Sfp Gene Research Articles

Unleashing Bacillus species as versatile antagonists: Harnessing the biocontrol potentials of the plant growth-promoting rhizobacteria to combat Macrophomina phaseolina infection in Gloriosa superba

Charcoal rot caused by Macrophomina phaseolina is one of the most devastating diseases that cause severe yield loss in Gloriosa superba cultivation. Plant growth-promoting rhizobacteria (PGPR) are extensively harnessed as biocontrol agents due to their effectiveness in combating a wide array of plant pathogens through a multifaceted approach. The present study delved into the mechanisms underlying its ability to inhibit root rot pathogen and its capacity to promote plant growth in G. superba, commonly known as glory lily. PGPR isolated from the rhizosphere of glory lily were subjected to in vitro assessments using the dual plate technique. The isolated Bacillus subtilis BGS-10 and B. velezensis BGS-21 showed higher mycelial inhibition (61%) against M. phaseolina. These strains also promote plant growth by producing indole-3-acetic acid, siderophore, ammonia, amylase, cellulase, pectinase, xylanase, and lipase chemicals. Genome screening of BGS-10 and BGS-21 revealed the presence of antimicrobial peptide genes such as Iturin (ituD gene), surfactin (srfA and sfp genes) along with the mycolytic enzyme β-1,3-glucanase. Further, the presence of secondary metabolites in the bacterial secretome was identified through gas chromatography-mass spectrometry (GC/MS) analysis. Notably, pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl), 9 H-pyrido[3,4-b] indole and L-leucyl-D-leucine exhibited the highest docking score against enzymes responsible for pathogen growth and plant cell wall degradation. Under glasshouse conditions, tuber treatment and soil application of talc-based formulation of B. subtilis BGS-10 and B. velezensis BGS-21 suppress the root rot incidence with a minimal disease incidence of 27.78% over untreated control. Concurrently, there was a notable induction of defense-related enzymes, including peroxidase (PO), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL), in glory lily. Therefore, it can be concluded that plant growth-promoting Bacillus strains play a significant role in fortifying the plant's defense mechanisms against the root rot pathogen.

The evolution of seminal fluid gene expression and postmating reproductive isolation in Drosophila.

Seminal fluid protein (Sfp) genes show, in general, a higher rate of sequence divergence than genes from other categories, which is often attributed to forms of postcopulatory sexual selection or sexual conflict. Recently, the relaxation of selective constraints has been proposed as an alternative explanation for the rapid sequence evolution of Sfps and other genes with sex-limited expression. The expression of Sfp genes is a likely target of selection, but the evolution of differences in their expression levels is less understood. Here, we explore both polymorphism and divergence in Sfp gene expression between Drosophila melanogaster and Drosophila simulans, how selection might have influenced their expression, and whether changes in expression might trigger the evolution of reproductive isolating barriers. In our analysis, Sfp genes showed higher divergence, but not higher polymorphism, in expression than the average male reproductive glands gene. Sfp genes with reproductive-tissue-specific expression were enriched for both directional and stabilizing selection, while relaxed selection was the predominant mode of evolution among Sfp genes with any other nonreproductive tissue-specific or nontissue-specific expression. The knockdown of single genes known to affect intraspecific sperm competition, and with patterns of expression divergence and polymorphism suggestive of directional selection, was not enough to break down postmating reproductive isolation barriers between species. Our results identify the expression of male-specific Sfp genes as an enriched target of selection and suggest a complex molecular relationship between postcopulatory sexual selection on a single gene's expression and its effect on the onset of speciation.

