Immunity-Guided Identification of Threonyl-tRNA Synthetase as the Molecular Target of Obafluorin, a β-Lactone Antibiotic

Article abstract of DOI:10.1021/acschembio.9b00590

To meet the ever-growing demands of antibiotic discovery, new chemical matter and antibiotic targets are urgently needed. Many potent natural product antibiotics which were previously discarded can also provide lead molecules and drug targets. One such example is the structurally unique β-lactone obafluorin, produced by Pseudomonas fluorescens ATCC 39502. Obafluorin is active against both Gram-positive and -negative pathogens; however, the biological target was unknown. We now report that obafluorin targets threonyl-tRNA synthetase, and we identify a homologue, ObaO, which confers immunity to the obafluorin producer. Disruption of obaO in P. fluorescens ATCC 39502 results in obafluorin sensitivity, whereas expression in sensitive E. coli strains confers resistance. Enzyme assays demonstrate that E. coli threonyl-tRNA synthetase is fully inhibited by obafluorin, whereas ObaO is only partly susceptible, exhibiting a very unusual partial inhibition mechanism. Altogether, our data highlight the utility of an immunity-guided approach for the identification of an antibiotic target de novo and will ultimately enable the generation of improved obafluorin variants.

Full text of DOI:10.1021/acschembio.9b00590

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Article
Immunity guided identification of threonyl-tRNA synthetase
as the molecular target of obafluorin, a beta-lactone antibiotic
Thomas A Scott, Sibyl F. D. Batey, Patrick Wiencek, Govind
Chandra, Silke Alt, Christopher S. Francklyn, and Barrie Wilkinson
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00590 • Publication Date (Web): 01 Nov 2019
Downloaded from pubs.acs.org on November 2, 2019
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Immunity guided identification of threonyl-tRNA synthetase as the
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molecular target of obafluorin, a β-lactone antibiotic
Thomas A. Scott,ac† Sibyl F. D. Batey,a† Patrick Wiencek,b Govind Chandra,a Silke Alt,a
Christopher S. Franklynb* and Barrie Wilkinsona*
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a Department of Molecular Microbiology, John Innes Centre, Norwich Research Park,
Norwich NR4 7UH, UK
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b Department of Biochemistry, College of Medicine, University of Vermont, Burlington, VT
05405, USA
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c Current address: Institute of Microbiology, Eidgenössische Technische Hochschule (ETH)
Zürich, Vladimir-Prelog-Weg 4, 8093 Zürich, Switzerland
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† These authors contributed equally to this work.
*Email: barrie.wilkinson@jic.ac.uk
Abstract
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To meet the ever-growing demands of antibiotic discovery, new chemical matter and
antibiotic targets are urgently needed. Many potent natural product antibiotics which
were previously discarded can also provide lead molecules and drug targets. One
such example is the structurally unique β-lactone obafluorin, produced by
Pseudomonas fluorescens ATCC 39502. Obafluorin is active against both Gram-
positive and -negative pathogens, however the biological target was unknown. We
now report that obafluorin targets threonyl-tRNA synthetase and we identify a
homologue, ObaO, which confers immunity to the obafluorin producer. Disruption of
obaO in P. fluorescens ATCC 39502 results in obafluorin sensitivity, whereas
expression in sensitive E. coli strains confers resistance. Enzyme assays demonstrate
that E. coli threonyl-tRNA synthetase is fully inhibited by obafluorin, whereas ObaO is
only partly susceptible, exhibiting a very unusual partial inhibition mechanism.
Altogether, our data highlight the utility of an immunity-guided approach for the
identification of an antibiotic target de novo and will ultimately enable the generation
of improved obafluorin variants.
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31 Introduction
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Natural products, especially those produced by microorganisms, account for
approximately 80% of all antibiotics in clinical use.1 Moreover, numerous other natural
products have been identified with potent antibacterial activity but have failed to
progress into clinical use, or even investigation, due to confounding activities such as
off target effects, poor bioavailability, or a lack of broad-spectrum activity. Given recent
predictions that infectious disease will become the biggest killer of humans by 2050
due to the alarming increase in antimicrobial resistance,2 reevaluation of these
previously discarded natural products is timely. By focusing on structurally unique
molecules we hypothesize that new targets and modes of action might be uncovered
and provide an opportunity to avoid cross resistance with existing clinical classes. One
such molecule is the β-lactone antibiotic obafluorin (1).
