Cyclic Lipopeptide Research Articles

Metabolic engineering of Streptomyces roseosporus for increased production of clinically important antibiotic daptomycin.

Daptomycin (DAP), a novel cyclic lipopeptide antibiotic produced by Streptomyces roseosporus, is clinically important for treatment of infections caused by multidrug-resistant Gram-positive pathogens, but the low yield hampers its large-scale industrial production. Here, we describe a combination metabolic engineering strategy for constructing a DAP high-yielding strain. Initially, we enhanced aspartate (Asp) precursor supply in S. roseosporus wild-type (WT) strain by separately inhibiting Asp degradation and competitive pathway genes using CRISPRi and overexpressing Asp synthetic pathway genes using strong promoter kasOp*. The resulting strains all showed increased DAP titre. Combined inhibition of acsA4, pta, pyrB, and pyrC increased DAP titre to 167.4 μg/mL (73.5% higher than WT value). Co-overexpression of aspC, gdhA, ppc, and ecaA led to DAP titre 168 μg/mL (75.7% higher than WT value). Concurrently, we constructed a chassis strain favourable for DAP production by abolishing by-product production (i.e., deleting a 21.1 kb region of the red pigment biosynthetic gene cluster (BGC)) and engineering the DAP BGC (i.e., replacing its native dptEp with kasOp*). Titre for the resulting chassis strain reached 185.8 μg/mL. Application of our Asp precursor supply strategies to the chassis strain further increased DAP titre to 302 μg/mL (2.1-fold higher than WT value). Subsequently, we cloned the engineered DAP BGC and duplicated it in the chassis strain, leading to DAP titre 274.6 μg/mL. The above strategies, in combination, resulted in maximal DAP titre 350.7 μg/mL (2.6-fold higher than WT value), representing the highest reported DAP titre in shake-flask fermentation. These findings provide an efficient combination strategy for increasing DAP production and can also be readily applied in the overproduction of other Asp-related antibiotics.

L-asparaginase-driven antibiosis in Pseudomonas fluorescens EK007: A promising biocontrol strategy against fire blight

Fire blight, caused by Erwinia amylovora, is a destructive bacterial disease affecting pear and apple trees. The biocontrol ability of Pseudomonas fluorescens EK007 suppresses E. amylovora through competitive exclusion. In this study, EK007 was isolated from the pear phylloplane and characterized as an effective biological agent through antibacterial compounds. To identify the mechanisms underlying EK007's biocontrol activity, physiological tests, transposon insertion mutant libraries, allelic exchange, and whole-genome sequencing were performed. A transposon mutation in the massC homolog gene, part of the massetolide A lipopeptide biosynthesis cluster, reduced the biocontrol efficiency. Allelic exchange confirmed cyclic lipopeptide (CLP) as part of the mechanism. Additionally, a gacA mutant isolated by transposon mutagenesis showed deficient inhibition activity. Culture conditions and nutritional sources clearly influenced EK007's antimicrobial activity against E. amylovora. Growth yield generally correlated with antibiotic production, with amino acids and iron affecting production. Asparagine and aspartate shut down biocontrol activity. This study presents preliminary findings on a novel CLP that may contribute to EK007's antibacterial activity against E. amylovora. While EK007 shows promise as a biocontrol candidate compared to related strains, these results are based solely on in vitro studies, highlighting the need for further investigations to evaluate its efficacy in natural environments.

Discovery and characterization of novel lipopeptides produced by Virgibacillus massiliensis with biosurfactant and antimicrobial activities

