Investigation of the potential of Brevibacillus spp. for the biosynthesis of nonribosomally produced bioactive compounds by combination of genome mining with MALDI-TOF mass spectrometry.

The biosynthetic potential of 11 Brevibacillus spp. strains was investigated by combination of genome mining with mass spectrometric analysis using MALDI-TOF mass spectrometry. These endophytic, plant associated Brevibacillus strains were isolated from crop plants, such as coffee and black pepper, in Vietnam. Draft genomes of these strains were available. They were classified (a) by comparison with type strains and a collection of genome-sequenced Brevibacillus spp. deposited in the NCBI data base as well as (b) by construction of a phylogenetic tree from the core sequences of publicly available genomes of Brevibacillus strains. They were identified as Brevibacillus brevis (1 strain); parabrevis (2 strains); porteri (3 strains); and 5 novel Brevibacillus genomospecies. Our work was specifically focused on the detection and characterization of nonribosomal peptides produced by these strains. Structural characterization of these compounds was performed by LIFT-MALDI-TOF/TOF mass spectrometric sequence analysis. The highlights of our work were the demonstration of the tyrocidines, a well-known family of cyclodecapeptides of great structural variability, as the main products of all investigated strains and the identification of a novel class of pentapeptides produced by B. brevis; B. schisleri; and B. porteri which we designate as brevipentins. Our biosynthetic studies demonstrate that knowledge of their biosynthetic capacity can efficiently assist classification of Brevibacillus species.

Plant-Associated Representatives of the Bacillus cereus Group Are a Rich Source of Antimicrobial Compounds.

Seventeen bacterial strains able to suppress plant pathogens have been isolated from healthy Vietnamese crop plants and taxonomically assigned as members of the Bacillus cereus group. In order to prove their potential as biocontrol agents, we perform a comprehensive analysis that included the whole-genome sequencing of selected strains and the mining for genes and gene clusters involved in the synthesis of endo- and exotoxins and secondary metabolites, such as antimicrobial peptides (AMPs). Kurstakin, thumolycin, and other AMPs were detected and characterized by different mass spectrometric methods, such as MALDI-TOF-MS and LIFT-MALDI-TOF/TOF fragment analysis. Based on their whole-genome sequences, the plant-associated isolates were assigned to the following species and subspecies: B. cereus subsp. cereus (6), B. cereus subsp. bombysepticus (5), Bacillus tropicus (2), and Bacillus pacificus. These three isolates represent novel genomospecies. Genes encoding entomopathogenic crystal and vegetative proteins were detected in B. cereus subsp. bombysepticus TK1. The in vitro assays revealed that many plant-associated isolates enhanced plant growth and suppressed plant pathogens. Our findings indicate that the plant-associated representatives of the B. cereus group are a rich source of putative antimicrobial compounds with potential in sustainable agriculture. However, the presence of virulence genes might restrict their application as biologicals in agriculture.

Two plant-associated Bacillus velezensis strains selected after genome analysis, metabolite profiling, and with proved biocontrol potential, were enhancing harvest yield of coffee and black pepper in large field trials.

Elimination of chemically synthesized pesticides, such as fungicides and nematicides, in agricultural products is a key to successful practice of the Vietnamese agriculture. We describe here the route for developing successful biostimulants based on members of the Bacillus subtilis species complex. A number of endospore-forming Gram-positive bacterial strains with antagonistic action against plant pathogens were isolated from Vietnamese crop plants. Based on their draft genome sequence, thirty of them were assigned to the Bacillus subtilis species complex. Most of them were assigned to the species Bacillus velezensis. Whole genome sequencing of strains BT2.4 and BP1.2A corroborated their close relatedness to B. velezensis FZB42, the model strain for Gram-positive plant growth-promoting bacteria. Genome mining revealed that at least 15 natural product biosynthesis gene clusters (BGCs) are well conserved in all B. velezensis strains. In total, 36 different BGCs were identified in the genomes of the strains representing B. velezensis, B. subtilis, Bacillus tequilensis, and Bacillus. altitudinis. In vitro and in vivo assays demonstrated the potential of the B. velezensis strains to enhance plant growth and to suppress phytopathogenic fungi and nematodes. Due to their promising potential to stimulate plant growth and to support plant health, the B. velezensis strains TL7 and S1 were selected as starting material for the development of novel biostimulants, and biocontrol agents efficient in protecting the important Vietnamese crop plants black pepper and coffee against phytopathogens. The results of the large-scale field trials performed in the Central Highlands in Vietnam corroborated that TL7 and S1 are efficient in stimulating plant growth and protecting plant health in large-scale applications. It was shown that treatment with both bioformulations resulted in prevention of the pathogenic pressure exerted by nematodes, fungi, and oomycetes, and increased harvest yield in coffee, and pepper.

