Genetic insights into pyralomicin biosynthesis in Nonomuraea spiralis IMC A-0156

Article abstract of DOI:10.1021/np400159a

The biosynthetic gene cluster for the pyralomicin antibiotics has been cloned and sequenced from Nonomuraea spiralis IMC A-0156. The 41 kb gene cluster contains 27 ORFs predicted to encode all of the functions for pyralomicin biosynthesis. This includes nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) required for the formation of the benzopyranopyrrole core unit, as well as a suite of tailoring enzymes (e.g., four halogenases, an O-methyltransferase, and an N-glycosyltransferase) necessary for further modifications of the core structure. The N-glycosyltransferase is predicted to transfer either glucose or a pseudosugar (cyclitol) to the aglycone. A gene cassette encoding C₇-cyclitol biosynthetic enzymes was identified upstream of the benzopyranopyrrole-specific ORFs. Targeted disruption of the gene encoding the N-glycosyltransferase, prlH, abolished pyralomicin production, and recombinant expression of PrlA confirms the activity of this enzyme as a sugar phosphate cyclase involved in the formation of the C₇-cyclitol moiety.

Full text of DOI:10.1021/np400159a

Article
pubs.acs.org/jnp
Genetic Insights into Pyralomicin Biosynthesis in Nonomuraea
spiralis IMC A‑0156
Patricia M. Flatt,† Xiumei Wu, Steven Perry, and Taifo Mahmud*
Department of Pharmaceutical Sciences, Oregon State University, Corvallis, Oregon 97331-3507, United States
S
* Supporting Information
ABSTRACT: The biosynthetic gene cluster for the pyralomicin
antibiotics has been cloned and sequenced from Nonomuraea
spiralis IMC A-0156. The 41 kb gene cluster contains 27 ORFs
predicted to encode all of the functions for pyralomicin
biosynthesis. This includes nonribosomal peptide synthetases
(NRPS) and polyketide synthases (PKS) required for the
formation of the benzopyranopyrrole core unit, as well as a suite
of tailoring enzymes (e.g., four halogenases, an O-methyl-
transferase, and an N-glycosyltransferase) necessary for further
modifications of the core structure. The N-glycosyltransferase is
predicted to transfer either glucose or a pseudosugar (cyclitol)
to the aglycone. A gene cassette encoding C7-cyclitol biosynthetic enzymes was identified upstream of the benzopyranopyrrole-
specific ORFs. Targeted disruption of the gene encoding the N-glycosyltransferase, prlH, abolished pyralomicin production, and
recombinant expression of PrlA confirms the activity of this enzyme as a sugar phosphate cyclase involved in the formation of the
C7-cyclitol moiety.
he pyralomicins are a group of antibiotics isolated from
the soil bacterium Nonomuraea spiralis IMC A-0156 that
agent acarbose, the antitumor agent mitomycin C, and the
sunscreen compounds mycosporine-like amino acids.10−12
Detailed BLAST and maximum likelihood analysis of protein
sequences from the superfamily of SPCs has revealed that while
they uniformly share a number of highly conserved motifs, each
family of SPC has a unique signature of altered binding-pocket
residues.12 Thus, the primary sequence can be used as a tool to
predict the enzymatic function and class of natural products
that will be produced by any given SPC or used to develop
degenerate primers to amplify specific SPC subclasses.
In this study, we have utilized the signature sequences within
the SPC superfamily to design degenerate primers specific for
the 2-epi-5-epi-valiolone synthase family of enzymes. The
degenerate primers were then used to PCR amplify a 2-epi-5-
epi-valiolone synthase homologue from the pyralomicin
producer N. spiralis IMC A-0156. Recombinant expression
and biochemical characterization of the homologue PrlA have
confirmed the activity of the enzyme as a 2-epi-5-epi-valiolone
synthase. The prlA gene sequence was subsequently used as a
homologous probe to screen a genomic DNA library from N.
spiralis, identifying a 41 kb DNA sequence containing the gene
cluster required for pyralomicin biosynthesis. Disruption of the
gene encoding the N-glycosyltransferase PrlH abrogated
pyralomicin production, confirming the critical function of
PrlH in pyralomicin biosynthesis.