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β-Lactone rings occur infrequently in Nature but are constituents of several different
natural product classes.3 Structurally similar to β-lactam rings, they are effective
electrophiles able to form covalent linkages with nucleophilic residues of target
proteins4 and possess significant therapeutic value as hydrolase inhibitors.5 Prominent
examples include the hybrid polyketide-non-ribosomal peptide (PK-NRP)
Salinosporamide A (marizomib), which is a potent 20S proteasome inhibitor that is
currently in Phase III clinical trials as a treatment for glioblastoma,6 and
tetrahydolipstatin (orlistat), a semi-synthetic derivative of the PK-NRP lipstatin
approved by the US Food and Drug Administration for the treatment of obesity due to
its powerful lipase inhibitory activity.7 Further validated targets of β-lactone inhibitors
include serine hydrolase and polyketide synthase enzymes associated with mycolic
acid biosynthesis, inhibition of which is lethal for Mycobacterium tuberculosis,8 as well
as esterases, cutinases, homoserine transacetylase, cathespin A, ClpP and N-
acylethanolamine acid amidase.9, 10
Due to their therapeutic potential, much effort has been put into elucidating the
genetic and biochemical basis for β-lactone ring formation during natural product
biosynthesis.11 Recent work in our lab12 and by others13 characterized the biosynthetic
pathway to the β-lactone antibiotic 1 from Pseudomonas fluorescens ATCC 39502. 1
is assembled by ObaI, a bimodular non-ribosomal peptide synthetase which catalyzes
amide bond formation between 2,3-dihydroxybenzoic acid (2), and the
nonproteinogenic amino acid (2S,3R)-2-amino-3-hydroxy-4-(4-nitrophenyl)butanoate
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(3), the product of a rare L-threonine transaldolase (Figure 1).12 Critically, it is this
unusual β-hydroxy-α-amino acid precursor that comprises the functional groups which
give rise to the β-lactone ring of 1, following cyclization catalyzed by a rare type I
thioesterase domain present in ObaI, which possesses a noncanonical active site Cys
residue.
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Despite undergoing facile hydrolysis and ring opening in the presence of
nucleophiles, 1 exhibits potent antibacterial activity against a range of Gram-positive
bacteria and is also active against Gram-negative bacteria including E. coli and various
Pseudomonas aeruginosa strains (Table 1 and Supplementary Figure 1).14, 15 When
dosed systemically, it protects mice challenged with Streptococcus pyogenes (ED50 =
50 mg kg-1) and is also reported to cause an unusual cell-elongation phenotype in E.
coli cells exposed to sub-lethal doses. These various observations suggest that 1 acts
in a specific manner rather than as a general acylating agent, yet, despite this unusual
activity profile, the molecular target for 1 remained obscure. Given the urgent need for
new antibacterial targets for antibiotic development, we turned our attention to
identifying the molecular target of 1.
NH2
H
ObaI
ATP
N
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OH O
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Figure 1 Biosynthesis of obafluorin (1) in P. fluorescens ATCC 39502 from 2,3-
dihydroxybenzoic acid (2) and (2S,3R)-2-amino-3-hydroxy-4-(4-
nitrophenyl)butanoate (3).
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87 RESULTS AND DISCUSSION
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Bioinformatic analysis identifies a putative 1 immunity gene. We set out to
identify the target of 1 using an immunity-guided approach, combining genetic
analyses with chemical complementation, bioassays, and in vitro enzyme assays. We
began our investigation by examining the genomic neighborhood surrounding the oba
biosynthetic gene cluster (BGC) (MiBIG accession: BGC0001437; GenBank
accession: KX931446.2) in search of immunity gene candidates. Genes encoding
immunity determinants commonly co-occur with the biosynthetic loci required for the
production of natural product antibiotics,16 a phenomenon that has inspired recent
genome-mining efforts to identify novel scaffolds with known targets.17-19 Comparative
genomic analysis of the oba BGC characterized in P. fluorescens ATCC 39502 with
other putative 1 BGCs present in the genomes of two P. fluorescens soil isolates,
several Burkholderia species and Chitiniphilus shinanonensis DSM 23277
(Supplementary Table 1) allowed the identification of a minimal set of genes
conserved across all clusters (Figure 2). In addition to those previously ascribed
biosynthetic or regulatory functions,12 only one additional gene, encoding a putative
threonyl-tRNA synthetase (ThrRS), was present in all putative 1 BGCs; we have
named this gene obaO (GenBank accession: KX931446.2).
