Enhancing surfactin production in B. velezensis Bs916 combined cumulative mutagenesis and expression key enzymes.

Surfactin is a lipopeptide which has attracted massive attention due to its versatile bioactive properties, although it has less commercial application due to its low yield in wild strains. The B. velezensis Bs916 has enable commercial production of surfactin due to its outstanding capacity to synthesize lipopeptides and amenable to genetically engineering. In this study, 20 derivatives with high surfactin production were obtained firstly by transposon mutagenesis and knockout techniques, and the surfactin yield of the derivative H5 (△GltB) was increased approximately 7-folds, reaching to 1.48 g/L. The molecular mechanism of high yielding surfactin in △GltB was investigated by the transcriptomic and KEGG pathway analysis. The results indicated that △GltB enhanced its ability to synthesize surfactin mainly by promoting transcription of the srfA gene cluster and inhibiting degradation of some key precursors such as fatty acid. Secondly, we obtained a triple mutant derivative BsC3 by cumulative mutagenesis of the negative genes GltB, RapF, and SerA, and it could increase the surfactin titer by twofold, reaching to 2.98 g/L. Thirdly, we achieved overexpression of two key rate-limiting enzyme genes, YbdT, and srfAD, and the derivative BsC5 which further increased the surfactin titer by 1.3-fold, reaching to 3.79 g/L. Finally, the yield of surfactin by derivatives was significantly increased under the optimal medium, particularly the BsC5 increased the surfactin titer to 8.37 g/L. To the best of our knowledge, this is one of the highest yields that have been reported. Our work may pave way for large scale production of surfactin by B. velezensis Bs916. KEY POINTS: • Elucidation of the molecular mechanism of surfactin high-yielding transposon mutant. • Genetically engineering of B. velezensis Bs916 surfactin titer to 8.37 g/L for large scale preparation.

Genome Sequencing and Genetic Engineering Reveal the Contribution of Bacitracin Produced by Bacillus paralicheniformis CPL618 to Anti-Staphylococcus aureus Activity.

Staphylococcus aureus is one of the important pathogens causing human diseases, especially its treatment has great challenges due to its resistance to methicillin and vancomycin. The Bacillus strains are known to be major sources of second metabolites that can function as drugs. Therefore, it is of great value to excavate metabolites with good inhibitory activity against S. aureus from Bacillus strains. In this study, a strain Bacillus paralicheniformis CPL618 with good antagonistic activity against S. aureus was isolated and genome analysis showed that the size was 4,447,938 bp and contained four gene clusters fen, bac, dhb, and lch which are potentially responsible for four cyclic peptides fengycin, bacitracin, bacillibactin, and lichenysin biosynthesis, respectively. These gene clusters were knockout by homologous recombination. The bacteriostatic experiment results showed that the antibacterial activity of ∆bac decreased 72.3% while Δfen, Δdhb, and ΔlchA did not significantly changed as that of wild type. Interestingly, the maximum bacitracin yield was up to 92 U/mL in the LB medium, which was extremely unusual in wild type strains. To further improve the production of bacitracin, transcription regulators abrB and lrp were knocked out, the bacitracin produced by ΔabrB, Δlrp, and ΔabrB + lrp was 124 U/mL, 112 U/mL, and 160 U/ml, respectively. Although no new anti-S. aureus compounds was found by using genome mining in this study, the molecular mechanisms of high yield of bacitracin and anti-S. aureus in B. paralicheniformis CPL618 were clarified. Moreover, B. paralicheniformis CPL618 was further genetically engineered for industrial production of bacitracin.

Urea impregnation into fungus pretreated corn stover to perform pyrolysis for production of nitrogen-containing bio-oil and nitrogen-doped biochar.

Urea was introduced into the fungal pretreated corn stover and then the urea soaked materials were subjected to pyrolysis for the production of nitrogen-containing bio-oil and nitrogen-doped biochar. The urea soaking effectively realized the enrichment of nitrogen-containing compounds in the bio-oil and the maximal content of the nitrogen-containing compounds in bio-oils reached up to 66.32% under 4 wt% urea concentration. Among the nitrogen-containing compounds, amines were the most dominant component with the maximal content of 41.17%. The higher urea concentration is beneficial to make more nitrogen be fixed in the biochar. The nitrogen content of the biochar reached up to 12.86 wt% under 8 wt% urea concentration. Nitrogen on the biochar surface existed in the form of pyrrolic-N, pyridinic-N and graphite-N. In conclusion, urea simple soak on fungus pretreated biomass to perform pyrolysis is a promising approach to obtain high value-added nitrogen-containing chemicals and nitrogen-doped biochar with high nitrogen content.

