Involvement of Peptidoglycan Receptor Proteins in Mediating the Growth-Promoting Effects of Bacillus pumilus TUAT1 in Arabidopsis thaliana.

Bacillus pumilus TUAT1 acts as plant growth-promoting rhizobacteria for various plants like rice and Arabidopsis. Under stress conditions, B. pumilus TUAT1 forms spores with a thick peptidoglycan (PGN) cell wall. Previous research showed that spores were significantly more effective than vegetative cells in enhancing plant growth. In Arabidopsis, lysin motif proteins, LYM1, LYM3 and CERK1, are required for recognizing bacterial PGNs to mediate immunity. Here, we examined the involvement of PGN receptor proteins in the plant growth promotion (PGP) effects of B. pumilus TUAT1 using Arabidopsis mutants defective in PGN receptors. Root growth of wild-type (WT), cerk1-1, lym1-1 and lym1-2 mutant plants was significantly increased by TUAT1 inoculation, but this was not the case for lym3-1 and lym3-2 mutant plants. RNA-seq analysis revealed that the expression of a number of defense-related genes was upregulated in lym3 mutant plants. These results suggested that B. pumilus TUAT1 may act to reduce the defense response, which is dependent on a functional LYM3. The expression of the defense-responsive gene, WRKY29, was significantly induced by the elicitor flg-22, in both WT and lym3 mutant plants, while this induction was significantly reduced by treatment with B. pumilus TUAT1 and PGNs in WT, but not in lym3 mutant plants. These findings suggest that the PGNs of B. pumilus TUAT1 may be recognized by the LYM3 receptor protein, suppressing the defense response, which results in plant growth promotion in a trade-off between defense and growth.

[The Proteome of Extracellular Membrane Vesicles from Bacillus pumilus 3-19].

Production of extracellular membrane vesicles plays an important role in communication in bacterial populations and in bacteria-host interactions. Vesicles as carriers of various regulatory and signaling molecules may be potentially used as disease biomarkers and promising therapeutic agents, including vaccine preparations. The composition of membrane vesicles has been deciphered for a limited number of Gram-negative and Gram-positive bacteria. In this work, for the first time, extracellular membrane vesicles of a streptomycin-resistant strain Bacillus pumilus 3-19, a producer of extracellular guanyl-preferring ribonuclease binase, are isolated, visualized, and characterized by their genome and proteome composition. It has been established that there is no genetic material in the vesicles and the spectrum of the proteins differs depending on the phosphate content in the culture medium of the strain. Vesicles from a phosphate-deficient medium carry 49 unique proteins in comparison with 101 from a medium with the high phosphate content. The two types of vesicles had 140 mutual proteins. Flagellar proteins, RNase J, which is the main enzyme of RNA degradosomes, phosphatases, peptidases, iron transporters, signal peptides, were identified in vesicles. Antibiotic resistance proteins and amyloid-like proteins whose genes are present in B. pumilus 3-19 cells are absent. Phosphate deficiency-induced binase was found only in vesicles from a phosphate-deficient medium.

Transcriptome analysis reveals the regulatory effects of Bacillus amyloliquefaciens and Bacillus pumilus on immune and digestive related genes in the spleen of weanling black goats.

The aim of the present study was to evaluate the effects of Bacillus amyloliquefaciens fsznc-06 and Bacillus pumilus fsznc-09 on the expressions of spleen genes in weanling Jintang black goats. Bacillus amyloliquefaciens fsznc-06 (BA-treated group) and Bacillus pumilus fsznc-09 (BP-treated group) were directly fed to goats, and the spleens were harvested for transcriptome analysis. The KEGG pathway analysis showed that the differentially expressed genes (DEGs) in BA-treated vs CON group were mainly involved in digestive system and immune system, while those in BP-treated vs CON group were mainly involved in immune system, and those in BA-treated vs BP-treated group were mainly involved in digestive system. In conclusion, Bacillus amyloliquefaciens fsznc-06 might promote the expressions of genes related to immune system and digestive system, reduce the expressions of disease genes related to digestive system and might promote mutual accommodation of some immune genes in weanling black goat. Bacillus pumilus fsznc-09 might promote the expressions of genes related to immune system and mutual accommodation of some immune genes in weanling black goat. Bacillus amyloliquefaciens fsznc-06 has advantages over Bacillus pumilus fsznc-09 in promoting the expressions of genes related to digestive system and mutual accommodation of some immune genes.

