Exploiting the Sensitivity of Nutrient Transporter Deletion Strains in

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Exploiting the sensitivity of nutrient transporter deletion strains in discovery of natural product antimetabolites Eric D Brown, Sebastian S Gehrke, Garima Kumar, Nicole A Yokubynas, JeanPhilippe Côté, Wenliang Wang, Shawn French, Craig R MacNair, and Gerard D Wright ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00149 • Publication Date (Web): 25 Oct 2017 Downloaded from http://pubs.acs.org on October 30, 2017

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ACS Infectious Diseases

Title: Exploiting the sensitivity of nutrient transporter deletion strains in discovery of natural product antimetabolites

Authors: Sebastian S. Gehrkeǂ, Garima Kumarǂ, Nicole A. Yokubynasǂ, Jean-Philippe Côté, Wenliang Wang, Shawn French, Craig R. MacNair, Gerard D. Wright, Eric D. Brown* ǂ Authors contributed equally to this work * Corresponding author

Affiliations Michael G. DeGroote Institute of Infectious Disease Research, Department of Biochemistry and Biomedical Science, McMaster University, 1200 Main Street West, Hamilton Ontario L8N 3ZS, Canada

Correspondence: Dr. Eric D. Brown – [email protected]

ACS Paragon Plus Environment


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Actinomycete secondary metabolites are a renowned source of antibacterial chemical scaffolds. Herein, we present a target-specific approach that increases the detection of antimetabolites from natural sources by screening actinomycete-derived extracts against nutrient transporter deletion strains. Based on the growth rescue patterns of a collection of 22 E. coli auxotrophic deletion strains representative of the major nutrient biosynthetic pathways, we demonstrate that antimetabolite detection from actinomycetederived extracts prepared using traditional extraction platforms is masked by nutrient supplementation. In particular, we find poor sensitivity for the detection of antimetabolites targeting vitamin biosynthesis. To circumvent this and as a proof of principle, we exploit the differential activity of actinomycete extracts against E. coli ∆yigM, a biotin transporter deletion strain versus wildtype E. coli. We achieve more than a 100-fold increase in antimetabolite sensitivity using this method, and demonstrate a successful bioassayguided purification of the known biotin antimetabolite, amiclenomycin. Our findings provide a unique solution to uncover the full potential of naturally derived antibiotics. Keywords: Antimicrobial Resistance, Natural Products; Drug discovery; Screen; Antimetabolites; Nutrient metabolism; Nutrient transport; Auxotrophy; Transporter Deletion; Escherichia coli


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Since the dawn of antimicrobial discovery, natural products isolated from soil-dwelling actinomycetes have proven to be a prominent and privileged source of antimicrobial chemical matter. Comprising more than 70% of clinically used antibiotics, they include reputable agents such as glycopeptides, β-lactams, tetracyclines, and, aminoglycosides.1-4 However, within the last few decades, uncovering novel antimicrobials from actinomycetes has become increasingly difficult.5 The stark insufficiency of new antimicrobial agents, concomitant with the overwhelming increase in the occurrence of antibiotic resistant pathogen infections places the inability to control bacterial infections as a paramount threat to modern medicine worldwide.6, 7 The predicted repercussions of a post-antibiotic era are fearsome, making the discovery of new antibacterial scaffolds critical. The current void in novelty from natural products is commonly linked with the repeated use of standard natural product screening and isolation protocols. Despite previous successes, these strategies frequently result in molecule re-discovery and the detection of chemically and functionally similar compounds that target a very limited spectrum of bacterial-specific cellular processes.5, 8, 9 Inherently, the key question still remains: have we previously harnessed the majority of nature’s antibacterial moieties and exhausted this resource, or have we merely scraped the surface of nature’s potential? Remarkably, recent genetic analysis supports that our current collection of natural product derived antibiotics represents only a fraction of the secondary metabolite producing capacity of each strain of actinomycetes; thus signifying a potential reservoir of untapped antibiotic compounds that have yet to be discovered.10-14 Significant pressure is thus consequently placed on the continuous development of novel approaches to mine the unexplored and expedite the identification of these novel compounds. Success has arisen from modern natural product drug discovery platforms including environmental, chemical and genetic stimulation of silent biosynthetic gene clusters, high-throughput screening of natural product extract libraries, isolation of actinomycetes from unique environments, combinatorial techniques, and more; however, these approaches are still unable to meet modern day demands.5, 13



