No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
The identification of bacillaene, the product of the PksX megacomplex in Bacillus subtilis
Abstract
The ≈80-kb pksX gene cluster in Bacillus subtilis encodes an unusual hybrid polyketide/nonribosomal peptide synthase that has been linked to the production of the uncharacterized antibiotic bacillaene. Multiple copies of this synthase, each similar in size to the ribosome, assemble into a single organelle-like complex with a mass of tens to hundreds of megadaltons. The resource requirements of the assembled megacomplex suggest that bacillaene has an important biological role. By coupling a differential NMR spectroscopic technique with genetically manipulated strains of B. subtilis, we were able to characterize the structure of this unusual secondary metabolite, which could not be predicted by using bioinformatic analysis. We report that bacillaene is a linear molecule with two amide bonds: the first links an α-hydroxy carboxylic acid to a ω-amino carboxylic acid containing a conjugated hexaene, and the second links the hexaene-containing carboxylic acid to an (ω-1) amino carboxylic acid containing a conjugated triene. Knowledge of bacillaene's structure has enabled us to annotate the pksX gene cluster and should facilitate the study of bacillaene's biosynthesis as well as its biological role in B. subtilis.
• NMR overlay
• nonribosomal peptide
• polyketide
• β-methyl
... Notably, in B. subtilis the giant bacillaene biosynthesis machinery forms an organelle-like complex that can be observed through cryoelectron microscopy [13]. The structure elucidation of "bacillaene" through extensive NMR spectroscopic methods revealed the presence of two major compounds, bacillaene (1) and dihydrobacillaene (2) (Scheme 1), and allowed for a detailed biosynthetic model [14]. As is typical for trans-AT systems, the bacillaene PKS-NRPS contains several irregular features such as split modules and duplicate ACP domains. ...
... As is typical for trans-AT systems, the bacillaene PKS-NRPS contains several irregular features such as split modules and duplicate ACP domains. Because of the absence of a ketoreductase (KR) domain in module 1, the starter unit was initially suggested to be α-hydroxyisocaproate [14], but later it was shown that the KR of module 3 acts twice, in the reduction of the β-ketoacyl intermediate of the elongation step of module 3 and in the reduction of the α-ketoisocaproate starter unit with introduction of an S configured stereocentre (highlighted in red in Scheme 1) [15]. The domain organisation of module 3 containing no enoylreductase (ER) domain furthermore suggests the formation of an α,β-unsaturated intermediate, and not a full reduction at this stage, in agreement with the presence of a double bond between C22 and C23 in 1. ...
... Further interesting features are the presence of a β-branching cassette for the installation of a β-methyl group at the stage of the module 7 intermediate (highlighted by the black dot) [14,16], and split modules between BaeJ and BaeL (module 5), as well as between BaeL and BaeM (module 9). These modules are suggested not to catalyse elongations, but the KS domains of modules 5 and 9 may only act in the translocation of the intermediate from the ACP of the previous module to the ACP of modules 5 and 9, respectively. ...
An isotopic labelling method was developed to investigate substrate binding by ketosynthases, exemplified by the second ketosynthase of the polyketide synthase BaeJ involved in bacillaene biosynthesis (BaeJ-KS2). For this purpose, both enantiomers of a ¹³ C-labelled N -acetylcysteamine thioester (SNAC ester) surrogate of the proposed natural intermediate of BaeJ-KS2 were synthesised, including an enzymatic step with glutamate decarboxylase, and incubated with BaeJ-KS2. Substrate binding was demonstrated through ¹³ C NMR analysis of the products against the background of various control experiments.
... Bacillaene is a multifunctional polyketide molecule produced by B. subtilis first reported in 1995 (Patel et al., 1995). The stereochemical structure of bacillaene is unstable and decomposes rapidly when exposed to light, oxygen, or room temperature (Rebecca et al., 2017). Bacillus spp. ...
... Chen, Tian, et al., 2018a) and Fusarium oxysporum (Xu et al., 2014). A series of glycosylated bacillaenes Chen et al., 2006;Rebecca et al., 2017;Straight et al., 2007). ...
... The pksX gene cluster consists of 16 genes encoding 13 polyketide synthases (PKSs) and 3 nonribosomal peptide synthetases (NRPSs), while the bae gene cluster consists of 14 genes (X. H. Chen et al., 2006;Rebecca et al., 2017). It was reported that the transcription of the pksX gene cluster was influenced by regulators Spo0A, AbrB, CodY, DegU, ComA, and ScoC, all of which were related to the regulation of bacterial biofilm formation (Comella et al., 2005;Carol et al., 2014;Hamon et al., 2004;Murray et al., 2009;Sonenshein et al., 2005). ...
Bacillus species act as plant growth-promoting rhizobacteria (PGPR) that can produce a large number of bioactive metabolites. Bacillaene, a linear polyketide/nonribosomal peptide produced by Bacillus strains, is synthesized by the trans-acyltransferase polyketide synthetase. The complexity of the chemical structure, particularity of biosynthesis, potent bioactivity, and the important role of competition make Bacillus an ideal antibiotic weapon to resist other microbes and maintain the optimal rhizosphere environment. This review provides an updated view of the structural features, biological activity, biosynthetic regulators of biosynthetic pathways, and the important competitive role of bacillaene during Bacillus survival.
... The genome analysis of the studied strains provided a total of 85 biological control compounds ( Table 2). The most prevalent was the siderophore Bacillibactin, which Catechol-type Siderophore Antifungal Antibiotic Antifungal Antifungal Siderophore Sporulation Antifungal Antimicrobial Luciana, 2014;Ley-López, 2022;Basso et al., 2018;Moldenhauer et al., 2010;Pedraza-Herrera et al., 2020;Wang et al., 2022;Wieland et al., 2009;Butcher et al., 2007. ...
... Bacillaene (100%) Bacitracin (88%) Mersacidin (100%) Antifungal Antibiotic Antibiótic Moldenhauer et al., 2010;Fira et al., 2018;Diabankana et al., 2022;Butcher et al., 2007. is a catechol siderophore [an organic compound with the formula C 6 H 4 (OH) 2 ]. It participates in the chelation of ferric iron (Fe 3+ ) from the surrounding environment and is subsequently transferred to the bacterial cytoplasm through the use of transporters (Valenzuela-Ruiz et al., 2022). ...
Objective/Background. Bacillus is a cosmopolitan bacterial genus with a great genome diversity. Thus, by exploring its genome background, it is possible to understand more about the physiological and biochemical traits involved in its biological control against phytopathogens. The objective of this work was to correlate the phylogenomic relationships of the type species of the genus Bacillus with the presence of gene clusters associated with biological control of plant pathogens, through genome mining.
Materials and Methods. Based on the literature, 336 species belonging to the genus Bacillus have been reported; however, after re classification, a total of 123 type species have been recognized, and curated genomes were found in the EzBioCloud platform (http://www.ezbiocloud.net/). The overall genome relatedness indices (OGRIs) were used for this work, which indicate how similar two sequences of a genome are. Then, the Realphy platform was used to create the phylogenomic tree 1.13 (Action-based phylogeny constructor reference). Finally, the prediction of biosynthetic gene clusters (BGC) associated with the biological control of phytopathogens was carried out using antiSMASH v6.0 (https://antismash. secondarymetabolites.org/).
Results. The present strategy allowed us to correlate and predict the biological control capacity of the Bacillus species under study based on their taxonomic affiliation since at a shorter evolutionary distance from Bacillus subtilis a high potential capacity to produce biological control compounds was observed. However, the possibility that they acquire the ability to produce new biocontrol compounds during their evolutionary separation is not ruled out.
Conclusion. This work validates the correlation between the taxonomic affiliation of the studied Bacillus species and their biological control capacity, which is useful in the bioprospecting stage to design promising biopesticides.
... Selection of potential antibiotic-producing strains of Bacillus spp., the individual gene had been targeted to design primer for detection like iturin. (6)], and bacillaene [34]. In this study, we targeted three polyketide antibiotic synthase genes viz., macrolactin, difficidin and bacillaene for developing a multiplex-PCR protocol to detect polyketide antibiotic-producing strains of Bacillus spp. ...
... including B. amyloliquefaciens, B. subtilis, B. cereus, B. pumilus, and B. licheniformis using multiplex-PCR and among them, the Isolates of B. amyloliquefaciens contained macrolactin, bacillaene and difficidin polyketide synthase antibiotic genes, while B. subtilis contained difficidin and macrolactin synthase antibiotic genes. In contrast to our results, Butchner et al. [34] reported that approximately 80 kb pksX gene cluster in B. subtilis encode an unusual hybrid polyketide that has been linked to the production of the uncharacterized antibiotic bacillaene. ...
... Selection of potential antibiotic-producing strains of Bacillus spp., the individual gene had been targeted to design primer for detection like iturin. (6)], and bacillaene [34]. In this study, we targeted three polyketide antibiotic synthase genes viz., macrolactin, difficidin and bacillaene for developing a multiplex-PCR protocol to detect polyketide antibiotic-producing strains of Bacillus spp. ...
... including B. amyloliquefaciens, B. subtilis, B. cereus, B. pumilus, and B. licheniformis using multiplex-PCR and among them, the Isolates of B. amyloliquefaciens contained macrolactin, bacillaene and difficidin polyketide synthase antibiotic genes, while B. subtilis contained difficidin and macrolactin synthase antibiotic genes. In contrast to our results, Butchner et al. [34] reported that approximately 80 kb pksX gene cluster in B. subtilis encode an unusual hybrid polyketide that has been linked to the production of the uncharacterized antibiotic bacillaene. ...
Bacillus spp. has the potential to control bacterial and fungal diseases of crops. In vitro study,
Bacillus amyloliquefaciens DSBA-11 showed best to inhibit the growth of Ralstonia pseudosolanacearum
as compared to Bacillus cereus JHTBS-7, B. pumilus MTCC-7092, B. subtilis DTBS-5 and B. licheniformis
DTBL-6.Three primers sets from nucleotide sequences of polyketide antibiotic synthase genes viz.,
macrolactin, difficidin and bacillaene of B. amyloliquefaciens FZB42 were designed and standardized
protocol for simultaneous detection of polyketide antibiotics-producing strains of Bacillus spp. by
multiplex—PCR with products size of 792 bp, 705 bp and 616 bp respectively. All the strains of B. amy�loliquefaciens contained three polyketide antibiotic synthase genes, and B. subtilis possessed difficidin
and macrolactin, whereas B. cereus JHTBS-7, B. pumilus MTCC-7092 and B. licheniformis DTBL-6 did
not contain any polyketide antibiotic genes. By using this technique, polyketide-producing strains
of Bacillus spp. were screened within a short period with high accuracy. These polyketide synthase
genes were cloned by using a T&A vector to study the role of these genes in producing antibiotics
that suppressed the growth of R. pseudosolanacearum under both in vitro and in vivo conditions.
Bio-efficacy of cloned products of these genes macrolactin, bacillaene, and difficidin along with parent
strain B. amyloliquefaciens DSBA-11 inhibited the growth of R. pseudosolanacearum and formed 1.9 cm2
,
1.9 cm2
, 1.7 cm2 and 3.3 cm2
inhibition area under in vitro conditions respectively. Minimum bacterial
wilt disease intensity (29.3%) with the highest biocontrol efficacy (57.72%) was found in tomato cv.
Pusa Ruby (susceptible to wilt disease) was treated with B. amyloliquefaciens DSBA-11 followed by
cloned products of difficidin and macrolactin under glasshouse conditions. Hence, the developed
multiplex protocol might be helpful for screening polyketide antibiotics producing potential strains
of Bacillus spp. from soil which can apply for managing the wilt disease of tomatoes. The polyketide
antibiotics produced by bacteria might have a significant role suppression of R. pseudosolanacearum
due to the disintegration of cells.
... One major class of antibiotics produced by bacteria are the Non-Ribosomal Peptides (NRPs) and Polyketides (PKS), which are synthesized by large multi-enzyme complexes of non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) 2,17,18 . B. subtilis contains three different NRP biosynthetic clusters, which are responsible for the biosynthesis of surfactin, bacilysin and plipastatin and a NRP/PKS hybrid biosynthetic cluster responsible for biosynthesis of bacillaene 2,[19][20][21][22] . In addition to NRPs/PKS antibiotics B. subtilis is also capable of producing potent lantibiotic subtilosin 23 and sublancin, an antimicrobial peptide belonging to the glycocin family 24 . ...
