Evaluation of Bacillus biocontrol potential through the lens of secondary metabolite diversity in genomic, enzymatic, and chemical space

Abstract

Global food security is under threat by plant-pathogenic oomycetes and fungi. The overuse and misuse of pesticides against such pathogens triggers fungicide resistance and long-term environmental contamination. This warrants exploration of sustainable and eco-friendly alternative strategies to chemical pesticides. Biological agents over synthetic pesticides have been considered an attractive alternative for plant and crop protection. In particular, bacteria inhabiting plant rhizospheres are well-suited as biopesticides and biofertilizers due to their ability to suppress root-associated plant pathogens and enhance plant growth through the production of various secondary metabolites, phytohormones, and lytic enzymes. To date, many bacteria-derived secondary metabolites have been shown to control plant pathogens and promote plant growth by direct or indirect mechanisms. Such secondary metabolites are produced by a tightly linked set of enzymes encoded by biosynthetic gene clusters (BGCs). Bacillus species, prolific producers of such metabolites, are commonly referred to as plant growth-promoting rhizobacteria (PGPR) for their ability to spur plant growth and exert disease biocontrol. The secondary metabolites imparting these properties are structurally and chemically diverse and include lipopeptides, bacteriocins, polyketides, siderophores, and terpenes. Previous studies have demonstrated that secondary metabolites within these classes directly contribute to Bacillus biological activity against plant pathogens. Accordingly, secondary metabolites with growth-promotional and antimicrobial potential continue to be of great interest for controlling plant diseases, promoting plant health, and developing drugs against infectious and chronic diseases. For more than a century, numerous antibiotics and antibiotic scaffolds have been discovered in bacteria, fungi, and plants. Bacillus species living in the soil and plant rhizosphere are a potential source of novel and industrially relevant antimicrobial compounds. The full potential of such natural products is only recently becoming known due to advances in genome sequencing, bioinformatics algorithms, and analytical instrumentation such as HPLC, LC-MS, and NMR. These advances have facilitated the identification of novel enzymes with primary and accessory roles in natural product biosynthesis, including their catalytic mechanisms and the regulatory systems associated with their activity. Studies have revealed a great diversity of biosynthetic accessory enzymes in soil-borne bacteria including Streptomyces, Pseudomonas, and Bacillus. In particular, cytochromes P450 are often found as accessory enzymes in natural product biosynthetic pathways. To date, more than 20 P450s have been identified in the biosynthesis of different classes of natural products, including macrolides (EryF and EryK), polyenes (PimD and CYP105P1), glycopeptides (OxyB, OxyC, and OxyD), polyketides (AurH, CalO2, and CYP158), alkaloids (StaP), and fatty acids (P450BioI and CYP74). These P450s have been reported to catalyze broad diversity of reactions, including hydroxylation, epoxidation, heterocyclization, and ring-coupling. Although these P450s have been characterized structurally and functionally, their physiological roles are far less understood. They are anticipated to be involved in either functionalization of structurally diverse natural products to establish/maintain a competing advantage or in the metabolic breakdown of antibiotic compounds to reduce cellular toxicity. Thus, investigations of accessory P450s for physiological, catalytic, and structural properties have continued to be of great interest. Previously, analysis of 128 Bacillus genomes identified 112 accessory P450 enzymes in the biosynthetic of various classes of secondary metabolites. However, little is known about the structural, catalytic, and physiological roles of these enzymes. The long-term goal of this research is to exploit Bacillus species as probiotics and biocontrol agents and develop their antimicrobial secondary metabolites as potential pesticides/fungicides. A deeper understanding of their biocontrol potential, through the lens of their secondary metabolite diversity in genomic, enzymatic, and chemical space, is a major step in that direction. Toward achieving these ends, the objective of this research is to identify Bacillus secondary metabolites, evaluate Bacillus biological activity against plant pathogens, and elucidate the functional roles of associated cytochrome P450 enzymes. In this