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Aminoacyl-tRNA synthetases (aaRSs) play an essential role in protein synthesis,
specifically activating and loading amino acids onto their cognate tRNAs.20 Although
ubiquitous, divergence in aaRS sequence structure has led to the evolution of natural
product (NP) antibiotics that selectively target those of competing organisms.21, 22
aaRS-targeting NPs have been applied in both crop protection and clinical use, with
the LeuRS inhibitor Agrocin TM84 used to treat crown gall disease23 and the IleRS
inhibitor mupirocin used topically in humans to treat skin infections.24 ThrRS is
targeted by the polyketide NP borrelidin, which acts by simultaneously occupying the
three catalytic subsites, along with a non-catalytic fourth subsite.25, 26 In several
instances, an immunity isoform of the target aaRS is encoded within the NP BGC as
an addition to the primary housekeeping copy encoded elsewhere in the genome of
the producing-organism.27-29 Indeed, the putative oba BGC-associated ThrRS
homologues all represent second copies in their respective genomes and, likewise, an
additional copy of ThrRS is only observed in the presence of the oba BGC amongst
these genera.
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Figure 2 Comparative analysis of obafluorin (1) biosynthetic gene clusters. Genes are
color-coded according to their function, with putative immunity colored in light blue. 4-
NPP = 4-Nitrophenylpyruvate, an intermediate in the biosynthesis of (2S,3R)-2-amino-
3-hydroxy-4-(4-nitrophenyl)butanoate (3).
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obaO confers immunity to 1 in the native producer. To confirm the ability of
ObaO to confer 1 immunity in the native producer, we attempted obaO inactivation
experiments using the pTS1 suicide vector generated previously12 to perform
knockouts in biosynthetic oba genes. However, following double crossover and
counter-selection, we observed that resulting colonies were always exclusively wild-
type (WT), consistent with a selection pressure against losing the immunity
determinant when the oba BGC is functional.
To circumvent this issue, we deleted obaO in a ΔobaL background in which 1
production is abolished but can be rescued by exogenous supplementation with 2. In
the absence of exogenous 2 the growing strains accumulate increased levels of the
shunt metabolites 4-nitrophenylethanol and 4-nitrophenylacetate, which is indicative
of an otherwise functional biosynthetic pathway.12 We were successful in generating
the ΔobaLΔobaO strain and resulting mutants were verified by PCR amplification
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across the newly deleted region, with subsequent sequencing of the amplicon. When
the ΔobaLΔobaO strain was grown in production medium supplemented with 2, growth
was strongly inhibited, in contrast to cultures of WT and ΔobaL supplemented with 2,
and un-supplemented controls (Figure 3). The ΔobaLΔobaO strain was
complemented genetically by ectopic expression of obaO using the pJH10TS vector
described previously,12 which restored WT-like growth and 1 production when the
strain was grown in 2-supplemented production medium (Figure 3). Taken together,
these observations are consistent with a role for ObaO in conferring immunity to 1 in
the native producer, P. fluorescens ATCC 39502.
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Figure 3 ObaO is the immunity determinant for 1 in P. fluorescens 39502. WT, ΔobaL,
ΔobaLΔobaO and ΔobaLΔobaO::obaO strains were grown ± 2,3-dihydroxybenzoic
acid (2,3-DHBA, 2) (0.2 mM). As shown previously,12 2 restores obafluorin (1)
production for ΔobaL, whereas the addition of 2 to ΔobaLΔobaO abolishes growth. a)
Aliquots of each strain after 14 h growth, with the purple coloration being indicative of
1 production. b) Log phase growth curves, showing complete absence of growth for
ΔobaLΔobaO + 2. Each data point is the average of three biological repeats and bars
show the standard error. c) Representative HPLC chromatograms at 270 nm for each
condition at 14 h; 1 elutes at 10.1 min (red dashed line) and the shunt metabolites 4-
nitrophenylethanol (blue dashed line) and 4-nitrophenylacetate (green dashed line)
elute at 7.7 min and 7.9 min respectively.