Characterization of pumilacidin, a lipopeptide biosurfactant produced from Bacillus pumilus NITDID1 and its prospect in bioremediation of hazardous pollutants.

Highly hydrophobic compounds like petroleum and their byproducts, once released into the environment, can persist indefinitely by virtue of their ability to resist microbial degradation, ultimately paving the path to severe environmental pollution. Likewise, the accumulation of toxic heavy metals like lead, cadmium, chromium, etc., in the surroundings poses an alarming threat to various living organisms. To remediate the matter in question, the applicability of a biosurfactant produced from the mangrove bacterium Bacillus pumilus NITDID1 (Accession No. KY678446.1) is reported here. The structural characterization of the produced biosurfactant revealed it to be a lipopeptide and has been identified as pumilacidin through FTIR, NMR, and MALDI-TOF MS. The critical micelle concentration of pumilacidin was 120 mg/L, and it showed a wide range of stability in surface tension reduction experiments under various environmental conditions and exhibited a high emulsification index of as much as 90%. In a simulated setup of engine oil-contaminated sand, considerable oil recovery (39.78%) by this biosurfactant was observed, and upon being added to a microbial consortium, there was an appreciable enhancement in the degradation of the used engine oil. As far as the heavy metal removal potential of biosurfactant is concerned, as much as 100% and 82% removal was observed for lead and cadmium, respectively. Thus, in a nutshell, the pumilacidin produced from Bacillus pumilus NITDID1 holds promise for multifaceted applications in the field of environmental remediation.

Tuning transcription factor DegU for developing extracellular protease overproducer in Bacillus pumilus.

BACKGROUND: Global transcription machinery engineering (gTME) is an effective approach employed in strain engineering to rewire gene expression and reshape cellular metabolic fluxes at the transcriptional level. RESULTS: In this study, we utilized gTME to engineer the positive transcription factor, DegU, in the regulation network of major alkaline protease, AprE, in Bacillus pumilus. To validate its functionality when incorporated into the chromosome, we performed several experiments. First, three negative transcription factors, SinR, Hpr, and AbrB, were deleted to promote AprE synthesis. Second, several hyper-active DegU mutants, designated as DegU(hy), were selected using the fluorescence colorimetric method with the host of the Bacillus subtilis ΔdegSU mutant. Third, we integrated a screened degU(L113F) sequence into the chromosome of the Δhpr mutant of B. pumilus SCU11 to replace the original degU gene using a CRISPR/Cas9 system. Finally, based on transcriptomic and molecular dynamic analysis, we interpreted the possible mechanism of high-yielding and found that the strain produced alkaline proteases 2.7 times higher than that of the control strain (B. pumilus SCU11) in LB medium. CONCLUSION: Our findings serve as a proof-of-concept that tuning the global regulator is feasible and crucial for improving the production performance of B. pumilus. Additionally, our study established a paradigm for gene function research in strains that are difficult to handle.

Regulating Pathways of Bacillus pumilus Adamalysin-like Metalloendopeptidase Expression.

The minor secreted proteinase of B. pumilus 3-19 MprBp classified as the unique bacillary adamalysin-like enzyme of the metzincin clan. The functional role of this metalloproteinase in the bacilli cells is not clear. Analysis of the regulatory region of the mprBp gene showed the presence of potential binding sites to the transcription regulatory factors Spo0A (sporulation) and DegU (biodegradation). The study of mprBp activity in mutant strains of B. subtilis defective in regulatory proteins of the Spo- and Deg-systems showed that the mprBp gene is partially controlled by the Deg-system of signal transduction and independent from the Spo-system.