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In recent years, antimetabolites have become increasingly attractive molecules for the development of new antimicrobial scaffolds. Numerous groups have linked nutrient biosynthetic processes to in vivo pathogen virulence.15-17 For example, infection models with knockout strains of Mycobacterium tuberculosis have identified limited levels of certain metabolites in vivo such as leucine, glutamine, biotin, pantothenate, proline, tryptophan, and pyridoxine; consequently reducing the pathogens’ capacity to establish an infection.16,

18-23

However, the detection of antimetabolites is

dependent on the contextual definition of gene essentiality, as their targets only become apparent when the cell is placed under considerable nutrient stress.24 For example, E. coli gains over 100 additional indispensable genes that are available as possible antibacterial targets when grown under nutrient limited conditions. Using this rationale, Zlitni et al., performed a synthetic compound screen against E. coli under nutrient limited conditions to successfully identify novel antibacterial inhibitors of vitamin and amino acid biosynthetic metabolism.25 Notably, the majority of antimetabolite antibiotics are discovered through chemical synthesis and synthetic compound screens.26 We questioned whether the observed lack of natural product antimetabolites might stem from traditional natural product screening methodologies. Conventionally, both the growth of the actinomycetes and the screening of their extracts for bioactive secondary metabolites have been conducted under various nutrient rich growth conditions.5, 27 As previously noted, the ability to detect antimetabolites is highly dependent on a nutrient-deprived environment.24,

25

We

hypothesized that nutrient carryover from the conventionally used nutrient-rich growth conditions to the crude extract during actinomycetes secondary metabolite isolation reduces the probability of detecting antimetabolites. Of note, antimetabolites are structurally very similar to the metabolites they interfere with.28 For example, the structure of sulfanilamide, one of the first identified antimetabolites, differs from its corresponding metabolite 4-aminobenzoic acid by one functional group, replacing the carboxylic acid moiety with a sulfonamide group. Chemically, this structure similarity further supports the likelihood for both the inhibitor and parent metabolite to be co-extracted simultaneously.


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To investigate if nutrient re-supplementation was indeed a factor limiting the discovery of natural product antimetabolites, we screened a collection of E. coli nutrient biosynthetic auxotrophs against biologically inactive actinomycete extracts derived from both nutrient-rich and nutrient-poor growth conditions. Growth of the nutrient biosynthetic auxotrophic E. coli strains upon the addition of the crude extracts derived from the actinomycete strains was observed to be both strain and nutrient specific. Of utmost concern, we report that the promising antimicrobial targets of the vitamin biosynthetic pathways including biotin, thiamine and pantothenate, displayed the most profound extent of nutrient resupplementation. Overall, our results indicate that a sizable percentage of antibacterial antimetabolites produced by actinomycetes are likely masked using conventional nutrient-rich based screening methodologies as a result of nutrient re-supplementation. Lastly, we describe a target-specific screening protocol to increase the detection of natural product antimetabolites by repressing nutrient resupplementation. Specifically, using E. coli ∆yigM, the biotin transport deletion strain, we demonstrate improved sensitivity to biotin antimetabolites present in actinomycete-derived crude extract. Furthermore, we

report

a

successful bioassay guided

purification of

amiclenomycin

(2s)-2-amino-4-(4-

aminocyclohexa-2,5-dien-1-yl)butanoic acid) (ACM), a known biotin antimetabolite, from crude extract, by exploiting its differential bioactivity against E. coli ∆yigM vs wildtype BW25113 strains. Overall, this represents a promising strategy to sidestep the limitations of nutrient re-supplementation and to improve the detection of natural product antimetabolites. RESULTS Varied Effect of Actinomycetes Growth Conditions on Nutrient Re-Supplementation To investigate the influence of nutrient re-supplementation on natural product antimetabolite detection, a collection of 22 minimal essential auxotrophic E. coli strains were systematically screened against a subset of 10 actinomycete strains from the Wright Actinomycete Collection (WAC). WAC is a collection of actinomycete strains mainly isolated from soil samples gathered from around the world. Table S1 lists the WAC identification numbers of the actinomycete strains chosen. The 10 selected