Novel Plant-Associated Brevibacillus and Lysinibacillus Genomospecies Harbor a Rich Biosynthetic Potential of Antimicrobial Compounds.

We have previously reported the draft genome sequences of 59 endospore-forming Gram-positive bacterial strains isolated from Vietnamese crop plants due to their ability to suppress plant pathogens. Based on their draft genome sequence, eleven of them were assigned to the Brevibacillus and one to the Lysinibacillus genus. Further analysis including full genome sequencing revealed that several of these strains represent novel genomospecies. In vitro and in vivo assays demonstrated their ability to promote plant growth, as well as the strong biocontrol potential of Brevibacilli directed against phytopathogenic bacteria, fungi, and nematodes. Genome mining identified 157 natural product biosynthesis gene clusters (BGCs), including 36 novel BGCs not present in the MIBiG data bank. Our findings indicate that plant-associated Brevibacilli are a rich source of putative antimicrobial compounds and might serve as a valuable starting point for the development of novel biocontrol agents.

Fusaricidins, Polymyxins and Volatiles Produced by Paenibacillus polymyxa Strains DSM 32871 and M1.

Paenibacilli are efficient producers of potent agents against bacterial and fungal pathogens, which are of great interest both for therapeutic applications in medicine as well as in agrobiotechnology. Lipopeptides produced by such organisms play a major role in their potential to inactivate pathogens. In this work we investigated two lipopeptide complexes, the fusaricidins and the polymyxins, produced by Paenibacillus polymyxa strains DSM 32871 and M1 by MALDI-TOF mass spectrometry. The fusaricidins show potent antifungal activities and are distinguished by an unusual variability. For strain DSM 32871 we identified numerous yet unknown variants mass spectrometrically. DSM 32871 produces polymyxins of type E (colistins), while M1 forms polymyxins P. For both strains, novel but not yet completely characterized polymyxin species were detected, which possibly are glycosylated. These compounds may be of interest therapeutically, because polymyxins have gained increasing attention as last-resort antibiotics against multiresistant pathogenic Gram-negative bacteria. In addition, the volatilomes of DSM 32781 and M1 were investigated with a GC-MS approach using different cultivation media. Production of volatile organic compounds (VOCs) was strain and medium dependent. In particular, strain M1 manifested as an efficient VOC-producer that exhibited formation of 25 volatiles in total. A characteristic feature of Paenibacilli is the formation of volatile pyrazine derivatives.

Complete genome sequence and epigenetic profile of Bacillus velezensis UCMB5140 used for plant and crop protection in comparison with other plant-associated Bacillus strains.

The application of biocontrol biopesticides based on plant growth-promoting rhizobacteria (PGPR), particularly members of the genus Bacillus, is considered a promising perspective to make agricultural practices sustainable and ecologically safe. Recent advances in genome sequencing by third-generation sequencing technologies, e.g., Pacific Biosciences' Single Molecule Real-Time (PacBio SMRT) platform, have allowed researchers to gain deeper insights into the molecular and genetic mechanisms of PGPR activities, and to compare whole genome sequences and global patterns of epigenetic modifications. In the current work, this approach was used to sequence and compare four Bacillus strains that exhibited various PGPR activities including the strain UCMB5140, which is used in the commercial biopesticide Phytosubtil. Whole genome comparison and phylogenomic inference assigned the strain UCMB5140 to the species Bacillus velezensis. Strong biocontrol activities of this strain were confirmed in several bioassays. Several factors that affect the evolution of active PGPR B. velezensis strains were identified: (1) horizontal acquisition of novel non-ribosomal peptide synthetases (NRPS) and adhesion genes; (2) rearrangements of functional modules of NRPS genes leading to strain specific combinations of their encoded products; (3) gain and loss of methyltransferases that can cause global alterations in DNA methylation patterns, which eventually may affect gene expression and regulate transcription. Notably, we identified a horizontally transferred NRPS operon encoding an uncharacterized polypeptide antibiotic in B. velezensis UCMB5140. Other horizontally acquired genes comprised a possible adhesin and a methyltransferase, which may explain the strain-specific methylation pattern of the chromosomal DNA of UCMB5140. KEY POINTS: • Whole genome sequence of the active PGPR Bacillus velezensis UCMB5140. • Identification of genetic determinants responsible for PGPR activities. • Role of methyltransferases and epigenetic mechanisms in evolution of bacteria.