... Bacillaene and dihydrobacillaene 19,37 are linear antimicrobial macrolides with two amide bonds and are synthesized by the PksC-R clusters mega-complex, which is composed of 13 PKS and three NRPS modules 37 . The expression of PKS genes requires the master regulator for biofilm formation, Spo0A, and promotes the competitiveness of biofilm communities 38 . ...
Microbial communities employ a variety of complex strategies to compete successfully against competitors sharing their niche, with antibiotic production being a common strategy of aggression. Here, by systematic evaluation of four non-ribosomal peptides/polyketide (NRPs/PKS) antibiotics produced by Bacillus subtilis clade, we revealed that they acted synergistically to effectively eliminate phylogenetically distinct competitors. The production of these antibiotics came with a fitness cost manifested in growth inhibition, rendering their synthesis uneconomical when growing in proximity to a phylogenetically close species, carrying resistance against the same antibiotics. To resolve this conflict and ease the fitness cost, antibiotic production was only induced by the presence of a peptidoglycan cue from a sensitive competitor, a response mediated by the global regulator of cellular competence, ComA. These results experimentally demonstrate a general ecological concept – closely related communities are favoured during competition, due to compatibility in attack and defence mechanisms.
... Bacillaene and dihydrobacillaene (Butcher et al., 2007;Straight et al., 2007) are polyketides synthesized by an enzymatic complex encoded in the pks gene cluster (Butcher et al., 2007;Straight et al., 2007). The 5′ UTR of pksC was found to be an element for induction of the pks operon. ...
... Bacillaene and dihydrobacillaene (Butcher et al., 2007;Straight et al., 2007) are polyketides synthesized by an enzymatic complex encoded in the pks gene cluster (Butcher et al., 2007;Straight et al., 2007). The 5′ UTR of pksC was found to be an element for induction of the pks operon. ...
Beneficial and probiotic bacteria play an important role in conferring immunity of their hosts to a wide range of bacterial, viral, and fungal diseases. Bacillus subtilis is a Gram-positive bacterium that protects the plant from various pathogens due to its capacity to produce an extensive repertoire of antibiotics. At the same time, the plant microbiome is a highly competitive niche, with multiple microbial species competing for space and resources, a competition that can be determined by the antagonistic potential of each microbiome member. Therefore, regulating antibiotic production in the rhizosphere is of great importance for the elimination of pathogens and establishing beneficial host-associated communities. In this work, we used B. subtilis as a model to investigate the role of plant colonization in antibiotic production. Flow cytometry and imaging flow cytometry (IFC) analysis supported the notion that Arabidopsis thaliana specifically induced the transcription of the biosynthetic clusters for the non-ribosomal peptides surfactin, bacilysin, plipastatin, and the polyketide bacillaene. IFC was more robust in quantifying the inducing effects of A. thaliana , considering the overall heterogeneity of the population. Our results highlight IFC as a useful tool to study the effect of association with a plant host on bacterial gene expression. Furthermore, the common regulation of multiple biosynthetic clusters for antibiotic production by the plant can be translated to improve the performance and competitiveness of beneficial members of the plant microbiome.
... B. subtilis strains secrete various types of antibiotics and toxins, some of which were detected in biofilms [7,21,22]. These antimicrobials include nonribosomal peptide and polyketide antibiotics, proteinderived peptide toxins, CDI toxins, and lantibiotics [7,[23][24][25][26]. The composition of antibiotic and toxin synthesis genes in B. subtilis genomes varies from strain to strain, and each B. subtilis strain often produces a distinct set of antimicrobials [7]. ...
... To compare the importance of other antimicrobials with LXG toxins in intraspecies competition, we analyzed the distribution of biosynthetic genes for 16 known and putative secreted antibiotics and toxins in 13 B. subtilis strains ( Table 2). These antimicrobials were the Rhs family of polymorphic toxin (WapA), protein-derived peptide toxins (SDP, YIT, SDP3, SDP4, and SKF), lantibiotics (sublancin, subtilosin, subtilin, and subtilomycin), non-ribosomally synthesized peptide and polyketide antibiotics (surfactin, plipastatin, mycosubtilin, bacilycin, and bacillaene), and an aminosugar antibiotic (kanosamine) [7,[23][24][25][26]59,60]. Note that these 16 antibiotics and toxins probably do not represent all the antimicrobials secreted by the 13 strains, and unknown antimicrobials likely exist. ...
Biofilms are multispecies communities, in which bacteria constantly compete with one another for resources and niches. Bacteria produce many antibiotics and toxins for competition. However, since biofilm cells exhibit increased tolerance to antimicrobials, their roles in biofilms remain controversial. Here, we showed that Bacillus subtilis produces multiple diverse polymorphic toxins, called LXG toxins, that contain N-terminal LXG delivery domains and diverse C-terminal toxin domains. Each B. subtilis strain possesses a distinct set of LXG toxin-antitoxin genes, the number and variation of which is sufficient to distinguish each strain. The B. subtilis strain NCIB3610 possesses six LXG toxin-antitoxin operons on its chromosome, and five of the toxins functioned as DNase. In competition assays, deletion mutants of any of the six LXG toxin-antitoxin operons were outcompeted by the wild-type strain. This phenotype was suppressed when the antitoxins were ectopically expressed in the deletion mutants. The fitness defect of the mutants was only observed in solid media that supported biofilm formation. Biofilm matrix polymers, exopolysaccharides and TasA protein polymers were required for LXG toxin function. These results indicate that LXG toxin-antitoxin systems specifically mediate intercellular competition between B. subtilis strains in biofilms. Mutual antagonism between some LXG toxin producers drove the spatial segregation of two strains in a biofilm, indicating that LXG toxins not only mediate competition in biofilms, but may also help to avoid warfare between strains in biofilms. LXG toxins from strain NCIB3610 were effective against some natural isolates, and thus LXG toxin-antitoxin systems have ecological impact. B. subtilis possesses another polymorphic toxin, WapA. WapA had toxic effects under planktonic growth conditions but not under biofilm conditions because exopolysaccharides and TasA protein polymers inhibited WapA function. These results indicate that B. subtilis uses two types of polymorphic toxins for competition, depending on the growth mode.
... One example from the holdout set that demonstrates this is the aurantinin cluster. 50 The closest match to aurantinin in the training set is the bacillaene cluster, 51 with a similarity score of 57%. Despite the high similarity, aurantinin and bacillaene have quite different molecular structures ( Figure 6B). ...
... not glycosylated, and contains peptides in addition to the main polyketide backbone. 51 Despite their different structures, aurantinin and bacillaene both have antibacterial activity. 50,52 The classifiers were able to successfully predict that aurantinin has antibacterial activity (60% average probability). ...
Research in natural products, the genetically encoded small molecules produced by organisms in an idiosyncratic fashion, deals with molecular structure, biosynthesis, and biological activity. Bioinformatics analyses of microbial genomes can successfully reveal the genetic instructions, biosynthetic gene clusters, that produce many natural products. Genes to molecule predictions made on biosynthetic gene clusters have revealed many important new structures. There is no comparable method for genes to biological activity predictions. To address this missing pathway, we developed a machine learning bioinformatics method for predicting a natural product’s antibiotic activity directly from the sequence of its biosynthetic gene cluster. We trained commonly used machine learning classifiers to predict antibacterial or antifungal activity based on features of known natural product biosynthetic gene clusters. We have identified classifiers that can attain accuracies as high as 80% and that have enabled the identification of biosynthetic enzymes and their corresponding molecular features that are associated with antibiotic activity.
... This gene is mutated in B. subtilis 168 (65) and might be responsible for the lack of any inhibitory effects on C. jejuni (Fig. 3). This functional phosphopantetheinyl transferase (sfp) transfers 49-phosphopantetheine from coenzyme A to carrier proteins during the early nonribosomal synthesis of antimicrobial compounds, such as surfactin, plipastatin, and bacillaene (67,(69)(70)(71)(72)(73). First, we tested whether the B. subtilis sfp gene mutation increases the survival of C. jejuni in direct coculture assays. ...
... In our search for metabolites affecting C. jejuni biofilm formation, we compared three different B. subtilis strains: the two highly related natural strains B. subtilis PS-216 and NCBI 3610, which show high genomic similarity (64), and the domesticated laboratory strain B. subtilis 168, which has lost important genetic biofilm determinants due to drastic UV and X-ray exposure (65). These include the sfp gene, which is involved in polyketide production and the nonribosomal peptide biosynthesis of surfactin, plipastatin, and bacillaene (67,68,71,82,83). Since B. subtilis 168 did not inhibit C. jejuni growth or C. jejuni biofilm formation, we took advantage of the genetic differences between B. subtilis 168 and PS-216/NCIB3610 to identify potential genetic determinants of PS-216 antagonism. ...
Biofilms are the predominant bacterial lifestyle and can protect microorganisms from environmental stresses. Multi-species biofilms can affect the survival of enteric pathogens that contaminate food products, and thus investigating the underlying mechanisms of multi-species biofilms is essential for food safety and human health. In this study, we investigated the ability of the natural isolate Bacillus subtilis PS-216 to restrain Campylobacter jejuni biofilm formation and adhesion to abiotic surfaces as well as to disrupt pre-established C. jejuni biofilms. Using confocal laser scanning microscopy and colony counts, we demonstrate that the presence of B. subtilis PS-216 prevents C. jejuni biofilm formation, decreases growth of the pathogen by 4.2 log 10 and disperses 26 h old pre-established C. jejuni biofilms. Furthermore, the co-inoculation of B. subtilis and C. jejuni interferes with the adhesion of C. jejuni to abiotic surfaces reducing it by 2.4 log 10 . We also show that contact-independent mechanisms contribute to the inhibitory effect of B. subtilis PS-216 on C. jejuni biofilm. Using B. subtilis mutants in genes coding for non-ribosomal peptides and polyketides revealed that bacillaene significantly contributes to the inhibitory effect of B. subtilis PS-216. In summary, we show a strong potential for the use of B. subtilis PS-216 against C. jejuni biofilm formation and adhesion to abiotic surfaces. Our research could bring forward novel applications of B. subtilis in animal production and thus contribute to food safety.
IMPORTANCE
Campylobacter jejuni is an intestinal commensal in animals (including broiler chickens), but also the most frequent cause of bacterial food-borne infection in humans. This pathogen forms biofilms which mend survival of C. jejuni in food processing and thus threaten human health. Probiotic bacteria represent a potential alternative in the prevention and control of food-borne infections. The beneficial bacterium, Bacillus subtilis has an excellent probiotic potential to reduce C. jejuni in the animal gastrointestinal tract. However, data on the effect of B. subtilis on C. jejuni biofilms are scarce. Our study shows that the B. subtilis natural isolate PS-216 prevents adhesion to the abiotic surfaces and the development of submerged C. jejuni biofilm during co-culture and destroys the pre-established C. jejuni biofilm. These insights are important for development of novel applications of B. subtilis that will reduce the use of antibiotics in human and animal health and increase productivity in animal breeding.
... NRPS were multienzyme complexes that bind malonyl derivatives and amino acids in a sequential manner ). NRPS enzymes utilized many different building blocks to produce a wide range of secondary metabolites with potential bioactive (Butcher et al. 2007). In the results of comparison with known gene clusters, six gene clusters showed 100% similarity with the clusters that have been reported, they were gene cluster #6, synthesized macrolactin H, gene cluster #7, synthesized bacillaene, gene cluster #8, synthesized fengycin, gene cluster #11, synthesized di cidin, gene cluster #12 synthesized bacillibactin, and gene cluster #13, synthesized bacilysin. ...