The study aimed to evaluate the biosurfactants (BSs) production by SM-23 strain of Virgibacillus identified by phenotypical and WGS analysis as Virgibacillus massiliensis. We first demonstrated the lipopeptides production by Virgibacillus massiliensis specie and studied their biochemical and molecular analysis as well as their biological potential. The GC–MS analysis indicated that methyl.2-hyroxydodecanoate was the major fatty acid compound with 33.22%. The maximum BSs production was obtained in LB medium supplemented by 1% olive oil (v/v) at 30 °C and 5% NaCl with 1.92 g/l. The obtained results revealed the significant biosurfactants/bioemulsifier potential compared to triton X100 with E24 of 100%, and an emulsification stability SE of 83%. The lipopeptides types were identified by FTIR analysis. A strong antimicrobial action was observed by the produced lipopeptides by the agar diffusion method against E.coli, K. pneumoniae, S. aureus, Fusarium sp, Alternaria sp, and Phytophtora sp. The complete genome sequencing showed genes involved in the synthesis of multiple compounds identified as amphipathic cyclic lipopeptides such as locillomycin/locillomycin B/locillomycin C and bacillibactin. Our results highlighted significant lipopeptides properties displayed by V. massiliensis that can be exploited to develop a novel strategy in the formulation of natural biocidal and fungicidal agents.

Design, synthesis, and bioactivity investigation of novel cyclic lipopeptide antibiotics targeting top-priority multidrug-resistant gram-negative bacteria

ObjectivesPolymyxins are the last-line therapy for top-priority multidrug-resistant (MDR) gram-negative bacteria. However, polymyxin nephrotoxicity impedes its clinical application. This study aimed to design, synthesize, and identify a novel and promising polymyxin derivative with high efficacy and low toxicity. MethodsTo design polymyxin derivatives, we reduced the hydrophobicity of the two hydrophobic domains (fatty acyl chain and D-Phe6-L-Leu7) and modified the positive charged L-2,4-diaminobutyric acid (Dab) residues. Twenty-five derivatives were synthesized, and their antibacterial activities in vitro and renal cytotoxicities were determined. The nephrotoxicity and pharmacokinetic parameters of compound 12 were examined in rats. Antibacterial efficacy in vivo was evaluated using a mouse systemic infection model. Surface plasmon resonance analysis, compound 12-rifampicin combination therapy, and scanning electron microscopy were used to study the mechanism of action of compound 12. ResultsThis research found a new compound, identified as compound 12, which showed similar or increased antibacterial activity against all tested sensitive and carbapenem-resistant gram-negative bacteria. It exhibited reduced renal cytotoxicity and nephrotoxicity, a favorable pharmacokinetic profile, and maintained or improved antibacterial efficacy in vivo. Importantly, its anti-Pseudomonas aeruginosa activity significantly improved. Compound 12, when combined with rifampicin, enhanced the activity of rifampin against gram-negative bacteria. Compound 12 also showed a high affinity for lipopolysaccharide and disrupted cell membrane integrity. ConclusionReducing the hydrophobicity of the two domains reduced renal cytotoxicity and nephrotoxicity. Shortening the side chain of Dab3 by one carbon maintained or increased its antibacterial activity both in vitro and in vivo. Furthermore, only the length of the side chain of Dab9 could be shortened by one carbon among the Dab1,5 and Dab8,9 residues. The bactericidal effects of compound 12 were related to the disruption of cell membrane integrity. Compound 12 may be a promising candidate for combating sensitive and carbapenem-resistant gram-negative bacterial infections, especially Pseudomonas aeruginosa.

Genome-Led Discovery of the Antibacterial Cyclic Lipopeptide Kutzneridine A and Its Silent Biosynthetic Gene Cluster from Kutzneria Species.

Genome analysis of Kutzneria sp. CA-103260 revealed a putative lipopeptide-encoding biosynthetic gene cluster (BGC) that was cloned into a bacterial artificial chromosome (BAC) and heterologously expressed in Streptomyces coelicolor M1152. As a result, a novel cyclic lipo-tetrapeptide containing two diaminopropionic acid residues and an exotic N,N-acetonide ring, kutzneridine A (1), was isolated and structurally characterized. Evaluation of the extraction conditions and isotope-labeling chemical modifications showed that the acetonide ring originated from acetone during isolation. The BGC was analyzed in silico and a biosynthetic pathway to 1 was proposed. Kutzneridine A displayed remarkable antibacterial activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci.