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actinomycete-derived crude extracts exhibit no antibacterial activity under traditional screening conditions. A schematic of the methodologies employed are depicted in Figure 1. Specifically, we investigated whether the extent of nutrient re-supplementation from the actinomycete crude extracts was responsible for masking the detection of antimetabolites produced by the actinomycetes. The 22 minimal essential genes were prioritized from the total collection of 119 genes known to be responsible for the growth in E. coli under nutrient-limited growth conditions (compared to rich microbiological media), based on the biosynthetic pathway of interest and the specific target location within each pathway.24 When applicable, one representative gene deletion from each of the major amino acid, nucleobase and vitamin biosynthetic pathways in E. coli was selected for analysis. Due to the inherent complexity of the E. coli metabolic network, often, additional salvage pathways and redundant enzymes exist for the biosynthesis of certain metabolites.29 As a result, a subset of metabolic targets including alanine, riboflavin, niacin, and 4-aminobenzoic acid could not be evaluated due to the lack of a fully auxotrophic phenotype when the deletion mutants were grown under minimal nutrient conditions. Nevertheless, we reasoned that this redundancy classifies these biosynthetic pathways as unappealing drug targets. Table S2 summarizes the representative auxotrophic mutants and the function of the deleted enzyme in each of the selected nutrient biosynthetic pathways.29 In the context of our assay, we reasoned that the gene deletion was analogous to an antimetabolite targeting the corresponding enzyme that the gene encodes for in the associated biosynthetic pathway.25, 30 Furthermore, we ensured that all of the auxotrophic mutants selected were unable to grow in nutrient-limited growth conditions and that this phenotype was reversed upon re-supplementation with the individual amino acid, vitamin or nucleobase of the target pathway of interest (Figure S1). Therefore, we concluded that any growth of the auxotrophic strains observed following the addition of crude extract indicates the presence of the target nutrient or an associated nutrient pre-cursor within the extract. Additionally, the use of non-antibacterial producing actinomycete strains ensured that the observed inability for an auxotrophic mutant to grow was attributed to the lack of nutrients present within the crude extract, and not due to the presence of a growth inhibitory compound. Due to the variable opaqueness between several of the actinomycete extracts, the growth of the


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auxotrophic mutants was measured using the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to detect the presence of viable cells post-incubation.31 This colorimetric assay served as both a qualitative and quantitative indicator of the ability of the auxotrophic mutant to grow in the presence of crude extract. The screen, in nutrient-limited media, of 22 auxotrophic E. coli strains against various actinomycete extracts demonstrated the capacity for nutrients within the actinomycete crude extract to be extracted simultaneously with the antibiotic secondary metabolites produced by the organism. Furthermore, we identified that the extent of nutrient re-supplementation is sufficient to rescue the growth of several of the nutrient biosynthetic auxotrophic mutants. Interestingly, we show that the observed resupplementation profiles are largely dependent on the representative metabolic pathway (Figure 2). Briefly, we identified three characteristic phenotypes: (1) nutrient targets displaying limited nutrient resupplementation (85% growth of the auxotrophic mutant). For example, the E. coli auxotrophic mutant ∆thrC was unable to grow in the presence of all actinomycete crude extracts tested, signifying negligible nutrient re-supplementation of threonine (Figure 2-A). In contrast, the auxotrophic mutant ∆bioD (Figure 2-C) exhibited nearly 100% growth relative to fully supplemented conditions, irrespective of whether the actinomycete extract was derived from nutrient-rich or nutrient-deprived growth conditions. This implies a high extent of nutrient re-supplementation of biotin from the crude extract, either from the nutrient rich growth conditions and/or excretion of biotin produced during actinomycete growth into the surrounding growth media. Lastly, the auxotrophic mutant ∆glyA displayed intermediate growth in the presence of crude extract (Figure 2-B) and thus a moderate extent of nutrient re-supplementation, principally from those actinomycete crude extracts derived from nutrient-rich growth conditions. As a result, we would predict increased difficulty detecting natural product glycine antimetabolites, though nearly not as challenging as vitamin biosynthetic inhibitors.