Transcriptome investigation on the multicellular behavior of Bacillus velezensis Bs916 anchoring surfactin.

AIMS: To provide valuable information for a comprehensive understanding of the multicellular behavior of Bacillus velezensis Bs916 regulated by surfactin and other natural signals by Transcriptome. METHODS AND RESULTS: Transcriptomics revealed a distinct effect on gene expression alterations caused by disruption of the surfactin gene cluster(Δsrf) and 100 µg/ml surfactin addition(Δsrf + SRF). A total of 1573 differential expression genes were identified among Bs916, Δsrf, and Δsrf + SRF and grouped into eight categories based on their expression profiles. RT-qPCR analysis of 30 candidate genes showed high consistency with those of transcriptome. Additionally, the expression of eight candidate genes regulated by surfactin in a dose-dependent manner was revealed by lacZ fusion. Based on the above evidence, we proposed that surfactin can act as an extracellular signal for monitoring biofilm formation in Bs916 by directly regulating the expression of AbrB, DegS-degU, and SinI-SinR, and indirectly regulating the phosphorylation of ComA and Spo0A. CONCLUSIONS: The biofilm of Δsrf was unable to restore significantly by surfactin addition, combined inclusion of surfactin (SRF), exopolysaccharide (EPS), and γ-poly-dl-glutamic acid (γ-PGA), results in significant restoration of Δsrf biofilm formation, thereby a preliminary model was presented about the molecular mechanism by which the signaling molecule surfactin regulates Bs916 multicellular behavior.

The synergy of thanatin and cathelicidin-BF-15a3 combats Escherichia coli O157:H7.

Escherichia coli O157:H7 is a pathogen that commonly causes foodborne illness and represents a health hazard to consumers. The combined use of synergistic antimicrobial peptides (AMPs) is a promising way to improve the microbiological safety of foods. In this study, we detected the synergistic interactions between thanatin and BF-15a3 to reduce their usage and obtain more efficient antibacterial activity. The minimal inhibitory concentrations (MICs) of thanatin and BF-15a3 against 49 E. coli O157:H7 strains were ranged from 2 to 8 µg/mL and 4-32 µg/mL, showed a general inhibitory effect on E. coli O157:H7 strains, respectively, even multidrug-resistant strains. Their fractional inhibitory concentration index (FICI) was 0.375, which suggested that their combination presented synergistic antibacterial effect against E. coli O157:H7. The killing kinetic curves indicated that the 0.25 × MIC combination had equivalent bactericidal effects to 1 × MIC thanatin or BF-15a3. When AMP combinations were used to treat eukaryotic cells to evaluate the hemolytic characteristics against rabbit erythrocytes and cytotoxicity against human embryonic kidney 293T (HEK-293T) cells and intestinal porcine enterocyte J2 (IPEC-J2) cells, no magnified adverse effects were observed, exhibiting higher specificity to bacteria and lower toxicity to eukaryotic cells. Compared with bacteriostasis of thanatin or BF-15a3 alone, the proportion of membrane-damaged bacteria treated with the synergetic combination did not appear a significant rise, interestingly the Zeta potential of them greatly decreased and their cell membrane permeability significantly increased. Besides, more release of ions and cytoplasm were detected, confirming a more severe loss of membrane integrity. These results suggested that the synergistic action mode of thanatin and BF-15a3 is likely attributed to damage aggravation to E. coli membrane. When applying in fresh-cut lettuce and cucumber, their combination allowed for 2.5 log CFU/piece reductions of E. coli O157:H7 in 24 h. In conclusion, the combination of thanatin and BF-15a3 showed excellent synthetic efficacy to kill E. coli O157:H7 in vitro under lower MICs than single use of them.