T
contain a unique benzopyranopyrrole chromophore linked to a
C7-cyclitol (pyralomicins 1a−1d) or glycosylated by glucose
(pyralomicins 2a−2c) (Figure 1).1,2 The aglycone region of the
molecule resembles pyoluteorin, the pyrrolomycins, the
marinopyrroles, TAN-876A, and TAN-876B (Figure 1) and is
predicted to house homologous enzymes within the bio-
synthetic pathway.3−7 The pyralomicins are active against
various bacteria, particularly strains of Micrococcus luteus, an
opportunistic Gram-positive bacterium that can cause endo-
carditis, intracranial suppuration, bacteraemia, and hepatic
abscess.1 The antibacterial activity of the pyralomicins appears
to be dependent upon the number and the position of chlorine
atoms within the molecules and the nature and methylation of
the glycone.1,2 Pyralomicin 1c, which contains the unmethy-
lated cyclitol as the glycone, exhibits more potent antibacterial
activity than its glucosyl analogue, pyralomicin 2c, and is also
more active than its 4′-methylated congener, pyralomicin 1a,
suggesting a role for the cyclitol moiety in the antimicrobial
activity.
Previous isotope-labeled feeding experiments have demon-
strated that the pyralomicins contain a polyketide-peptide-
derived core structure that is assembled from proline, two
acetates, and one propionate, and the cyclitol moiety is derived
from 2-epi-5-epi-valiolone, implicating a sugar phosphate cyclase
(SPC) enzyme in their biosynthetic pathway.8,9 SPCs represent
a superfamily of enzymes involved in the biosynthesis of a wide
array of primary and secondary natural products, including well-
known antibiotics such as rifamycin, neomycin, kanamycin, and
gentamicin, the antifungal agent validamycin, the antidiabetic
Received: February 23, 2013
© XXXX American Chemical Society and
American Society of Pharmacognosy
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Figure 1. Structures of the pyralomicins and related compounds.
valiolone; however, it cannot effectively cyclize other sugar
phosphate intermediates including DAHP, amino-DAHP,
glucose 6-phosphate, fructose 6-phosphate, mannose 6-
phosphate, and ribose 5-phosphate (Figure 2b−g). An
isotopically labeled authentic sample, 2-epi-5-epi-[6-2H2]-
valiolone,16 was used for comparison (Figure 2f,g). The
enzyme product was detected as the tetrakis-trimethylsilyl
derivative of 2-epi-5-epi-valiolone [m/z 480 (M+)] with major
fragment ions at m/z 335, 276, 217, 147, and 73 (Figure 2e).
This fragmentation pattern is consistent with that of the
authentic sample, which showed m/z 482 (M+), 335, 278, 217,
147, 73 (Figure 2g). The high specificity of PrlA as a 2-epi-5-
epi-valiolone synthase is strong evidence that this enzyme is
part of the pyralomicin biosynthetic gene cluster and is involved
in the formation of the C7-cyclitol functional group
incorporated into pyralomicins 1a−1d.
RESULTS AND DISCUSSION
■
Isolation of the Pyralomicin Biosynthetic Gene
Cluster. To identify the biosynthetic gene cluster of
pyralomicin, high molecular weight genomic DNA was isolated
from N. spiralis, end-filled and ligated into pCC1Fos,
CopyControl Fosmid (Epicenter), to yield a library of ∼5000
colonies. Degenerate primers were designed to homologous
regions within the 2-epi-5-epi-valiolone synthases, AcbC and
ValA, from the acarbose and validamycin pathways.13,14
Subsequent primers were used to PCR amplify a fragment of
a 2-epi-5-epi-valiolone synthase gene from the pyralomicin
producer. This sequence served as a probe for screening the
library. In addition, the pltA halogenase gene from the
pyoluteorin cluster was used as a second heterologous
probe.3 Results from the dual screening yielded eight
overlapping fosmids.
Sequencing and Characterization of the prl Gene
Cluster. With the function of PrlA confirmed, three of the
overlapping cosmids isolated in the screening process were
sequenced using a combination of pyrosequencing and
chromosome walking. A region of ∼41 kb of DNA revealed
27 ORFs predicted to be involved in the biosynthesis of the
pyralomicin aglycone and the cyclitol unit, as well as ORFs
predicted to function in the regulation and transport (Figure 3
and Table S1). The upstream sequence ends with prlZ, whose
gene product may be involved in the regulation of pyralomicin
biosynthesis,17 whereas the downstream sequence ends with
prlT, which encodes a halogenase protein. Additional
sequencing of 5 kb of DNA on either side of the putative
cluster did not reveal any additional open reading frames
related to secondary metabolite biosynthesis. Thus, we believe
that all of the genes required for pyralomicin biosynthesis are
housed within the gene cassette provided in Figure 3.