2-Keto-L-gulonic acid inhibits the growth of Bacillus pumilus and Ketogulonicigenium vulgare.

The typical vitamin C mixed-fermentation process's second stage involves bioconversion of L-sorbose to 2-keto-L-gulonic acid (2-KLG), using a consortium comprising Ketogulonicigenium vulgare and Bacillus spp. (as helper strain). The concentration of the helper strain in the co-fermentation system was closely correlated with K. vulgare cell growth and 2-KLG accumulation. To understand the tolerance and response of the helper strain and K. vulgare to 2-KLG, 2-KLG was added to the single-strain system of Bacillus pumilus and K. vulgare and the basic physiological and biochemical properties were determined. In this study, the addition of 1 mg/mL 2-KLG reduced the number of viable and spore cells, lowered the levels of intracellular reactive oxygen species (ROS), enhanced the intra- and extracellular total antioxidant capacity (T-AOC), and significantly affected the B. pumilus sporulation-related genes expression levels. Furthermore, the addition of 1 mg/mL 2-KLG increased the intracellular ROS levels, decreased the intra- and extracellular T-AOC, and downregulated the antioxidant enzyme-related genes and 2-KLG production enzyme-related genes of K. vulgare. These results suggested that 2-KLG could induce acidic and oxidative stress in B. pumilus and K. vulgare, which could be a guide for a greater understanding of the interaction between the microorganisms.

Pangenome analyses of Bacillus pumilus, Bacillus safensis, and Priestia megaterium exploring the plant-associated features of bacilli strains isolated from canola.

Previous genome mining of the strains Bacillus pumilus 7PB, Bacillus safensis 1TAz, 8Taz, and 32PB, and Priestia megaterium 16PB isolated from canola revealed differences in the profile of antimicrobial biosynthetic genes when compared to the species type strains. To evaluate not only the similarities among B. pumilus, B. safensis, and P. megaterium genomes but also the specificities found in the canola bacilli, we performed comparative genomic analyses through the pangenome evaluation of each species. Besides that, other genome features were explored, especially focusing on plant-associated and biotechnological characteristics. The combination of the genome metrics Average Nucleotide Identity and digital DNA-DNA hybridization formulas 1 and 3 adopting the universal thresholds of 95 and 70%, respectively, was suitable to verify the identification of strains from these groups. On average, core genes corresponded to 45%, 52%, and 34% of B. pumilus, B. safensis, and P. megaterium open pangenomes, respectively. Many genes related to adaptations to plant-associated lifestyles were predicted, especially in the Bacillus genomes. These included genes for acetoin production, polyamines utilization, root exudate chemoreceptors, biofilm formation, and plant cell-wall degrading enzymes. Overall, we could observe that strains of these species exhibit many features in common, whereas most of their variable genome portions have features yet to be uncovered. The observed antifungal activity of canola bacilli might be a result of the synergistic action of secondary metabolites, siderophores, and chitinases. Genome analysis confirmed that these species and strains have biotechnological potential to be used both as agricultural inoculants or hydrolases producers. Up to our knowledge, this is the first work that evaluates the pangenome features of P. megaterium.

An antimicrobial peptide specifically active against Listeria monocytogenes is secreted by Bacillus pumilus SF214.

BACKGROUND: Members of the Bacillus genus produce a large variety of antimicrobial peptides including linear or cyclic lipopeptides and thiopeptides, that often have a broad spectrum of action against Gram-positive and Gram-negative bacteria. We have recently reported that SF214, a marine isolated strain of Bacillus pumilus, produces two different antimicrobials specifically active against either Staphylococcus aureus or Listeria monocytogenes. The anti-Staphylococcus molecule has been previously characterized as a pumilacidin, a nonribosomally synthesized lipopetide composed of a mixture of cyclic heptapeptides linked to fatty acids of variable length. RESULTS: Our analysis on the anti-Listeria molecule of B. pumilus SF214 indicated that it is a peptide slightly smaller than 10 kDa, produced during the exponential phase of growth, stable at a wide range of pH conditions and resistant to various chemical treatments. The peptide showed a lytic activity against growing but not resting cells of Listeria monocytogenes and appeared extremely specific being inactive also against L. innocua, a close relative of L. monocytogenes. CONCLUSIONS: These findings indicate that the B. pumilus peptide is unusual with respect to other antimicrobials both for its time of synthesis and secretion and for its strict specificity against L. monocytogenes. Such specificity, together with its stability, propose this new antimicrobial as a tool for potential biotechnological applications in the fight against the dangerous food-borne pathogen L. monocytogenes.