Antimicrobial cyclic lipopeptides from Bacillus mojovensis B1302 against wheat root rot

Bipolaris sorokiniana is the main pathogen affecting wheat, which can cause major disasters in wheat planting areas. In this study, cyclic lipopeptides (CLP) were extracted from Bacillus mojovensis B1302 and had antimicrobial activity on Bipolaris sorokiniana and other four pathogenic fungi. CLP could inhibit the growth of B. sorokiniana and made mycelia swelling and deformed. In addition, trypan blue staining indicated the distortion of mycelia and DAPI staining showed mycelial DNA damage in the CLP treatment group. Moreover, CLP were effective in controlling wheat root rot with the efficacy of 80.32%, which was not significantly different from that of commercial fungicide (carbendazim). Next, in the oil spreading test, the diffusion diameter of 40 μL CLP (10 mg/mL) reached 8.37 cm with a stable spreading ring, indicating CLP had surface activity. And also, CLP had strong antioxidant capacity and DPPH scavenging activity. Furthermore, CLP had better temperature tolerance at 20–40 °C and pH tolerance at 4–8, respectively, still maintained a high inhibition rate after 3h treatment under UV irradiation, and were stable under trypsin treatment. However, CLP were sensitive to pepsin and metal ions. In conclusion, CLP had antimicrobial activity, antioxidant capacity and environmental tolerance, which had good potential for development into biocontrol agents against wheat root rot.

Real-World Clinical Characteristics and Outcomes with Daptomycin Use in Pediatric Patients: A Retrospective Case Series.

Daptomycin (DAP) is a cyclic lipopeptide that exhibits potent in vitro activity against many drug-resistant gram-positive organisms, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Despite substantial reports evaluating the clinical outcomes of DAP within the adult population, real-world data are lacking in children. The primary goal of this evaluation was to describe the clinical characteristics and outcomes of DAP use in pediatric patients across a wide range of infections. This retrospective evaluation included patients < 18 years of age who were treated with DAP from January 2014 to May 2023. The primary objective was to evaluate the composite clinical success, which was defined as a 30-day survival, the lack of a 30-day microbiological recurrence, and the resolution of signs and symptoms of an acute infection without therapy modifications based on clinical failures. Secondary objectives included adverse effects potentially attributable to DAP and reasons for DAP utilization. Forty patients were included, which were predominately male (62.5%) and white (52.5%), with a median age of 8.7 [IQR, 4.4-16.0] years. DAP was used for a wide range of infections, including central line-associated bloodstream infections (CLABSIs; 32.5%), infective endocarditis (15.0%), surgical-site infections (12.5%), and osteomyelitis (12.5%). The most common pathogen isolated was MRSA (37.5%), and most patients were bacteremic (60.0%). The median DAP dose was 8 [IQR, 6-10] mg/kg, and the median duration of the DAP therapy was 11.5 [IQR, 4.8-18.8] days. Most patients achieved composite clinical success (75.0%). An adverse effect occurred in 5.0% of the patients. DAP was prescribed the most for its ease of use/ability to facilitate discharge (40.0%) and/or for issues with alternative therapies (37.5%). Most pediatric patients that received DAP demonstrated clinical success with a low incidence of adverse effects. Larger, real-world studies of DAP use are necessary to further assess clinical outcomes.

ActO, a positive cluster-situated regulator for actinomycins biosynthesis in Streptomyces antibioticus ZS

Actinomycins are a class of cyclic lipopeptide antibiotics produced by Streptomyces, which have rich biological activities and demonstrate great potential value. Among them, actinomycin D is currently the effective drug for some malignant tumor diseases. Although the chemical properties, biological activities and biosynthesis of actinomycins have been extensively studied, the regulation of their biosynthesis remains poorly understood. Streptomyces antibioticus ZS isolated from deep-sea corals is a producer of actinomycin D and actinomycin V. Here, we reported the characterization of a cluster-situated regulator ActO in actinomycins biosynthetic gene cluster (act cluster) of S. antibioticus ZS, which belongs to LmbU family. Deletion of actO completely blocked the synthesis of actinomycins. Overexpression of actO increased the yields of actinomycin D and actinomycin V by 4.4 fold and 2.6 fold, respectively. The result of RT-qPCR showed that ActO activates the transcription of all genes in act cluster. However, no specific binding of His6-ActO to the promoters of target genes was observed after electrophoretic mobility shift assay (EMSA). These results proved that ActO serves as a positive regulator involved in the biosynthesis of actinomycins, affecting the transcription of all genes related to the synthesis of intermediates, skeleton modification and extracellular transportation of final products. Moreover, we demonstrated that overexpression of actO is a novel strategy to increase the yields of actinomycins.