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obaO confers high-level resistance to 1-sensitive E. coli strains. To further
validate our hypothesis, we introduced the obaO gene into 1-sensitive bacterial strains
to investigate its ability to confer resistance to those strains when challenged with 1.
Given that E. coli was previously identified as being sensitive to 1,14 we tested the
reference strain E. coli ATCC 25922, and E. coli NR698 which has increased
permeability to antibiotics due to compromised outer-membrane lipopolysaccharide
assembly.30 Off-target reactions of 1 with culture medium components precluded the
use of microbroth assays, so we turned to disc-diffusion based approaches. We found
that 1 binds to paper discs, as reported previously,31 so we instead employed a spot-
on-lawn approach by directly applying a solution of 1 dissolved in acetonitrile to the
surface of agar bioassay plates. Using this method, 1 was shown to have a minimum
inhibitory concentration (MIC) of 256 μg mL-1 for E. coli 25922, and 4 μg mL-1 for E.
coli NR698 (Figure 4, Table 1 and Supplementary Figure 1). However, when obaO
was expressed ectopically in both strains using the pJH10TS vector, both became
resistant to 1 up to the highest concentration tested of 2000 μg mL-1. This represents
increases in the MIC of 1 of at least 8- and 250-fold for E. coli ATCC 25922 and E. coli
NR698, respectively, relative to empty vector controls. Therefore, obaO confers
transferable high-level resistance to 1.
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To determine whether simple overexpression of an additional copy of ThrRS is
sufficient to provide resistance to 1, we turned our attention to the primary
housekeeping versions of ThrRS present in P. fluorescens ATCC 39502 (PfThrRS)
and E. coli (EcThrRS; GenBank accession: WP_001144202.1). The ΔobaLΔobaO
strain was complemented by ectopic expression of the genes encoding either PfThrRS
or EcThrRS (amplified from E. coli BL21 (DE3)) using the pJH10TS vector12 and was
then grown in production medium supplemented with 2 to activate 1 biosynthesis. We
observed that culture growth plateaued after 5 h and that the production of 1 was not
restored in both cases (Figure 5, panel a). A similar phenotype was observed in the
IleRS mupirocin immunity gene knock out and attributed to the stringent response,
resulting from amino acid starvation.32 Thus, we hypothesize that 1 is initially produced
in growing cells but reaches localized levels that inhibit the elevated levels of ThrRS
leading to accumulation of uncharged tRNAThr and induction of the stringent response.
This results in transcriptional shutdown of non-essential genes and is consistent with
the observed growth inhibition, and the lack of accumulated shunt metabolites and 1
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(Figure 5, panel a). In contrast, the un-supplemented control suffers minimal growth
defects and retains the ability to accumulate shunt metabolites. Similarly, we found
that when E. coli bioassay strains expressing either an additional copy of their native
EcThrRS or PfThrRS were challenged with 1, there was only a modest increase in 1-
resistance for E. coli NR698, with a 1 MIC of 16 μg mL-1 (2-fold increase versus control).
The MIC of 1 against E. coli 25922 was unchanged at 256 μg mL-1 (Figure 5, panel b
and Table 1). This suggested that ObaO functions as the primary 1 resistance
determinant.
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Figure 4 ObaO confers transferable immunity to obafluorin (1)-sensitive E. coli strains.
1 spot-on-lawn assays with reference strain E. coli 25922 and permeable strain E. coli
NR698, with zones of clearing indicating growth inhibition. Numbers indicate 1
concentrations (in µg mL-1), with MeCN as a negative control and kanamycin (50 µg
mL-1) as a positive control. ObaO confers resistance to >2000 µg mL-1 1, in contrast
to the WT strains and empty vector controls. Images are representative of three
biological repeats for each strain.
