Metabolic flexibility enables Bacillus sp. G2112 to survive phenazine-1-carboxylic acid toxicity

Abstract

Plant pathogens cause severe crop losses and thus threaten global food supply. Although pesticides can control most pathogens, fast development of resistance to them and their toxicity calls for alternatives. Microbial symbionts of plants constitute a promising source for so-called biocontrol agents because they can efficiently protect plants without posing environmental risks. Microorganisms of the genera Bacillus and Pseudomonas usually occur together in soils and can promote plant growth and control pathogens. Many pseudomonads release the antibiotic phenazine-1-carboxylic acid (PCA) in the soil to gain a competitive advantage. PCA inhibits several plant pathogens, and can effectively control the take-all disease of wheats. In my doctoral thesis, I evaluated how the potential biocontrol organism Bacillus sp. G2112 isolated from cucumber (Cucumis sativus) adapted to phenazine-1-carboxylic acid (PCA), the major antibiotic compound produced by a co-isolate, Pseudomonas sp. G124. Bacillus sp. G2112 detoxifies phenazine-1-carboxylic acid by N5 glucosylation Both Bacillus sp. G2112 and Pseudomonas sp. G124 inhibited Erwinia and Fusarium, which are pathogens of cucumber, highlighting their potential for biocontrol. However, Pseudomonas sp. G124 produces PCA (1) which kills Bacillus sp. G2112 at concentrations above 40 µg/mL. Bacillus sp. G2112 survives this antagonism by converting PCA (1), which is yellow, to red pigments. Two of such pigments having a quasimolecular ion [M+H]+ at m/z 402 were purified by reversed phase chromatography, and identified by high-resolution mass spectrometry, NMR, and chemical degradation as N5-glucosylated phenazine derivatives: 7-imino-5N-(1′β-D-glucopyranosyl)-5,7-dihydro¬phenazine-1-carboxylic acid (2) and 3-imino-5N-(1′β-D-glucopyranosyl)-3,5-dihydrophenazine-1-carboxylic acid (3). The pigments did not inhibit Bacillus sp. G2112, proving that their production is a resistance mechanism. Glucose availability determines the adaptive strategy of Bacillus sp. G2112 biofilms to long-term phenazine-1-carboxylic acid exposure How Bacillus sp. G2112 adapts to long term PCA (1) exposure with and without exogenous glucose supply in its late growth stage was investigated. Under both nutrient conditions, Bacillus sp. G2112 produced red pigments, imino-N5-glucopyranosyl PCA derivatives, in the early growth stages. With glucose added, PCA (1) was transformed to 1-hydroxyphenazine (1-HP, 2) in addition to an unidentified labile compound that may be an oxygenated phenazine-1-carboxylic acid. Without glucose, phenzine-1-carboxylic acid methyl ester (PCAMe, 3) was obtained in the late stationary phase cultures. 1-HP (2) was highly active against the plant pathogens Erwinia tracheiphila, Streptomyces turgidiscabies and Fusarium graminearum. Thus, Bacillus sp. G2112 could potentiate the scope of phenazine antibiotics from pseudomonads if glucose could be provided by the host plant, e.g. in root exudates. PCAMe (3) did not inhibit the tested plant pathogens. Surprisingly, 1-HP (2) was also active against Bacillus sp. G2112, and PCAMe (3) produced inhibition zones after being initially inactive, indicating that it was further modified by Bacillus sp. G2112 cells growing at earlier growth phase to an active compound. The bioactivities of 1-HP (2) and PCAMe (3) suggest that they are either detoxification intermediates destined for further modifications or are active metabolites generated by Bacillus sp. G2112 biofilms to control population growth in resource-depleted environments. Diverse detoxification products reflect metabolic flexibility in conversion of phenazine-1-carboxylic acid by Bacillus sp. G2112 A time-course assay was performed in order to identify how Bacillus sp. G2112 detoxifies PCA (1). Upon exposure, Bacillus sp. G2112 instantly glucosylates PCA (1) yielding two phenazine derivatives with quasimolecular ions at m/z 387 and m/z 389. If glucose was exogenously provided, this glucosylation reaction reaches maximum within 2 h but takes up to 1 d if glucose was not added in the culture. MS/MS analyses identified the compounds with m/z 387 and m/z 389 as 1-carboxy-5N-(1'β-glucospyranosyl)phenazin-5-ium (3) and 5N-(1'β-glucosyl)-5,10-dihydro-phenazine-1-carboxylic acid (2), respectively. 5N-(1'β-glucosyl)-5,10-dihydrophenazine-1-carboxylic acid was partially purified and its identity was confirmed by NMR. Rapid decomposition of the glycosylated phenazine derivatives from the 4th day released PCA (1) back into the culture, indicating that PCA (1) was only temporarily inactivated by glycosylation. However, a portion of the glycosylated PCA derivatives was further modified with imino and oxo substituents at positions 3 and 7 of the PCA heterocycle to yield several red pigments including 3-imino-5N-(1′β-D-glucopyranosyl)-3,5-dihydrophenazine-1-carboxy-lic acid (5) and 7-imino-5N-(1′β-D-glucopyranosyl)-5,7-dihydrophenazine-1-carboxylic acid (6). In addition, the more complex 2-(((N-acetyl-γ-aminobutyroyl)thio)methyl)-4-carboxy-N10-(1'β-D-glucopyranosyl)-2,3-dihydro-1H-pyrrolo[2,3-b]phenazin-10-ium (11) with a quasimolecular ion at m/z 601 was formed. The complex structure of the latter compound with several modifications suggests that it may possess other functions than simply serve as detoxification product. Bacillus sp. G2112 produces a large diversity of nobilamide peptides which induce biofilm formation in pseudomonads and mycobacteria Bacillus sp. G2112 produces surfactins, iturins and fengycins which are commonly produced by bacilli. In addition, it also produces several nobilamide peptides including A-3302-B known for its antistaphylococcal, antiviral, cancer-suppressing and TRPV-1 pain receptor-antagonizing activities. Twenty-one related nobilamides, sixteen of which are previously unknown, were identified using high resolution mass spectrometry, tandem mass spectrometry, Marfey's derivatization and NMR. The diversity of the new peptides, named nobilamide J – Y, resulted from incorporation of different acyl groups, amino acid substitutions, positional swapping of amino acid moieties, lactone ring opening and sequence truncation in relation to the original depsipeptide A-3302-B. This diversity highlights the highly relaxed substrate specificity of the nobilamide biosynthetic enzymes in Bacillus sp. G2112 compared to previously reported nobilamide peptide producers. Antimicrobial assays with some of the nobilamide peptides revealed that only A-3302-B was active at 7 µg/hole against Lysinibacillus sphaericus and Staphylococcus aureus. However, all five tested compounds (A-3302-B, nobilamide A, I, T and Y) promoted biofilm formation in Mycobacterium aurum and two Pseudomonas spp. Variations in the amino acid sequence length and molecular structure did not cause any clear differences in biofilm-inducing activity.