Staphylococcus aureus peptide deformylase with an IC50 value of 7.5 microM and also showed antibacterial activity against Escherichia coli and S. aureus.     

Effect of the combination of bio-organic fertiliser with Bacillus amyloliquefaciens NJN-6 on the control of banana Fusarium wilt disease,crop production and banana rhizosphere culturable microflora

Soil amended with organic amendments has been suggested to be a strategy for managing the Fusarium wilt disease which severely hindered the banana production. The effects of four fertilisation regimes, including chemical fertiliser, manure composts and bio-organic fertiliser (BIO) containing Bacillus amyloliquefaciens NJN-6 for 2-year continuous application on the banana Fusarium wilt disease incidence, crop yield and rhizosphere culturable microbial community were investigated. To explore the soil microflora, plate counting method, in vitro screening method for antagonism, eco-physiological index and culture-dependent denaturing gradient gel electrophoresis method (CD DGGE) were used. The highest banana yield, culturable bacteria, actinobacteria and Bacillus populations, culturable bacteria to fungi (B/F) value, antagonistic Bacillus ratio and lowest Fusarium wilt disease incidence were observed in the BIO treatment. Based on CD DGGE results, the BIO application significantly altered the soil bacteria structure and showed highest richness and diversity. The phylogenetic analysis of the selected bands showed that the most abundant phyla were Proteobacteria and Bacteroidetes and BIO application enriched the genera Comamonas, Chitinophaga, the species Bacillus flexus and uncultured Bacillus. All the results showed that 2-year continuous application of BIO containing B. amyloliquefaciens NJN-6 more effectively controlled Fusarium wilt disease and improved fruit yields under field conditions and modulated banana rhizosphere microflora.     

A new member of the surfactin family produced by Bacillus subtilis with low toxicity on erythrocyte

A new member of the surfactin family of microbial lipopeptides has been isolated from the cell broth of Bacillus subtilis TD 7 by reverse phase high performance liquid chromatography (RP-HPLC), and identified by FT-IR, GC–MS, LC–MS/MS, ¹H-NMR and ¹³C-NMR. The surfactin-C₁₁ consists of a heptapeptide with the sequence of N - Glu - Leu - Leu - Val - Asp - Leu - Leu - C, and a β-hydroxy fatty acid chain with 11 carbons, which is a new member with the shortest hydrophobic chain among the surfactin family produced by B. subtilis. The critical micelle concentration (CMC) of the surfactin-C₁₁ is 5.59 × 10⁻⁵ mol/L in 10 mmol/L phosphate buffer solution (PBS, pH 7.4) at 25 °C, and the surface tension at CMC (γ_CMC) is 34.42 mN/m. The hemolytic activities of the surfactin-C₁₁ on 2 % defibrinated sheep blood showed a HC₅₀ of 448.51 μmol/L. Surfactin with short fatty acid chain has low toxicity on erythrocyte, which implies potential applications in biotechnological and pharmaceutical fields.     

A Bacillus subtilis 168 mutant with increased xylose uptake can utilize xylose as sole carbon source

Abstract Bacillus subtilis 168 is unable to effectively utilize xylose as sole carbon source. We demonstrate here that this strain cannot actively transport xylose into the cell. After leaving B. subtilis 168 for a few days on minimal plates with xylose as sole carbon source large colonies arise with a frequency of 1 × 10⁻⁶/cell. These mutants grow well on xylose and efficiently take up that sugar. This new property is not inducible by xylose, indicating that the mutation is neither in the xyl nor in the xyn operon.     

Enhanced beta-galactosidase production by high cell-density culture of recombinant Bacillus subtilis with glucose concentration control

Cell concentration, recombinant protein (beta-galactosidase) level, and the specific enzyme expression level were increased from 19 to 184 g/L, 18.3 to 129 U/mL, and 3.2 to 5.7 U/mg protein, respectively, in fed-batch culture of recombinant Bacillus subtilis when glucose concentration was controlled at 1 g/L as compared with those of conventional fed-batch culture. Glucose concentration of the culture broth was monitored by an automatic on-line glucose analyzer and controlled with a moving identification combined with optimal control (MICOC) strategy. When glucose concentrations were controlled at 10, 1, and 0.2 g/L, accumulated propionic acid concentrations and specific enzyme activities were 18.5, 4.4, and 0.6 g/L and 2.9, 5.7, and 7.1 U/mg protein, respectively. The addition of various concentrations of sodium propionate to the growth medium in batch cultures resulted in a drastic decrease in the growth rate with respect to propionate concentration. The propionic acid was shown to be responsible for cell growth inhibition and enzyme activity reduction in fed-batch culture. (c) 1992 John Wiley & Sons, Inc.     