Profiling for Bioactive Peptides and Volatiles of Plant Growth Promoting Strains of the Bacillus subtilis Complex of Industrial Relevance.

Plant growth promoting rhizobacteria attain increasing importance in agriculture as biofertilizers and biocontrol agents. These properties significantly depend on the formation of bioactive compounds produced by such organisms. In our work we investigated the biosynthetic potential of 13 industrially important strains of the Bacillus subtilis complex by mass spectrometric methodology. Typing of these organisms was performed with MALDI-TOF mass spectrometry followed by comprehensive profiling of their bioactive peptide products. Volatiles were determined by gas chromatography-mass spectrometry. Representative products of the members of the B. subtilis complex investigated in detail were: the surfactin familiy (surfactins, lichenysins, pumilacidins); the iturin family (iturins, mycosubtilins and bacillomycins); plantazolicin and the dual lantibiotics lichenicidins, as well as a wide spectrum of volatiles, such as hydrocarbons (alkanes/alkenes), alcohols, ketones, sulfur-containing compounds and pyrazines. The subcomplexes of the B. subtilis organizational unit; (a) B. subtilis/Bacillus atrophaeus; (b) B. amyloliquefaciens/B. velezensis; (c) B. licheniformis, and (d) B. pumilus are equipped with specific sets of these compounds which are the basis for the evaluation of their biotechnological and agricultural usage. The 13 test strains were evaluated in field trials for growth promotion of potato and maize plants. All of the implemented strains showed efficient growth stimulation of these plants. The highest effects were obtained with B. velezensis, B. subtilis, and B. atrophaeus strains.

Genetic, Epigenetic and Phenotypic Diversity of Four Bacillus velezensis Strains Used for Plant Protection or as Probiotics.

Bacillus velezensis strains are applied as ecologically safe biopesticides, plant growth promoting rhizobacteria (PGPR), and in veterinary probiotics. They are abundant in various environments including soil, plants, marine habitats, the intestinal micro-flora, etc. The mechanisms underlying this adaptive plasticity and bioactivity are not well understood, nor is it clear why several strains outperform other same species isolates by their bioactivities. The main objective of this work was to demonstrate versatility of bioactivities and lifestyle strategies of the selected B. velezensis strains suitable to serve as model organisms in future studies. Here, we performed a comparative study of newly sequenced genomes of four B. velezensis isolates with distinct phenotypes and isolation origin, which were assessed by RNA sequencing under the effect of root exudate stimuli and profiled by epigenetic modifications of chromosomal DNA. Among the selected strains, UCMB5044 is an oligotrophic PGPR strain adapted to nutrient poor desert soils. UCMB5113 and At1 are endophytes that colonize plants and require nutrient rich media. In contrast, the probiotic strain, UCMB5007, is a copiotroph, which shows no propensity to colonize plants. PacBio and Illumina sequencing approaches were used to generate complete genome assemblies, tracing epigenetic modifications, and determine gene expression profiles. All sequence data was deposited at NCBI. The strains, UCMB5113 and At1, show 99% sequence identity and similar phenotypes despite being isolated from geographically distant regions. UCMB5007 and UCMB5044 represent another group of organisms with almost identical genomes but dissimilar phenotypes and plant colonization propensity. The two plant associated strains, UCMB5044 and UCMB5113, share 398 genes putatively associated with root colonization, which are activated by exposure to maize root exudates. In contrast, UCMB5007 did not respond to root exudate stimuli. It was hypothesized that alterations in the global methylation pattern and some other epigenetic modifications enable adaptation of strains to different habitats and therefore may be of importance in terms of the biotechnological applicability of these bacteria. Contrary, the ability to grow on root exudates as a sole source of nutrients or a strong antagonism against phytopathogens showed by the strains in vitro cannot be considered as good predictors of PGPR activities.