Transcriptome dynamics uncovers long non-coding RNAs response to salinity stress in Chenopodium quinoa.

Chenopodium quinoa is a crop with outstanding tolerance to saline soil, but long non-coding RNAs (LncRNAs) expression profile driven by salt stress in quinoa has rarely been observed yet. Based on the high-quality quinoa reference genome and high-throughput RNA sequencing (RNA-seq), genome-wide identification of LncRNAs was performed, and their dynamic response under salt stress was then investigated. In total, 153,751 high-confidence LncRNAs were discovered and dispersed intensively in chromosomes. Expression profile analysis demonstrated significant differences between LncRNAs and coding RNAs. Under salt stress conditions, 4,460 differentially expressed LncRNAs were discovered, of which only 54 were differentially expressed at all the stress time points. Besides, strongly significantly correlation was observed between salt-responsive LncRNAs and their closest neighboring genes (r = 0.346, p-value < 2.2e-16). Furthermore, a weighted co-expression network was then constructed to infer the potential biological functions of LncRNAs. Seven modules were significantly correlated with salt treatments, resulting in 210 hub genes, including 22 transcription factors and 70 LncRNAs. These results indicated that LncRNAs might interact with transcription factors to respond to salinity stress. Gene ontology enrichment of the coding genes of these modules showed that they were highly related to regulating metabolic processes, biological regulation and response to stress. This study is the genome-wide analysis of the LncRNAs responding to salt stress in quinoa. The findings will provide a solid framework for further functional research of salt responsive LncRNAs, contributing to quinoa genetic improvement.

Extracellular lipopeptide bacillomycin L regulates serial expression of genes for modulating multicellular behavior in Bacillus velezensis Bs916.

In wild strains of Bacillus, a handful of extracellular natural products act as signals that can regulate multicellular behavior, but relatively little is known about molecular mechanisms' detail. We proposed a previously unreported molecular mechanism for triggering multicellularity in B. velezensis Bs916 by an endogenous cyclic lipopeptide, bacillomycin L. The genome-wide effect on gene expression was caused by the disruption of bacillomycin L gene cluster, and 100 µg/mL bacillomycin L was revealed by quantitative transcriptomics. A total of 878 differentially expressed genes among Bs916, Δbl, and Δbl + 100BL were identified and grouped into 9 functional categories. The transcription levels of 40 candidate genes were further evaluated by RT-qPCR analysis. The expression of eight candidate genes regulated by bacillomycin L in a dose-dependent manner was revealed by LacZ fusion experiment. Although the addition of bacillomycin L could not completely restore the expression levels of the differentially regulated genes in △bl, our results strongly suggest that bacillomycin L acts as a tuning signal of swarming motility and complex biofilm formation by indirectly regulating the expression levels of some two-component systems (TCSs) connector genes, particularly including several Raps that potentially regulate the phosphorylation levels of three major regulators ComA, DegU, and Spo0A.Key points• Proposed model for bacillomycin L regulation in B. velezensis Bs916.• Bacillomycin L can act as an extracellular signal to regulate the phosphorylation levels of three major regulators, ComA, DegU, and Spo0A and control the multicellular processes of vegetative growth, competent, motility, matrix production, sporulation, and autolysis.

Fundamental to achieving fast separations with high efficiency: A review of chromatography with superficially porous particles.

Types of particles have been fundamental to LC separation technology for many years. Originally, LC columns were packed with large-diameter (>100 µm) calcium carbonate, silica gel, or alumina particles that prohibited fast mobile-phase speeds because of the slow diffusion of sample molecules inside deep pores. During the birth of HPLC in the 1960s, superficially porous particles (SPP, ≥30 µm) were developed as the first high-speed stationary-phase support structures commercialized, which permitted faster mobile-phase flowrates due to the fast movement of sample molecules in/out of the thin shells. These initial SPPs were displaced by smaller totally porous particles (TPP) in the mid-1970s. But SPP history repeated when UHPLC emerged in the 2000s. Stationary-phase support structures made from sub-3-µm SPPs were introduced to chromatographers in 2006. The initial purpose of this modern SPP was to enable chromatographers to achieve fast separations with high efficiency using conventional HPLCs. Later, the introduction of sub-2-µm SPPs with UHPLC instruments pushed the separation speed and efficiency to a very fast zone. This review aims at providing readers a comprehensive and up-to-date view on the advantages of SPP materials over TPPs historically and theoretically from the material science angle.