Molecular identification and biological control of Ralstonia solanacearum from wilt of papaya by natural compounds and Bacillus subtilis: An integrated experimental and computational study

Ralstonia solanacearum is a harmful pathogen that causes severe wilt disease in several vegetables. In the present study, we identified R. solanacearum from wilt of papaya by 16S rRNA PCR amplification. Virulence ability of R. solanacearum was determined by amplification of approximately 1500 bp clear band of hrpB gene. Further, in-vitro seed germination assay showed that R. solanacearum reduced the germination rate up to 26.21%, 34% and 33.63% of cucumber, bottle guard and pumpkin seeds, respectively whereas shoot and root growth were also significantly decreased. Moreover, growth inhibition of R. solanacearum was recorded using antibacterial compound from medicinal plant and antagonistic B. subtilis. Petroleum ether root extract of Rauvolfia serpentina showed highest 22 ± 0.04 mm diameter of zone of inhibition where methanolic extract of Cymbopogon citratus and ethanolic extract of Lantana camara exhibited 20 ± 0.06 mm and 20 ± 0.01 mm zone of inhibition against R. solanacearum, respectively. In addition, bioactive compounds of B. subtilis inhibited R. solanacearum growth by generating 17 ± 0.09 mm zone of inhibition. To unveil the inhibition mechanism, we adopted chemical-protein interaction network and molecular docking approaches where we found that, rutin from C. citratus interacts with citrate (Si)-synthase and dihydrolipoyl dehydrogenase of R. solanacearum with binding affinity of −9.7 kcal/mol and −9.5 kcal/mol while quercetin from B. subtillis interacts with the essential protein F0F1 ATP synthase subunit alpha of the R. solancearum with binding affinity of −6.9 kcal/mol and inhibit the growth of R. solanacearum. Our study will give shed light on the development of eco-friendly biological control of wilt disease of papaya.     

A new polypeptide associated with RNA polymerase from Bacillus subtilis during late stages of vegetative growth

DNA-dependent RNA polymerase has been purified from $\text{Bacillus subtilis}$ at various stages of vegetative cell growth. Polymerase isolated from cultures approaching the end of the logarithmic growth phase was associated with a 60,000-dalton polypeptide and was only 10–20% as active as polymerase isolated from rapidly growing cells. Appearance of this new polypeptide and the change in template activity occur prior to stage 0 of sporulation.     

N3ICD with the transmembrane domain can effectively inhibit EMT by correcting the position of tight/adherens junctions

EMT allows a polarized epithelium to lose epithelial integrity and acquire mesenchymal characteristics. Previously, we found that overexpression of the intracellular domain of Notch3 (N3ICD) can inhibit EMT in breast cancer cells. In this study, we aimed to elucidate the influence of N3ICD or N3ICD combined with the transmembrane domain (TD+N3ICD) on the expression and distribution of TJs/AJs and polar molecules. We found that although N3ICD can upregulate the expression levels of the above-mentioned molecules, TD+N3ICD can inhibit EMT more effectively than N3ICD alone. TD+N3ICD overexpression upregulated the expression of endogenous full-length Notch3 and contributed to correcting the position of TJs/AJs molecules and better acinar structures formation. Co-immunoprecipitation results showed that the upregulated endogenous full-length Notch3 could physically interact with E-ca in MDA-MB-231/pCMV-(TD+N3ICD) cells. Collectively, our data indicate that overexpression of TD+N3ICD can effectively inhibit EMT, resulting in better positioning of TJs/AJs molecules and cell-cell adhesion in breast cancer cells.