Genome Mining of the Lipopeptide Biosynthesis of Paenibacillus polymyxa E681 in Combination with Mass Spectrometry: Discovery of the Lipoheptapeptide Paenilipoheptin.

Paenibacillus polymyxa strains are qualified for agro-biotechnological uses such as plant growth promotion and for biocontrol strategies against deleterious phytopathogenic competitors in the soil depending on their attractive arsenal of bioactive compounds. Moreover, they are potent producers of antibiotics for medical applications. To identify new products of such organisms, genome mining strategies in combination with mass spectrometry are the methods of choice. Herein, we performed such studies with the Paenibacillus strain E681. Bioinformatic evaluation of its genome sequence revealed four gene clusters A-D encoding nonribosomal peptide synthetases (NRPSs). Accordingly, four lipopeptide families were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Clusters A and D codify the well known fusaricidins and polymyxins. A yet-unknown lipoheptapeptide was discovered and structurally characterized by de novo sequencing by using MALDI-LIFT-TOF/TOF MS. It was designated as paenilipoheptin. From structure predictions we infer that the production of this agent is encoded by gene cluster C. Gene cluster B encodes the synthesis of tridecaptins, a family of open-chain lipotridecapeptides. Strain E681 produces new subspecies of such compounds (tridecaptins E) showing variations both in their fatty-acid part as well as in their peptide part.

Ketonization of Proline Residues in the Peptide Chains of Actinomycins by a 4-Oxoproline Synthase.

X-type actinomycins (Acms) contain 4-hydroxyproline (Acm X0 ) or 4-oxoproline (Acm X2 ) in their ß-pentapeptide lactone rings, whereas their α ring contains proline. We demonstrate that these Acms are formed through asymmetric condensation of Acm half molecules (Acm halves) containing proline with 4-hydroxyproline- or 4-oxoproline-containing Acm halves. In turn, we show-using an artificial Acm half analogue (PPL 1) with proline in its peptide chain-their conversion into the 4-hydroxyproline- and 4-oxoproline-containing Acm halves, PPL 0 and PPL 2, in mycelial suspensions of Streptomyces antibioticus. Two responsible genes of the Acm X biosynthetic gene cluster of S. antibioticus, saacmM and saacmN, encoding a cytochrome P450 monooxygenase (Cyp) and a ferredoxin were identified. After coexpression in Escherichia coli, their gene products converted PPL 1 into PPL 0 and PPL 2 in vivo as well as in situ in permeabilized cell of the transformed E. coli strain in conjunction with the host-encoded ferredoxin reductase in a NADH (NADPH)-dependent manner. saAcmM has high sequence similarity to the Cyp107Z (Ema) family of Cyps, which can convert avermectin B1 into its keto derivative, 4''-oxoavermectin B1. Determination of the structure of saAcmM reveals high similarity to the Ema structure but with significant differences in residues decorating their active sites, which defines saAcmM and its orthologues as a distinct new family of peptidylprolineketonizing Cyp.

Fusaricidins from Paenibacillus polymyxa M-1, a family of lipohexapeptides of unusual complexity-a mass spectrometric study.

Paenibacillus polymyxa are rhizobacteria with a high potential to produce natural compounds of biotechnological and medical interest. Main products of P. polymyxa are fusaricidins, a large family of antifungal lipopeptides with a 15-guanidino-3-hydroxypentadecanoic acid (GHPD) as fatty acid side chain. We use the P. polymyxa strain M-1 as a model organism for the exploration of the biosynthetic potential of these rhizobacteria. Using matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) about 40 new fusaricidins were detected which were fractionated by reversed-phase (rp) HPLC. Their structure was determined by MALDI-LIFT-TOF/TOF fragment analysis. The dominant fragment in the product ion spectra of fusaricidins appeared at m/z 256.3, 284.3 and 312.4, respectively, indicating variations in their fatty acid part. Two new subfamilies of fusaricidins were introduced which contain guanidino-3-hydroxyhepta- and nonadecanoic acid as fatty acid constituents. Apparently, the end-standing guanidine group is not modified as shown by direct infusion nano-electrospray ionization mass spectrometry (nano-ESI MS). The results of this study suggest that advanced mass spectrometry is the method of choice for investigating natural compounds of unusual diversity, like fusaricidins. Copyright © 2016 John Wiley & Sons, Ltd.