A gas chromatography-flame ionization detection method for direct and rapid determination of small molecule volatile organic compounds in aqueous phase.

It is still difficult to directly detect low content of volatile organic compounds (VOCs) in water samples by gas chromatography (GC) because when water is the only solvent, it would result in the instability and poor repeatability of peak retention time and peak shape. The adverse effects of water on direct GC analysis of VOCs cannot be significantly reduced or eliminated by simply changing the detection condition of GC. However, it was found that the addition of methanol in samples to a certain final proportion, such as 50 or 75% (v/v), could greatly reduce or eliminate the adverse effects of water. By using 75% (v/v) methanol as a solvent, the standard curves of ethanol, acetic acid, acetone, and isopropanol with correlation coefficient (R 2) over 0.99 were successfully plotted by gas chromatography-flame ionization detection (GC-FID) in a certain concentration range, respectively. The results showed that the retention time and peak shape stability of ethanol, acetic acid, acetone, and isopropanol in aqueous solution were greatly improved by the addition of methanol to final concertation of 75% (v/v). To verify the practical application potential of this method, the method was applied to the detection of components in isopropanol fermentation wastewater. The results showed that the method has well applicability and reliability. The key points in the application of this method were also summarized. This GC analysis method would have a wider and better application prospect in the detection of water-soluble organic matters.

Mortalin restricts porcine epidemic diarrhea virus entry by downregulating clathrin-mediated endocytosis.

Clathrin-mediated endocytosis is a mechanism used for the invasion of cells by a variety of viruses. Mortalin protein is involved in a variety of cellular functions and plays a role in viral infection. In this study, we found that mortalin significantly inhibited the replication of porcine epidemic diarrhea virus (PEDV) through restricting virus entry. Mechanistically, a biochemical interaction between the carboxyl terminus of mortalin and clathrin heavy chain (CLTC) was been found, and mortalin could induce CLTC degradation through the proteasomal pathway, thereby inhibiting the clathrin-mediated endocytosis of PEDV into host cells. In addition, artificial changes in mortalin expression affected the cell entry of transferrin, further confirming the above results. Finally, we confirmed that this host-mounted antiviral mechanism was broadly applicable to other viruses, such as vesicular stomatitis virus (VSV), rotavirus (RV), and transmissible gastroenteritis virus (TGEV), which use the same clathrin-mediated endocytic to entry. These results reveal a new function of mortalin in inhibiting endocytosis, and provide a novel strategy for treating PEDV infections.

Knockout of acetoacetate degradation pathway gene atoDA enhances the toxicity tolerance of Escherichia coli to isopropanol and acetone.

Isopropanol and acetone are important chemical products and potential high-quality new fuels. Both of them are metabolites of isopropanol synthesis pathway, but they are toxic to most bacteria. In this study, toxicity tolerance of Escherichia coli strains was evaluated by detecting their growth rates under different concentrations of isopropanol and acetone. It was showed that isopropanol was more toxic to E. coli than acetone, and the native strain MG1655 had better tolerance over DH5α to either acetone or isopropanol of 300 mM. Key genes of ethanol synthesis pathway, acetic acid metabolism pathway, and acetoacetic acid degradation pathway, including adhE, ackA-pta, and atoDA, were knocked out in MG1655 to form mutants MGΔadhE, MGΔackA-pta, and MGΔatoDA. The tolerance performances of the mutants to isopropanol and acetone were determined under various concentrations including 300 mM, 500 mM, and 700 mM, respectively. The mutant MGΔatoDA exhibited excellent tolerance to both acetone and isopropanol of 500 mM, and MGΔackA-pta could tolerate acetone at 500 mM rather than isopropanol, while the deletion of adhE in MGΔadhE resulted in a severe cell growth defection. Although isopropanol and acetone at 700 mM caused severe growth inhibition on each strain, cell growth could be restored to varying degrees with the prolongation of culture time. This phenomenon was suggested to be related to the volatilization of isopropanol and acetone based on volatilization tests. It was envisioned that MG1655 was a suitable host strain for isopropanol metabolic engineering research, and the acetoacetic acid degradation pathway gene atoDA, was probably the key optimizing point for isopropanol production.