study, a total of 288 novel PGPR Bacillus strains were evaluated as commercially viable biological agents against root-associated plant pathogens. A comprehensive discovery framework incorporating genome mining, antibiosis, chemical, and phenotypic screening was established to evaluate Bacillus biocontrol potential. In addition, 1,562 Bacillus genome sequences were analyzed to identify BGC-affiliated P450s that may be involved in functional modification of secondary metabolites. The P450s were further studied in silico for structural and functional features and a representative CYP102A2 predicted to be associated with plantazolicin biosynthesis was further investigated in vitro towards elucidating its catalytic role. Our antibiosis screening of 288 Bacillus strains against a plant-pathogenic oomycete, Phytophthora nicotianae showed 59 strains with strong antibiosis activity, whereas 41 and 188 strains were weak and non-inhibitors, respectively. Importantly, fifty-six out of 59 strong-inhibitory strains were distributed within only five species: B. velezensis, B. subtilis, B. pumilus, B. safensis, and B. altitudinis. The high concentration of anti-P. nicotianae activity among five Bacillus species suggested these species may carry common factors responsible for this antibiosis activity. In order to investigate the breadth of antibiosis activity, the most promising 59 P. nicotianae-inhibitory Bacillus strains were further evaluated against three root-associated plant-pathogenic fungi: Fusarium oxysporum, F. graminearum, and Rhizoctonia solani. All strains from B. velezensis and all but one B. subtilis strain exhibited strong antibiosis activity against all three fungi; however, strains from B. pumilus, B. safensis, and B. altitudinis exhibited no or weak inhibition against these fungi. Accounting for the breadth of inhibition, B. velezensis and B. subtilis were classified as “generalists” while B. pumilus, B. safensis, and B. altitudinis were classified as specialists for their relatively narrow inhibitory properties. BGC analysis of all 288 Bacillus strains showed that highly conserved antimicrobial peptide and polyketide-producing BGCs were distributed among the generalists and specialists but virtually absent among non-inhibitory species. In addition, generalists invariably carried 2 or 3 lipopeptide BGCs that were predicted to produce fengycin and surfactin or iturin, fengycin, and surfactin, respectively. Consistent with BGC predictions, chemical analyses of extracts from the culture media of representative strains showed that iturin/bacillomycin L, fengycin, and surfactin were produced by B. velezensis while fengycin and surfactin were produced by B. subtilis. As a contrast, only surfactin among these three was produced by B. pumilus, B. safensis, and B. altitudinis. Evaluation of purified bacillomycin L (an iturin), fengycin, and surfactin for antibiosis activity against P. nicotianae and F. oxysporum in plate-based and 96-well plate microtiter-based assays showed each compound exerted antibiosis activity against the target pathogens. Eighteen strains from the most promising antifungal/anti-oomycete species, Bacillus velezensis, were further compared for their abilities to exert intraspecies inhibition, mount intraspecies resistance, and in addition, to show antifungal activity, biofilm formation, and extent of antimicrobial secondary metabolite production. Interestingly, a wide range of intraspecies inhibition and resistance responses were observed. The AB01 and JJ951 strains showed the most robust antibacterial activity against other B. velezensis strains, while AB01, JJ951, AP46, and JJ747 showed the greatest resistance to inhibition by other strains. Phylogenetic analyses based on 16S rRNA sequence from all 18 strains indicated that conserved regulatory/resistance factors may be responsible for these observed intraspecies interactions. Further, evaluation of antibiosis activity of each B. velezensis strain against F. oxysporum, F. graminearum, and R. solani showed a variable inhibitory response depending on the fungus being evaluated. Accounting for overall antifungal activity, AP215 exhibited more robust inhibition than any of the other strains evaluated, but substantial antifungal inhibition was also observed for AB01, JJ1284, AP52, JM204, AP81, AP202, JM199, and JJ747. Comparison of the production of three polyketides (bacillaene, difficidin, and macrolactin W) and three lipopeptides (iturin/bacillomycin L, fengycin, and surfactin) showed that AB01 and JJ951 produced larger quantities of these antimicrobial compounds than any other strains. From mammals to bacteria, cytochromes P450 typically play one (or both) of