Investigation of Metabolites Isolated from Sphingomonas Egypticus DM, A Rhizosphere of Datura Metel

Using naturally occurring bioactive compounds has been crucial in progressing contemporary medical practices. The rhizosphere, which refers to the narrow region of soil surrounding plant roots, serves as a highly dynamic environment for soil bacteria owing to the substantial quantity of organic compounds released into the soil by plant roots. This paper outlines the fermentation and subsequent processing of rhizosphere Sphingomonas egypticus DM. The culture media of Sphingomonas was cultivated and subsequently subjected to propagation. The ethyl acetate extract was then obtained and subjected to fractionation and purification utilizing various chromatographic techniques. This process led to the isolation of a compound under investigation that exhibited a distinct spot on thin-layer chromatography (TLC) plates, with a calculated retention factor (Rf = 0.56) using a mobile phase composed of methylene chloride and methanol (9:1). Upon spraying with anisaldehyde/sulphuric reagent and subsequent heating for a brief period, the compound displayed a violet colour. This compound was investigated in vitro to assess its antimicrobial and minimum inhibitory concentration (MIC) capabilities. The results demonstrated a significant inhibitory effect against phytopathogenic fungi, specifically Rhizoctonia solani (21.3 mm) and Alternaria alternate (18.3 mm). Additionally, a moderate inhibitory effect was observed against Pseudomonas aeruginosa (12.7 mm), whereas a somewhat less inhibitory effect was shown against Streptococcus mutans (9.3 mm). The study also demonstrated that (MIC) against Fusarium oxysporum was seen at a concentration of 125 µg/mL compared to various conventional antibiotics. Finally, the strain underwent PCR screening to detect PKS and lipopeptide 4'-phosphopantetheinyl transferase sfp genes. The PCR amplification assay demonstrated the presence of genes encoding the KS domain and Surfactin. Furthermore, the sequences of Sphingomonas egypticus DM have been officially recorded in the NCBI GenBank database and can be accessed using the accession codes OR469907 and OR499756.

Construction of lipopeptide mono-producing Bacillus strains and comparison of their antimicrobial activity

Bacillus amyloliquefaciens generally secretes a mixture of multiple cyclic lipopeptides, including iturin, surfactin, and plipastatin, and it is challenging to discriminate each cyclic lipopeptide's antimicrobial activity directly. In this study, a chassis strain Bacillus subtilis 1A751 WR without cyclic lipopeptides operons was constructed by CRISPR/Cas9. The iturin gene cluster of B. amyloliquefaciens HYM-12 was captured and assembled with sfp and degQ genes using transformation-associated recombination (TAR). It was then integrated into the chassis strain to obtain an iturin mono-producing strain that could synthesize six iturin isoforms. The frameshift sfp gene of strain 1A751 Δpps was repaired by a scarless gene knock-in method to generate a surfactin mono-producing strain. Antimicrobial activity experiments indicated that the extracts of iturin, surfactin, and our previously constructed plipastatin mono-producing strains exhibited powerful inhibitory effects toward pathogenic bacteria. Interestingly, iturin showed significant antifungal activity, but surfactin and plipastatin exhibited weak antifungal activity. Compared with amphotericin B, iturin has a solid and durable antifungal activity. This study demonstrates that TAR cloning is efficient for cloning large gene clusters, proves the differences in the antimicrobial activity of three cyclic lipopeptides, and lays a theoretical basis for further elucidating their antimicrobial mechanism.

Improvement of lipopeptide production in Bacillus subtilis HNDF2-3 by overexpression of the sfp and comA genes

Bacillus subtilis HNDF2-3 can produce a variety of lipopeptide antibiotics with lower production. To improve its lipopeptide production, three genetically engineered strains were constructed. The results of real-time PCR showed that the highest transcriptional levels of the sfp gene in F2-3sfp, F2-3comA and F2-3sfp-comA were 29.01, 6.65 and 17.50 times of the original strain, respectively, while the highest transcriptional levels of the comA gene in F2-3comA and F2-3sfp-comA were 10.44 and 4.13 times of the original strain, respectively. The results of ELISA showed that the malonyl-CoA transacylase activity of F2-3comA was the highest, reaching 18.53 IU/L at 24 h, the data was 32.74% higher than that of the original strain. The highest total lipopeptide production of F2-3sfp, F2-3comA and F2-3sfp-comA induced by IPTG at optimal concentration were 33.51, 46.05 and 38.96% higher than that of the original strain, respectively. The results of HPLC showed that iturin A production of F2-3sfp-comA was the highest, which was 63.16% higher than that of the original strain. This study laid the foundation for further construction of genetically engineered strains with high lipopeptide production.