Discovering more novel antimicrobial compounds has become an keen research problem. In this study, YA215 genome was sequenced by the Illumina HiSeq + PacBio sequencing platform. Genome assembly was performed by Unicycler software and the gene clusters responsible for secondary metabolite biosynthesis were predicted by antiSMASH. The genome comprised 3976514 bp and had a 46.56% G + C content. 3809 coding DNA sequences, 27 rRNAs, 86 tRNAs genes and 79 sRNA were predicted. Strain YA215 was re-identified as Bacillus velezensis based on ANI and OrthoANI analysis. In the COG database, 23 functional groups from 3090 annotations were predicted. In the GO database, 2654 annotations were predicted. 2486 KEGG annotations linked 41 metabolic pathways. Glycosyl transferases, polysaccharide lyases, auxiliary activities, glycoside hydrolases, carbohydrate esterases and carbohydrate-binding modules were predicted among the 127 annotations in the CAZy database. AntiSMASH analysis predicted that strain B. velezensis YA215 boasted 13 gene clusters involved in synthesis of antimicrobial secondary metabolites including surfactin, fengycin, macrolactin H, bacillaene, difficidin, bacillibactin, bacilysin, and plantazolicin. Three of the gene clusters (gene cluster 5, gene cluster 9 and gene cluster 10) have the potential to synthesize unknown compounds. The research findings have the potential to contribute significantly to the development of natural novel compounds with antimicrobial activity in B. velezensis YA215.
... Consequently, E. coli colonies lost completely their complex morphology and at the microscale cells lost their protective matrix "shield" that consists of a basket-like structure that curli fibers form around each cell. We identified bacillaene, a NRP/PK and Sfpdependent metabolite secreted by B. subtilis 21,30 , as the effector molecule and could demonstrate that bacillaene exerts its inhibition by directly impeding the assembly of the CsgB/CsgA subunits into amyloid fibers. Various experimental evidences support this mode of action: (i) bacillaene does not downregulate transcription of curli structural or regulatory genes (expression of csgBAC and csgDEFG operons actually increases in cells experiencing inhibition of curli production), (ii) in the presence of bacillaene, E. coli cells within the macrocolony produce and secrete the curli subunits, which, however, instead of remaining associated to the biofilm cells in the form of CR-stainable amyloid fibers -as occurs in the absence of bacillaene-, diffuse away into the underlying agar, (iii) in CsgA polymerization assays in vitro bacillaene directly abolishes the accumulation of CsgA amyloid fibers. ...
The propensity of bacteria to grow collectively in communities known as biofilms and their ability to overcome clinical treatments in this condition has become a major medical problem, emphasizing the need for anti-biofilm strategies. Antagonistic microbial interactions have extensively served as searching platforms for antibiotics, but their potential as sources for anti-biofilm compounds has barely been exploited. By screening for microorganisms that in agar-set pairwise interactions could antagonize Escherichia coli’s ability to form macrocolony biofilms, we found that the soil bacterium Bacillus subtilis strongly inhibits the synthesis of amyloid fibers –known as curli-, which are the primary extracellular matrix (ECM) components of E. coli biofilms. We identified bacillaene, a B. subtilis hybrid non-ribosomal peptide/polyketide metabolite, previously described as a bacteriostatic antibiotic, as the effector molecule. We found that bacillaene combines both antibiotic and anti-curli functions in a concentration-dependent order that potentiates the ecological competitiveness of B. subtilis , highlighting bacillaene as a metabolite naturally optimized for microbial inhibition. Our studies revealed that bacillaene inhibits curli by directly impeding the assembly of the CsgB and CsgA curli subunits into amyloid fibers. Moreover, we found that curli inhibition occurs despite E. coli attempts to reinforce its protective ECM by inducing curli genes via a RpoS-mediated competition sensing response trigged by the threatening presence of B. subtilis . Overall, our findings illustrate the relevance of exploring microbial interactions not only for finding compounds with unknown and unique activities, but for uncovering additional functions of compounds previously categorized as antibiotics.
... Additionally, Bacillus spp. also produces hybrid polyketide-nonribosomal peptide metabolites with antimicrobial activity, such as bacillaene produced by B. subtilis that inhibits a broad spectrum of bacteria (Butcher et al., 2007;Patel et al., 1995). Bacillus strains can engage hydrolases, such as chitinase, cellulase, xylanase, glucanase, and protease, to attack the glycosidic bonds of the fungal pathogen's main cell wall components (e.g., chitin, glucan, and protein) to inhibit their growth (Bardin et al., 2015;Jamali et al., 2020). ...
Species of the genus Bacillus have been widely used for the biocontrol of plant diseases in the demand for sustainable agricultural development. New mechanisms underlying Bacillus biocontrol activity have been revealed with the development of microbiome and microbe‐plant interaction research. In this review, we first briefly introduce the typical Bacillus biocontrol mechanisms, such as the production of antimicrobial compounds, competition for niches/nutrients, and induction of systemic resistance. Then, we discussed in detail the new mechanisms of pathogen quorum sensing interference and reshaping of the soil microbiota. The “cry for help” mechanism was also introduced, in which plants can release specific signals under pathogen attack to recruit biocontrol Bacillus for root colonization against invasion. Finally, two emerging strategies for enhancing the biocontrol efficacy of Bacillus agents, including the construction of synthetic microbial consortia and the application of rhizosphere‐derived prebiotics, were proposed.
... Both PKSs and NRPSs are multi-enzyme complexes with the purpose of combining malonyl derivatives and amino acids in a sequential fashion, respectively. These tailoring enzymes make use of a number of different building blocks in order to produce a wide range of secondary metabolites that have potential of bioactivity applications (Butcher et al., 2007). The results demonstrate that the genome of strain VR-34 predominantly harbors gene clusters associated with the synthesis of secondary metabolites such as difficidin, aurantinin, elansolid, basiliskamide, macrobrevin, kalimantacin, sowinholide A, bacillaene, calyculin, and oocydin. ...
Peyronellaea arachidicola, a fungus that specifically targets the foliage of peanut plants, causes web blotch disease,
which has an impact on the crop’s yield and quality. In the present study, Whole-genome sequencing identified
and characterized Bacillus velezensis VR-34, as a biocontrol antagonistic strain. In vitro assay showed that VR-34
had a significant antagonistic effect on P. arachidicola with an inhibition zone of 90.39 mm, which was higher
than the inhibition zone of 80.45 mm for Ck. In greenhouse assay, in the pre-inoculated treatment, it was
observed that the VR-34 strain exhibited a biocontrol efficacy of 93.52% against peanut web blotch disease,
whereas the control group showed a biocontrol efficacy of 95.64%.. However, in the post-inoculated treatment, it
was observed that the VR-34 strain exhibited a biocontrol efficacy of 89.89% against peanut web blotch disease,
whereas the control group showed a biocontrol efficacy of 88.71%. The SEM analysis revealed that VR-34 caused
damaged the hyphal surfaces and broken of spores of P. arachidicola. Whole-genome sequencing revealed twenty
functional groups from 2833 annotations in the COG database, most of which were involved in transcription,
secondary metabolite biosynthesis, amino acid transport and metabolism, and carbohydrate metabolism. 1694
KEGG annotations link 33 metabolic pathways. Glycoside hydrolases, glycosyl transferases, carbohydrate es�terases, auxiliary activities, carbohydrate-binding modules, and polysaccharide lyases were among the 172 an�notations in the CAZy database. antiSMASH analysis predicted that strain VR-34 can produce the secondary
metabolites difficidin, aurantinin, elansolid, basiliskamide macrobrevin, kalimantacin, sowinholide A, bacil�laene, and calyculin. In vitro, and greenhouse studies revealed that VR-34 controlled peanut web blotch disease.
The whole genome study showed the VR-34 strain is B. velezensis and could synthesize antifungal secondary
metabolites.
... The bacterial biocontrol organism B. subtilis synthesizes a variety of biologically active compounds with a broad range of activity against phytopathogens (Shafi et al., 2017) and is capable of inducing host systemic resistance (Aliye et al., 2008;Bais et al., 2004;Butcher et al., 2007). It has the potential of forming multicellular structures and biofilms (Bais et al., 2004;Branda et al., 2001) and can also compete for iron and nutrients through siderophore synthesis (de Boer et al., 2003;Saha et al., 2015). ...
There is little information on the effect of temperature, relative humidity (RH) and vapour pressure deficit (VPD) on the viable populations of two commercial biocontrol strains, Bacillus subtilis QST 713 (recently classified as B. velezensis) and Gliocladium catenulatum J1446 (syn. Clonostachys rosea). The PMAxx™‐qPCR molecular assay was used to quantify the viable BCA populations on fully extended lettuce and strawberry leaves under a range of temperature and relative humidity combinations. Overall, there was a small decline in the population size of viable cells for the two biocontrol organisms on strawberry and lettuce leaves within 10 days of application. However, for most experimental runs which contained general UK agronomy climates, such a decline was not statistically significant. Moreover, for a few runs, the viable populations increased significantly in optimal BCA growth temperatures with high RH. Only temperature (ambient) and dew point significantly affected the rate of temporal changes in the viable biocontrol population size. Thus, an increasing temperature led to decreased daily mortality. It should be noted that much of the variability in the estimated daily mortality rates remains unaccounted for, thus unless under extreme conditions, the biocontrol efficacy in practice is less likely to be affected by the survival of biocontrol microbes but more likely to be affected by other factors that influence the density of biocontrol cells in the phyllosphere: especially dilution due to rapid host leaf expansion, spray coverage, and rain wash‐off.
... In particular, B. subtilis, B. amyloliquefaciens, B. pumilus, and B. cereus have been reported to produce novel compounds in co-culture that act as antimicrobials (Trischman et al., 2004;Peterson et al., 2006;Straight et al., 2006;Benitez et al., 2011;Schneider et al., 2012;Moree et al., 2013;Aghcheh and Kubicek, 2015), biosurfactants (Dusane et al., 2011) and inhibitors against fungi and bacteria (Dusane et al., 2011;Wu et al., 2018;Li et al., 2020). Research to date points out that Bacillus species produce a wide range of these bioactive substances when in co-culture with Cytophaga-Flavobacterium (Peterson et al., 2006), Streptomyces (Straight et al., 2006;Butcher et al., 2007;Schneider et al., 2012), fungi (Dusane et al., 2011;Moree et al., 2013), Pseudomonas (Andrić et al., 2021), E. coli (Benitez et al., 2011;Chanos and Mygind, 2016), as well as other Bacillus species (Trischman et al., 2004;Dusane et al., 2011). Moreover, this species is also used as a challenge strain, inducing strong metabolic production in competitive species (Watsuji et al., 2007;Shah et al., 2008;Hoefler et al., 2012;Ge et al., 2014;Akone et al., 2016;Chanos and Mygind, 2016;Ebrahim et al., 2016;Sung et al., 2017;Yu et al., 2017;Shin et al., 2018;El-Sayed et al., 2021;Bagheri et al., 2022;Sun et al., 2022). ...
In natural product research, microbial metabolites have tremendous potential to provide new therapeutic agents since extremely diverse chemical structures can be found in the nearly infinite microbial population. Conventionally, these specialized metabolites are screened by single-strain cultures. However, owing to the lack of biotic and abiotic interactions in monocultures, the growth conditions are significantly different from those encountered in a natural environment and result in less diversity and the frequent re-isolation of known compounds. In the last decade, several methods have been developed to eventually understand the physiological conditions under which cryptic microbial genes are activated in an attempt to stimulate their biosynthesis and elicit the production of hitherto unexpressed chemical diversity. Among those, co-cultivation is one of the most efficient ways to induce silenced pathways, mimicking the competitive microbial environment for the production and holistic regulation of metabolites, and has become a golden methodology for metabolome expansion. It does not require previous knowledge of the signaling mechanism and genome nor any special equipment for cultivation and data interpretation. Several reviews have shown the potential of co-cultivation to produce new biologically active leads. However, only a few studies have detailed experimental, analytical, and microbiological strategies for efficiently inducing bioactive molecules by co-culture. Therefore, we reviewed studies applying co-culture to induce secondary metabolite pathways to provide insights into experimental variables compatible with high-throughput analytical procedures. Mixed-fermentation publications from 1978 to 2022 were assessed regarding types of co-culture set-ups, metabolic induction, and interaction effects.