Metabolite profiling and molecular characterization of NBAIR BSWG1: A potential strain of Bacillus subtilis against Fusarium oxysporium f. sp. udum

To address the fungal wilt of pigeon pea caused by Fusarium oxysporium f. sp. udum, farmers currently rely on chemical fungicides, despite their harmful effects. However, there is a growing need for safer alternatives like green pesticides. Bacterial biocontrol agents and their derivatives serve as potential green pesticides in the management of plant pathogens. In the present study, we aimed to identify indigenous Bacillus subtilis strains effective against F. oxysporium f. sp. udum. We used PCR and MALDI-TOF analysis to identify the active components responsible for the efficiency of efficient strain. Biochemical studies of cell-free extracts extracted from B. subtilis strains demonstrated the highest biosurfactant activity in NBAIR BSWG1, with an oil displacement of 2 cm and an emulsification index of 60 %. Molecular characterization confirmed the presence of surfactin, fengycin, and iturin coding genes in the B. subtilis strains, among them, NBAIR BSWG1 showed the highest number of lipopeptide-producing genes. Meanwhile, NBAIR BSWG1 showed inhibition of 79.84 % against F. oxysporium f. sp. udum using cell-free extract. Further metabolite profiling of NBAIR BSWG1 using MALDI-TOF analysis further confirmed surfactin, fengycin, and iturin in the purified cell-free extract of NBAIR BSWG1. Two peaks with m/z of 923.77 and 1149.92 were identified as novel lipopeptide compounds which need further characterization. The present study identified NBAIR BSWG1 as an efficient bacterial strain for the inhibition of F. oxysporium f. sp. udum and its antifungal properties are mainly due to the production of cyclic lipopeptides.

Eco-friendly Management of Plant Pathogens through Secondary Metabolites Released by Fluorescent Pseudomonads

Concerning sustainable agriculture, plant growth promoting rhizobacteria (PGPR), which are a subgroup of “fluorescent pseudomonads,” are crucial. They are widely known for supporting plant health through a variety of methods. The use of fluorescent pseudomonads in agri-biotechnology has gained traction due to their potential for safeguarding plants from a variety of phytopathogens. Fluorescent pseudomonads being commercialized as bioinoculants for the treatment of various plant diseases is currently regarded as highly successful on a global scale. Fluorescent pseudomonads are being employed as efficient bio-control agents (BCAs) against an array of phytopathogens. Due to their capacity to generate a wide range of secondary metabolites, they offer enormous promise as BCA. Thus, this review’s goal is to outline and evaluate the functions of fluorescent pseudomonads’ secondary metabolites in reducing phytopathogens and improving plant health. Prominent secondary metabolites linked to biocontrol through fluorescent pseudomonads include phenazines (PHZ), 2, 4-diacetylphloroglucinol (DAPG), pyoluteorin (PLT), pyrrolnitrin (PRN), cyclic lipopeptides (CLPs), and volatile organic compounds (VOCs), including hydrogen cyanide (HCN). The antifungal, antibacterial, antiviral, antitumor, and antinematicidal effects of these metabolites are well-established.

Combining cyclic lipopeptides and cinnamon extract enhance antifungal activity against Fusarium oxysporum strains pathogenic to banana and delay Fusarium wilt under greenhouse conditions