Bacillus velezensis SQR9 inhibition to fungal denitrification responsible for decreased N2O emissions from acidic soils

Tropical and subtropical acidic soils are hotspots of global terrestrial nitrous oxide (N2O) emissions, with N2O produced primarily through denitrification. Plant growth-promoting microbes (PGPMs) may effectively mitigate soil N2O emissions from acidic soils, achieved through differential responses of bacterial and fungal denitrification to PGPMs. To test this hypothesis, we conducted a pot experiment and the associated laboratory trials to gain the underlying insights into the PGPM Bacillus velezensis strain SQR9 effects on N2O emissions from acidic soils. SQR9 inoculation significantly reduced soil N2O emissions by 22.6–33.5 %, dependent on inoculation dose, and increased the bacterial AOB, nirK and nosZ genes abundance, facilitating the reduction of N2O to N2 in denitrification. The relative contribution of fungi to the soil denitrification rate was 58.4–77.1 %, suggesting that the N2O emissions derived mainly from fungal denitrification. The SQR9 inoculation significantly inhibited the fungal denitrification and down-regulated fungal nirK gene transcript, dependent on the SQR9 sfp gene, which was necessary for secondary metabolite synthesis. Therefore, our study provides new evidence that decreased N2O emissions from acidic soils can be due to fungal denitrification inhibited by PGPM SQR9 inoculation.

Genetically engineered Blue silkworm capable of synthesizing natural blue pigment

Synthetic biology is an eco-friendly and sustainable approach for the production of compounds, particularly used when the production processes involve toxic reagents. In this study, we used the silk gland of silkworm to produce indigoidine, a valuable natural blue pigment that cannot be synthesized naturally in animals. We genetically engineered these silkworms by integrating the indigoidine synthetase (idgS) gene from S. lavendulae and the PPTase (Sfp) gene from B. subtilis into the silkworm genome. In the resulting Blue silkworm, indigoidine was detected at a high level in the posterior silk gland (PSG), spanning all developmental stages from larvae to adults, without affecting silkworm growth or development. This synthesized indigoidine was secreted from the silk gland and subsequently stored in the fat body, with only a small fraction being excreted by the Malpighian tubule. Metabolomic analysis revealed that Blue silkworm efficiently synthesized indigoidine by upregulating l-glutamine, the precursor of indigoidine, and succinate, which is related to energy metabolism in the PSG. This study represents the first synthesis of indigoidine in an animal and therefore opens a new avenue for the biosynthesis of natural blue pigments and other valuable small molecules.

Bacillus thuringiensis PM25 ameliorates oxidative damage of salinity stress in maize via regulating growth, leaf pigments, antioxidant defense system, and stress responsive gene expression.

Soil salinity is the major abiotic stress that disrupts nutrient uptake, hinders plant growth, and threatens agricultural production. Plant growth-promoting rhizobacteria (PGPR) are the most promising eco-friendly beneficial microorganisms that can be used to improve plant responses against biotic and abiotic stresses. In this study, a previously identified B. thuringiensis PM25 showed tolerance to salinity stress up to 3 M NaCl. The Halo-tolerant Bacillus thuringiensis PM25 demonstrated distinct salinity tolerance and enhance plant growth-promoting activities under salinity stress. Antibiotic-resistant Iturin C (ItuC) and bio-surfactant-producing (sfp and srfAA) genes that confer biotic and abiotic stresses were also amplified in B. thuringiensis PM25. Under salinity stress, the physiological and molecular processes were followed by the over-expression of stress-related genes (APX and SOD) in B. thuringiensis PM25. The results detected that B. thuringiensis PM25 inoculation substantially improved phenotypic traits, chlorophyll content, radical scavenging capability, and relative water content under salinity stress. Under salinity stress, the inoculation of B. thuringiensis PM25 significantly increased antioxidant enzyme levels in inoculated maize as compared to uninoculated plants. In addition, B. thuringiensis PM25-inoculation dramatically increased soluble sugars, proteins, total phenols, and flavonoids in maize as compared to uninoculated plants. The inoculation of B. thuringiensis PM25 significantly reduced oxidative burst in inoculated maize under salinity stress, compared to uninoculated plants. Furthermore, B. thuringiensis PM25-inoculated plants had higher levels of compatible solutes than uninoculated controls. The current results demonstrated that B. thuringiensis PM25 plays an important role in reducing salinity stress by influencing antioxidant defense systems and abiotic stress-related genes. These findings also suggest that multi-stress tolerant B. thuringiensis PM25 could enhance plant growth by mitigating salt stress, which might be used as an innovative tool for enhancing plant yield and productivity.