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Figure 5 PfThrRS and EcThrRS do not confer immunity to obafluorin (1). a)
Complementation of P. fluorescens ΔobaLΔobaO with ThrRS genes. ΔobaLΔobaO
was complemented with PfThrRS or EcThrRS in pJH10TS and grown ± 2,3-
dihydroxybenzoic acid (2,3-DHBA, 2) (0.2 mM). In contrast to obaO (Figure 3) growth
plateaued after ~5 h for PfThrRS and EcThrRS strains fed with 2, and 1 production
was not restored. Aliquots of cultures after 14 h growth, log phase growth curves and
representative HPLC chromatograms are shown, analogous to Figure 3. Each growth
curve data point is the average of three biological repeats, with bars showing the
standard error and HPLC analysis was carried out on every biological repeat. 1 is
initially produced in growing cells but reaches localized levels that inhibit the elevated
levels of ThrRS, leading to accumulation of uncharged tRNAThr and induction of the
stringent response. This is consistent with the observed growth inhibition and the lack
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of accumulated shunt metabolites and 1. b) 1 spot-on-lawn assays as in Figure 4, with
reference strain E. coli 25922 and permeable strain E. coli NR698 overexpressing
either PfThrRS or an additional copy of EcThrRS: in contrast to ObaO, there is no
difference in the MIC value for strain E. coli 25922 and a modest 2-fold increase in
MIC for strain E. coli NR698. Images are representative of three biological repeats for
each strain.
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Strain
1 MIC (µg mL-1)
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B. subtilis
4
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MRSA
E. coli ATCC 25922
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>2000
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E. coli ATCC 25922 pJH10TS
E. coli ATCC 25922 pJH10TS-obaO
E. coli ATCC 25922 pJH10TS-EcThrRS
E. coli ATCC 25922 pJH10TS-PfThrRS
E. coli NR698
E. coli NR698 pJH10TS
E. coli NR698 pJH10TS-obaO
E. coli NR698 pJH10TS- EcThrRS
E. coli NR698 pJH10TS- PfThrRS
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>2000
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Table 1. Obafluorin (1) is active against both Gram-positive and Gram-negative
bacteria. 1 MICs were determined by the spot-on-lawn method, as shown in Figures
4, Figure 5 and Supplementary Figure 1. Experiments were performed in triplicate for
each strain. Further details on each strain can be found in Supplementary Table 2.
1 is potent inhibitor of EcThrRS and a partial inhibitor of ObaO. To confirm that
1 is a bona fide inhibitor of ThrRS, we examined the formation of Thr-tRNAThr by
EcThrRS in the presence of 0–5 μM 1. The rate of aminoacylation of tRNA decreased
progressively over this concentration range, with complete inhibition occurring around
1 μM (Figure 6, panel a). Fitting of the data to a dose response equation returned an
IC50 of 92  21 nM, confirming that 1 is a potent inhibitor of EcThrRS (Figure 6, panel
c). Aminoacylation assays with ObaO indicated that, in the absence of 1, ObaO
exhibits activity comparable to EcThrRS. When ObaO was challenged with increasing
concentrations of 1, aminoacylation was initially decreased but not completely
inhibited at the highest concentrations of 1 (Figure 6, panel b). In order to fit the data
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and derive an IC50, a modified dose response equation was employed that includes a
term denoting residual enzyme activity at saturating inhibitor concentrations.33 Fitting
the data to this equation gave an IC50 of 50  25 nM and a fractional residual activity
of 0.35  0.07 (Figure 6, panel d), signifying a partial inhibition mechanism. This is a
rarely reported phenomenon, whereby the activity of an enzyme cannot be driven to
zero, even at the highest concentrations of inhibitor. It is often attributed to
experimental artefacts, such as limited compound solubility at high concentrations,33
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Figure 6 Complete and partial Inhibition of EcThrRS and ObaO, respectively, by
obafluorin (1). Progress curves for EcThrRS (a) and ObaO (b) in the presence of
varying (0–5 µM) concentrations of 1. Enzyme (10 nM) and varying concentrations of
1 were pre-incubated for 30 min prior to adding to a reaction containing saturating
substrates. Reactions (n = 3) included enzyme at 10 nM and saturating concentration
of tRNA, threonine, and ATP. The progress curves were fit to a linear equation to
derive initial rates. Error bars represent the standard error for each time point. Dose
response curves for EcThrRS (c) and ObaO (d) were calculated from the data in a
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and b by plotting the fractional velocity at each of seven different inhibitor
concentrations against log [1]. Details of the fitting routines are presented in the
Methods. CPM = counts per min.