AbstractFusarium wilt of banana (FWB) caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc) is a widely distributed disease that generates devastating losses in banana production. Foc belongs to the Fusarium oxysporum species complex (FOSC) which includes several evolutionary lineages. Nine of them are pathogenic to banana such as F. phialophorum, F. grosmichelli, F. duoseptatum and the most aggressive F. odoratissimum tropical race 4 (TR4). No control method has been successfully implemented to manage FWB, then enhancing the potential of management approaches can avoid or delay disease epidemics and reduce disease severity. Here we determined the antifungal effect of different plant-based extracts against Foc in vitro, and whether the combination of cinnamon (Cinnamomum zeylanicum) extract and Bacillus tequilensis EA-CB0015 cyclic lipopeptides had an additive effect against different Foc lineages in vitro and against FWB in banana plants in greenhouse. We found, from 17 plant-based natural extracts, that cinnamon was highly active against Foc strain IB (race 1). Furthermore, cinnamon and cyclic lipopeptides inhibited different strains of various evolutionary lineages of Foc belonging to race 1 and TR4, and their combination increased in 1.4-fold the effect of the single extracts in vitro. Our results showed that soil concentration of F. odoratissimum TR4-II5 decreased by 1000-fold when treated with the combination of 488 mg L−1 cinnamon and 128 mg L−1 lipopeptides in a soil microcosm system after 5 days of incubation, followed by a partial population recovery after 21 days. In greenhouse experiments, the combination reduced external but not internal FWB symptoms, and cinnamon extract had a significant impact on internal plant symptoms. Taken together, the effect of cyclic lipopeptides with cinnamon extract on Foc supports their function towards delaying the effect of disease progression and suggests that the combination enhances the effect of the single extracts.

Nanoencapsulation enhances the antimicrobial and antioxidant stability of cyclic lipopeptides for controlling Fusarium graminearum

Fusarium graminearum not only causes Fusarium head blight (FHB) on wheat but also produces fungal toxins that pose a serious threat to food safety. Biological control is one of the safe and most effective alternative methods. In this study, cyclic lipopeptides (CLPs) produced from Bacillus mojavensis B1302 were extracted and identified by LC-MS/MS. After preparing mesoporous silica nanoparticles-NH2 (MSNsN) and encapsulating CLPs, the characterization analysis showed that the interaction between CLPs and MSNsN enhanced the crystal structure of CLPs-MSNsN. The antimicrobial activity and antioxidant capacity of CLPs-MSNsN stored at 20 °C and 45 °C were decreased more slowly than those of free CLPs with increasing storage time, indicating the enhancement of the antimicrobial and antioxidant stability of CLPs. Moreover, the field control efficacy of long-term stored CLPs-MSNsN only decreased from 78.66% to 63.2%, but the efficacy of free CLPs decreased significantly from 84.34% to 26.01%. The deoxynivalenol (DON) content of wheat grains in the CLPs-MSNsN treatment group was lower than that in the free CLPs treatment group, which showed that long-term stored CLPs-MSNsN reduced the DON content in wheat grains. Further analysis of the action mechanism of CLPs-MSNsN on F. graminearum showed that CLPs-MSNsN could disrupt mycelial morphology, cause cell apoptosis, lead to the leakage of proteins and nucleic acids, and destroy the cell permeability of mycelia. This work puts a novel insight into the antimicrobial and antioxidant stability enhancement of CLPs-MSNsN through encapsulation and provides a potential fungicide to control F. graminearum, reduce toxins and ensure food safety.

Fungal membrane determinants affecting sensitivity to antifungal cyclic lipopeptides from Bacillus spp.

Bacillus spp. produce numerous antimicrobial metabolites. Among these metabolites, cyclic lipopeptides (CLP) including fengycins, iturins, and surfactins are known to have varying antifungal activity against phytopathogenic fungi. The differential activities of CLP have been attributed to diverse mechanisms of action on fungal membranes. However, the precise biochemical determinants driving their antifungal modes of action have not been conclusively identified. In this study, three plant pathogenic fungi of varying lipopeptide sensitivities, Alternaria solani, Cladosporium cucumerinum, and Fusarium sambucinum, were studied to determine how their cell membrane lipid compositions may confer sensitivity and/or tolerance to fengycin, iturin, and surfactin. Results indicated that sensitivity to all three lipopeptides correlated with lower ergosterol content and elevated phospholipid fatty acid unsaturation. Fungal sensitivity to surfactin was also notably different than fengycin and iturin, as surfactin was influenced more by lower phosphatidylethanolamine amounts, higher levels of phosphatidylinositol, and less by phospholipid fatty acyl chain length. Results from this study provide insight into the fungal membrane composition of A. solani, F. sambucinum, and C. cucumerinum and the specific membrane characteristics influencing the antifungal effectiveness of fengycin, iturin, and surfactin. Understanding of these determinants should enable more accurate prediction of sensitivity-tolerance outcomes for other fungal species exposed to these important CLP.