Plant growth-promoting and antibacterial activities of cultivable bacteria alive in tobacco field against Ralstonia solanacearum.

Bacterial wilt disease caused by Ralstonia solanacearum leads to decrease of crops yield. Investigation of cultivable bacteria diversity provides more microbial species for screening antagonistic bacteria. In the present study, a variety of cultivation methods were used to investigate the diversity of cultivable bacteria alive in tobacco field. A total of 441 bacterial strains were obtained that belonged to four phyla, 49 genera and 146 species. Actinobacteria and Proteobacteria were the dominant phyla. Agrobacterium, Arthrobacter, Bacillus, Klebsiella, Paenarthrobacter, Pseudomonas and Pseudarthrobacter were the dominant genera. Some rare genera were discovered including Bosea, Cedecea, Delftia and Dyella. Diversity, species and abundances of bacteria altered under different cultivation conditions. One hundred three bacterial strains showed plant growth-promoting attributes. Twenty Bacillus strains showed high antibacterial activity against R. solanacearum. In field experiments, individual strain and consortia of Bacillus subtilis, B. siamensis and B. vallismortis effectively inhibited bacterial wilt. The core genes that controlled synthesis of secondary metabolites were knocked out in B. vallismortis SSB-10. Difficidin, which was synthesized by dif operon and controlled by sfp gene, was the antibacterial substance produced by SSB-10. Difficidin destroyed cell wall and cell membrane of R. solanacearum and inhibited its motility, production of extracellular polysaccharides and cellulase activity.

Bacillus mycoides PM35 Reinforces Photosynthetic Efficiency, Antioxidant Defense, Expression of Stress-Responsive Genes, and Ameliorates the Effects of Salinity Stress in Maize.

Soil salinity is one of the abiotic constraints that imbalance nutrient acquisition, hampers plant growth, and leads to potential loss in agricultural productivity. Salt-tolerant plant growth-promoting rhizobacteria (PGPR) can alleviate the adverse impacts of salt stress by mediating molecular, biochemical, and physiological status. In the present study, the bacterium Bacillus mycoides PM35 showed resistance up to 3 M NaCl stress and exhibited plant growth-promoting features. Under salinity stress, the halo-tolerant bacterium B. mycoides PM35 showed significant plant growth-promoting traits, such as the production of indole acetic acid, siderophore, ACC deaminase, and exopolysaccharides. Inoculation of B. mycoides PM35 alleviated salt stress in plants and enhanced shoot and root length under salinity stress (0, 300, 600, and 900 mM). The B. mycoides PM35 alleviated salinity stress by enhancing the photosynthetic pigments, carotenoids, radical scavenging capacity, soluble sugars, and protein content in inoculated maize plants compared to non-inoculated plants. In addition, B. mycoides PM35 significantly boosted antioxidant activities, relative water content, flavonoid, phenolic content, and osmolytes while reducing electrolyte leakage, H2O2, and MDA in maize compared to control plants. Genes conferring abiotic stress tolerance (CzcD, sfp, and srfAA genes) were amplified in B. mycoides PM35. Moreover, all reactions are accompanied by the upregulation of stress-related genes (APX and SOD). Our study reveals that B. mycoides PM35 is capable of promoting plant growth and increasing agricultural productivity.