two roles in metabolism, the derivatization of metabolites to specialize their function (e.g., steroid hormone biosynthesis) or to facilitate detoxification/excretion of xenobiotic compounds. Both functions could play an important role in secondary metabolite biosynthesis and resistance to toxicity exerted by the same. In order to investigate the diversity of antimicrobial secondary metabolite scaffolds, how that influences the variation in their bioactivity, and to what extent this is mediated by cytochromes P450, a comprehensive analysis was carried out using 1,562 Bacillus genomes. From a total of 5,051 cytochrome P450 genes, we identified 614 integrated within biosynthetic gene clusters (BGCs) as “accessory genes”. The most common BGC-affiliated P450 families were CYP113, CYP134, CYP109, CYP107, and CYP102 and these were associated with the secondary metabolite BGCs of difficidin, cyclodipeptide, bacillibactin, bacillaene, and plantazolicin, respectively. Interestingly, amino acid sequence conservation of enzymes within each P450 family showed CYP113 and CYP134 were the most highly conserved, whereas CYP107 and CYP109 were the least conserved. This observation indicated that phylogenetically-related amino acid sequence conservation of each P450 family may be linked with substrate specificity with respective biosynthetic pathway. High-resolution homology models of a representative enzyme from each BGC-affiliated P450 family showed distinct structural features at the active site that may be related to substrate specificity with the respective biosynthetic pathway. Molecular docking simulations of each P450 with candidate substrates showed favorable binding of CYP113, CYP134, CYP109, CYP107, and CYP102 with the putative substrates difficidin, cyclodipeptide, dihydroxybenzoate, bacillaene, and plantazolicin, respectively. To facilitate elucidation of a functional role for a CYP102 in plantazolicin biosynthesis, a representative CYP102A2 (P450BM3) protein from Bacillus amyloliquefaciens FZB42 (BaCYP102A2) (NCBI accession: WP_012117030) was synthesized, cloned into a pET21 vector, and used to transform E. coli (BL21-[DE3]) for expression. The enzyme was purified using Ni-NTA affinity, anion-exchange, and size-exclusion chromatographies. The identity of full-length BaCYP102A2 enzyme was confirmed by the presence of a major band at ~120 kDa in SDS-PAGE gel, a heme-based Soret band at 418.5 nm in UV-vis spectra, along with the type-I spectral shift upon substrate binding and sigmoidal and hyperbolic kinetic responses of NADPH oxidation by BaCYP102A2 in the presence of the archetypal substrates sodium dodecyl sulfate (SDS) and oleic acid (OA), respectively. Fitting of substrate titration curves and steady-state kinetic responses of BaCYP102A2 using the substrates SDS, myristic acid (MA), and palmitic acid (PA) produced sigmoidal responses while OA produced a hyperbolic response. In summary, the research of this dissertation has revealed that Bacillus antibiosis against P. nicotianae is highly conserved within five Bacillus species (B. velezensis, B. subtilis, B. pumilus, B. safensis, and B. altitudinis). B. velezensis and B. subtilis exhibited broad antibiosis activity against P. nicotianae as well as multiple fungal pathogens. Comparison of genomic, antibiosis, and chemical profiles showed that the production of phylogenetically conversed lipopeptides correlates with these observed antibiotic abilities. Further quantitative evaluation of 18 B. velezensis strains showed wide range of intraspecies inhibition activity, resistance to such inhibition, and antifungal activity that may be linked with the expression of regulatory/resistance factors and/or production of antimicrobial compounds. Strains AB01 and JJ951 exhibited robust intraspecies inhibition activity, and strains AB01, JJ951, AP46, and JJ747 exhibited the greatest resistance to inhibition by other B. velezensis strains. Strain AP215 exhibited the most robust antifungal activity. Evaluation of phylogeny and amino acid sequence conservation of 614 BGC-affiliated Bacillus P450s showed enzymes from each P450 family may be linked with specific secondary metabolite biosynthesis. In silico structural and substrate-binding evaluation of a representative enzyme from five BGC-affiliated P450 families showed distinct structural features that matched with favorable substrate binding associated with the respective secondary metabolite biosynthesis. Finally, a representative BaCYP102A2 enzyme predicted to be involved in plantazolicin biosynthesis was expressed, purified, and further evaluated for substrate binding titration and steady-state kinetic responses with typical CYP102 substrates.