A Family of Cyclic Lipopeptides Found in Human Isolates of Bacillus Ameliorates Acute Colitis via Direct Agonism of Toll-Like Receptor 2 in a Murine Model of Inflammatory Bowel Disease

BackgroundThe Bacillus-derived cyclic lipopeptides (surfactin, iturin, and fengycin) form potent Heterogeneous Lipopeptide Micelle (HeLM) complexes. HeLM is a small molecule that has been shown to have immunomodulatory effects. However, how HeLM regulates inflammation is not clear, moreover its application to Inflammatory Bowel Disease (IBD), specifically Ulcerative Colitis (UC), has not been tested before.AimsTo use a murine model of IBD and determine the effects of HeLM and related molecular mechanisms of action.MethodsColitis was induced in mice by administration of 4% Dextran Sodium Sulfate. Three preparations were tested against negative and positive controls: Purified HeLM, the wild-type strain that produces it, and an isogenic mutant that does not produce HeLM. Clinical, biochemical, and histological scoring systems were used to assess the severity of colitis. RT-qPCR and cell cultures were used to determine the levels of molecular signaling. Fecal samples were processed for metagenomic analysis.ResultsPurified HeLM, and the wild-type strain, significantly decreased the severity of colitis as determined by the disease activity index (DAI), mouse colitis histology index (MCHI), fecal calprotectin, and colonic length. This effect was not seen in the mutant. HeLM was found to be an agonist to TLR-2, seemingly activating the Toll-Like Receptor 2/IL-10 pathway, with subsequent downregulation of inflammatory cytokines (TNF-α, IL-1β, and IL-6). At higher concentrations HeLM inhibited lipopolysaccharide ligands from activating TLR-4. The reduction in colitis was not due to microbiome modulation, as had previously been hypothesized.ConclusionOur results indicate that HeLM ameliorates colitis by TLR-2-induced IL-10 production and possibly via the inhibition of lipopolysaccharide.

Solid-phase synthesis of amphiphilic cyclic peptides as a surfactin mimetic

The cyclic lipopeptide surfactin, an anionic biosurfactant, has received increased interest owing to its unique characteristics and promising applications. Although surfactin is known to exhibit distinctive surface activities and self-assembly ability, the role of the cyclic peptide moiety in its physico-chemical properties requires further investigation. Here, we report the solid-phase peptide synthesis of surfactin mimics such as cyclic heptapeptides with the same amino acid sequence as surfactin (CSF) and cyclic octapeptides with dodecyl chains (CSF12). The structure–property relationships of the cyclic peptides were then examined. The acidic forms of CSF and CSF12 were poorly soluble in water, but the addition of 1.5 or 2.0 equiv. of NaOH increased their solubility in aqueous solutions. The disodium salt of CSF (CSF-2Na) reduced the surface tension of water to 39.0 mN/m despite its absence of a long alkyl chain, indicating that the cyclic peptide moiety plays a crucial role in the surface activity of the surfactin mimic. In the case of the disodium salt of CSF12 (CSF12–2Na), the introduction of a dodecyl chain to the cyclic peptide moiety led to an increase in molecular orientation such that saturation at the air–water interface was observed. Thus, a long alkyl chain enhances the molecular assembly of the cyclic peptide at the air–water interface. The introduction of a long alkyl chain also enhanced the solubilisation of phospholipids. For instance, CSF12–1.5Na induced the solubilisation of L-α-dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles in phosphate-buffered saline (PBS). These findings greatly contribute to the fundamental understanding of the broad spectrum of physico-chemical properties and biological activities of bio-derived cyclic peptides. Furthermore, these insights can also be helping design libraries for developing cyclic peptides for challenging therapeutics in skin disease or as cosmeceuticals to enhance skin appearance.