Condition‐dependent seminal fluid gene expression and intergenerational paternal effects on ejaculate quality

AbstractThere is increasing evidence to show that the environment experienced by fathers can have intergenerational paternal effects on the phenotype of offspring. Paternal effects can be mediated by ejaculate traits beyond the genes carried by sperm, for example through methylation of DNA during spermatogenesis, or non‐sperm components of the ejaculate such as small RNAs or seminal fluid proteins (sfps).Theory suggests that ejaculate‐mediated paternal effects can have important evolutionary consequences, especially if they exhibit condition‐dependent expression. Here we use the nutritional geometry approach to explore condition dependence in ejaculate traits and intergenerational paternal effects in the cricketTeleogryllus oceanicus.We fed males one of 24 chemically defined diets that varied in macronutrient concentration and the ratio of protein (P) to carbohydrate (C). We measured paternal expenditure on testes and accessory gland growth, and the expression of three sfp genes that are known to impact sperm viability and the survival of embryos to hatching. We also measured the hatching success and reproductive phenotypes of adult offspring.We found linear effects of P and C on testes and accessory gland mass, and linear and correlated effects of P and C on the expression of two sfp genes. Paternal diet had no effect on the survival of embryos to hatching.There were significant intergenerational effects of paternal diet on the viability of sperm produced by sons, but no paternal effects on the expression of sfp genes in sons or fecundity in daughters.Our data suggest that ejaculate quality can have a significant non‐genetic component determined by the paternal environment, which holds important implications for the evolutionary maintenance of variation in heritable male fitness.A freePlain Language Summarycan be found within the Supporting Information of this article.

Bacillus amyloliquefaciens-Derived Lipopeptide Biosurfactants Inhibit Biofilm Formation and Expression of Biofilm-Related Genes of Staphylococcus aureus.

Biosurfactants (BSs) are surface-active compounds produced by diverse microorganisms, including the genus Bacillus. These bioactive compounds possess biological activities such as antiadhesive, antimicrobial and antibiofilm effects that can lead to important applications in combating many infections. Based on these findings, we decided to investigate the antibiofilm activity of BSs from the marine Bacillus amyloliquefaciens against Staphylococcus aureus CCM 4223. Expression of biofilm-related genes was also evaluated using qRT-PCR. Isolated and partially purified BSs were identified and characterized by molecular tools and by UHPLC-DAD and MALDI-TOF/MS. Bacillus amyloliquefaciens 3/22, that exhibited surfactant activity evaluated by oil spreading assay, was characterized using the 16S rRNA sequencing method. Screening by PCR detected the presence of the sfp, srfAA, fenD and ituD genes, suggesting production of the lipopeptides (LPs) surfactin, fengycin and iturin. The above findings were further supported by the results of UHPLC-DAD and MALDI-TOF/MS. As quantified by the crystal violet method, the LPs significantly (p < 0.001) reduced biofilm formation of S. aureus in a dose-dependent manner and decreased expression of biofilm-related genes fnbA, fnbB, sortaseA and icaADBC operon. Data from our investigation indicate a promising therapeutic application for LPs isolated from B. amyloliquefaciens toward prevention of S. aureus biofilm infections.

Increasing fengycin production by strengthening the fatty acid synthesis pathway and optimizing fermentation conditions

In the present work, Bacillus subtilis 168 was engineered as a cell factory for fengycin production by activating its dormant native fengycin synthetase. Initially, the sfp gene from Bacillus amyloliquefaciens FZB42 was introduced into B. subtilis 168 to activate fengycin biosynthesis. Then, the original promoter of the degQ gene was replaced with the P43 promoter, resulting in strain 168DS. The fengycin production of 168DS increased to 10.40 mg/L in flask culture, realizing significant fengycin synthesis starting from zero. Subsequently, the birA, acs and accACD genes were overexpressed in the engineered strain 168DS to enhance the yield of fengycin, resulting in strain 168DSABA. The fengycin production of 168DSABA increased to 24.70 mg/L in flask culture, representing a 137% increase over the parental strain 168DS. Additionally, we also attempted to increase fengycin production by optimizing the medium components and fermentation conditions. This optimization strategy increased the fengycin production from 24.70 to 130.10 mg/L. Subsequent metabolomic analysis revealed the underlying reasons for the increase of fengycin production. This strategy provides the possibility to increase the industrial production of fengycin and promote its applications by reducing its price.