Characterization of Novel Fusaricidins Produced by Paenibacillus polymyxa-M1 Using MALDI-TOF Mass Spectrometry.

Paenibacillus polymyxa-M1 is a potent producer of bioactive compounds, such as lipopeptides, polyketides, and lantibiotics of biotechnological and medical interest. Genome sequencing revealed nine gene clusters for nonribosomal biosynthesis of such agents. Here we report on the investigation of the fusaricidins, a complex of cyclic lipopeptides containing 15-guanidino-3-hydroxypentadecanoic acid (GHPD) as fatty acid component by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). More than 20 variants of these compounds were detected and characterized in detail. Mass spectrometric sequence analysis was performed by MALDI-LIFT-TOF/TOF fragment analysis. The obtained product ion spectra show a specific processing in the fatty acid part. GHPD is cleaved between the α- and ß-position yielding two fragments a and b, one bearing the end-standing guanidine group and another one comprising the residual two C-atoms of GHPD with the attached peptide moiety. The complete sequence of all fusaricidins was derived from sets of bn- and yn-ions. The fusaricidin complex can be divided into four lipopeptide families, three of them showing variations of the amino acid in position 3, Val or Ile for the first and Tyr or Phe for families 2 and 3, respectively. A collection of novel fusaricidins was detected differing from those of families 1-3 by an additional residue of 71 Da (family 4). LIFT-TOF/TOF fragment spectra of these species imply that in their peptide moiety, an Ala-residue is attached by an ester bond to the free hydroxyl group of Thr4. More than 10 novel fusaricidins were characterized mass spectrometrically.

MALDI-TOF mass spectrometry, an efficient technique for in situ detection and characterization of actinomycins.

An extensive study of actinomycins was performed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS). Actinomycins represent a well-known family of peptidolactone chromopeptides with potent cytostatic and antibiotic properties. Using five well-characterized streptomycete strains, we introduced MALDI-TOF MS as an efficient technique for rapid in situ detection of actinomycins in surface extracts of cells picked from agar plates. By this procedure, actinomycin complexes can be investigated with high sensitivity and accuracy in a minimum of time. These studies were complemented by mass spectrometric investigation of actinomycins obtained from culture filtrate extracts and purified by high-performance liquid chromatography to detect yet unknown actinomycin species. By feeding experiments, C-demethyl-actinomycins from Streptomyces chrysomallus and Streptomyces parvulus as well as hemi-actinomycins from Streptomyces antibioticus lacking one of the two pentapeptide lactone rings were isolated and characterized as novel variants for structure-activity relationship studies. Structural characterization of the investigated actinomycins was performed by post source decay MALDI-TOF MS. The specific features of the fragmentation patterns of the protonated and cationized forms of selected actinomycins were investigated in detail.

Amylocyclicin, a novel circular bacteriocin produced by Bacillus amyloliquefaciens FZB42.

Bacillus amyloliquefaciens FZB42 is a Gram-positive plant growth-promoting bacterium with an impressive capacity to synthesize nonribosomal secondary metabolites with antimicrobial activity. Here we report on a novel circular bacteriocin which is ribosomally synthesized by FZB42. The compound displayed high antibacterial activity against closely related Gram-positive bacteria. Transposon mutagenesis and subsequent site-specific mutagenesis combined with matrix-assisted laser desorption ionization-time of flight mass spectroscopy revealed that a cluster of six genes covering 4,490 bp was responsible for the production, modification, and export of and immunity to an antibacterial compound, here designated amylocyclicin, with a molecular mass of 6,381 Da. Peptide sequencing of the fragments obtained after tryptic digestion of the purified peptide revealed posttranslational cleavage of an N-terminal extension and head-to-tail circularization of the novel bacteriocin. Homology to other putative circular bacteriocins in related bacteria let us assume that this type of peptide is widespread among the Bacillus/Paenibacillus taxon.

Polymyxin P is the active principle in suppressing phytopathogenic Erwinia spp. by the biocontrol rhizobacterium Paenibacillus polymyxa M-1.