... It is encoded by a hybrid PKS-NRPS biosynthetic gene cluster known as bacillaene PksX synthase ( Figure 6). The pksX mega gene cluster in B. subtilis 168 genome consisted of 5 open reading frames named pksJ, pksL, pksM, pksN, and pksR [104,129]. The first two adenylation domains of pksJ incorporate glycin and α-hydroxy-isocaproic acid. ...
Despite their remarkable biosynthetic potential, Bacillus subtilis have been widely overlooked. However, their capability to withstand harsh conditions (extreme temperature, Ultraviolet (UV) and γ-radiation, and dehydration) and the promiscuous metabolites they synthesize have created increased commercial interest in them as a therapeutic agent, a food preservative, and a plant-pathogen control agent. Nevertheless, the commercial-scale availability of these metabolites is constrained due to challenges in their accessibility via synthesis and low fermentation yields. In the context of this rising in interest, we comprehensively visualized the antimicrobial peptides produced by B. subtilis and highlighted their prospective applications in various industries. Moreover, we proposed and classified these metabolites produced by the B. subtilis group based on their biosynthetic pathways and chemical structures. The biosynthetic pathway, bioactivity, and chemical structure are discussed in detail for each class. We believe that this review will spark a renewed interest in the often disregarded B. subtilis and its remarkable biosynthetic capabilities.
... Here, it was found that polyene antibiotic bacillaene was the antibacterial substance produced by B. velezensis E9. Butcher et al. (22) reported that bacillaene is active against a broad spectrum of bacteria by inhibiting prokaryotic protein synthesis. Here, we report for the first time that bacillaene produced by Bacillus inhibited growth of the plant pathogen R. solanacearum. ...
Bacterial wilt disease causes heavy yield losses in many crops. Endophytic microbiomes play important roles in control of plant diseases.
... We have shown that B. subtilis PS-216 antagonism is mediated by the polyketide antibiotic bacillaene, as PS-216 which lacks the pks operon, responsible for bacillaene synthesis, completely loses its ability to inhibit S. Typhimurium growth and adhesion to polystyrene surfaces. This diffusible polyketide antibiotic (12,17,63) inhibits the growth of various bacteria when purified or present in spent media (13). However, its influence on competition in a mixed-species biofilm has been less studied (17,(19)(20)(21)46) and has not yet been addressed for B. subtilis-Salmonella interactions. ...
Probiotic bacteria represent an alternative for controlling foodborne disease caused by Salmonella enterica , which constitutes a serious concern during food production due to its antibiotic resistance and resilience to environmental stress. Bacillus subtilis is gaining popularity as a probiotic, but its behavior in biofilms with pathogens such as Salmonella remains to be elucidated. Here, we show that the antagonism of B. subtilis is mediated by the polyketide bacillaene and that the production of bacillaene is a highly dynamic trait which depends on environmental factors such as nutrient availability and the presence of competitors.
... Bacilysin is a non-ribosomally synthesized dipeptide antibiotic that inhibits Gram-negative foodborne pathogens [107][108][109]. Bacillaene is a polyene antibiotic that can accelerate biofilm formation and has activity against a broad spectrum of bacteria, including S. aureus and E. coli [110][111][112][113]. It functions by inhibiting bacterial protein synthesis, but it cannot inhibit eukaryotic protein synthesis. ...
Despite the commercial rise of probiotics containing Bacillaceae spp., it remains important to assess the safety of each strain before clinical testing. Herein, we performed preclinical analyses to address the safety of Bacillus subtilis BS50. Using in silico analyses, we screened the 4.15 Mbp BS50 genome for genes encoding known Bacillus toxins, secondary metabolites, virulence factors, and antibiotic resistance. We also assessed the effects of BS50 lysates on the viability and permeability of cultured human intestinal epithelial cells (Caco-2). We found that the BS50 genome does not encode any known Bacillus toxins. The BS50 genome contains several gene clusters involved in the biosynthesis of secondary metabolites, but many of these antimicrobial metabolites (e.g., fengycin) are common to Bacillus spp. and may even confer health benefits related to gut microbiota health. BS50 was susceptible to seven of eight commonly prescribed antibiotics, and no antibiotic resistance genes were flanked by the complete mobile genetic elements that could enable a horizontal transfer. In cell culture, BS50 cell lysates did not diminish either Caco-2 viability or monolayer permeability. Altogether, BS50 exhibits a robust preclinical safety profile commensurate with commercial probiotic strains and likely poses no significant health risk to humans.
... To begin with, most Bacillus are known as safe bacteria that naturally inhabit the phyllosphere and the soil, often in association with the roots of higher plants. As a result, they have evolved to become "microbial factories": they produce biologically active molecules that can be beneficial for plants (Pérez-García et al., 2011;Radhakrishnan et al., 2017;Schallmey et al., 2004;Stein, 2005), in terms of inhibiting phytopathogens or inducing host systemic resistance (Butcher et al., 2007;Nagórska et al., 2007;Ongena et al., 2007;Ongena & Jacques, 2008). They can also antagonize pathogens on the surface of the leaves by outcompeting them for nutrients or space (Sivakumar et al., 2020). ...
Maize (Zea mays L.), a major crop in Argentina and a staple food around the world, is affected by the emergence and re‐emergence of foliar diseases. Agrochemicals are the main control strategy nowadays, but they can cause resistance in insects and microbial pathogens and have negative effects on the environment and human health. An emerging alternative is the use of living organisms, i.e. microbial biocontrol agents, to suppress plant pathogen populations. This is a risk‐free approach when the organisms acting as biocontrol agents come from the same ecosystem as the foliar pathogens they are meant to antagonize. Some epiphytic microorganisms may form biofilm by becoming aggregated and attached to a surface, as is the case of spore‐forming bacteria from the genus Bacillus. Their ability to sporulate and their tolerance to long storage periods make them a frequently used biocontrol agent. Moreover, the biofilm that they create protects them against different abiotic and biotic factors and helps them to acquire nutrients, which ensures their survival on the plants they protect. This review analyzes the interactions that the phyllosphere‐inhabiting Bacillus genus establishes with its environment through biofilm, and how this lifestyle could serve to design effective biological control strategies.
... The majority of the BGCs found in B. subtilis STRP31 and B. velezensis SPL51 are already known (Fig. 4B). The known BGCs encoded in the genomes are: anabaenopeptin [37], bacilysin [38], bacillibactin [39], difficidin [40], fengycin [41], bacillaene [42], macrolactin H [43], plantazolicin [44], surfactin [45], subtilomycin [46], subtilosin A [47], paeninodin [48] (additional file S2). The highest number of the unknown BCGs belong to strain Paenibacillus sp. ...
Background
Biocontrol agents are sustainable eco-friendly alternatives for chemical pesticides that cause adverse effects in the environment and toxicity in animals including humans. An improved understanding of the phyllosphere microbiology is of vital importance for biocontrol development. Most studies have been directed towards beneficial plant-microbe interactions and ignore the pathogens that might affect humans when consuming vegetables. In this study we extended this perspective and investigated potential biocontrol strains isolated from tomato and lettuce phyllosphere that can promote plant growth and potentially antagonize human pathogens as well as plant pathogens. Subsequently, we mined into their genomes for discovery of antimicrobial biosynthetic gene clusters (BGCs), that will be further characterized.
Results
The antimicrobial activity of 69 newly isolated strains from a healthy tomato and lettuce phyllosphere against several plant and human pathogens was screened. Three strains with the highest antimicrobial activity were selected and characterized ( Bacillus subtilis STRP31, Bacillus velezensis SPL51, and Paenibacillu s sp. PL91). All three strains showed a plant growth promotion effect on tomato and lettuce. In addition, genome mining of the selected isolates showed the presence of a large variety of biosynthetic gene clusters. A total of 35 BGCs were identified, of which several are already known, but also some putative novel ones were identified. Further analysis revealed that among the novel BGCs, one previously unidentified NRPS and two bacteriocins are encoded, the gene clusters of which were analyzed in more depth.
Conclusions
Three recently isolated strains of the Bacillus genus were identified that have high antagonistic activity against lettuce and tomato plant pathogens. Known and unknown antimicrobial BGCs were identified in these antagonistic bacterial isolates, indicating their potential to be used as biocontrol agents. Our study serves as a strong incentive for subsequent purification and characterization of novel antimicrobial compounds that are important for biocontrol.
... Bacillaene is a polyene antibiotic with a two-amide bond linear structure (Butcher et al., 2007). It is mainly produced by B. subtilis (Patel et al., 1995), B. amyloliquefaciens, and B. velezensis (Aleti et al., 2015;Chen et al., 2006). ...
... Here we report that in addition to their structural role, manifested by their necessity for B. subtilis to generate pellicles (floating biofilms) with itself [62] and with a related bacterium, B. atrophaeus (Fig. 1), the exopolysaccharides act to induce the production of bacillaene, a polyketide antibiotic [33,[63][64][65] which was recently shown to be essential for the elimination of phylogenetically distinct Bacillus species [41,66]. Our results indicate that this non-structural role of the exopolysaccharides is manifested by a capacity of B. subtilis to repel the phylogenetically distinct competitor, B. mycoides from the pellicle and its surrounding media. ...
In nature, bacteria frequently reside in differentiated communities or biofilms. These multicellular communities are held together by self-produced polymers that allow the community members to adhere to the surface as well as to neighbor bacteria. Here, we report that exopolysaccharides prevent Bacillus subtilis from co-aggregating with a distantly related bacterium Bacillus mycoides, while maintaining their role in promoting self-adhesion and co-adhesion with phylogenetically related bacterium, Bacillus atrophaeus. The defensive role of the exopolysaccharides is due to the specific regulation of bacillaene. Single cell analysis of biofilm and free-living bacterial cells using imaging flow cytometry confirmed a specific role for the exopolysaccharides in microbial competition repelling B. mycoides. Unlike exopolysaccharides, the matrix protein TasA induced bacillaene but inhibited the expression of the biosynthetic clusters for surfactin, and therefore its overall effect on microbial competition during floating biofilm formation was neutral. Thus, the exopolysaccharides provide a dual fitness advantage for biofilm-forming cells, as it acts to promote co-aggregation of related species, as well as, a secreted cue for chemical interference with non-compatible partners. These results experimentally demonstrate a general assembly principle of complex communities and provides an appealing explanation for how closely related species are favored during community assembly. Furthermore, the differential regulation of surfactin and bacillaene by the extracellular matrix may explain the spatio-temporal gradients of antibiotic production within biofilms.
... Although, cellulase has been analysed from bacterial sources but their number is lesser compared to their abundance in nature. The bacterial cellulase has been studied and purified from Bacillus subtilis [162,163] Paenibacillus sp. [ ...
Cellulase enzyme complex is comprised of three enzymes namely exo-glucanase, endo-glucanase and β-glucosidase which act synergistically to deconstruct cellulosic biomass in order to produce fermentable sugars. The enzymes are produced naturally by the living organisms such as bacteria, fungi and algae. The majority of microorganisms that live in extreme environments including hot/cold springs, rumen stomach, deep ocean trench, acidic/alkaline pH environment, have been regarded as appealing producers of cellulase. Cellulases produced by microorganisms have enormous applications in different industries such as agriculture, food and feed production, brewing, textile, laundry and biofuel production. Scientists as well as industry researchers consider cellulases as a prospective candidate for further studies due to the intricacy of the enzyme system and massive industrial potential. Scientific belief in its production and further studies challenges are receiving greater attention these days, notably in the intent of decreasing its production cost at the industrial scale. In this review, future possibilities of using cellulase for various industrial applications are also addressed.
... Examples of non-amino acid starter units can be found in the cahuitamycin (2), bacillaene (18), and rapamycin (19) pathways. These three pathways also demonstrate that initiation modules containing NRPS A domains are present at the beginning of NRPS, PKS and hybrid PKS/NRPS pathways. ...