Bacillus pumilus proteome changes in response to 2,4,6-trinitrotoluene-induced stress.

2,4,6-Trinitrotoluene (TNT) is the most widely used nitroaromatic compound and is highly resistant to degradation. Most aerobic microorganisms reduce TNT to amino derivatives via formation of nitroso- and hydroxylamine intermediates. Although pathways of TNT degradation are well studied, proteomic analysis of TNT-degrading bacteria was done only for some individual Gram-negative strains. Here, we isolated a Gram-positive strain from TNT-contaminated soil, identified it as Bacillus pumilus using 16S rRNA sequencing, analyzed its growth, the level of TNT transformation, ROS production, and revealed for the first time the bacillary proteome changes at toxic concentration of TNT. The transformation of TNT at all studied concentrations (20-200 mg/L) followed the path of nitro groups reduction with the formation of 4-amino-2,6-dinitrotoluene. Hydrogen peroxide production was detected during TNT transformation. Comparative proteomic analysis of B. pumilus showed that TNT (200 mg/L) inhibited expression of 46 and induced expression of 24 proteins. Among TNT upregulated proteins are those which are responsible for the reductive pathway of xenobiotic transformation, removal of oxidative stress, DNA repair, degradation of RNA and cellular proteins. The production of ribosomal proteins, some important metabolic proteins and proteins involved in cell division are downregulated by this xenobiotic.

Isolation and Characterization of Two Novel Siphoviruses Novomoskovsk and Bolokhovo, Encoding Polysaccharide Depolymerases Active against Bacillus pumilus.

This study describes two novel bacteriophages infecting members of the Bacillus pumilus group. Even though members of the group are not recognized as pathogenic, several strains belonging to the group have been reported to cause infectious diseases in plants, animals and humans. Bacillus pumilus group species are highly resistant to ultraviolet radiation and capable of forming biofilms, which complicates their eradication. Bacteriophages Novomoskovsk and Bolokhovo were isolated from soil samples. Genome sequencing and phylogenetic analysis revealed that the phages represent two new species of the genus Andromedavirus (class Caudoviricetes). The phages remained stable in a wide range of temperatures and pH values. A host range test showed that the phages specifically infect various strains of B. pumilus. The phages form clear plaques surrounded by halos. Both phages Novomoskovsk and Bolokhovo encode proteins with pectin lyase domains-Putative depolymerases. Obtained in a purified recombinant form, the proteins produced lysis zones on the lawn of a B. pumilus strain. This suggests that Novomoskovsk and Bolokhovo may be effective for the eradication of B. pumilus biofilms.

Characterization and engineering of two new GH9 and GH48 cellulases from a Bacillus pumilus isolated from Lake Bogoria.

OBJECTIVES: To search for new alkaliphilic cellulases and to improve their efficiency on crystalline cellulose through molecular engineering RESULTS: Two novel cellulases, BpGH9 and BpGH48, from a Bacillus pumilus strain were identified, cloned and biochemically characterized. BpGH9 is a modular endocellulase belonging to the glycoside hydrolase 9 family (GH9), which contains a catalytic module (GH) and a carbohydrate-binding module belonging to class 3 and subclass c (CBM3c). This enzyme is extremely tolerant to high alkali pH and remains significantly active at pH 10. BpGH48 is an exocellulase, belonging to the glycoside hydrolase 48 family (GH48) and acts on the reducing end of oligo-ß1,4 glucanes. A truncated form of BpGH9 and a chimeric fusion with an additional CBM3a module was constructed. The deletion of the CBM3c module results in a significant decline in the catalytic activity. However, fusion of CBM3a, although in a non native position, enhanced the activity of BpGH9 on crystalline cellulose. CONCLUSIONS: A new alkaliphilic endocellulase BpGH9, was cloned and engineered as a fusion protein (CBM3a-BpGH9), which led to an improved activity on crystalline cellulose.