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however, the contrasting EcThrRS and ObaO profiles with the same 1 concentration
ranges, clearly exclude that possibility in this case. Previous examples of partial
inhibition mechanisms include the inhibition of reverse transcriptase by the non-
nucleoside inhibitor tetrahydro-benzodiazepine34 and inhibition of tyrosinase by
substituted benzaldehydes.35
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In the case of mupirocin, differences in resistant and susceptible IleRSs can be
pinpointed to amino acid polymorphisms in the binding site, with resistance associated
with eukaryotic-like enzymes.36 For EcThrRS and ObaO all amino acid side chains
that interact with tRNA, the catalytic Zn2+ ion and the Thr-AMP substrate intermediate
in the E. coli enzyme are conserved (Supplementary Figure 2).37 To investigate the
origin of 1-resistant ThrRS homologues we carried out a phylogenetic analysis of
ObaO, homologues from other putative 1 BGCs, and primary copies of ThrRS from a
range of organisms. Consistent with the conserved nature of this essential
housekeeping protein we observed low bootstrap values at basal nodes of the tree,
making it difficult to draw any firm conclusions about the origin of the 1 immunity gene.
However, the resulting tree does suggest that the resistance isoforms are more closely
related to one another than to housekeeping copies from the same species.
302 Conclusion
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Using a combination of bioinformatics, mutational analyses, bioassays, and in vitro
enzyme assays we identified the gene product ObaO as the immunity determinant of
1, which functions as a resistant isoform of the 1 target ThrRS. Immunity guided natural
product target discovery has been the subject of much interest in recent years,18, 38
however there are still relatively few examples in which a hitherto unknown target has
been identified de novo from its immunity determinant.39, 40 Our data demonstrate the
utility of this approach, especially in cases such as ours, when the inherent lability of
the compound in aqueous solution precluded the generation of spontaneous mutants.
We established that while expression of an extra copy of the native PfThrRS or the E.
coli EcThrRS could restore some growth in the obaO deficient P. fluorescens strain,
only complementation with ObaO restored WT-like growth and 1 production. We also
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showed that expression of EcThRS and PfThrRS did not significantly alter the MIC for
1 in E. coli strains. These data indicate that ObaO does not provide immunity by a
simple increase in levels of a functional ThrRS. In this preliminary study, the details of
the specific mechanism of inhibition, and its competitive or non-competitive basis,
have not been fully resolved. However, the data reported here do appear to rule out
the simple model in which ObaO has acquired resistance to 1 owing to absence of
inhibitor binding. Instead, the partial inhibition of ObaO by 1 suggests the possibility
that 1 binds to a region of the active site that does not completely overlap with the site
of threonine or ATP binding, but nevertheless influences the ability of ObaO to execute
conformational changes associated with catalysis. Indeed, a similar mechanism for
the ThrRS inhibitor borrelidin was proposed as part of the determination of the X-ray
structure of the ThrRS:borrelidin complex.26 One prediction of such a model is time
dependent inhibitor behavior, which will be addressed in future experiments.
Our findings set the scene for future study on the mechanism by which 1 inhibits
sensitive ThrRS, and into the resistance determinants of ObaO. In turn, this will enable
rational drug design to generate improved versions of obafluorin.
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330 METHODS
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General Methods. All primers, plasmids and strains are reported in Supplementary
Tables 2–3. All strains were maintained on lysogeny broth (LB) medium with
appropriate selection at 37 °C for E. coli strains or 28 °C for P. fluorescens ATCC
39502 strains.
Construction of knockout, complementation and expression plasmids.
Plasmids were constructed using standard ligation methods or using a Gibson
Assembly® Cloning Kit (NEB) according to the manufacturer’s instructions. Gene
sequences were amplified from genomic DNA templates and vector backbones were
prepared by either digestion with restriction endonucleases or PCR-amplification from
plasmid DNA.
ΔobaO strains were generated using the suicide vector pTS1, constructed
previously.12 Primers were designed to amplify 800–1,200 bp flanking regions of the
obaO protein coding sequence (PCS) for cloning into pTS1 between XbaI and AvrII
sites, and AvrII and BmtI sites. Flanking regions were designed to comprise 10–50
PCS codons at either end of the obaO gene to minimize polar effects, leaving a
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truncated chromosomal copy of the gene with an in-frame deletion and internal AvrII
site cloning artefact following double homologous recombination.
For genetic complementation, the obaO, EcThrRS and PfThrRS PCSs were cloned
from start to stop either as BmtI-KpnI fragments (obaO and EcThrRS) or as a NdeI-
XbaI fragment (PfThrRS). These were ligated into pJH10TS12 for introduction and
ectopic expression in the ΔobaLΔobaO strain. The obaO and EcThrRS PCSs were
also cloned into pET28a(+) as NdeI-XhoI fragments, for expression of N-terminally
hexahistidine tagged proteins.