Iturin and surfactin from the endophyte Bacillus amyloliquefaciens strain RKEA3 exhibits antagonism against Staphylococcus aureus

Bacillus amyloliquefaciens strain RKEA3 , an isolated endophytic strain from Avicennia officinalis, was found to be shown antagonistic activity against Staphylococcus aureus ATCC25923 , Streptococcus pyogens ATCC19615, Staphylococcus aureus ATCC 9219 , clinical strains of Staphylococcus aureus and Cryptococcus neoformans. The antimicrobial lipopeptide produced by this strain was isolated by acid precipitation followed by dialysis, lyophilization, and methanol extraction. The influence of various parameters on the antibacterial activity of isolated lipopeptide was studied, and it was found that they could survive a wide temperature and pH range. Proteolytic enzymes, metals, surfactants, and organic solvents had no effect on its action. A typical lipopeptide absorption peak and a band of peptide bonds and ester groups were visible in the UV and FTIR spectra of the isolated lipopeptide. The Rf values of bioactive lipopeptide on developed TLC plate against Staphylococcus aureus ATCC25923 were calculated as 0.82 and 0.75 by bioautographic assay. The existence of ituD, ituD1, sfp, and sur3 genes in the genome and mass spectra of isolated lipopeptide revealed that Bacillus amyloliquefaciens strain RKEA3 could produce an array of iturin and surfactin-like peptides. The antibacterial effectiveness of isolated lipopeptide against several bacterial strains and its substantially lower hemolytic activity suggest that it could be developed as a medicine. • Antibiotic resistance of S.aureus is a major concern around the world. • Bacillus amyloliquefaciens strain RKEA3 showed antagonism against S.aureus . • Stability of isolated lipopeptides under various parameters are investigated. • Characterization and gene identification revealed existence of iturin and surfactin. • Bioactive lipopeptides showing reduced hemolysis were isolated.

Bacillus amyloliquefaciens YN201732 Produces Lipopeptides With Promising Biocontrol Activity Against Fungal Pathogen Erysiphe cichoracearum.

Bacillus amyloliquefaciens YN201732 is an endophytic bacteria with high biocontrol efficiency and broad-spectrum antimicrobial activities. In order to clarify the main active ingredients and their antifungal mechanisms against powdery mildew of tobacco, this study is focused on lipopeptide obtained through acid precipitation and organic solvent extraction. HPLC and LCMS-IT-TOF were used to separate and identify antimicrobial lipopeptides. Findings revealed that bacillomycin D plays an important role against surrogate fungal pathogen Fusarium solani. Synthetic pathways of sfp, bacillomycin D, and fengycin were separately disrupted. The sfp gene knockout mutant B. amyloliquefaciens YN201732M1 only showed minor antagonistic activity against F. solani. While Erysiphe cichoracearum spore germination was inhibited and pot experiments displayed a significant decrease in tobacco powdery mildew. The spore inhibition rate of YN201732M1 was only 30.29%, and the pot experiment control effect was less than 37.39%, which was significantly lower than that of the wild type. The inhibitory effect of mutant YN201732M2 (deficient in the production of bacillomycin D) and mutant YN201732M3 (deficient in the production of fengycin) on the spore germination of E. cichoracearum were 50.22% and 53.06%, respectively, suggesting that both fengycin and bacillomycin D had potential effects on spore germination of powdery mildew. Interestingly, in a greenhouse assay, both B. amyloliquefaciens YN201732M2 and YN201732M3 mutants displayed less of a control effect on tobacco powdery mildew than wild type. The results from in vitro, spore germination, and greenhouse-pot studies demonstrated that antimicrobial lipopeptides especially bacillomycin D and fengycin may contribute to the prevention and control of tobacco powdery mildew. In addition, gene mutation related to lipopeptide synthesis can also affect the biofilm formation of strains.