Engineered biosynthesis of cyclic lipopeptide locillomycins in surrogate host Bacillus velezensis FZB42 and derivative strains enhance antibacterial activity.

Locillomycins are cyclic lipononapeptides assembled by a nonlinear hexamodular NRPS and have strong antibacterial activity. In this study, we genetically engineered Bacillus velezensis FZB42 as a surrogate host for the heterologous expression of the loc gene cluster for locillomycins. The fosmid N13 containing whole loc gene cluster was screened from the B. velezensis 916 genomic library. Subsequently, a spectinomycin resistance cassette, and the cassette fused with an IPTG inducible promoter Pspac, was introduced in the fosmid N13 using λ Red recombination system, respectively. The resulting fosmids, designated N13+Spec and N13+PSSpec, were used for the transformation of B. velezensis FZB42 to obtain derivative strains FZBNPLOC and FZBPSLOC. RT-PCR and qRT-PCR results revealed the efficient heterologous expression of the loc gene cluster in both derivative strains. Particularly, there was positive correlation between the derivative FZBPSLOC strain and the enhanced production of locillomycins upon addition of the inducer IPTG with the highest production of locillomycins at 15-fold more than that of B. velezensis 916. This overproduction of locillomycins was also related to the enhancement of antibacterial activity against methicillin-resistant Staphylococcus aureus, and exhibited moderate changes in its hemolytic activity. Together our findings demonstrate that the nonlinear hexamodular NRPS, encoded by the loc gene cluster from B. velezensis 916, is sufficient for the biosynthesis of cyclic lipononapeptide locillomycins in the surrogate host B. velezensis FZB42. Moreover, the FZBPSLOC strain will also be useful for further development of novel locillomycins derivatives with improved antibacterial activity.

Rhizosphere Microbiomes from Root Knot Nematode Non-infested Plants Suppress Nematode Infection.

Root knot nematodes (RKN, Meloidogyne spp.) are serious pathogens of numerous crops worldwide. Understanding the roles plant rhizosphere soil microbiome play during RKN infection is very important. The current study aims at investigating the impacts of soil microbiome on the activity of RKN. In this study, the 16S rRNA genes of the bacterial communities from nematode-infested and non-infested rhizosphere soils from four different plants were sequenced on the Illumina Hi-Seq platform. The soil microbiome effects on RKN infection were tested in a greenhouse assay. The non-infested soils had more microbial diversity than the infested soils from all plant rhizospheres, and both soil types had exclusive microbial communities. The inoculation of the microbiomes from eggplant and cucumber non-infested soils to tomato plants significantly alleviated the RKN infection, while the microbiome from infested soil showed increased the RKN infection. Furthermore, bacteria Pseudomonas sp. and Bacillus sp. were screened out from non-infested eggplant soil and exhibited biocontrol activity to RKN on tomato. Our findings suggest that microbes may regulate RKN infection in plants and are involved in biocontrol of RKN.

Microwave pyrolysis of moso bamboo for syngas production and bio-oil upgrading over bamboo-based biochar catalyst.

Microwave pyrolysis of moso bamboo over bamboo-based biochar catalyst was conducted to achieve the bio-oil upgrading and high quality syngas production. The influence of the biochar on bamboo pyrolysis involving the temperature rise, product yield, and bio-oil and gas compositions was studied. The gas production was facilitated by the biochar mainly at the cost of the bio-oil, indicating the biochar had an excellent activity for the bio-oil cracking. The main compositions in bio-oil were acetic acid and phenol with the total contents ranging from 73.145% to 82.84% over the biochar catalysts, suggesting the upgrading of the bio-oil were achieved. The biochar exerted a positive effect on the syngas (CO + H2) production with the maximum content reaching up to 65.13 vol% at the 20 wt% addition amount of biochar under microwave condition. The biochar became more effective on the bio-oil upgrading and syngas production under microwave heating than conventional heating.

Loss of GltB Inhibits Biofilm Formation and Biocontrol Efficiency of Bacillus subtilis Bs916 by Altering the Production of γ-Polyglutamate and Three Lipopeptides.