Lipid lysination by MprF contributes to hemolytic pigment retention in group B Streptococcus

Group B Streptococcus (GBS) is the leading cause of neonatal sepsis and meningitis. A major virulence factor is a pigmented beta-haemolytic/cyto-lysin (β-h/c) toxin with an ornithine rhamnolipid structure. We initially observed that absence of MprF enzyme altered pigmentation and haemolytic activity in GBS. Next, we showed that MprF-dependent lipid lysination contributes to the retention of the ornithine rhamnolipid within GBS membrane. Furthermore, cationic lipidation by MprF altered membrane properties contributing to resistance to the cyclic lipopeptide daptomycin and to acidic pH. This study highlights the importance of cationic lipids in cell envelope homeostasis and in modulating β-h/c activity.

Effects of branched-chain amino acids on surfactin structure and antibacterial activity in Bacillus velezensis YA215.

Antibiotics are essential for combating pathogens; however, their misuse has led to increased resistance, necessitating the search for effective, low-toxicity alternatives. Surfactin, a cyclic lipopeptide with a C12-C17 β-hydroxy fatty acid chain, exhibits significant antibacterial activity and resists resistance, making it a research focus. Nonetheless, the effects of branched-chain amino acids (BCAAs) on surfactin's structure and activity are not well understood. This study examines the influence of BCAAs (L-valine, L-leucine, and L-isoleucine) on the lipopeptide (surfactin) produced by Bacillus velezensis YA215. Process optimization shows that adding 1g/L of L-Leu and L-Ile, and 0.5g/L of L-Val, maximized surfactin production to 18.59%, 19.23%, and 20.64%, respectively. Surfactin content peaked at 36h with L-Val and L-Ile, yielding 19.72% and 11.37%. In contrast, L-Leu addition peaked at 24h, yielding 11.33%. Notably, L-Val supplementation resulted in the highest relative surfactin content. Antimicrobial testing demonstrated that BCAAs significantly enhance the antibacterial effects of lipopeptides against Escherichia coli and Staphylococcus aureus, with Val showing the most pronounced effect. The addition of BCAAs notably altered the composition of surfactin fatty acid chains. Specifically, Val increased the proportions of iso C14 and iso C16 β-hydroxy fatty acids from 13.3% and 4.216-23.803% and 8.31%, respectively. Additionally, the amino acid composition at the 7th position of the peptide chain changed significantly, especially with Val addition, which increased the proportion of C14 [Val 7] surfactin by 3.29 times. These structural changes are likely associated with the enhanced antibacterial activity of surfactin. These findings provide valuable insights into the roles of BCAAs in microbial fermentation, underscoring their importance in metabolic engineering to enhance the production of bioactive compounds.

Biocontrol efficacy of Pseudomonas mediterranea PVCT 3C against Plenodomus tracheiphilus: In vitro and in planta mechanisms at early disease stages

In this study, we investigated the biocontrol activity of the P. mediterranea strain PVCT 3C against Mal secco, a severe disease of citrus caused by the vascular fungus Plenodomus tracheiphilus. In vitro, bacterial diffusible compounds, volatile organic compounds and culture filtrates produced by PVCT 3C reduced the mycelial growth and conidial germination of P. tracheiphilus, also affecting the mycelial pigmentation. The application of bacterial suspensions by leaf-spraying before the inoculation with the pathogen on plants of the highly susceptible species sour orange and lemon led to an overall reduction in incidence and disease index, above all during the early disease stage. PVCT 3C genome was subjected to whole-genome shotgun sequencing to study the molecular mechanisms of action of this strain. In silico annotation of biosynthetic gene clusters for secondary metabolites revealed the presence of numerous clusters encoding antimicrobial compounds (e.g. cyclic lipopeptides, hydrogen cyanide, siderophores) and candidate novel products. During the asymptomatic disease phase (seven days post-inoculation), bacterial treatments interfered with the expression of different fungal genes, as assessed with an NGS and de novo assembly RNA-seq approach. These results suggest that P. mediterranea PVCT 3C or its secondary metabolites may offer a potential effective and sustainable alternative to contain P. tracheiphilus infections via integrated management.

Decreased susceptibility to viscosin in Streptococcus pneumoniae.