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Mutagenesis and complementation experiments. pTS1-obaO knockout
constructs were introduced into P. fluorescens ATCC 39502 strains via conjugation
from E. coli S17-1 pir and single-crossover mutants were selected for on LB
supplemented with 50 µg mL-1 tetracycline (Tc50). Positive colonies were cultured
overnight in antibiotic-free medium to allow time for a second cross-over event to occur.
sacB counter-selection could then be performed by plating culture dilutions on LB
supplemented with 7% (w/v) sucrose to select against retention of the pTS1 vector
backbone. Colony PCR was then performed to distinguish double cross-over mutants
from WT colonies, with subsequent Sanger sequencing (Eurofins Genomics) to
confirm the expected deletion.
For the WT strain, all of the resulting colonies tested retained the obaO gene,
presumably due to selection against losing the 1 immunity gene. Consistent with this
hypothesis, it was possible to delete obaO in the previously reported ΔobaL strain,12
in which 1 biosynthesis is abolished. The resulting ΔobaLΔobaO strain was
subsequently complemented with pJH10TS-obaO, pJH10TS-EcThrRS and pJH10TS-
PfThrRS constructs, or a pJH10TS empty vector control, via conjugation and positive
clones were selected for on LB Tc50. Clones were screened by colony PCR and
confirmed by sequencing.
Analysis of growth and metabolite production. WT and recombinant P.
fluorescens ATCC 39502 strains were grown in 1 Production Medium (OPM)
comprising: yeast extract 0.5% (w/v), D-glucose 0.5% (w/v), MgSO4.7H2O 0.01%
(w/v), and FeSO4 0.01% (w/v), dissolved in Milli-Q (Merck Millipore) filtered water. A
toothpick was used to inoculate 100 mL of OPM seed culture (250 mL Erlenmeyer
flask) from a single colony, with subsequent growth for 24 h at 25 °C, 300 rpm. 1 mL
of this culture was used to inoculate 100 mL (500 mL Erlenmeyer flask) OPM
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production cultures, which were supplemented with either 2 in DMSO to a final
concentration of 0.2 mM or DMSO only (final concentration of 0.2% DMSO in both
cases).
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For growth curves, production cultures were grown at 25 °C, 300 rpm and OD600
measurements relative to an OPM blank were recorded every hour for 10 h. For
metabolite analysis, after 14 h of growth 1 mL of culture broth was extracted with an
equal volume of ethyl acetate by mixing at 1,400 rpm for 15 min. Samples were then
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centrifuged (15,682
g for 15 min), and the organic phase was collected and
×
evaporated. The resulting extract was dissolved in MeCN (250 µL) and centrifuged
(15,682 g for 20 min) to remove any remaining cell debris, before HPLC analyses.
×
Analytical HPLC. Samples were analyzed on an Agilent 1100 system using a
Gemini 3 µm NX-C18 110 Å, 150 x 4.6 mm column (Phenomenex) with a gradient
elution: MeCN/0.1% (v/v) TFA (H2O) gradient from 10/90 to 100/0 0–15 min, 100/0 for
15–16 min, gradient to 10/90 16–16.50 min and 10/90 for 16.50–23 min. The flow rate
was 1 mL min-1 and the injection volume of each sample 10 µL. Chromatograms were
recorded at 270 nm, the λmax for 1. The identity of 1 peaks were confirmed by
comparison with an authentic standard. Retention times were: 1 10.1 min, 4-
nitrophenylethanol 7.7 min and 4-nitropenylacetate 7.9 min, the latter two compounds
being shunt metabolites which are elevated in non-complemented ΔobaL strains.
Antibacterial assays. For agar diffusion bioassays, test strains were grown for 16-
18 h in 5 mL LB cultures, containing appropriate selection. 500 µL of each culture was
used to inoculate 50 mL LB cultures, which were incubated at 37 °C with 250 rpm until
OD600 = 0.3–0.4. Cultures were diluted 1:10 with molten soft nutrient agar (SNA),
before pouring into appropriately sized petri dishes to set. Serial dilutions of 1 were
prepared in MeCN and 4 µL of each dilution was applied directly onto the SNA surface.