High-level expression of a ß-mannanase (manB) in Pichia pastoris GS115 for mannose production with Penicillium brevicompactum fermentation pretreatment of soybean meal.

An endo-1,4-ß-mannanase gene (manB) from a Bacillus pumilus Nsic-2 grown in a stinky tofu emulsion was cloned and expressed in Pichia pastoris GS115. After characterized, the endo-1,4-ß-mannanase (manB) show maximum activity at pH 6.0 and 50 °C with LBG as substrate and perform high stability at a range of pH 6-8. After applying for a shake flask fermentation, the specific activity of manB reached 3462 U/mg. To produce mannose, the soybean meal (SBM) was pretreated by biological fermentation for 11 days with Penicillium brevicompactum, and then hydrolyzed by manB. As a result, mannose yield reached 3.58 g per 1 kg SBM which indicated that 0.358% SBM was converted into mannose after hydrolyzation, and mean a total 20% mannan of SBM converting into mannose, while the control group demonstrated only 1.78% conversion. An effective ß-mannanase for the bioconversion of mannan-rich biomasses and an efficient method to produce mannose with soybean meal were introduced.

Mediation of induced systemic resistance by the plant growth-promoting rhizobacteria Bacillus pumilus S2-3-2.

Plant-rhizobacteria interaction and co-evolution developed adaptive strategies which may help the plant survive in nature. Plant rhizosphere soil isolates were analyzed to investigated the effects of rhizobacteria for promoting plant growth and suppress plant disease. Bacterial strains which isolated from plant rhizosphere soil were screened for elicitation of induced systemic resistance (ISR) on tobacco. Strain S2-3-2 results in significant reduction of disease severity on tobacco, it was identified as Bacillus pumilus by multilocus sequence analysis (MLSA). Strain S2-3-2 was deeper studied for pepper plant growth promotion and biological control activity against pepper bacterial spot disease. It was found that the pepper disease severity was decreased when the roots were drenched with strain S2-3-2, and the pepper plants had a higher weight and chlorophyll content, as compared with the mock-treated plants. Transcriptional expression of pathogenesis-related (PR) protein genes in pepper was analyzed by real-time PCR, gene expressions of CaPR1, CaPR4, and CaPR10 were increased when the plants were treated with strain S2-3-2. Moreover, strain S2-3-2 was tested for the production of indole-3-acetic acid (IAA), and it was determined to emit volatiles that enhance the growth of the tobacco plants. Interesting, heat-killed S2-3-2 enhance the pepper root growth, increase the gene expressions of CaPR4 and CaPR10 after pathogen challenge for 6 h, but limited to suppress the pepper bacterial spot disease as compare to the mock-treated plants. Strain S2-3-2 can be a potential biological control agent on the plant root for plant growth promoting and disease suppression.

A novel DNA methylation motif identified in Bacillus pumilus BA06 and possible roles in the regulation of gene expression.

Single-molecule real-time (SMRT) sequencing can be used to identify a wide variety of chemical modifications of the genome, such as methylation. Here, we applied this approach to identify N6-methyl-adenine (m6A) and N4-methyl-cytosine (m4C) modification in the genome of Bacillus pumilus BA06. A typical methylation recognition motif of the type I restriction-modification system (R-M), 5'-TCm6AN8TTGG-3'/3'-AGTN8m6AACC-5', was identified. We confirmed that this motif was a new type I methylation site using REBASE analysis and that it was recognized by a type I R-M system, Bpu6ORFCP, according to methylation sensitivity assays in vivo and vitro. Furthermore, we found that deletion of the R-M system Bpu6ORFCP induced transcriptional changes in many genes and led to increased gene expression in pathways related to ABC transporters, sulfur metabolism, ribosomes, cysteine and methionine metabolism and starch and sucrose metabolism, suggesting that the R-M system in B. pumilus BA06 has other significant biological functions beyond protecting the B. pumilus BA06 genome from foreign DNA.