Production and Characterization of Recombinant
PrlA. Chromosome walking within the region of the 2-epi-5-
epi-valiolone synthase gene revealed the complete ORF (open
reading frame) of prlA. The PrlA homologue contains 57% and
54% identity with the ValA and SalQ 2-epi-5-epi-valiolone
synthases from the validamycin (Streptomyces hygroscopicus
subsp. jinggangensis) and salbostatin (Streptomyces albus) gene
clusters, respectively, whereas PrlA demonstrated only 22%
identity with the AroB dehydroquinate synthase from
Emericella nidulans, suggesting that sedoheptulose 7-phosphate
is the native substrate for the enzyme.13−15 To test this
hypothesis, the prlA gene was cloned into pRSET B expression
plasmid (Invitrogen). Expression of the gene yielded a soluble
6XHis-PrlA fusion protein that was purified using nickel
agarose chromatography (Figure 2a). Biochemical assays of the
enzyme demonstrate that it is capable of catalyzing the
cyclization of sedoheptulose 7-phosphate to 2-epi-5-epi-
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Figure 2. Recombinant expression and characterization of PrlA. (a) Heterologously expressed and purified PrlA: lane 1, soluble fraction of PrlA; lane
2, Ni-NTA agarose-purified PrlA. (b) TLC analysis of PrlA enzyme activity: lane 1, boiled PrlA with sedoheptulose 7-phosphate; lane 2, cell-free
extract of E. coli harboring empty vector with sedoheptulose 7-phosphate; lane 3, PrlA with sedoheptulose 7-phosphate; lane 4, 2-epi-5-epi-valiolone
standard. (c) TLC analysis of the substrate specificity of PrlA. Enzyme reaction mixtures contained 5 mM of the following substrates: lane 1,
aminoDAHP; lane 2, DAHP; lane 3, glucose 6-phosphate; lane 4, fructose 6-phosphate; lane 5, mannose 6-phosphate; lane 6, ribose 5-phosphate;
lane 7, sedoheptulose 7-phosphate; and lane 8, 2-epi-5-epi-valiolone standard. (d−g) GC-MS profiles of silylated PrlA reaction product and silylated
2-epi-5-epi-[6-2H2]valiolone standard. (d) GC trace of silylated PrlA reaction product. (e) MS fragmentation pattern of the major peak eluting at
8.65−8.75 min from the silylated PrlA reaction product [m/z 480 (M+)]. (f) GC trace of silylated 2-epi-5-epi-[6-2H2]valiolone standard. (g) MS
fragmentation pattern of the major peak eluting at 8.65−8.75 min from the silylated 2-epi-5-epi-[6-2H2]valiolone standard [m/z 482 (M+)].
Formation of the C7-Cyclitol Unit. Production of the C7-
cyclitol is proposed to be mediated through the actions of
several enzymes within the pathway (Scheme 1). These include
the 2-epi-5-epi-valiolone synthase PrlA, a putative phosphomu-
tase (PrlB), a cyclitol kinase (PrlU), two cyclitol dehydro-
genases (PrlV and PrlW), and a 2-epi-5-epi-valiolone phosphate
epimerase (PrlX). This cassette of genes shares high homology
with the cyclitol biosynthetic genes from the salbostatin and
acarbose gene clusters (Figure 3, Table S1).15,18 The gene prlF
encodes an enzyme homologous to RebM from the
rebeccamycin biosynthetic gene cluster.19 RebM is predicted
to function as a glucose O-methyltransferase during rebecca-
mycin biosynthesis.20,21 Similarly, the cyclitol moiety in
pyralomicins 1a and 1b contains O-methyl groups at the C-4
position, suggesting a role for PrlF. The PrlG protein has high
similarity to galactose/hexose 1-phosphate uridylyltransferases
including GalT from the milbemycin-producing S. bingcheng-
gensis (63% identity/76% similarity), suggesting a role for PrlG
in the activation of the cyclitol moiety to the nucleotidyl
derivative.22,23
acyl-CoA dehydrogenase (PrlJ), four halogenases (PrlM, PrlN,
PrlO, and PrlT), two polyketide synthases (PrlP and PrlQ), an
FAD reductase (PrlL), a stand-alone PCP domain (PrlS), and a
stand-alone type II thioesterase (PrlR).
Biosynthesis of the aglycone is thought to be initiated
through the activation of a proline residue via PrlK and covalent
linkage to the small stand-alone PCP domain, PrlS (Scheme 2).