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Upon re-supplementation of the 22 auxotrophic mutants, it was evident that growth rescue was both nutrient and actinomycete strain specific (Figure 3). Overall, the auxotrophic mutants displaying restricted growth in the presence of crude extract included ∆asnA∆asnB, ∆cysK, ∆glnA, ∆hisD, ∆serB, ∆thrC, ∆ilvC, ∆leuA, ∆purC and ∆pyrB. Therefore, the biosynthetic targets we would predict to have relatively limited interference from nutrient re-supplementation include asparagine, cysteine, glutamine, histidine, serine, threonine, branched chain amino acids and nucleobases. The auxotrophic mutants ∆argC, ∆glyA, ∆metB, ∆proA, ∆pheA, ∆tyrA, ∆trpB and ∆pdxH displayed partial growth in the presence of crude extract; thus, the biosynthetic targets displaying moderate nutrient re-supplementation and a reduced probability of antimetabolite detection include: arginine, glycine, methionine, proline, aromatic amino acids, and pyridoxine. For these metabolites, nutrient re-supplementation was predominantly observed for those extracts derived from a nutrient-rich growth background. Therefore, the lack of nutrient re-supplementation from the actinomycete strains derived from minimal nutrient conditions supports that a major contributor to the observed re-supplementation is the nutrient-rich media used for actinomycete growth. Lastly, and of most concern, the growth of auxotrophic mutants ∆bioD, ∆thiE and ∆panC was fully rescued upon the addition of actinomycete crude extract irrespective of growth conditions. Therefore, the biosynthetic targets displaying the highest extent of nutrient re-supplementation included biotin, pantothenate and thiamine. With the exception of glutamine, all auxotrophic mutants grew in the presence of media-only extracts derived from the nutrient-rich Bennett’s medium and not from the media-only extracts derived from nutrient-limited M9 media, thus indicating that indeed standard extraction protocols co-extract nutrients and nutrient precursors present within the growth media. Interestingly, the broad-scale analysis of nutrient re-supplementation revealed the crude extracts produced by certain strains of actinomycete had heightened proportions of nutrient re-supplementation. For example, the extracts derived from the strain 7874 consistently displayed augmented growth for the majority of auxotrophic mutants tested when compared to other actinomycete strains analyzed (Figure 3).


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Therefore, we conclude that the detectability of various antimetabolites from natural products is also strain-dependent. We additionally examined the implications of nutrient re-supplementation by comparing the foldshift of the minimum inhibitory concentration (MIC) of known antimetabolites in the presence of actinomycete crude extract. DL-3-Hydroxynorvaline (2-amino-3-hydroxypentanoic acid), L-norleucine ((2S)-2-aminohexanoic

acid),

and,

MAC13772

((2-(2-Nitrophenyl)acetohydrazide),

antimetabolites of threonine, methionine, and biotin, respectively.25,

32, 33

are

known

Aligned with our previous

results, in the presence of 2.5% crude actinomycete extract, there was a negligible difference in the MIC curve for the threonine antimetabolite DL-3-hydroxynorvaline (Figure S2-A), a moderate decrease in the MIC for the methionine biosynthetic inhibitor L-norleucine , (Figure S2-B), and a significant decrease in the MIC for MAC13772 , the biotin biosynthesis inhibitor (Figure S2-C). Moreover, as demonstrated by L-norleucine, at higher concentrations of antimetabolite, antibacterial activity can be detected despite the interference from crude extract nutrient re-supplementation. This implies that only very potent molecules or high concentrations of a specific antimetabolite within a crude extract would likely be identified using conventional nutrient-rich screening methods. This concentration dependence may also explain why certain antimetabolites have been isolated previously using conventional nutrient-rich platforms. Overall, our data validate the premise of reduced antimetabolite detection in the presence of actinomycete crude extracts. Screening with Transporter Deletion Mutants Aids in the Detection of Antimetabolites The vitamin auxotrophic E. coli mutants ∆panC, ∆bioD, and ∆thiE displayed nearly 100% growth in the presence of all tested crude extracts, irrespective of nutrient-rich or nutrient-poor actinomycete growth conditions (Figure 3). We therefore prioritized how to circumvent the issue of nutrient resupplementation in hopes to improve the detection of antimetabolites targeting these pathways from natural sources.



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Recently, Coté et al., demonstrated the vital connection between the essentiality of nutrient stress genes and nutrient acquisition.34 Briefly, genes previously identified as indispensable only in nutrientlimited growth conditions can become essential in nutrient-rich growth conditions if the transporter of the specific nutrient of interest is deleted. We therefore speculated that screening for natural product antimetabolites against specific transporter deletion mutants would successfully reduce nutrient resupplementation, and introduce a target-specific approach for detecting antimetabolites from natural products (Figure 4A). Using this approach, we tested the sensitivity of the known E. coli BioA inhibitor MAC13772 in the presence of 2.5% actinomycete crude extract against wildtype E. coli BW25113 and the biotin transporter mutant strain ∆yigM. As anticipated, the sensitivity to MAC13772 was significantly improved using the E. coli ∆yigM mutant as evidenced by a more than 100-fold increase in potency (Figure 4B). A Novel Target-Specific Screen Employing Transporter Deletion E. coli Mutants The remarkably high sensitivity of the E. coli ∆yigM mutant assay with MAC13772 suggests that indeed transporter deletion strains can be used to detect specific nutrient antimetabolites, especially those present in lower concentrations or with reduced potency, to mitigate the consequences of nutrient supplementation. In order to test the efficacy of the transporter deletion screening methodology, we screened natural product crude extracts from the WAC against wildtype E. coli BW25113 and E. coli ∆yigM in M9 minimal nutrient conditions (Figure 5).35 Of the 960 extracts screened, we obtained 22 hits with the ∆yigM mutant and 10 hits with the wildtype E. coli BW25113. The 10 hits detected using wildtype E. coli BW25113 were common among both strains. Due to the increased sensitivity of the assay using the E. coli ∆yigM mutant, the 12 hits unique to this strain were predicted to inhibit the biotin biosynthesis pathway. The WAC identification numbers of these 12 hits are listed in Table S3. The biotin biosynthetic pathway involves the conversion of pimeloyl-CoA and alanine to biotin in a four step process catalyzed by the enzymes BioF, BioA, BioD and BioB.36 In an attempt to elucidate the specific target of the antimetabolites produced by the 12 actinomycete strains, we tested the suppression