Enhancing the decolorization activity of Bacillus pumilus W3 CotA-laccase to Reactive Black 5 by site-saturation mutagenesis.

Reactive Black 5 (RB5) is a typical refractory azo dye. Widespread utilization of RB5 has caused a variety of environmental and health problems. The enzymatic degradation of RB5 can be a promising solution due to its superiority as an eco-friendly and cost-competitive process. Bacterial CotA-laccase shows great application prospect to eliminate hazardous dyes from wastewater. However, efficient decolorization of RB5 CotA-laccase generally requires the participation of costly, toxic mediators. In the present study, we modified the amino acids Thr415 and Thr418 near the type 1 copper site and the amino acid Gln442 at the entrance of the substrate-binding pocket of Bacillus pumilus W3 CotA-laccase to boost its RB5 decolorization activity based on molecular docking analysis and site-saturation mutagenesis. Through the strategies, two double site mutants T415D/Q442A and T418K/Q442A obtained demonstrated 43.94 and 52.64% RB5 decolorization rates in the absence of a mediator at pH 10.0, respectively, which were about 3.70- and 4.43-fold higher compared with the wild-type CotA-laccase. Unexpectedly, the catalytic efficiency of the T418K/Q442A to ABTS was enhanced by 5.33-fold compared with the wild-type CotA-laccase. The mechanisms of conferring enhanced activity to the mutants were proposed by structural analysis. In summary, the mutants T415D/Q442A and T418K/Q442A have good application potentials for the biodegradation of RB5. KEY POINTS: • Three amino acids of CotA-laccase were manipulated by site-saturation mutagenesis. • Decolorization rate of two mutants to RB5 was enhanced 3.70- and 4.43-fold, respectively. • The mechanisms of awarding enhanced activity to the mutants were supposed.

RNA sequencing reveals small RNAs in Bacillus pumilus under different growth phases of the protease fermentation process.

Bacillus pumilus, an endospore-forming soil bacterium, produces a wide array of extracellular proteins, such as proteases, which are already applied in the chemical, detergent and leather industries. Small noncoding regulatory RNAs (sRNAs) in bacteria are important RNA regulators that act in response to various environmental signals. Here, an RNA-seq-based transcriptome analysis was applied to B. pumilus SCU11, a strain that produces extracellular alkaline protease, across various growth phases of the protease fermentation process. Through bioinformatics screening of the sequencing data and visual inspection, 84 putative regulatory sRNAs were identified in B. pumilus, including 21 antisense sRNAs and 63 sRNAs in intergenic regions. We experimentally validated the expression of 48 intergenic sRNAs by quantitative RT-PCR (qRT-PCR). Meanwhile, the expression of 6 novel sRNAs was confirmed by northern blotting, and the expression profiles of 5 sRNAs showed close correlation with the growth phase. We revealed that the sRNA Bpsr137 was involved in flagellum and biofilm formation in B. pumilus. The identification of a global set of sRNAs increases the inventory of regulatory sRNAs in Bacillus and implies the important regulatory roles of sRNA in B. pumilus. These findings will contribute another dimension to the optimization of crucial metabolic activities of B. pumilus during a productive fermentation process.

Site-directed mutagenesis of coenzyme-independent carotenoid oxygenase CSO2 to enhance the enzymatic synthesis of vanillin.