BACKGROUND: Nine gene clusters dedicated to nonribosomal synthesis of secondary metabolites with possible antimicrobial action, including polymyxin and fusaricidin, were detected within the whole genome sequence of the plant growth-promoting rhizobacterium (PGPR) Paenibacillus polymyxa M-1. To survey the antimicrobial compounds expressed by M-1 we analyzed the active principle suppressing phytopathogenic Erwinia spp. RESULTS: P. polymyxa M-1 suppressed the growth of phytopathogenic Erwinia amylovora Ea 273, and E. carotovora, the causative agents of fire blight and soft rot, respectively. By MALDI-TOF mass spectrometry and reversed-phase high-performance liquid chromatography (RP-HPLC), two antibacterial compounds bearing molecular masses of 1190.9 Da and 1176.9 Da were detected as being the two components of polymyxin P, polymyxin P1 and P2, respectively. The active principle acting against the two Erwinia strains was isolated from TLC plates and identified by postsource decay (PSD)-MALDI-TOF mass spectrometry as polymyxin P1 and polymyxin P2. These findings were corroborated by domain structure analysis of the polymyxin (pmx) gene cluster detected in the M-1 chromosome which revealed that corresponding to the chemical structure of polymyxin P, the gene cluster is encoding D-Phe in position 6 and L-Thr in position 7. CONCLUSIONS: Identical morphological changes in the cell wall of the bacterial phytopathogens treated with either crude polymyxin P or culture supernatant of M-1 corroborated that polymyxin P is the main component of the biocontrol effect exerted by strain M-1 against phytopathogenic Erwinia spp.

Occurrence and biosynthesis of C-demethylactinomycins in actinomycin-producing Streptomyces chrysomallus and Streptomyces parvulus.

Streptomyces chrysomallus and Streptomyces parvulus produce novel C-demethylactinomycins besides their normal actinomycins when fed with 3-hydroxyanthranilic acid (3-HA). The 3-HA is incorporated into pentapeptide lactone precursors in competition with the regular precursor 4-methyl-3-hydroxyanthranilic acid (4-MHA). The resultant 3-HA pentapeptide lactones can condense with each other, as well as with the continuously formed 4-MHA pentapeptide lactones giving C-demethylactinomycins lacking one or both methyl groups in their phenoxazinone chromophores. In case of C-demethylactinomyins lacking one methyl group, the condensation was shown to be regiospecific directing the 3-HA portion almost exclusively to the α-side of the phenoxazinone chromophore. As 3-HA is a weaker substrate for the 4-MHA-incorporating enzyme actinomycin synthetase I than 4-MHA, C-demethylactinomycins never exceeded 7-8% of total actinomycin formed. Surprisingly, C-demethylactinomycins (up to 0.8%) were also found in the actinomycin mixtures of unsupplemented streptomycete cultures after longer cultivation times, indicating the natural presence of 3-HA. Feeding with 3-hydroxykynurenine (3-HK) induced also formation of C-demethylactinomycins indicating that 3-HK is source of 3-HA. Analysis of tryptophan metabolites in the intracellular pools of the streptomycetes using 5-(3)H-tryptophan as radiotracer revealed formation of 4-MHA, but not of 3-HA. This indicates that intracellular 3-HK is almost exclusively converted to 3-hydroxy-4-methylkynurenine (4-MHK), which has been identified previously as direct precursor of 4-MHA. However, small amount of 3-HK leaking out from the 4-MHA pathway can be prematurely converted to 3-HA all along the cultivation of the streptomycetes resulting in the formation of natural C-demethylactinomycins.

Genome sequence of the plant growth promoting strain Bacillus amyloliquefaciens subsp. plantarum B9601-Y2 and expression of mersacidin and other secondary metabolites.