Natural products are a rich source of diverse chemical compounds, many with pharmaceutical potential. Structural and biochemical investigations into the initiation steps of three natural product biosynthetic pathways were conducted. These findings contribute to the adaptation of natural products for industrial applications or for improved pharmaceutical properties as well as the discovery of novel enzymatic chemistry. The olefin synthase from the marine cyanobacterium Synechococcus sp. PCC7002 converts stearic acid to 1-nonadecene, a terminal olefin. Biologically generated length-controlled olefins have potential applications as lubricants, surfactants, chemical feedstocks, and biofuels. The fatty acid-ACP ligase (FAAL) domain of olefin synthase selects stearic acid for conversion to 1-nonadecene, dictating the length of olefin produced. Crystal structures of the SynFAAL guided the creation of a number of variants that led to the discovery of an electrostatic fatty acid selection mechanism and a better understanding of the basis of fatty acid length selectivity. The cahuitamycin pathway of Streptomyces gandocaensis natively produces three cahuitamycin variants, which are all potent inhibitors of Acinetobacter baumannii biofilm formation. Variation originates in the starter unit selected by aryl-AMP ligase CahJ. Crystal structures of several CahJ-substrate complexes provided a structure-based explanation of substrate selectivity in the creation of natural cahuitamycins. Together with a comprehensive set of substrate selectivity data, the structure provides a roadmap for the creation of novel cahuitamycin congeners for future investigation as biofilm inhibitors. Apratoxin A from the marine cyanobacterium Moorea bouillonii contains a rare t-butyl group. The steps for t-butyl biosynthesis likely involve novel enzymatic reactions by domains within AprA, the first protein of the biosynthetic pathway. A di-domain of AprA containing a type I S-adenosylmethionine dependent methyltransferase (MT) domain was shown to possess both acyltransfer and methylation activity. The crystal structure of the di-domain indicated that the MT domain performs both reactions and that the other domain is vestigial. The initiation steps are an important source of chemical diversity in natural product biosynthesis. Understanding the mechanisms of substrate recognition used by initiation enzymes could allow for the rational redesign of substrate specificity leading to biological products with improved characteristics.
... Thus, we explored the B. cabrialesii TE3 T genome to identify and establish potential metabolite candidates to be involved in B. sorokiniana biocontrol. We identified 7 BGCs in B. cabrialesii TE3 T genome employing a genome-mining strategy (antiSMASH/BiGSCAPE), which previously have been reported to be involved in the biosynthesis of bioactive metabolites with wide spectrum antibacterial (bacilysin, bacillaene, subtilosin A, and bacillibactin) and antifungal activity (surfactin, fengycin, and rhizocticin A) in another Bacillus species (Butcher et al., 2007;Hertlein et al., 2014;Ö zcengiz and Ög ülür, 2015;Rapp et al., 1988;Villarreal-Delgado et al., 2018). This suggests that B. cabrialesii TE3 T could have biocontrol activity against other fungi and bacteria, opening the door to further explore its biocontrol activity and mechanisms of action against other economically important phytopathogens (Córdova-Albores et al., 2021). ...
Bipolaris sorokiniana is an important biotic constraint for global wheat production, causing spot blotch disease. In this work, we present a comprehensive characterization of the cell-free culture filtrate (CF) and precipitated fraction (PF) of Bacillus cabrialesii TE3T showing an effective inhibition of spot blotch. Our results indicated that CF produced by B. cabrialesii TE3T inhibits the growth of B. sorokiniana through stable metabolites (after autoclaving and proteinase K treatment). Antifungal metabolites in CF and PF were explored by an integrated genomic-metabolomic approach. Genome-mining revealed that strain TE3T contains the biosynthetic potential to produce wide spectrum antifungal (surfactin, fengycin, and rhizocticin A) and antibacterial metabolites (bacillaene, bacilysin, bacillibactin, and subtilosin A), and through bioactivity-guided LC-ESI-MS/MS approach we determined that a lipopeptide complex of surfactin and fengycin homologs was responsible for antifungal activity exhibited by B. cabrialesii TE3T against the studied phytopathogen. In addition, our results demonstrate that i) a lipopeptide complex inhibits B. sorokiniana by disrupting its cytoplasmatic membrane and ii) reduced spot blotch disease by 93 %. These findings show the potential application of metabolites produced by strain TE3T against B. sorokiniana and provide the first insight into antifungal metabolites produced by the novel Bacillus species, Bacillus cabrialesii.
... First, we identified an uncharacterized iModulon that contains genes responsible for capsular polyglutamate synthesis, biofilm components, and synthesis of the peptide/polyketide antibiotic bacillaene 39 (Figure 3a). This iModulon is activated in early biofilm production and stationary phase (Figure 3b), and the activities are correlated with the Since B. subtilis is a model organism for biofilm formation, and no single regulator is known to control all of these processes, this iModulon presents a compelling foundation for the potential discovery of a novel transcriptional regulator central to biofilm formation. ...
We are firmly in the era of biological big data. Millions of omics datasets are publicly accessible and can be employed to support scientific research or build a holistic view of an organism. Here, we introduce a workflow that converts all public gene expression data for a microbe into a dynamic representation of the organism's transcriptional regulatory network. This five-step process walks researchers through the mining, processing, curation, analysis, and characterization of all available expression data, using Bacillus subtilis as an example. The resulting reconstruction of the B. subtilis regulatory network can be leveraged to predict new regulons and analyze datasets in the context of all published data. The results are hosted at https://imodulondb.org/, and additional analyses can be performed using the PyModulon Python package. As the number of publicly available datasets increases, this pipeline will be applicable to a wide range of microbial pathogens and cell factories.
... than the isolation media. Many reports have revealed the existence of competition between bacteria, such as the genera Bacillus and Streptomyces (Butcher et al., 2007;Straight et al., 2007;Vargas-Bautista et al., 2014). In our study, the SCP strategy containing four different inhibitors significantly reduced the number of clones of fast-growing bacteria, represented by the genus Bacillus (Fig. S5). ...
Streptomycetes have been, for over 70 years, one of the most abundant sources for the discovery of new antibiotics and clinic drugs. However, in recent decades, it has been more and more difficult to obtain new phylotypes of the genus Streptomyces by using conventional samples and culture strategies. In this study, we combined culture-dependent and culture-independent approaches to better explore the Streptomyces communities in desert sandy soils. Moreover, two different culture strategies termed Conventional Culture Procedure (CCP) and Streptomycetes Culture Procedure (SCP) were employed to evaluate the isolation efficiency of Streptomyces spp. with different intensities of selectivity. The 16S rRNA gene amplicon analysis revealed a very low abundance (0.04−0.37%, average 0.22%) of Streptomyces in all the desert samples, conversely the percentage of Streptomyces spp. obtained by the culture-dependent method was very high (5.20−39.57%, average 27.76%), especially in the rhizospheric sand soils (38.40−39.57%, average 38.99%). Meanwhile, a total of 1589 pure cultures were isolated successfully, dominated by Streptomyces (29.52%), Microvirga (8.06%) and Bacillus (7.68%). In addition, 400 potential new species were obtained, 48 of which belonged to the genus Streptomyces. More importantly, our study demonstrated the SCP strategy which had highly selectivity could greatly expand the number and phylotypes of Streptomyces spp. by almost 4-fold than CCP strategy. These results provide insights on the diversity investigation of desert Streptomyces, and it could be reference for researchers to bring more novel actinobacteria strains from the environment into culture.
... Bacillaene has an unusual, linear polyene structure consisiting of of six conjugated carbon-carbon double bonds and two amide bonds. Its complex structure is synthesized by three giant polyketide synthases that form an enzymatic complex of the size of ribosomes [124,125]. ...
The fundamental feature of “active honeys” is the presence and concentration of antibacterial compounds. Currently identified compounds and factors have been described in several review papers without broader interpretation or links to the processes for their formation. In this review, we indicate that the dynamic, antagonistic/competitive microbe–microbe and microbe–host interactions are the main source of antibacterial compounds in honey. The microbial colonization of nectar, bees and honey is at the center of these interactions that in consequence produce a range of defence molecules in each of these niches. The products of the microbial interference and exploitive competitions include antimicrobial peptides, antibiotics, surfactants, inhibitors of biofilm formation and quorum sensing. Their accumulation in honey by horizontal transfer might explain honey broad-spectrum, pleiotropic, antibacterial activity. We conclude that honey is an ecological reservoir of antibacterial compounds produced by antagonistic microbial interactions in plant nectars, honey and honey bee. Thus, refocusing research on secondary metabolites resulting from these microbial interactions might lead to discovery of new antibacterial compounds in honey that are target-specific, i.e., acting on specific cellular components or inhibiting the essential cellular function.
... Bacilysin is one of the simplest peptide antibiotics secreted by certain species of Bacillus [32]. Bacillaene is a polyene antibacterial substance secreted by Bacillus [33]. Surfactin and fengycin are broad-spectrum antibacterial substances present in several Bacillus species, and their antibacterial activity has been widely reported [34]. ...
An endophytic bacterium Bacillus velezensis BY6 was isolated from the wood stems of healthy Populus davidiana × P. alba var. pyramidalis (PdPap). The BY6 strain can inhibit pathogenic fungus Alternaria alternate in PdPap and promote growth of PdPap seedlings. In the present study, we used the Pacific Biosciences long-read sequencing platform, a single-molecule real-time (SMRT) technology for strain BY6, to perform complete genome sequencing. The genome size was 3,898,273 bp, the number of genes was 4,045, and the average GC content was 47.33%. A complete genome of strain BY6 contained 110 secondary metabolite gene clusters. Nine of the secondary metabolite gene clusters exhibited antifungal activity and promoted growth functions primarily involved in the synthesis of surfactin, bacteriocins, accumulated iron ions, and related antibiotics. Gene clusters provide genetic resources for biotechnology and genetic engineering, and enhance understanding of the relationship between microorganisms and plants.
... In addition, compounds with either α-β double bonds or β-γ double bonds are very common in polyketides, such as rhizoxin 9 and bacillaene. 10 Therefore, DHs are notable for their catalytic activities in polyketide biosynthesis. ...
Dehydratase (DH), a domain in polyketide synthase (PKS) modules, can catalyze the dehydration of β-hydroxy to an α, β-unsaturated acyl intermediate. As the first dual-function (dehydratase/isomerase) DH domain accessible in PDB database, gephyronic acid (GphF) DH1 domain from the PKS biosynthetic pathway attracts great concerns of researchers. However, the mechanisms of dehydration and isomerization in type I PKS still remain unclear. In this study, MD simulations and QM/MM calculations were combined to elucidate the molecular mechanism of GphF DH1. The results indicated that GphF DH1 has better recognition effect towards (2R,3R)-substrate and prefers forming the α-β double bond in advance to the β-γ double bond directly. By calculating the binding energy, some key residues near the active pocket were highlighted. Umbrella sampling results showed that nonmethylated substrates could form intra-molecular hydrogen bonds more easily than α-methyl substrates. The QM/MM calculations with M062X/6-311+G**//M062X/6-31G* method and SMD solvation correction supported one-base dehydration and one-base isomerization mechanism with energy barriers of 27.0 kcal/mol and 17.2 kcal/mol, respectively. These results can encourage future studies for the comprehensive understanding of the catalytic mechanism of PKS DHs and for the rational design of typical DHs.
... 1−4 Since the first discovery in 1995, 1 their structures have not been identified for a decade because of chemical instability, until the arduous task was completed by Clardy and Walsh groups in 2007. 2 According to the literature studies, bacillaenes are considered as antibiotic weapons for Bacillus to resist other environmental microbes. 1,5−7 However, from the perspective of evolution, it does not seem to be enough because the bacillaene-producing Bacillus strains encode a variety of structurally stable antibiotics. ...
Bacillaenes are a class of poly-unsaturated enamines produced by Bacillus strains that are notoriously unstable toward light, oxygen, and normal temperature. Herein, in an in-depth study of this highly unstable chemotype, the stability and biological function of bacillaenes were investigated. The structure change of the bacillaene scaffold was tracked by time-course ¹H NMR data analysis coupled with the differential analysis of 2D-NMR spectra method, which was demonstrated to be a “domino” effect triggered by 4′,5′-cis (2 and 3) configuration rearranged to trans (2a and 3a). These findings provide the possibility for stabilizing the bacillaene scaffold by chemical modification of its trigger points. In the biofilm assay, compounds 1 and 2 accelerated self-biofilm formation in Bacillus methylotrophicus B-9987 at low concentrations of 1.0 and 0.1 μg/mL. Interestingly, bacillaenes play dual roles as antibiotic and biofilm enhancers in a dose-dependent manner, both of which serve in the self-protection of Bacillus.