Kanamycin (50 µg mL-1) was used as a positive control, and MeCN as a negative
control. Plates were incubated at 25 °C for 16–18 h. The MIC was defined as the
lowest concentration of compound that resulted in a zone of inhibition. Experiments
were carried out in at least triplicate for each strain.
Protein expression and purification. E. coli NiCo21(DE3) (NEB) carrying pLysS
and either pET28a(+)-EcThrRS or pET28a(+)-obaO were used to express and purify
proteins using immobilized metal affinity chromatography (IMAC) and size exclusion
chromatography (SEC), as described previously.12 Proteins were purified in a buffer
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containing 25 mM Tris/HCl at pH 8.0 with NaCl (300 mM), MgCl2 (10 mM) and glycerol
(10% v/v), with the addition of 250 mM imidazole for IMAC elution Protein identities
were confirmed by excising bands from SDS-PAGE gels, subjecting them to tryptic
digest according to standard procedures adapted from Shevchenko et al.41 and
LCMS/MS analysis of peptide fragments with an Orbitrap-FusionTM mass
spectrometer (Thermo Fisher).
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In vivo tRNAThr transcription and purification. To obtain purified tRNAThr for
kinetics and aminoacylation assays, tRNAThr was overexpressed in E. coli and purified
by gel electrophoresis and electroelution as described in previous work.42 E. coli
tRNAThr was expressed in BL21 E. coli cells and purified via phenol chloroform
extraction. The tRNAThr was then precipitated overnight in 2.5x volume EtOH and 0.1x
volume sodium acetate and subjected to centrifugation. After washing the pellet with
75% v/v EtOH it was resuspended in 10 mM HEPES pH 6.0. This sample was then
mixed with 6x blue loading dye and loaded into a large urea gel (6.5% polyacrylamide
(19:1 acrylamide:bisacrylamide), 8 M urea, and 0.5 M sodium acetate pH 5.0). The gel
was subject to 50 W until the dye front almost ran off the gel, at which point the gel
was imaged via a UV light box and the tRNAThr band was identified. This band was
excised, chopped and placed into an electroeluter apparatus (Whatman/Schleicher &
Schuell) overnight. After electroelution, the purified sample was again precipitated with
2.5x volume EtOH and 0.5x volume sodium acetate and then resuspended in TE pH
6.0 buffer.
Aminoacylation Assay. To measure the effects of 1 on ThrRS canonical tRNA
charging activity, an assay modified from Ruan et al.43 was used, where active enzyme
was incubated with its necessary substrates and its activity measured usng 14C labeled
Thr and a liquid scintillation counter. Purified ThrRS protein (10 nM) was pre-incubated
with varying concentrations of 1 for 10 min. After pre-incubation, ThrRS and 1 were
added to a master reaction mixture with the final concentrations of 100 mM HEPES
pH 7.0, 4 mM ATP, 10 mM MgCl2, 50 M 14C labeled Thr (Moravek), and 5 µM tRNAThr.
This mixture was then incubated for 10 min, with time points being taken at 1, 2.5, 5,
and 10 min. At each time point, three 5 µL aliquots were spotted onto 5% (v/v)
trichloroacetic acid (TCA) presoaked 3MM Whatman paper (Sigma-Aldrich). After
letting the spots dry, the Whatman paper was washed three times with 5% TCA, and
once with 95% v/v EtOH. Whatman paper was dried and the counts on each square
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of paper analyzed with a liquid scintillation counter using Hydrofluor Liquid Scintillation
Fluid (National Diagnostics). To calculate IC50, fractional initial velocities (velocity of
inhibited reaction/velocity in the absence of inhibitor) were plotted against the log of 1
concentration and then fitted to equation 1.32
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(1)
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For ObaO, a modified version of the equation was used to take into account the
observed partial inhibition:
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(2)
455 Conflicts of interest
There are no conflicts to declare
456
457 Acknowledgements
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This work was supported by the Biotechnology and Biological Sciences Research
Council via institute strategic program BBS/E/J/00PR9791 (to the John Innes Centre
(JIC), and research grant BB/P021506/1 to B.W. The contributions of P.W. and C.S.F.
were supported by National Institute of General Medical Sciences grant GM54899-20.
We thank L. Hill and G. Saalbach (JIC) for excellent metabolomics support.
Supporting Information Available: This material is available free of charge via the
Internet. Figures S1-S3, Tables S1-S3, and Gene and Protein Sequences.
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