The plant-associated Bacillus amyloliquefaciens subsp. plantarum strain B9601-Y2, isolated from wheat rhizosphere, is a powerful plant growth-promoting rhizobacterium. Its relative large genome size of 4.24Mbp, exceeding that of other representatives of the B. amyloliquefaciens subsp. plantarum taxon, is mainly due to the presence of 18 DNA-islands containing remnants of phages, a unique restriction modification system, a gene cluster for mersacidin synthesis, and an orphan gene cluster devoted to non-ribosomal synthesis of an unidentified peptide. Like other members of the taxon, the Y2 genome contains giant gene clusters for non-ribosomal synthesis of the polyketides macrolactin, difficidin, and bacillaene, the antifungal lipopeptides bacillomycin D, and fengycin, the siderophore bacillibactin, and the dipeptide bacilysin. A gene cluster encoding enzymes for a degradative pathway with 2-keto-3-deoxygluconate and 2-keto-3-deoxy-phosphogluconate as intermediates was explored by genome mining and found as being a unique feature for representatives of the plantarum subspecies. A survey of the Y2 genome against other B. amyloliquefaciens genomes revealed 130 genes only occurring in subsp. plantarum but not in subsp. amyloliquefaciens. Notably, the surfactin gene cluster is not functional due to a large deletion removing parts of the Srf synthetases B and C. Expression of polyketides, lipopeptides, mersacidin, and of the growth hormone indole-3-acetic acid in Y2 was demonstrated by matrix-assisted laser desorption ionization-time of flight mass spectroscopy and high-performance liquid chromatography, respectively.

Plantazolicin A and B: structure elucidation of ribosomally synthesized thiazole/oxazole peptides from Bacillus amyloliquefaciens FZB42.

The structures of the ribosomally synthesized peptide antibiotics from Bacillus amyloliquefaciens FZB42, plantazolicin A and B, have been elucidated by high resolving ESI-MSMS, 2D (1)H-(13)C-correlated NMR spectroscopy as well as (1)H-(15)N-HMQC/(1)H-(15)N-HMBC NMR experiments. (15)N-labeling prior to the experiments facilitated the structure determination, unveiling a hitherto unusual number of thiazoles and oxazoles formed from a linear 14mer precursor peptide. This finding further extends the number of known secondary metabolites from B. amyloliquefaciens and represents a new type of secondary metabolites from the genus Bacillus.

Efficient colonization of plant roots by the plant growth promoting bacterium Bacillus amyloliquefaciens FZB42, engineered to express green fluorescent protein.

A single copy of the gfp gene linked with the P(spac) promoter and flanked by the terminal FZB42 amyE sequences was stably integrated into the chromosome of plant growth promoting bacterium Bacillus amyloliquefaciens FZB42 via homologous recombination. A spontaneous mutant, FB01mut, emitting bright fluorescence was detected among the transformants and found suitable for colonization experiments performed with Zea mays, Arabidopsis thaliana and Lemna minor. Real-time RT-PCR revealed that FB01mut expressed 2.5 times more of the gfp transcript than the original GFP-labeled strain. Confocal laser scanning microscopy of plant roots infected with gfp+ tagged FZB42 revealed that the bacterium behaves different in colonizing surfaces of plant roots of different species. In contrast to maize, FZB42 colonized preferentially root tips when colonizing Arabidopsis. FZB42 colonized heavily Lemna fronds and roots by forming biofilms consisting of extracellular matrix and cells with altered morphology. Surfactin, but no other lipopeptide or polyketide synthesized by FZB42 under laboratory conditions, was detected in extracts of Lemna plantlets colonized by FZB42. Due to its stable and long-lasting emission of bright fluorescence without antibiotic pressure FB01mut is an excellent tool for studying plant colonization under competitive, environmental conditions.

Plantazolicin, a novel microcin B17/streptolysin S-like natural product from Bacillus amyloliquefaciens FZB42.

Here we report on a novel thiazole/oxazole-modified microcin (TOMM) from Bacillus amyloliquefaciens FZB42, a Gram-positive soil bacterium. This organism is well known for stimulating plant growth and biosynthesizing complex small molecules that suppress the growth of bacterial and fungal plant pathogens. Like microcin B17 and streptolysin S, the TOMM from B. amyloliquefaciens FZB42 undergoes extensive posttranslational modification to become a bioactive natural product. Our data show that the modified peptide bears a molecular mass of 1,335 Da and displays antibacterial activity toward closely related Gram-positive bacteria. A cluster of 12 genes that covers ∼10 kb is essential for the production, modification, export, and self-immunity of this natural product. We have named this compound plantazolicin (PZN), based on the association of several producing organisms with plants and the incorporation of azole heterocycles, which derive from Cys, Ser, and Thr residues of the precursor peptide.