Once tethered to the PCP domain through a thioester linkage,
the proline residue is proposed to be modified by the PrlJ
dehydrogenase and halogenated by PrlT to yield a chlorinated
pyrrole intermediate. This is similar and consistent with the
mechanism of proline activation and halogenation shown by
Walsh and co-workers for pyoluteorin biosynthesis.25 PrlK
shares high identity with other known NRPS adenylation
domains including the Pyr8 (64% identity) and PltF (51%
identity) acyl-CoA ligases from the pyrrolomycin and
pyoluteorin pathways, respectively, and with the acyl-CoA
ligase CouN4 from the coumermycin pathway.3,24,26
PrlT shares higher identity with Pyr17 (73% identity; from
the pyrrolomycin pathway) and PltA (59% identity; from the
pyoluteorin cluster) than with any of the other halogenases
within these clusters, suggesting that this enzyme will function
in the halogenation of the PCP-bound pyrrolyl intermediate.
Extension of the halogenated pyrrolyl intermediate is predicted
to occur via the action of three PKS modules housed in PrlP
and PrlQ. The nascent aglycone would then be released from
Formation of the Core Unit Benzopyranopyrrole. The
biosynthesis of the aglycone portion of pyralomicin is proposed
to take place via a suite of genes that share high identity with
both the pyrrolomycin and pyoluteorin biosynthetic genes
(Figure 3, Table S1).3,24 These include genes encoding a
putative nonribosomal peptide synthetase (NRPS) (PrlK), an
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Figure 3. Genetic organization of the pyralomicin gene cluster (prl) compared with partial gene clusters of salbostatin (sal), acarbose (acb),
pyrrolomycin (pyr), and pyoluteorin (plt): (a) salbostatin cluster, (b) acarbose cluster, (c) pyralomicin cluster, (d) pyrrolomycin cluster, (e)
pyoluteorin cluster.
Scheme 1. Proposed Mode of Formation of the Cyclitol Moiety
the enzyme-bound intermediate via the type II thioesterase
PrlR (Scheme 2). The remaining prl halogenases, PrlM, PrlN,
and PrlO, demonstrate high homology with the pyrrolomycin
halogenases Pyr11 (53% identity/67% similarity), Pyr17 (53%
identity/71% similarity), and Pyr16 (47% identity/65%
similarity) respectively, and are proposed to mediate the
halogenation of the PKS-derived phenyl ring (Scheme 2).
Although the core structure of pyralomicin is similar to
pyoluteorin and pyrrolomycin, it differs from the latter
compounds in that a unique cyclization reaction involving the
rearrangement of the proline residue occurs to yield a
benzopyranopyrrole core. Interestingly, comparison of the
pyralomicin biosynthetic gene cluster with the pyoluteorin and
pyrrolomycin clusters has not revealed a novel enzyme
candidate to mediate the proline rearrangement and ring
cyclization. However, the pyralomicin cluster contains a total of
four ORFs encoding putative halogenase enzymes, despite the
pyralomicins described to date containing only two or three
halogen atoms. Therefore, it is possible that one of those
putative halogenase enzymes is involved in the formation of the
benzopyranopyrrole core structure. There is precedence in the
literature for cryptic halogenation pathways that lead to the
formation of novel biosynthetic features. For example, in
coronamic acid biosynthesis the CmaB halogenase mediates the
chlorination of allo-isoleucine, which is further modified by an
intramolecular gamma-elimination of the chlorine atom to yield
cyclopropyl ring formation.27 A similar reaction has been
shown to occur during the biosynthesis of the kutzneride family
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Scheme 2. Proposed Mode of Formation of the NRPS-PKS-Derived Backbone and Attachment of the C7-Cyclitol Unit
of hexapeptidolactones.28 Within this pathway, KtzD mediates a
cryptic halogenation that is further modified to a cyclopropyl
intermediate by KtzA. Thus, during pyralomicin biosynthesis
we predict that a FADH2-dependent halogenase is involved in
proline ring rearrangement and the cyclization leading to the
formation of the benzopyranopyrrole core structure (Scheme
S1).
The co-occurrence of another benzopyranopyrrole antibiotic,
TAN-876A, and closely related TAN-876B (both contain no
sugar or cyclitol) in Streptomyces sp. C-70899 suggests that the
cyclitol attachment takes place at a late stage in pyralomicin
biosynthesis.7 This reaction may be catalyzed by the putative N-
glycosyltransferase enzyme PrlH. PrlH has high homology
(63% identity/73% similarity) with RebG, the N-glycosyl-
transferase from the rebeccamycin pathway.19 RebG is
responsible for transferring D-glucose to an indolocarbazole
aglycone core, which retains structural features that are similar
to the benzopyranopyrrole core of pyralomicin (Figure 1).