AIMS: This study examined the contribution of GltB on biofilm formation and biocontrol efficiency of B. subtilis Bs916. METHODS AND RESULTS: The gltB gene was identified through a biofilm phenotype screen and a bioinformatics analysis of serious biofilm formation defects, and then a gltB single knockout mutant was constructed using homologous recombination. This mutant demonstrated severe deficits in biofilm formation and colonisation along with significantly altered production ofγ-polyglutamate (γ-PGA) and three lipopeptide antibiotics (LPs) as measured by a transcriptional analysis of both the wild type B. subtilis Bs916 and the gltB mutant. Consequently, the mutant strain retained almost no antifungal activity against Rhizoctonia solani and exhibited decreased biocontrol efficiency against rice sheath blight. Very few gltB mutant cells colonised the rice stem, and they exhibited no significant nutrient chemotaxis compared to the wild type B. subtilis Bs916. The mechanism underlying these deficits in the gltB mutant appears to be decreased significantly in production of γ-PGA and a reduction in the production of both bacillomycin L and fengycin. Biofilm restoration of gltB mutant by additionγ-PGA in the EM medium demonstrated that biofilm formation was able to restore significantly at 20 g/L. CONCLUSIONS: GltB regulates biofilm formation by altering the production ofγ-PGA, the LPs bacillomycin L and fengcin and influences bacterial colonisation on the rice stem, which consequently leads to poor biocontrol efficiency against rice sheath blight. SIGNIFICANCE AND IMPACT OF STUDY: This is the first report of a key regulatory protein (GltB) that is involved in biofilm regulation and its regulation mechanism and biocontrol efficiency by B. subtilis.

Unusual Biosynthesis and Structure of Locillomycins from Bacillus subtilis 916.

Three families of Bacillus cyclic lipopeptides--surfactins, iturins, and fengycins--have well-recognized potential uses in biotechnology and biopharmaceutical applications. This study outlines the isolation and characterization of locillomycins, a novel family of cyclic lipopeptides produced by Bacillus subtilis 916. Elucidation of the locillomycin structure revealed several molecular features not observed in other Bacillus lipopeptides, including a unique nonapeptide sequence and macrocyclization. Locillomycins are active against bacteria and viruses. Biochemical analysis and gene deletion studies have supported the assignment of a 38-kb gene cluster as the locillomycin biosynthetic gene cluster. Interestingly, this gene cluster encodes 4 proteins (LocA, LocB, LocC, and LocD) that form a hexamodular nonribosomal peptide synthetase to biosynthesize cyclic nonapeptides. Genome analysis and the chemical structures of the end products indicated that the biosynthetic pathway exhibits two distinct features: (i) a nonlinear hexamodular assembly line, with three modules in the middle utilized twice and the first and last two modules used only once and (ii) several domains that are skipped or optionally selected.

Nonribosomal peptide synthase gene clusters for lipopeptide biosynthesis in Bacillus subtilis 916 and their phenotypic functions.

Bacillus cyclic lipopeptides (LPs) have been well studied for their phytopathogen-antagonistic activities. Recently, research has shown that these LPs also contribute to the phenotypic features of Bacillus strains, such as hemolytic activity, swarming motility, biofilm formation, and colony morphology. Bacillus subtilis 916 not only coproduces the three families of well-known LPs, i.e., surfactins, bacillomycin Ls (iturin family), and fengycins, but also produces a new family of LP called locillomycins. The genome of B. subtilis 916 contains four nonribosomal peptide synthase (NRPS) gene clusters, srf, bmy, fen, and loc, which are responsible for the biosynthesis of surfactins, bacillomycin Ls, fengycins, and locillomycins, respectively. By studying B. subtilis 916 mutants lacking production of one, two, or three LPs, we attempted to unveil the connections between LPs and phenotypic features. We demonstrated that bacillomycin Ls and fengycins contribute mainly to antifungal activity. Although surfactins have weak antifungal activity in vitro, the strain mutated in srfAA had significantly decreased antifungal activity. This may be due to the impaired productions of fengycins and bacillomycin Ls. We also found that the disruption of any LP gene cluster other than fen resulted in a change in colony morphology. While surfactins and bacillomycin Ls play very important roles in hemolytic activity, swarming motility, and biofilm formation, the fengycins and locillomycins had little influence on these phenotypic features. In conclusion, B. subtilis 916 coproduces four families of LPs which contribute to the phenotypic features of B. subtilis 916 in an intricate way.