Vanillin is a popular flavoring compound and an important food additive. Owing to the consumer preference for inexpensive natural aroma flavors, vanillin production through a biotechnological pathway has become of great interest and commercial value in recent years. In this study, an enzymatic synthetic system for vanillin using a coenzyme-independent decarboxylase (FDC) and oxygenase (CSO2) cascade was reconstituted and optimized. This system produces a slightly higher production yield (40.20%) than the largest yield reported for immobilized FDC and CSO2 (35.00%) with ferulic acid as a substrate. It was previously reported that the low catalytic activity and thermal instability of CSO2 restrict the overall productivity of vanillin. In present study, site-directed mutagenesis was applied to rate-limiting oxygenase CSO2 to generate positive mutants. The production yields of mutants A49P (58.44%) and Q390A (65.29%) were 1.45- and 1.62-fold that of CSO2 wild type, respectively. The potential mechanism for enhanced vanillin production using A49P involved increased thermostability and catalytic efficiency, while that using Q390A was probably associated with a better thermostable performance and increased catalytic efficiency resulting from a larger entrance channel.

Screening for Candidate Genes Associated with Biocontrol Mechanisms of Bacillus pumilus DX01 Using Tn5 Transposon Mutagenesis and a 2-DE-Based Comparative Proteomic Analysis.

A total of 1467 mutants of the biocontrol bacterium Bacillus pumilus DX01 were obtained by Tn5 insertional mutagenesis and subjected to the determination of antagonistic capabilities. Compared with the wild-type strain DX01, the mutant M25 was identified to have the most significant reduction in antagonistic capability against the phytopathogen Bipolaris maydis and extracellular proteinase activity. The integration site of the exogenous T-DNA in the genome of mutant M25 was revealed in the coding region of malony CoA-ACP transacylase (MCAT) gene (mcat), which belongs to a polyketide synthase (PKS) gene cluster, DX01pks of B. pumilus DX01. Furthermore, the whole DX01pks gene cluster was cloned using Illumina Solexa sequencing technology, and it has a modular framework different from the other two gene clusters involved in polyketide synthesis in B. amyloliquefaciens FZB42 (pks1) and B. subtilis 168 (pksX). Finally, in order to gain more insights into the molecular mechanisms of biocontrol of B. pumilus DX01, the changes in the relative level of expression of total proteins between the original strain DX01 and the mutant M25 were detected by 2-DE-based proteomic analysis. A total of twenty differentially expressed proteins were identified upon the mcat gene transposition mutagenesis. Of these proteins, seven proteins were up-regulated in expression level and the other proteins were down-regulated. Taken together, the results in this study showed that Tn5 transposon mutagenesis of B. pumilus DX01 can lead to a significant change of antiphytopathogen ability, and the DX01pks gene cluster possibly play a potential role in the biocontrol processes of this bacterium.

Characterization of a Protease Hyper-Productive Mutant of Bacillus pumilus by Comparative Genomic and Transcriptomic Analysis.

Bacillus pumilus BA06 has great potential for the production of alkaline proteases. To improve the protease yield, classical mutagenesis to combine the physical and chemical mutagens was performed to obtain a protease hyper-productive mutant SCU11. The full genome sequences of BA06 and SCU11 strains were assembled through DNA sequencing using the PacBio sequencing platform. By comparative genomics analysis, 147 SNPs and 15 InDels were found between these two genomes, which lead to alternation of coding sequence in 15 genes. Noticeable, the gene (kinA) encoding sporulation kinase A is interrupted by introducing a stop codon in its coding region in BA06. Interestedly, this gene is reversely corrected in SCU11. Furthermore, comparative transcriptome analysis revealed that kinA and two positive regulatory genes (DegU and Spo0A) were upregulated in transcription in SCU11. In terms of the transcriptional data, upregulation of a phosphorylation cascade starting with KinA may enhance Spo0A phosphorylation, and thus activate expression of the gene aprE (encoding major extracellular protease) through repression of AbrB (a repressor of aprE) and activation of SinI, an antagonist of SinR (a repressor of aprE). In addition, the other genes involved in various metabolic pathways, especially of membrane transport and sporulation, were altered in transcription between these two strains. Conclusively, our transcriptome data suggested that upregulation degU and spo0A, as well as kinA, may at least partially contribute to the high production of alkaline protease in SCU11.