Efficient secretory expression of Bacillus stearothermophilus α/β-cyclodextrin glycosyltransferase in Bacillus subtilis

Bacillus stearothermophilus α/β-cyclodextrin glycosyltransferase (α/β-CGTase) is an excellent transglycosylase with broad potential for food application, but its expression level is low in Bacillus subtilis. In this study, the optimal signal peptide for α/β-CGTase expression was screened from 173 signal peptides in B. subtilis WS11. The α/β-CGTase activity in a 3-L fermentor reached 151.93 U⋅ mL−1, but substantial amounts of inclusion bodies were produced. The N-terminal 12 amino acids of α/β-CGTase were then replaced with the N-terminal 15 amino acids of a β-CGTase from the same family that has a high percentage of disorder-promoting amino acids. As a result, the inclusion bodies were significantly reduced, and the enzyme activity increased to 249.35 U mL−1, 2.3 times that of the strain constructed previously. Finally, the ppsE and sfp genes of B. subtilis WS11, which are related to lipopeptide biosurfactant synthesis, were knocked out to produce B. subtilis WS13. When B. subtilis WS13 was used to produce α/β-CGTase in a 3-L fermentor, 70 % less defoaming agent was required than with B. subtilis WS11. Furthermore, enzyme production and growth of WS13 were equivalent to those of WS11. This study is of great significance for future research to efficiently scale-up production of α/β-CGTase.

Construction and description of a constitutive plipastatin mono-producing Bacillus subtilis

BackgroundPlipastatin is a potent Bacillus antimicrobial lipopeptide with the prospect to replace conventional antifungal chemicals for controlling plant pathogens. However, the application of this lipopeptide has so far been investigated in a few cases, principally because of the yield in low concentration and unknown regulation of biosynthesis pathways. B. subtilis synthesizes plipastatin by a non-ribosomal peptide synthetase encoded by the ppsABCDE operon. In this study, B. subtilis 3NA (a non-sporulation strain) was engineered to gain more insights about plipastatin mono-production.ResultsThe 4-phosphopantetheinyl transferase Sfp posttranslationally converts non-ribosomal peptide synthetases from inactive apoforms into their active holoforms. In case of 3NA strain, sfp gene is inactive. Accordingly, the first step was an integration of a repaired sfp version in 3NA to construct strain BMV9. Subsequently, plipastatin production was doubled after integration of a fully expressed degQ version from B. subtilis DSM10T strain (strain BMV10), ensuring stimulation of DegU-P regulatory pathway that positively controls the ppsABSDE operon. Moreover, markerless substitution of the comparably weak native plipastatin promoter (Ppps) against the strong constitutive promoter Pveg led to approximately fivefold enhancement of plipastatin production in BMV11 compared to BMV9. Intriguingly, combination of both repaired degQ expression and promoter exchange (Ppps::Pveg) did not increase the plipastatin yield. Afterwards, deletion of surfactin (srfAA-AD) operon by the retaining the regulatory comS which is located within srfAB and is involved in natural competence development, resulted in the loss of plipastatin production in BMV9 and significantly decreased the plipastatin production of BMV11. We also observed that supplementation of ornithine as a precursor for plipastatin formation caused higher production of plipastatin in mono-producer strains, albeit with a modified pattern of plipastatin composition.ConclusionsThis study provides evidence that degQ stimulates the native plipastatin production. Moreover, a full plipastatin production requires surfactin synthetase or some of its components. Furthermore, as another conclusion of this study, results point towards ornithine provision being an indispensable constituent for a plipastatin mono-producer B. subtilis strain. Therefore, targeting the ornithine metabolic flux might be a promising strategy to further investigate and enhance plipastatin production by B. subtilis plipastatin mono-producer strains.

Heterologous Expression of Sfp-Type Phosphopantetheinyl Transferase is Indispensable in the Biosynthesis of Lipopeptide Biosurfactant.