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of their inhibitory activity against E. coli ∆yigM mutant strain by supplementing the media with the intermediates and final product of the biotin biosynthetic pathway; 7-keto-8-aminopelargonate (KAPA), 7,8-diaminopelargonate (DAPA), dethiobiotin (DTB), and biotin. The growth inhibition in minimal nutrient conditions that was previously observed in all 12 extracts was fully suppressed by the addition of the downstream metabolites of BioA, BioD, and BioB (DAPA, DTB, and biotin, respectively). The addition of KAPA, on the other hand, had no effect on the bioactivity of any of the extracts. We note here that the KAPA concentrations selected for supplementation were sufficient to rescue the growth of a ∆bioF mutant (Figure S3). These suppression results strongly suggested that the target of the antimetabolites produced by all 12 strains of actinomycete was BioA, also known as 7,8diaminopelargonic acid synthase, the enzyme catalyzing the conversion of KAPA to DAPA.

Bioassay Guided Isolation of ACM Dipeptides, a BioA Inhibitor, using E. coli ∆yigM Mutant Strain From the E. coli ∆yigM mutant screen we found 12 strains of actinomycete producing biotin antimetabolites. Since all of the crude extracts were identified as targeting the BioA enzyme, we hypothesized that the 12 actinomycete strains isolated were likely producing the same antimetabolite. We therefore selected one of the 12 strains, WAC5950, to isolate and purify the antimetabolite from the crude extract using chromatography coupled with bioassay-guided fractionation. The fractions were screened for their biological activity against both the wildtype E. coli BW25113 and the E. coli ∆yigM mutant strains. We were interested in the fractions that showed significant growth inhibition against the ∆yigM mutant compared to the wildtype E. coli BW25113 strain, as it indicated that these fractions contained the biotin antimetabolite. The fractions were subsequently analyzed using thin layer chromatography (TLC). The biologically active fractions showing similar spotting patterns on the TLC plates were pooled together and further purified until the TLC plates showed one unanimous spot, representing purified product. Two bioactive compounds were isolated, each as a yellowish-brown solid. These compounds were identified as methyl-valine-ACM and methyl-isoleucine-ACM using a LTQ OrbiTrap XL MS (Figure S4). The peak at m/z 293.1861 corresponds to [M + H]+-NH3 for methyl-valine-ACM (calculated



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for C16H25N2O3, 293.1865), and m/z 307.2018 corresponds to [M + H]+-NH3 for methyl-isoleucine-ACM (calculated for C17H27N2O3, 307.2022). ACM is a well characterized natural product biotin biosynthesis inhibitor first reported in 1974.37 ACM has been isolated from various strains of actinomycete as a single amino acid, or a di/tri-peptide unit, all of which exhibit antibacterial properties.38, 39 ACM is known to inhibit the BioA enzyme which catalyzes the conversion of KAPA to DAPA.40 Unfortunately, due to low yields of our isolated compounds, structure confirmation by 1H-NMR was not possible. However, the mass over charge ratios, and the suppression of growth inhibition in the presence of DAPA, DTB, biotin, but not KAPA, reaffirms that these purified compounds are indeed ACM dipeptides.38, 40 DISCUSSION In this work, we demonstrate through the growth of nutrient biosynthetic auxotrophic E. coli mutants in the presence of various actinomycete-derived crude extracts that nutrient re-supplementation from the use of conventional nutrient-rich screening platforms disguises the activity of many antimetabolites produced by actinomycetes. Indeed, this result is consistent with the overall lack of discovered antimetabolites from natural products. Interestingly, our results highlight that certain metabolic targets, noted as displaying moderate and high proportions of nutrient re-supplementation, appear to be more prone to this limitation. Importantly, this does not equate that the detection of inhibitors of these biosynthetic pathways is impossible; but rather it implies that the probability of finding inhibitors of these particular pathways is significantly reduced by a lack of assay sensitivity. Specifically, based on the high growth of the auxotrophic E. coli mutants ∆bioD, ∆thiE and ∆panC, we underline that targets of vitamin biosynthetic metabolism, including biotin, thiamine, and pantothenate, appear to be exceptionally susceptible to nutrient re-supplementation. Alarmingly, these biosynthetic metabolites have commonly been identified as the antimetabolite targets with the highest therapeutic potential.15,