Growing numbers of infections caused by antibiotic-resistant Streptococcus pneumoniae strains are a major concern for healthcare systems that will require new antibiotics for treatment as well as preventative measures that reduce the number of infections. Lipopeptides are antimicrobial molecules, of which some are used as antibiotics, including the last resort antibiotics daptomycin and polymyxins. Here we have studied the antimicrobial effect of the cyclic lipopeptide viscosin on S. pneumoniae growth and morphology. Most lipopeptides function as surfactants that create pores in membrane layers, which is regarded as their main antimicrobial activity. We show that viscosin can inhibit growth of S. pneumoniae without disintegration of the cytoplasmic membrane. Instead, the cells developed abnormal shapes and misplaced new division sites. The cell wall of these bacteria appeared less dense in electron microscopy images, suggesting that viscosin interfered with normal cell wall synthesis. Corroborating this observation, a luciferase reporter assay was used to show that the two-component systems LiaFSR and CiaRH, which are known to be activated upon cell wall stress, were strongly induced by viscosin. Furthermore, a mutant displaying 1.8-fold decreased susceptibility to viscosin was generated by sequential exposure to increasing concentrations of the lipopeptide. The mutant suffered from significant fitness loss and had mutations in genes involved in fatty acid synthesis, teichoic acid synthesis, and cell wall synthesis as well as transcription and translation. How these mutations might be linked to decreased viscosin susceptibility is discussed.IMPORTANCEStreptococcus pneumoniae is a leading cause of bacterial pneumonia, sepsis, and meningitis in children, and the incidence of infections caused by antibiotic-resistant strains is increasing. Development of new antibiotics is therefore necessary to treat these types of infections in the future. Here, we have studied the activity of the antimicrobial lipopeptide viscosin on S. pneumoniae and show that in addition to having the typical membrane destabilizing activity of lipopeptides, viscosin inhibits pneumococcal growth by obstructing normal cell wall synthesis. This suggests a more specific mode of action than just the surfactant activity. Furthermore, we show that S. pneumoniae does not easily acquire resistance to viscosin, which makes it a promising molecule to explore further, for example, by synthesizing less toxic derivates that can be tested for therapeutic potential.

Biological control of the shot-hole disease in flowering cherry tree using antimicrobial compounds produced by Bacillus velezensis 8–2

BackgroundEffective control of shot-hole disease in flowering cherries is challenging because of multiple causative pathogens (bacteria and fungi). Bacillus species are well-known for their ability to control plant pathogens; therefore, biological control potential of a Bacillus isolate, B. velezensis 8–2, against SH disease on flowering cherry trees was investigated.ResultsThis study revealed strong antimicrobial activity of Bacillus velezensis 8–2 against various plant pathogenic bacteria and fungi, particularly focusing on Xanthomonas arboricola pv. pruni (Xap) and Mycosphaerella cerasella (Mc), which cause shot-hole (SH) disease in flowering cherry trees. In vitro assays showed that the fermentation filtrate of B. velezensis 8–2 inhibited bacterial and fungal growth with minimum inhibitory concentrations of 1.25–10% and 2.5–10%, respectively. UPLC-Q–Orbitrap–MS analysis revealed that B. velezensis 8–2 produced antagonistic compounds, including polyketides (difficidin and oxydifficidin) and cyclic lipopeptides (iturin A, fengycin, and surfatin). To enhance antimicrobial activity, fermentation parameters for optimal production of two antibacterial and three antifungal compounds were investigated in a 5 L jar fermenter. By regulating the agitation speed to sustain the state of vegetative cells, the production period was extended by 20 h at 400 rpm, resulting in maximum yields of 86.6 μg/mL for difficidin and 150.0 μg/mL for oxydifficidin within a 72 h fermentation period. In a field trial, a 500-fold diluted 10% suspension concentrate formulation of B. velezensis 8–2 effectively inhibited the development of SH disease, demonstrating 66.6% disease control and a 90.2% disease symptoms reduction.ConclusionsThis is the first report to assess the disease control efficacy of B. velezensis for the biocontrol of SH disease in the field. These results suggest that the application of B. velezensis 8–2 could serve as a practical alternative for managing various bacterial and fungal diseases, including the management of SH disease in flowering cherry trees.Graphical