Bacterial multimodular polyketide synthases (PKSs) are giant enzymes that generate a wide range of therapeutically important but synthetically challenging natural products. Diversification of polyketide structures can be achieved by engineering these enzymes. However, notwithstanding successes made with textbook cis -acyltransferase ( cis -AT) PKSs, tailoring such large assembly lines remains challenging. Unlike textbook PKSs, trans -AT PKSs feature an extraordinary diversity of PKS modules and commonly evolve to form hybrid PKSs. In this study, we analyzed amino acid coevolution to identify a common module site that yields functional PKSs. We used this site to insert and delete diverse PKS parts and create 22 engineered trans -AT PKSs from various pathways and in two bacterial producers. The high success rates of our engineering approach highlight the broader applicability to generate complex designer polyketides.
Bacillus halotolerans F29-3, a Gram-positive bacterium, is recognized for its synthesis of the antifungal substance fengycin. This announcement introduces the complete genome sequence and provides insights into the genetic products related to antibiotic secondary metabolites, including non-ribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and NRPS/PKS combination.
Peptide natural products have a wide range of useful applications as pesticides, veterinary agents, pharmaceuticals, and bioproducts. To discover new natural products, manipulate them for analog generation, and to harness the potential of these bioactive compounds for synthetic biology, it is necessary to develop robust methods for the expression of biosynthetic genes. Cell-free synthetic biology is emerging as an important complementary approach because it is highly desirable to express protein on a more rapid timescale and does not rely upon the genetic tractability of a strain thus improving the throughput of design-build-test-learn cycles. Additionally, generating metabolites outside the cell can overcome issues such as cellular toxicity which can hamper applications like antibiotic development. In this review, we focus on the cell-free production of peptide natural products generated by non-ribosomal peptide synthetase. Nonribsomal peptides are biosynthesized by non-ribosomal peptide synthetases which are large “mega” enzymes that provide specific challenges to heterologous expression. First, we summarize NRPSs and their corresponding peptide metabolites that are expressed in cell-free systems. With that, we discuss the requirements and challenges to express such large proteins in cell-free protein synthesis as well as host machineries that have been developed for cell-free protein synthesis that could be particularly relevant to generating non-ribosomal peptide metabolites in the future. The development of cell-free systems can then be used for prototyping to accelerate efforts towards engineered biosynthesis of these complex pathways.
Covering: 2000 to 2023The kingdom Fungi has become a remarkably valuable source of structurally complex natural products (NPs) with diverse bioactivities. Since the revolutionary discovery and application of the antibiotic penicillin from Penicillium, a number of fungi-derived NPs have been developed and approved into pharmaceuticals and pesticide agents using traditional "activity-guided" approaches. Although emerging genome mining algorithms and surrogate expression hosts have brought revolutionary approaches to NP discovery, the time and costs involved in developing these into new drugs can still be prohibitively high. Therefore, it is essential to maximize the utility of existing drugs by rational design and systematic production of new chemical structures based on these drugs by synthetic biology. To this purpose, there have been great advances in characterizing the diversified biosynthetic gene clusters associated with the well-known drugs and in understanding the biosynthesis logic mechanisms and enzymatic transformation processes involved in their production. We describe advances made in the heterogeneous reconstruction of complex NP scaffolds using fungal polyketide synthases (PKSs), non-ribosomal peptide synthetases (NRPSs), PKS/NRPS hybrids, terpenoids, and indole alkaloids and also discuss mechanistic insights into metabolic engineering, pathway reprogramming, and cell factory development. Moreover, we suggest pathways for expanding access to the fungal chemical repertoire by biosynthesis of representative family members via common platform intermediates and through the rational manipulation of natural biosynthetic machineries for drug discovery.
Bacillus velezensis strain GB03 is a Gram-positive rhizosphere bacterium known for its ability to promote plant growth and immunity. This review provides a comprehensive overview of the research on GB03 from its initial discovery in Australian wheat fields in 1971 to its current applications. Recognized as a model plant growth-promoting rhizobacterium (PGPR), GB03 has exhibited outstanding performance in enhancing the growth and protection of many crop plants including cucumber, pepper, wheat, barley, soybean, and cotton. Notably, GB03 has been reported to elicit plant immune response, referred to as induced systemic resistance (ISR), against above-ground pathogens and insect pests. Moreover, a pivotal finding in GB03 was the first-ever identification of its bacterial volatile compounds, which are known to boost plant growth and activate ISR. Research conducted over the past five decades has clearly demonstrated the potential of GB03 as an eco-friendly substitute for conventional pesticides and fertilizers. Validating its safety, the U.S. Environmental Protection Agency endorsed GB03 for commercial use as Kodiak ® in 1998. Subsequently, other compounds, such as BioYield™, were released as a biological control agent against soil-borne pathogens and as a biofertilizer, utilizing a durable spore formulation. More recently, GB03 has been utilized as a keystone modulator for engineering the rhizosphere microbiome and for eliciting microbe-induced plant volatiles. These extensive studies on GB03 underscore its significant role in sustainable agriculture, positioning it as a safe and environmentally-friendly solution for crop protection.
The emergence of drug‐resistant pathogens necessitates the development of new countermeasures. In this regard, the introduction of probiotics to directly attack or competitively exclude pathogens presents a useful strategy. Application of this approach requires an understanding of how a probiotic and its target pathogen interact. A key means of probiotic‐pathogen interaction involves the production of small molecules called natural products (NPs). Here, we report the use of whole‐cell matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐ToF) mass spectrometry to characterize NP production by candidate probiotics (mouse airway microbiome isolates) when co‐cultured with the respiratory pathogen Burkholderia. We found that a Bacillus velezensis strain inhibits growth of and elicits NP production by Burkholderia thailandensis. Dereplication of known NPs detected in the metabolome of this B. velezensis strain suggests that a previously unannotated bioactive compound is involved. Thus, we present the use of whole‐cell MALDI as a broadly applicable method for screening the NP composition of microbial co‐cultures; this can be combined with other ‐omics methods to characterize probiotic‐pathogen and other microbe‐microbe interactions.
The genome of an antibiotic-producing bacterium, Bacillus velezensis H208, was sequenced. Strain H208 was isolated from ginger rhizosphere in Laifeng County, China. The genome consisted of 3,929,792 bp, with a GC content of 46.5%, and contained 3,773 protein-coding genes and 118 noncoding RNA genes.
The soil microbiome is involved in the processes of microbial corrosion, in particular, by the formation of biofilm. It has been proposed that an environmentally friendly solution to this corrosion might be through biological control. Bacillus velezensis NUChC C2b, Streptomyces gardneri
ChNPU F3 and S. canus NUChC F2 were investigated as potentially ‘green’ biocides to prevent
attachment to glass as a model surface and the formation of heterotrophic bacterial biofilm
which participates in the corrosion process. Results showed high antagonistic and antibiofilm
properties of S. gardneri ChNPU F3; which may be related to the formation of secondary antimicrobial metabolites by this strain. B. velezensis NUChC C2b and S. gardneri ChNPU F3 could be
incorporated into green biocides – as components of antibiofilm agents that will protect material from bacterial corrosion or as agents that will prevent historical heritage damage.
https://www.tandfonline.com/eprint/BFJVTVEGND4JJJ5FCFTX/full?target=10.1080/08927014.2022.2151362
Entomopathogenic microorganisms have potential for biological control of insect pests. Their main secondary metabolites include polyketides, nonribosomal peptides, and polyketide-nonribosomal peptide (PK-NRP) hybrids. Among these secondary metabolites, polyketides have mainly been studied for structural identification, pathway engineering, and for their contributions to medicine. However, little is known about the function of polyketides in insect virulence. This review focuses on the role of bacterial and fungal polyketides, as well as PK-NRP hybrids in the insect infection and killing. We also discuss gene distribution and evolutional relationships among different microbial species. Further, the role of microbial polyketides and the hybrids in modulating insect-microbial symbiosis is also explored. Understanding the mechanisms of polyketides in insect pathogenesis, how compounds moderate the host-fungus interaction, and the distribution of PKS genes across different fungi and bacteria will facilitate the discovery and development of novel polyketide-derived bio-insecticides.
This study is the primary initiative to identify Bacillus velezensis HNA3 whole genome sequence and reveal its genomic properties as an effective biocontrol agent against plant pathogens and a plant growth stimulator. HNA3 genetic profile can be used as a reference for future studies that can be applied as a highly effective biofertilizer and bio fungicide inoculum to improve agriculture productivity.
The agricultural industry utilizes antibiotic growth promoters to promote livestock growth and health. However, the World Health Organization has raised concerns over the ongoing spread of antibiotic resistance transmission in the populace, leading to its subsequent ban in several countries, especially in the European Union. These restrictions have translated into an increase in pathogenic outbreaks in the agricultural industry, highlighting the need for an economically viable, non-toxic, and renewable alternative to antibiotics in livestock. Probiotics inhibit pathogen growth, promote a beneficial microbiota, regulate the immune response of its host, enhance feed conversion to nutrients, and form biofilms that block further infection. Commonly used lactic acid bacteria probiotics are vulnerable to the harsh conditions of the upper gastrointestinal system, leading to novel research using spore-forming bacteria from the genus Bacillus. However, the exact mechanisms behind Bacillus probiotics remain unexplored. This review tackles this issue, by reporting antimicrobial compounds produced from Bacillus strains, their proposed mechanisms of action, and any gaps in the mechanism studies of these compounds. Lastly, this paper explores omics approaches to clarify the mechanisms behind Bacillus probiotics.
Background
Biocontrol agents are sustainable eco-friendly alternatives for chemical pesticides that cause adverse effects in the environment and toxicity in animals including humans. An improved understanding of the phyllosphere microbiology is of vital importance for biocontrol development. Most studies have been directed towards beneficial plant-microbe interactions and ignore the pathogens that might affect humans when consuming vegetables. In this study we extended this perspective and investigated potential biocontrol strains isolated from the tomato and lettuce phyllosphere that can promote plant growth and antagonize mammalian pathogens as well as plant pathogens. Subsequently, we mined into their genomes for discovery of antimicrobial biosynthetic gene clusters (BGCs), several of which are good candidates to produce protectants against microbial plant and mammalian pathogens.
Results
The antimicrobial activity of 69 newly isolated strains from a healthy tomato and lettuce phyllosphere against several plant and mammalian pathogens was determined with plates assays. Three strains with the highest antimicrobial activity against the relevant pathogens were selected and characterized (Bacillus subtilis STRP31, Bacillus velezensis SPL51, and Paenibacillus sp. PL91). All three strains showed a plant growth promotion effect by the production of volatile compounds (VOCs) on tomato and lettuce. In addition, genome mining of these isolates showed the presence of a large variety of biosynthetic gene clusters. A total of 39 BGCs were identified, of which several are already known, such as bacilysin, bacillibactin, surfactin, subtilomycin, etc., but also several novel ones. Further analysis revealed that among the novel BGCs, one NRPS and two bacteriocins are encoded which were analyzed in more depth.
Conclusions
Several antimicrobial BGCs were found in the selected strains, including the rediscovery of known ones, but also the discovery of novel ones. Our study serves as support for subsequent examination and characterization of novel antimicrobial metabolites, and the possibility of developing biocontrol agents.
Covering: up to 2020
Polyketides form a large group of bioactive secondary metabolites that usually contain one or more double bonds. Although most of the double bonds found in polyketides are trans or E-configured, several cases are known in which cis or Z-configurations are observed. Double bond formation by polyketide synthases (PKSs) is widely recognised to be catalysed by ketoreduction and subsequent dehydration of the acyl carrier protein (ACP)-tethered 3-ketoacyl intermediate in the PKS biosynthetic assembly line with a specific stereochemical course in which the ketoreduction step determines the usual trans or more rare cis double bond configuration. Occasionally, other mechanisms for the installation of cis double bonds such as double bond formation during chain release or post-PKS modifications including, amongst others, isomerisations or double bond installations by oxidation are observed. This review discusses the peculiar mechanisms of cis double bond formation in polyketide biosynthesis.