Abbreviations: EMT: Epithelial-mesenchymal transition; TJs: Tight junctions; AJs: Adherens junctions; aPKC: Atypical protein kinase C; Crb: Crumbs; Lgl: Lethal (2) giant larvae; LLGL2: lethal giant larvae homolog 2; PAR: Partitioning defective; PATJ: Pals1-associated TJ protein     

A revised model for the control of fatty acid synthesis by master regulator Spo0A in Bacillus subtilis

In starving Bacillus subtilis cells, the accDA operon encoding two subunits of the essential acetyl‐CoA carboxylase (ACC) has been proposed to be tightly regulated by direct binding of the master regulator Spo0A to a cis element (0A box) in the promoter region. When the 0A box is mutated, biofilm formation and sporulation have been reported to be impaired. Here, we present evidence that two 0A boxes, one previously known (0A‐1) and another newly discovered (0A‐2) in the accDA promoter region are positively and negatively regulated by Spo0A～P respectively. Cells with mutated 0A boxes experience slight delays in sporulation, but eventually sporulate with high efficiency. In contrast, cells harboring a single mutated 0A‐2 box are deficient for biofilm formation, while cells harboring either a mutated 0A‐1 box or both mutated 0A boxes form biofilms. We further show that the essential ACC enzyme localizes on or near the cell membrane by directly observing a functional GFP fusion to one of the enzyme's subunits. Collectively, we propose a revised model in which accDA is primarily transcribed by a major σ^A‐RNA polymerase, while Spo0A～P plays an additional role in the fine‐tuning of accDA expression upon starvation to support proper biofilm formation and sporulation.     

A novel structural fold in polysaccharide lyases: Bacillus subtilis family 11 rhamnogalacturonan lyase YesW with an eight-bladed beta-propeller

Rhamnogalacturonan (RG) lyase produced by plant pathogenic and saprophytic microbes plays an important role in degrading plant cell walls. An extracellular RG lyase YesW from saprophytic Bacillus subtilis is a member of polysaccharide lyase family 11 and cleaves glycoside bonds in polygalacturonan as well as RG type-I through a beta-elimination reaction. Crystal structures of YesW and its complex with galacturonan disaccharide, a reaction product analogue, were determined at 1.4 and 2.5 A resolutions with final R-factors of 16.4% and 16.6%, respectively. The enzyme is composed of an eight-bladed beta-propeller with a deep cleft in the center as a basic scaffold, and its structural fold has not been seen in polysaccharide lyases analyzed thus far. Structural analysis of the disaccharide-bound YesW and a site-directed mutagenesis study suggested that Arg-452 and Lys-535 stabilize the carboxyl group of the acidic polysaccharide molecule and Tyr-595 makes a stack interaction with the sugar pyranose ring. In addition to amino acid residues binding to the disaccharide, one calcium ion, which is coordinated by Asp-401, Glu-422, His-363, and His-399, may mediate the enzyme activity. This is, to our knowledge, the first report of a new structural category with a beta-propeller fold in polysaccharide lyases and provides structural insights into substrate binding by RG lyase.     

Regulation of pho regulon gene expression by the carbon control protein A, CcpA, in Bacillus subtilis

Bacterial regulons involved in carbon, nitrogen and phosphorus metabolism must interact for purposes of coordination, but the mechanisms involved are not understood. We here report that the carbon control pro-tein-A (CcpA) of Bacillus subtilis, primarily concerned with carbon metabolism, influences expression of various phosphorus (pho) regulon genes including the two alkaline phosphatase structural genes, phoA and phoB. The directions and magnitudes of the effects of glucose and the loss of CcpA on these two genes depend on growth conditions, but they always correlate inversely. Absolute expression levels of phoA and phoB depend on a rich nitrogen source, and gene activation by a fermentable substrate such as glucose depends on the presence of a respiratory substrate such as succinate. We show that these CcpA-dependent glucose effects can be explained by the effects of glucose and CcpA acting on the phoPR operon. Although a good CcpA-binding site (CRE) is found in the control region of the phoPR operon, direct regulation of phoPR gene expression by CcpA via this CRE could not account for the effects of glucose and CcpA on phoA and phoB gene expression. We conclude that CcpA exerts indirect control over the pho regulon by a mechanism that involves CcpA and PhoRP but does not involve the phoPR operon CRE.