16, 41, 42

In contrast with some of the other biosynthetic targets, such as serine

(which has similar enzymatic counterparts in human biosynthetic metabolism),43 mammals lack the ability to biosynthesize biotin, thiamine, and, pantothenate.16, 41, 42 Additionally, the enzymes involved in these


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biosynthetic pathways are largely conserved among bacterial pathogens, making them attractive targets for new therapies. In vivo pathogenicity models support these biosynthetic pathways as ideal candidates for novel antibacterial scaffolds.16, 41, 42, 44 Not surprisingly, despite the ideality of these targets, uncovering natural product compounds inhibiting vitamin biosynthetic pathways has proven to be extremely difficult.45 For example, a brief examination of the literature has confirmed only two previously identified natural product pantothenate antimetabolites,26, 44, 46 and only one thiamine antimetabolite.47-49 Of note, despite the apparent relative abundance of biotin biosynthetic inhibitors, a deeper investigation reveals that the majority of these scaffolds are simply variations of ACM, the first identified natural biotin-targeting antimetabolite. Moreover, Bayer et al., detail a unique synergistic mechanism associated specifically with some ACMproducing Streptomyces strains involving the co-production of biotin sequestering proteins, which may explain their previous identification from natural sources.50 Nevertheless, the mere existence of these few antimetabolites demonstrates, in general, that nature supports the selection of vitamin antimetabolites as antibacterial targets. Therefore, our results favorably suggest that there are likely additional inhibitors of these and other biosynthetic pathways that have yet to be identified. Our results demonstrate that the extent of nutrient re-supplementation is often dependent on the specific strain of actinomycetes. Several studies have previously emphasized the impact of media composition on actinomycete phenotype, antibiotic production, nutrient availability, and the onset and intensity of secondary metabolite production.12,

51-56

More specifically, the uptake and production of

nutrients required for ideal secondary metabolite production is strain dependent, which may explain the observed variations in the magnitude of nutrient re-supplementation for a specific nutrient between multiple strains.57 Therefore, we further speculate that this variability may also play a role in explaining why in previous instances, naturally derived antimetabolites targeting those metabolic targets displaying a higher susceptibility to nutrient re-supplementation have been identified. Lastly, the reduction of nutrient re-supplementation detected from those extracts derived from nutrient-poor growth conditions indicates



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that the growth of actinomycete strains in minimal nutrient conditions also represents a possible viable alternative for future isolation of natural product antimetabolites. However, as seen with several of the vitamin biosynthetic pathways, even minimal nutrient growth conditions were not enough to prevent nutrient re-supplementation, stressing the development of novel non-traditional screening approaches. The increased sensitivity of MAC13772 observed in the presence of crude extracts against the biotin transporter deletion strain E. coli ∆yigM, (Figure 4) provides a concrete example of an inventive and practical strategy that can be employed to increase antimetabolite detection from natural sources. In this work, we presented a screening protocol as a proof of principle that successfully exploits this elevated sensitivity for detecting antimetabolites using transporter deletion strains. Our preliminary screen of 960 actinomycete-derived crude extracts against wildtype E. coli BW25113 and ∆yigM strains nicely illustrates the increased antimetabolite detection in transporter deletion strains by evading nutrient resupplementation. We also demonstrate that the differential sensitivity of E. coli BW25113 and ∆yigM strains against biotin antimetabolites was conducive in a bioassay-guided isolation of ACM, a BioA inhibitor, from actinomycete crude extracts. Based on the success of these initial screens, we therefore propose the use of transporter deletion mutants for the detection of other antimetabolites from natural products. By increasing the sensitivity for antimetabolite detection using transporter deletions, we can further broaden the search for antibacterial chemical matter from natural product extracts. We believe that future application of this tool will greatly enhance the detection of antimetabolite from natural products. There are, however, a few limitations to this strategy that must be acknowledged. The intrinsic complexity of metabolism limits the utility of this tool specifically to those biosynthetic targets, such as pantothenate and biotin, which have one core nutrient-transporter into the cell. For example, many metabolites, such as branched chain amino acids, can be imported by multiple transport systems.58 As a result, evading nutrient re-supplementation may not be practicable if these shared transport systems are responsible for the influx of other critical molecules into the cell. Additionally, the detection of antimetabolites using this strategy is contingent upon the parent metabolite and antimetabolite using