Myxococcus xanthus cells coordinate cellular motility, biofilm formation, and development through the use of cell signaling pathways. In an effort to understand the mechanisms underlying these processes, the inner membrane (IM) and outer membrane (OM) of strain DK1622 were fractionated to examine protein localization. Membranes were enriched from spheroplasts of vegetative cells and then separated into three peaks on a three-step sucrose gradient. The high-density fraction corresponded to the putative IM, the medium-density fraction corresponded to a putative hybrid membrane (HM), and the low-density fraction corresponded to the putative OM. Each fraction was subjected to further separation on discontinuous sucrose gradients, which resulted in discrete protein peaks for each major fraction. The purity and origin of each peak were assessed by using succinate dehydrogenase (SDH) activity as the IM marker and reactivities to lipopolysaccharide core and O-antigen monoclonal antibodies as the OM markers. As previously reported, the OM markers localized to the low-density membrane fractions, while SDH localized to high-density fractions. Immunoblotting was used to localize important motility and signaling proteins within the protein peaks. CsgA, the C-signal-producing protein, and FibA, a fibril-associated protease, were localized in the IM (density, 1.17 to 1.24 g cm(-3)). Tgl and Cgl lipoproteins were localized in the OM, which contained areas of high buoyant density (1.21 to 1.24 g cm(-3)) and low buoyant density (1.169 to 1.171 g cm(-3)). FrzCD, a methyl-accepting chemotaxis protein, was predominantly located in the IM, although smaller amounts were found in the OM. The HM peaks showed twofold enrichment for the type IV pilin protein PilA, suggesting that this fraction contained cell poles. Two-dimensional polyacrylamide gel electrophoresis revealed the presence of proteins that were unique to the IM and OM. Characterization of proteins in an unusually low-density membrane peak (1.072 to 1.094 g cm(-3)) showed the presence of Ta-1 polyketide synthetase, which synthesizes the antibiotic myxovirescin A.
Specimen preparation methods based on high-pressure freezing and freeze-substitution have enabled significant advances in the quality of morphological preservation of biological samples for electron microscopy. However, visualization of a subset of cellular membranes, particularly the endoplasmic reticulum and cis Golgi, is often impaired by a lack of contrast. By contrast, some efforts to increase membrane staining may lead to excessively granular staining. No one freeze-substitution method has emerged that both overcomes these limitations and is suitable for all types of analysis. However, one or more of the following protocols, perhaps with minor modifications, should yield satisfactory results in most cases. Freeze-substitution in glutaraldehyde and uranyl acetate in acetone, followed by embedding in Lowicryl HM20, generates samples suitable for both immunolocalization and high-resolution structural studies. Membranes are typically lightly stained but very well defined. Initial freeze-substitution in tannic acid and glutaraldehyde in acetone prior to exposure to osmium tetroxide significantly enhanced contrast on mammalian cellular membranes. Finally, initial trials indicate that freeze-substitution in potassium permanganate in acetone can provide strong contrast on endoplasmic reticulum and Golgi as well as other membranes. The tendency of permanganate to degrade cytoskeletal elements and other proteins when employed in aqueous solutions at room temperature is apparently curtailed when it is used as a freeze-substitution reagent.
A screening program for bioactive compounds from marine cyanobacteria led to the isolation of jamaicamides A-C. Jamaicamide A is a novel and highly functionalized lipopeptide containing an alkynyl bromide, vinyl chloride, beta-methoxy eneone system, and pyrrolinone ring. The jamaicamides show sodium channelblocking activity and fish toxicity. Precursor feeding to jamaicamide-producing cultures mapped out the series of acetate and amino acid residues and helped develop an effective cloning strategy for the biosynthetic gene cluster. The 58 kbp gene cluster is composed of 17 open reading frames that show an exact colinearity with their expected utilization. A novel cassette of genes appears to form a pendent carbon atom possessing the vinyl chloride functionality; at its core this contains an HMG-CoA synthase-like motif, giving insight into the mechanism by which this functional group is created.
Analysis of the genome of Streptomyces aizunensis NRRL B-11277 indicated its potential to produce a compound of novel and highly predictable structure. The structure was predicted with sufficient accuracy to allow straightforward detection of the specific metabolite in HPLC profiles of fermentation extracts and hence to guide the isolation. The spectroscopic work was reduced to a confirmation of structure rather than a first principle determination. The compound, ECO-02301 (1), demonstrated potent antifungal activity. This work exemplifies not only the discovery of novel antibiotics from well-characterized organisms but also the utility of genomics as a further tool, complementary to spectroscopy, to enable rapid determination of complex structures.
Although bacterial polyketides are of considerable biomedical interest, the molecular biology of polyketide biosynthesis in
Bacillus spp., one of the richest bacterial sources of bioactive natural products, remains largely unexplored. Here we assign for
the first time complete polyketide synthase (PKS) gene clusters to Bacillus antibiotics. Three giant modular PKS systems of the trans-acyltransferase type were identified in Bacillus amyloliquefaciens FZB 42. One of them, pks1, is an ortholog of the pksX operon with a previously unknown function in the sequenced model strain Bacillus subtilis 168, while the pks2 and pks3 clusters are novel gene clusters. Cassette mutagenesis combined with advanced mass spectrometric techniques such as matrix-assisted
laser desorption ionization-time of flight mass spectrometry and liquid chromatography-electrospray ionization mass spectrometry
revealed that the pks1 (bae) and pks3 (dif) gene clusters encode the biosynthesis of the polyene antibiotics bacillaene and difficidin or oxydifficidin, respectively.
In addition, B. subtilis OKB105 (pheA sfp0), a transformant of the B. subtilis 168 derivative JH642, was shown to produce bacillaene, demonstrating that the pksX gene cluster directs the synthesis of that polyketide.
A 3D look at 2D spectra: Two previously unreported indole alkaloids could be rapidly identified from a library of unfractionated fungal extracts by using a newly developed protocol for the differential analysis of arrays of 2D NMR spectra (see picture). The technique thus represents an effective tool for the non-discriminatory characterization of secondary-metabolite mixtures. (Figure Presented).
Nonribosomal peptide synthetases are large enzyme complexes that synthesize a variety of peptide natural products through a thiotemplated mechanism. Assembly of the peptides proceeds through amino acid loading, amide-bond formation and chain translocation, and finally thioester lysis to release the product. The final products are often heavily modified, however, through methylation, epimerization, hydroxylation, heterocyclization, oxidative cross-linking and attachment of sugars. These activities are the province of specialized enzymes (either embedded in the multidomain nonribosomal peptide synthetase structure or standalone).
Bacillaene, a novel polyene antibiotic, was discovered and isolated from fermentation broths of a strain of Bacillus subtilis. The novel antibiotic has a nominal molecular weight of 580 and an empirical formula of C35H48O7. Bacillaene is active against a broad spectrum of bacteria in agar-plate diffusion assays. Studies in vitro indicate that the antibiotic inhibits prokaryotic protein synthesis but not eukaryotic protein synthesis. Cell survival studies performed with strains of Escherichia coli indicate that the antibiotic is a bacteriostatic agent.
A putative catalytic triad consisting of tyrosine, serine, and lysine residues was identified in the ketoreductase (KR) domains of modular polyketide synthases (PKSs) based on homology modeling to the short chain dehydrogenase/reductase (SDR) superfamily of enzymes. This was tested by constructing point mutations for each of these three amino acid residues in the KR domain of module 6 of the 6-deoxyerythronolide B synthase (DEBS) and determining the effect on ketoreduction. Experiments conducted in vitro with the truncated DEBS Module 6+TE (M6+TE) enzyme purified from Escherichia coli indicated that any of three mutations, Tyr --> Phe, Ser --> Ala, and Lys --> Glu, abolish KR activity in formation of the triketide lactone product from a diketide substrate. The same mutations were also introduced in module 6 of the full DEBS gene set and expressed in Streptomyces lividans for in vivo analysis. In this case, the Tyr --> Phe mutation appeared to completely eliminate KR6 activity, leading to the 3-keto derivative of 6-deoxyerythronolide B, whereas the other two mutations, Ser --> Ala and Lys --> Glu, result in a mixture of both reduced and unreduced compounds at the C-3 position. The results support a model analogous to SDRs in which the conserved tyrosine serves as a proton donating catalytic residue. In contrast to deletion of the entire KR6 domain of DEBS, which causes a loss in substrate specificity of the adjacent acyltransferase (AT) domain in module 6, these mutations do not affect the AT6 specificity and offer a potentially superior approach to KR inactivation for engineered biosynthesis of novel polyketides. The homology modeling studies also led to identification of amino acid residues predictive of the stereochemical nature of KR domains. Finally, a method is described for the rapid purification of engineered PKS modules that consists of a biotin recognition sequence C-terminal to the thioesterase domain and adsorption of the biotinylated module from crude extracts to immobilized streptavidin. Immobilized M6+TE obtained by this method was over 95% pure and as catalytically effective as M6+TE in solution.
Getting down to specifics: Key amino acid residues were found to correlate with ketoreductase domain stereospecificity in modular polyketide synthases. These residues may allow alcohol stereochemistry (see scheme; ACP, acyl carrier protein) in polyketides to be pridicted from ketoreductase sequences. The results also suggest that polyketide synthse dehydratase domains have a preference for 3-hydroxyacyl substrates with the same alcohol stereochemistry as the (3R)-hydroxyacyl chains used by dehydrates in fatty acid synthases.
Curacin A (1) is a potent cancer cell toxin obtained from strains of the tropical marine cyanobacterium Lyngbya majuscula found in Curaçao. Its structure is unique in that it contains the sequential positioning of a thiazoline and cyclopropyl ring, and it exerts its potent cell toxicity through interaction with the colchicine drug binding site on microtubules. A series of stable isotope-labeled precursors were fed to cultures of curacin A-producing strains and, following NMR analysis, allowed determination of the metabolic origin of all atoms in the natural product (one cysteine, 10 acetate units, two S-adenosyl methionine-derived methyl groups) as well as several unique mechanistic insights. Moreover, these incorporation experiments facilitated an effective gene cloning strategy that allowed identification and sequencing of the approximately 64 kb putative curacin A gene cluster. The metabolic system is comprised of a nonribosomal peptide synthetase (NRPS) and multiple polyketide synthases (PKSs) and shows a very high level of collinearity between genes in the cluster and the predicted biochemical steps required for curacin biosynthesis. Unique features of the cluster include (1) all but one of the PKSs are monomodular multifunctional proteins, (2) a unique gene cassette that contains an HMG-CoA synthase likely responsible for formation of the cyclopropyl ring, and (3) a terminating motif that is predicted to function in both product release and terminal dehydrative decarboxylation.
The endospore-forming rhizobacterium Bacillus subtilis- the model system for Gram-positive organisms, is able to produce more than two dozen antibiotics with an amazing variety of structures. The produced anti-microbial active compounds include predominantly peptides that are either ribosomally synthesized and post-translationally modified (lantibiotics and lantibiotic-like peptides) or non-ribosomally generated, as well as a couple of non-peptidic compounds such as polyketides, an aminosugar, and a phospholipid. Here I summarize the structures of all known B. subtilis antibiotics, their biochemistry and genetic analysis of their biosyntheses. An updated summary of well-studied antibiotic regulation pathways is given. Furthermore, current findings are resumed that show roles for distinct B. subtilis antibiotics beyond the "pure" anti-microbial action: Non-ribosomally produced lipopeptides are involved in biofilm and swarming development, lantibiotics function as pheromones in quorum-sensing, and a "killing factor" effectuates programmed cell death in sister cells. A discussion of how these antibiotics may contribute to the survival of B. subtilis in its natural environment is given.
Marine organisms are a rich source of secondary metabolites. They have yielded thousands of compounds with a broad range of biomedical applications. Thus far, samples required for preclinical and clinical studies have been obtained by collection from the wild, by mariculture, and by total chemical synthesis. However, for a number of complex marine metabolites, none of these options is feasible for either economic or environmental reasons. In order to proceed with the development of many of these promising therapeutic compounds, a reliable and renewable source must be found. Over the last twenty years, the study of microbial secondary metabolites has greatly advanced our understanding of how nature utilizes simple starting materials to yield complex small molecules. Much of this work has focused on polyketides and nonribosomal peptides, two classes of molecules that are prevalent in marine micro- and macroorganisms. The lessons learned from the study of terrestrial metabolite biosynthesis are now being applied to the marine world. As techniques for cloning and heterologous expression of biosynthetic pathways continue to improve, they may provide our greatest hope for bridging the gap between the promise and application of many marine natural products.