Phosphopantetheinyl transferases are of tremendous enthusiasm inferable from their fundamental parts in activating polyketide, fatty acid, and non-ribosomal peptide synthetase enzymes and additionally an increasing number of biotechnological applications. The present study reports the identification of sfp gene from the Paenibacillus sp. D9, which encompasses 693bp encoding a 230-amino acid protein with a molecular weight of 25.3kDa. The amino acid sequence Paenibacillus sp. D9 Sfp revealed more than 90% sequence identity to other Sfp proteins from other Paenibacillus. The sfp gene was cloned and recovered efficiently using affinity chromatography with maximal specific phosphopantetheinyl transferase activity at an optimal pH of 8.0 and temperature of 30°C. The enzyme also exhibited stability under a wide-ranging pH and temperature. The presence of Zn2+, Cu2+, and Fe2+ ions improved the enzymatic activity, while other metals such as Ni2+, Co2+, and Mg2+ had inhibitory effects. The introduction of EDTA also displayed no inhibition. Kinetic parameters were obtained having values of 4.52mg/mL, 35.33 U/mg, 3.64s-1, and 0.104mM-1 s-1 for Km, Vmax, kcat, and kcat/Km, respectively. The biosurfactant synthesized by the recombinant BioSp was found to be surface active, reducing the surface tension to 33.7 mN/m on the glucose substrate after 5days of incubation at 37°C. The recombinant Escherichia coli strain also exhibited an improvement in biosurfactant yield (1.11g/L) when contrasted with 0.52g/L from Paenibacillus sp. D9. High esterase activity of 2.55IU/mL using p-nitrophenyl acetate was observed on the recombinant strain, as the protein connected with the release of the biosurfactant was observed to be an esterase. The characteristics of improved biosurfactant and esterase synthesis by hyper-producing recombinant strain possess numerous values from biotechnology standpoint.

Genomic and molecular characterisation of Escherichia marmotae from wild rodents in Qinghai-Tibet plateau as a potential pathogen

Wildlife is a reservoir of emerging infectious diseases of humans and domestic animals. Marmota himalayana mainly resides 2800–4000 m above sea level in the Qinghai-Tibetan Plateau, and is the primary animal reservoir of plague pathogen Yersinia pestis. Recently we isolated a new species, Escherichia marmotae from the faeces of M. himalayana. In this study we characterised E. marmotae by genomic analysis and in vitro virulence testing to determine its potential as a human pathogen. We sequenced the genomes of the seven E. marmotae strains and found that they contained a plasmid that carried a Shigella-like type III secretion system (T3SS) and their effectors, and shared the same O antigen gene cluster as Shigella dysenterae 8 and E. coli O38. We also showed that E. marmotae was invasive to HEp-2 cells although it was much less invasive than Shigella. Thus E. marmotae is likely to be an invasive pathogen. However, E. marmotae has a truncated IpaA invasin, and lacks the environmental response regulator VirF and the IcsA-actin based intracellular motility, rendering it far less invasive in comparison to Shigella. E. marmotae also carried a diverse set of virulence factors in addition to the T3SS, including an IS1414 encoded enterotoxin gene astA with 37 copies, E. coli virulence genes lifA/efa, cif, and epeA, and the sfp gene cluster, Yersinia T3SS effector yopJ, one Type II secretion system and two Type VI secretion systems. Therefore, E. marmotae is a potential invasive pathogen.

Genome analysis of a Bacillus subtilis strain reveals genetic mutations determining biocontrol properties

Several Bacillus strains are used as biocontrol agents, as they frequently have strong antagonistic effects against microbial plant pathogens. Bacillus strain SZMC 6179J, isolated from tomato rhizosphere, was previously shown to have excellent in vitro antagonistic properties against the most important fungal pathogens of tomato (Alternaria solani, Botrytis cinerea, Phytophthora infestans and Sclerotinia sclerotiorum) as well as several Fusarium species. Taxonomic investigations revealed that it is a member of the B. subtilis subsp. subtilis group and very closely related with the reference type strain B. subtilis subsp. subtilis 168. The sequenced genome of strain SZMC 6179J contains the genes responsible for the synthesis of the extracellular antibiotics surfactin, fengycin and bacilysin. Compared to strain 168, a prophage-like region is missing from the genome of SZMC 6179J, while there are 106 single nucleotide polymorphisms and 23 deletion-insertion polymorphisms. The high biocontrol potential of strain SZMC 6179J may results from a single base deletion in the sfp gene encoding the transcription factor of the surfactin and fengycin operons. Hypermutated regions reflecting short-time evolutionary processes could be detected in SZMC 6179J. The deletion-insertion polymorphism in the sfp gene and the detected hypermutations can be suggested as genetic determinants of biocontrol features in B. subtilis.Graphical