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different modes of transport into the cell. Lastly, on a practical basis, we propose that any future targeted antimetabolite screens should focus on the biosynthetic targets that have the greatest potential for in vivo efficacy. CONCLUSIONS Antibiotic resistance has reached crisis proportions and action is desperately required to discover novel and effective antimicrobials. As a whole, actinomycetes represent the most dominant source for antibacterial compounds. However, antimetabolites represent only a very minor fraction of compounds so far characterized from this source. Herein, we present data showing that nutrient re-supplementation from conventionally used nutrient-rich screening platforms is responsible for masking the antibacterial activity of a sizable percentage of the available natural antimetabolites. Overall, these findings present a serious shortcoming hindering the discovery of novel targets of bacterial biosynthetic metabolism from natural sources. However, through the use of nutrient limited growth media and transporter deletion mutants, we have identified a new strategy to evade nutrient re-supplementation and enhance the detection of natural antimetabolites. The results of this research were critical to the development of this strategy, and are consequently vital to the optimization and design of additional innovative tactics for future discovery efforts.



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MATERIALS AND METHODS Actinomycetes Growth and Secondary Metabolite Extraction The actinomycete strains were obtained from the WAC. All of the actinomycete strains are referred to by their respective WAC identification number assigned by the Wright Laboratory. The identification number was modified with a “B” indicating culture growth in nutrient rich Bennett’s media, or an “M9” indicating culture growth in nutrient limited M9 minimal media. All the components for the media and the solvents were purchased from Fischer Scientific. Bennett’s media was prepared by adding 10 g potato starch, 2 g casamino acids, 1.8 g yeast, 2 mL Czapek mineral mix (10 g potassium chloride, 10 g magnesium sulfate, 12 g sodium nitrate, 0.2 g ferrous sulfate, 200 µL concentrated hydrochloric acid, 100 mL water, filter sterilized), to 1 L of deionized water and sterilized by autoclaving for 45 minutes. Nutrient limited M9 minimal media was prepared by adding 200 mL of 5 × M9 salts solution (64 g disodium hydrogen phosphate, 15 g potassium dihydrogen phosphate, 2.5 g sodium chloride, and 5 g ammonium chloride, 1 L deionized water, sterilize by autoclaving), 2 mL of 1 M magnesium sulfate, 0.1 mL of 1 M calcium chloride, and 20 mL of 20% glucose, to 780 mL of sterile deionized water. Seed cultures were inoculated from frozen stock from the WAC into 3 mL of Streptomyces Antibiotic Activity Media (SAM). SAM was prepared adding by 15 g glucose, 15 g soytone, 5 g sodium chloride, 1 g yeast, 1 g calcium carbonate, 2.5 mL glycerol, to 1 L of deionized water and sterilized by autoclaving for 45 minutes. The liquid bacterial cultures were incubated for 4-6 days at 30°C, shaking at 250 rpm. Following the initial incubation, each strain was spread to 100% confluency onto solid M9 minimal or Bennett’s media plates with 1.5% w/v agar. The bacterial strains incubated until sporulation (6-9 days) at 30 °C. The agar plates containing the sporulated actinomycete strains were crushed through a sterile 60 mL syringe and combined with 15 mL of 100% methanol solvent into 50 mL falcon tubes. Samples were then incubated for 24 h at 4 °C, shaking at 200 rpm. The agar was removed by filtering the samples through 6.5 inch Non Gauze Milk Filters (KenAG) into fresh 50 mL falcon tubes. The methanol extract was concentrated to 2 mL using the Heidolph™ Hei-Vap Rotary Evaporator Advantage and diluted to a final