Bacillomycin L and surfactin contribute synergistically to the phenotypic features of Bacillus subtilis 916 and the biocontrol of rice sheath blight induced by Rhizoctonia solani.

The antagonistic activity of lipopeptides in Bacillus subtilis 916 has been well documented, yet relatively little is known about their mechanism in biofilm formation and environmental colonization. This study sought to examine the interaction of B. subtilis 916 on Rhizoctonia solani-infected rice sheath to elucidate the mechanism of colonization on plant leaves. Results showed that the mutants Δbac, Δsrf, and Δsrf + bac of B. subtilis 916, deficient in bacillomycin L and surfactin production, respectively, not only altered colony morphology but also changed swarming motility, reduced antagonistic activity, and decreased biofilm formation. In particular, biofilm formation in mutant Δbac, not Δsrf or Δsrf + bac, were restored with addition of surfactin and bacillomycin L at 10 and 50 µg/mL, respectively. Moreover, surfactin and bacillomycin L were able to restore or enhance swarming motility in the corresponding mutants at 10 µg/mL, respectively. With the aid of green fluorescent protein tagging, it was demonstrated that B. subtilis 916 formed a robust biofilm on the rice sheath blight lesion and colonized well on R. solani-infected rice sheath, while its corresponding mutants performed poorly. These observations also correlated with the rice cultivar pot experiments, in which B. subtilis 916 exhibited greater biocontrol than its mutants. Our results suggest that surfactin and bacillomycin L contribute differently but synergistically to the biocontrol of rice sheath blight in B. subtilis 916 through its antifungal activity, biofilm formation, and colonization.

Genome sequence of the plant growth-promoting rhizobacterium Bacillus sp. strain 916.

Bacillus sp. strain 916, isolated from the soil, showed strong activity against Rhizoctonia solani. Here, we present the high-quality draft genome sequence of Bacillus sp. strain 916. Its 3.9-Mb genome reveals a number of genes whose products are possibly involved in promotion of plant growth or antibiosis.

Three non-aspartate amino acid mutations in the ComA Response regulator receiver motif severely decrease surfactin production, competence development and spore formation in Bacillus subtilis.

Bacillus subtilis strains produce a broad spectrum of bioactive peptides. The lipopeptide surfactin belongs to one wellknown class, which includes amphiphilic membrane-active biosurfactants and peptide antibiotics. Both the srfA promoter and the ComP-ComA signal transduction system are an important part of the factor that results in the production of surfactin. Bs-M49, obtained by means of low-energy ion implantation in wild-type Bs-916, produced significantly lower levels of surfactin, and had no obvious effects against R. solani. Occasionally, we found strain Bs- M49 decreased spore formation and the development of competence. Blast comparison of the sequences from Bs- 916 and M49 indicate that there is no difference in the srfA operon promoter PsrfA, but there are differences in the coding sequence of the comA gene. These differences result in three missense mutations within the M49 ComA protein. RT-PCR analyses results showed that the expression levels of selected genes involved in competence and sporulation in both the wild-type Bs-916 and mutant M49 strains were significantly different. When we integrated the comA ORF into the chromosome of M49 at the amyE locus, M49 restored hemolytic activity and antifungal activity. Then, HPLC analyses results also showed the comA-complemented strain had a similar ability to produce surfactin with wild-type strain Bs-916. These data suggested that the mutation of three key amino acids in ComA greatly affected the biological activity of Bacillus subtilis. ComA protein 3D structure prediction and motif search prediction indicated that ComA has two obvious motifs common to response regulator proteins, which are the Nterminal response regulator receiver motif and the Cterminal helix-turn-helix motif. The three residues in the ComA N-terminal portion may be involved in phosphorylation activation mechanism. These structural prediction results implicate that three mutated residues in the ComA protein may play an important role in the formation of a saltbridge to the phosphoryl group keeping active conformation to subsequent regulation of the expression of downstream genes.