Disorazoles are polyketides produced by the myxobacterium Sorangium cellulosum So ce12. Their mode of action is to inhibit tubulin polymerization and destabilize microtubules. Using transposon mutagenesis, two mutant strains were identified that produced no disorazoles. Sequencing the DNA flanking the insertions revealed a polyketide synthase gene cluster that would encode three polypeptides, DszA, DszB, and DszC, with DszC containing both nonribosomal peptide synthetase and polyketide synthase modules. The disorazole polyketide synthase modules lack an acyltransferase domain. Instead, a separate gene, dszD, encodes an AT protein, thus revealing that the disorazole gene cluster falls into the trans-AT Type I family of PKS enzymes.
Microorganisms produce an immense variety of natural products with useful biological activities. These compounds are often biosynthesized by multifunctional megasynthetases known as polyketide synthases and nonribosomal peptide synthetases. Recent literature on these natural product assembly lines suggests that they have a much greater mechanistic diversity than originally anticipated.
Analyses of microbial genome sequences reveal numerous examples of gene clusters encoding proteins typically involved in complex natural product biosynthesis but not associated with the production of known natural products. In Streptomyces coelicolor M145 there are several gene clusters encoding new nonribosomal peptide synthetase (NRPS) systems not associated with known metabolites. Application of structure-based models for substrate recognition by NRPS adenylation domains predicts the amino acids incorporated into the putative peptide products of these systems, but the accuracy of these predictions is untested. Here we report the isolation and structure determination of the new tris-hydroxamate tetrapeptide iron chelator coelichelin from S. coelicolor using a genome mining approach guided by substrate predictions for the trimodular NRPS CchH, and we show that this enzyme, which lacks a C-terminal thioesterase domain, together with a homolog of enterobactin esterase (CchJ), are required for coelichelin biosynthesis. These results demonstrate that accurate prediction of adenylation domain substrate selectivity is possible and raise intriguing mechanistic questions regarding the assembly of a tetrapeptide by a trimodular NRPS.
For screening a pool of potential substrates that load carrier domains found in nonribosomal peptide synthetases, large molecule mass spectrometry is shown to be a new, unbiased assay. Combining the high resolving power of Fourier transform mass spectrometry with the ability of adenylation domains to select their own substrates, the mass change that takes place upon formation of a covalent intermediate thus identifies the substrate. This assay has an advantage over traditional radiochemical assays in that many substrates, the substrate pool, can be screened simultaneously. Using proteins on the nikkomycin, clorobiocin, coumermycin A1, yersiniabactin, pyochelin, and enterobactin biosynthetic pathways as proof of principle, preferred substrates are readily identified from substrate pools. Furthermore, this assay can be used to provide insight into the timing of tailoring events of biosynthetic pathways as demonstrated using the bromination reaction found on the jamaicamide biosynthetic pathway. Finally, this assay can provide insight into the role and function of orphan gene clusters for which the encoded natural product is unknown. This is demonstrated by identifying the substrates for two NRPS modules from the pksN and pksJ genes that are found on an orphan NRPS/PKS hybrid cluster from Bacillus subtilis. This new assay format is especially timely for activity screening in an era when new types of thiotemplate assembly lines that defy classification are being discovered at an accelerating rate.
The pksX gene cluster from Bacillus subtilis is predicted to encode the biosynthesis of an as yet uncharacterized hybrid nonribosomal peptide/polyketide secondary metabolite. We used a combination of biochemical and mass spectrometric techniques to assign functional roles to the proteins AcpK, PksC, PksL, PksF, PksG, PksH, and PksI, and we conclude that they act to incorporate an acetate-derived β-methyl branch on an acetoacetyl-S-carrier protein and ultimately generate a Δ²-isoprenyl-S-carrier protein. This work highlights the power of mass spectrometry to elucidate the functions of orphan biosynthetic enzymes, and it details a mechanism by which single-carbon β-branches can be inserted into polyketide-like structures. This pathway represents a noncanonical route to the construction of prenyl units and serves as a prototype for the intersection of isoprenoid and polyketide biosynthetic manifolds in other natural product biosynthetic pathways.
• mass spectrometry
• orphan gene cluster
• hybrid nonribosomal peptide/polyketide
• polyketide methylation
This study describes the functional identification of a pair of mechanistically diverse enzymes that catalyze the successive dehydration (CurE ECH1) and decarboxylation (CurF ECH2) of (S)-HMG-ACP to generate a 3-methylcrotonyl-ACP intermediate, the presumed precursor of the cyclopropyl ring in curacin A. The reactions catalyzed by ECH1 and ECH2 are found in a broad cross-section of microbial natural product gene clusters and participate in the introduction of carbon chain branch points and functional group diversity as key steps in the HMG-CoA synthase mediated addition of C-2 from acetate to the beta-carbonyl group of polyketide chains.
Evidence suggests that polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes are organized in clusters. This basic knowledge of PKS and NRPS systems provides context for more efficient efforts in combinatorial biosynthesis to create collections of natural product variants with novel structure and function. In view of this information, an overview is given of the logic of assembly-line enzymology, enzyme machinery, assembly-line operations and mechanisms of PKS and NRPS assembly.
Several nonribosomal peptide natural products are composites of alpha-hydroxy acid and alpha-amino acid monomers. Cereulide, the emetic toxin from the human pathogen Bacillus cereus, and valinomycin, from Streptomyces spp., are closely related macrocyclic K+ ionophores. The macrocyclic core of each natural product contains alternating peptide (six) and ester (six) bonds, and their cyclododecadepsipeptide structures consist of a tetradepsipeptide unit repeated three times. Here we overexpress the cereulide NRPS alpha-hydroxy acid specifying modules from CesA and CesB and demonstrate that each contains an alpha-keto acid activating adenylation domain and a chiral alpha-ketoacyl-S-carrier protein reductase (alpha-KR). The logic used by the cereulide NRPS is likely at work in the valinomycin NRPS and may be the general strategy used in bacterial NRPSs to form alpha-hydroxy acid containing natural products.
Nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and hybrid NRPS/PKS are of particular interest, because they produce numerous therapeutic agents, have great potential for engineering novel compounds, and are the largest enzymes known. The predicted masses of known enzymatic assembly lines can reach almost 5 megadaltons, dwarfing even the ribosome (≈2.6 megadaltons). Despite their uniqueness and importance, little is known about the organization of these enzymes within the native producer cells. Here we report that an 80-kb gene cluster, which occupies ≈2% of the Bacillus subtilis genome, encodes the subunits of ≈2.5 megadalton active hybrid NRPS/PKS. Many copies of the NRPS/PKS assemble into a single organelle-like membrane-associated complex of tens to hundreds of megadaltons. Such an enzymatic megacomplex is unprecedented in bacterial subcellular organization and has important implications for engineering novel NRPS/PKSs.
• bacillaene
• nonribosomal peptide
• polyketide
• Streptomyces
- Z Chang
- N Sitachitta
- Jv Rossi
- Ma Roberts
- Pm Flatt
- J Jia
- Dh Sherman
- Wh Gerwick
Chang Z, Sitachitta N, Rossi JV, Roberts MA, Flatt PM, Jia J, Sherman DH,
Gerwick WH (2004) J Nat Prod 67:1356–1367.
- Pd Straight
- Ma Fischbach
- Ct Walsh
- Dz Rudner
- R Kolter
Straight PD, Fischbach MA, Walsh CT, Rudner DZ, Kolter R (2007) Proc Natl
Acad Sci USA 104:305–310.
- L Gu
- J Jia
- H Liu
- K Hakansson
- Wh Gerwick
- Dh Sherman
Gu L, Jia J, Liu H, Hakansson K, Gerwick WH, Sherman DH (2006) J Am
Chem Soc 128:9014–9015.
- Pc Dorrestein
- J Blackhall
- Pd Straight
- Ma Fischbach
- S Garneau-Tsodikova
- Dj Edwards
- S Mclaughlin
- M Lin
- Wh Gerwick
- R Kolter
Dorrestein PC, Blackhall J, Straight PD, Fischbach MA, Garneau-Tsodikova
S, Edwards DJ, McLaughlin S, Lin M, Gerwick WH, Kolter R, et al. (2006)
Biochemistry 45:1537–1546.
- Jl Fortman
- Dh Sherman
Fortman JL, Sherman DH (2005) ChemBioChem 6:960–978.
- P Caffrey
Caffrey P (2003) ChemBioChem 4:654–657.
- R Carvalho
- R Reid
- N Viswanathan
- H Gramajo
- B Julien
Carvalho R, Reid R, Viswanathan N, Gramajo H, Julien B (2005) Gene
359:91–98.
- Ct Calderone
- We Kowtoniuk
- Nl Kelleher
- Ct Walsh
- Pc Dorrestein
Calderone CT, Kowtoniuk WE, Kelleher NL, Walsh CT, Dorrestein PC (2006)
Proc Natl Acad Sci USA 103:8977–8982.
- Ct Walsh
- H Chen
- Ta Keating
- Bk Hubbard
- Hc Losey
- L Luo
- Cg Marshall
- Da Miller
- Hm Patel
Walsh CT, Chen H, Keating TA, Hubbard BK, Losey HC, Luo L, Marshall CG,
Miller DA, Patel HM (2001) Curr Opin Chem Biol 5:525–534.
- Xh Chen
- J Vater
- J Piel
- P Franke
- R Scholz
- K Schneider
- A Koumoutsi
- G Hitzeroth
- N Grammel
- Aw Strittmatter
Chen XH, Vater J, Piel J, Franke P, Scholz R, Schneider K, Koumoutsi A,
Hitzeroth G, Grammel N, Strittmatter AW, et al. (2006) J Bacteriol 188:4024–4036.
- Jb Mcalpine
- Bo Bachmann
- M Piraee
- S Tremblay
- Am Alarco
- E Zazopoulos
- Cm Farnet
McAlpine JB, Bachmann BO, Piraee M, Tremblay S, Alarco AM, Zazopoulos
E, Farnet CM (2005) J Nat Prod 68:493–496.
- Ma Fischbach
- Ct Walsh
Fischbach MA, Walsh CT (2006) Chem Rev 106:3468–3496.
- Sc Wenzel
- R Muller
Wenzel SC, Muller R (2005) Curr Opin Chem Biol 9:447–458.
- S Lautru
- Rj Deeth
- Lm Bailey
- Gl Challis
Lautru S, Deeth RJ, Bailey LM, Challis GL (2005) Nat Chem Biol 1:265–269.
- Ps Patel
- S Huang
- S Fisher
- D Pirnik
- C Aklonis
- L Dean
- E Meyers
- P Fernandes
- F Mayerl
Patel PS, Huang S, Fisher S, Pirnik D, Aklonis C, Dean L, Meyers E, Fernandes
P, Mayerl F (1995) J Antibiot (Tokyo) 48:997–1003.
- Dj Edwards
- Bl Marquez
- Lm Nogle
- K Mcphail
- De Goeger
- Ma Roberts
- Wh Gerwick
Edwards DJ, Marquez BL, Nogle LM, McPhail K, Goeger DE, Roberts MA,
Gerwick WH (2004) Chem Biol 11:817–833.
- Na Magarvey
- M Ehling-Schulz
- Ct Walsh
Magarvey NA, Ehling-Schulz M, Walsh CT (2006) J Am Chem Soc 128:10698–
10699.
- R Reid
- M Piagentini
- E Rodriguez
- G Ashley
- N Viswanathan
- J Carney
- Dv Santi
- Cr Hutchinson
- R Mcdaniel
Reid R, Piagentini M, Rodriguez E, Ashley G, Viswanathan N, Carney J, Santi
DV, Hutchinson CR, McDaniel R (2003) Biochemistry 42:72–79.