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working concentration of 50% DMSO. The extracts were incubated at room temperature for 24 h, followed by centrifugation for 10 minutes at 4000 rpm using the Thermo Scientific® Sorvall Legend RT centrifuge, and stored at 4 °C. Each of the crude actinomycete extracts was prepared in triplicate from each strain for both nutrient conditions. MTT Nutrient Re-Supplementation Auxotroph Assay The Keio library of E. coli K-12 strain BW25113 nutrient auxotroph deletion strains were obtained from the Nara Institute of Science and Technology, Japan24, with the exception of the L-asparagine auxotrophic E. coli strain, ER 4813 (ATCC 25287), which required a double-deletion and was obtained from the E. Coli Genetic Stock Center (CGSC), Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA. A subset of 22 strains from the Keio collection were prioritized for the purpose of this study to have one representative auxotrophic strain for each of the major amino acid, vitamin and nucleotide biosynthetic pathways. A summary of each of the genetic deletions analyzed and their role in biosynthetic metabolism is provided in the Table S1. Cultures were prepared by inoculating a single colony of the respective knockout strain into LB media supplemented with 50 µg/mL kanamycin overnight at 37 °C shaking at 250 rpm. Cells were pelleted and washed three times with 1X PBS (pH 7.4, sterile filtered at 0.2 µm) and re-suspended into 1 mL of 1X PBS to remove any excess nutrients. The cells were diluted to a final working concentration of 1:1000 in fresh M9 minimal media, with 5% crude extract, to a final volume of 100 µl in M9 minimal media in clear flat bottom 96-well plates for overnight incubation of 16 h at 37 °C. The minimum growth control was set as growth of each deletion strain in the presence of M9 minimal media with 50 µg/mL kanamycin; the maximum growth control was set as the growth of each deletion strain in the presence of M9 supplemented growth media with 50 µg/mL kanamycin. Following incubation, 10 µl of 0.5% MTT dye solution (Thiazolyl Blue Tetrazolium Bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), ≥98.0% (M 2128 Sigma Aldrich) solubilized in 1X PBS at pH 7.4, sterile filtered at 0.2 µm) was added to each well and the incubated for 1–2 h at 37°C. Next, 10% w/v SDS buffer was added to a final volume of 200 µl and incubated at room



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temperature for 4 hours. Images of the 96-well plates were captured using the EPSON PerfectionTM v750 Pro and bacterial growth was quantified with Fiji® software using the recorded integrated density values in each well. All experiments were performed in duplicate and all data were normalized for plate specific effects as described in the data collection and analysis section later. Bioactivity Assay Bioactivity assays were conducted using wildtype E. coli K-12 strain BW25113 or the Keio deletion strains. A mid-log subculture of the E. coli strain was diluted 103 fold into fresh M9 minimal media, and set up to a final volume of 100 µl in clear flat bottom 96-well plates with or without 2.5% crude actinomycete extract for overnight incubation for 16 h at 37 °C. For known antimetabolites, bioactivity was determined using a MIC assay from 2-fold serial dilutions of a range of 1-2x the MIC in minimal nutrient conditions including L-norleucine (Sigma Aldrich, CAS 327-57-1), DL-3-hydroxynorvaline (Sigma Aldrich, CAS 2280-42-4), and MAC13772 (3B Scientific Corporation, 847-281-9822). The 96well plates were incubated overnight at 37 °C for 16 h and bacterial growth was determined by measuring the OD600nm using the Tecan Infinite® M1000 Microplate reader. The OD600nm of the plates was read prior to incubation to account for background absorbance. The MIC was defined as the lowest concentration of compound that inhibits visible growth. All experiments were performed in duplicate and all data were normalized for plate specific effects as described in the data collection and analysis section below. Nutrient-Specific Transporter Deletion Screen for Natural Product Antimetabolite Detection in Minimal Media In order to screen the WAC for biotin specific antimetabolites, 960 actinomycete-derived natural product crude extracts were screened against wildtype E. coli BW25113 and ∆yigM mutant strains, separately. For each strain, 1 µL of crude extract (final working concentration of 2% v/v) from the master compound plates was dispensed into a clear flat bottom 384-well assay plate, followed by the addition of 49 µL of a 103 fold dilution of the mid-log subculture of the E. coli strain (BW25113 or ∆yigM). High and low


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controls for both the strains constituted of 2% (v/v) DMSO and 256 µg/mL MAC13772, respectively. All the liquid handling was performed using a Biomek FX liquid handler (Beckman Coulter Inc., Fullerton, CA) in the McMaster High Throughput Screening Laboratory. Upon mixing the bacterial cultures with the crude extracts, the OD600nm of the plate was read using the Envision (Perkin Elmer, Waltham, MA) to account for any background interference due to the color of the crude extracts. The plates were incubated overnight at 37 °C for 16 h in a stationary incubator before measuring their final OD600nm. All the set-ups were done in duplicates, and the growth data was analyzed as described in the data collection and analysis section below. The extracts that resulted in

Exploiting the Sensitivity of Nutrient Transporter Deletion Strains in

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