To test the involvement of prlH in pyralomicin biosynthesis,
we inactivated the gene in N. spiralis. A 697 bp BamHI-ApaI
fragment of the prlH gene was cloned into the pBsk vector. An
apramycin resistance cassette and the oriT loci from pOJ446
were subsequently cloned into the pBsk vector backbone to
give pKO3A4. Single crossover recombination of the prlH
deletion plasmid pKO3A4 was achieved using conjugation of N.
spiralis with E. coli ET12567 transformed with pKO3A4 and the
pUB307 conjugation helper plasmid. The mutant was
genotypically confirmed by PCR (Figure S1) and was grown
side-by-side with wild-type N. spiralis in the pyralomicin
production medium. Analysis of extracts from the prlH mutant
strain using LC-MS shows that the mutant no longer produces
the suite of pyralomicin analogues present in the wild-type
strain (Figure 4). This provides evidence that prlH is essential
for the biosynthesis of pyralomicin and that a single
glycosyltransferase is required for the addition of either the
glucose or cyclitol moiety into the pyralomicin aglycone core
structure. However, neither the aglycone nor the cyclitol
moiety could be detected in the culture broths of the mutant.
The hypothetical mechanism of these halogenases implies
that the chlorination involves formation of hypochlorous acid
(HOCl) or a more stable Lys-eNH-Cl (lysine chloramine)
from reaction of HOCl with the active site residue lysine. The
intramolecular migration of the carbonyl group of the pyrrole
ring and the benzopyranopyrrole ring formation in pyralomicin
and TAN-876A biosyntheses could well be initiated by the
same type of reaction catalyzed by the halogenases (Scheme
S1). Alternatively, the rearrangement reaction may be initiated
by epoxidation of the pyrrole ring. However, no putative
tailoring genes for such a reaction can be found within the
pyralomicin gene cluster, suggesting that the latter mechanism
is less likely. Overall, the ring cyclization event represents an
unprecedented reaction mechanism that has not previously
been described in other known biosynthetic pathways.
Attachment of the C7-Cyclitol Unit to the Benzopyr-
anopyrrole Core. The pyralomicins are unique in that they
are the only known natural products containing a pseudosugar
(C7-cyclitol) moiety together with a PKS-derived aglycone.
Many C7N-aminocyclitol-containing compounds, including
acarbose and validamycin A, resemble oligosaccharides. Thus,
the biosynthetic mechanism utilized by N. spiralis to
incorporate this unique pseudosugar into an aglycone core
structure is of interest.
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Nonomuraea spiralis IMC A-0156 was kindly provided by Drs.
Hiroshi Naganawa and Yoshikazu Takahashi at the Institute of
Microbial Chemistry, Japan. N. spiralis was maintained on BTT agar
(1% D-glucose, 0.1% yeast extract, 0.1% beef extract, 0.2% casitone,
and 1.5% Bacto-agar, pH 7.4 prior to sterilization) at 30 °C. Spore
formation in N. spiralis was induced by growth on modified minimal
medium agar (0.05% L-asparagine, 0.05% K2HPO4, 0.02%
MgSO4·7H2O, 1% D-glucose, 1.5% Bacto-agar, pH 7.4 prior to
sterilization) at 30 °C for 1 to 2 weeks. For the production of
pyralomicins, N. spiralis was grown in seed medium (2% D-galactose,
2% dextrin, 1% Bacto-soytone, 0.5% corn steep liquor, 0.2%
(NH4)2SO4, 0.2% CaCO3, a drop of silicone oil, pH 7.4 prior to
sterilization) at 30 °C for 5−7 days. The seed culture (10%) was then
inoculated into production medium (3% potato starch, 1.5% soy flour,
0.5% corn steep liquor, 0.2% yeast extract, 0.05% MgSO4·7H2O, 0.3%
NaCl, 0.001% CoCl2, 0.3% CaCO3, 1−2 drops silicone oil, pH 7.4
prior to sterilization), and the culture was shaken at 200 rpm at 30 °C
for an additional 5−7 days. Production of the pyralomicins
corresponded to a light pink color change in the bacterial mycelium
and a purple to wine coloration within the production medium.
Growth of N. spiralis in RARE3 medium (1% D-glucose, 0.4% yeast
extract, 1% malt extract, 0.2% Bacto-peptone, 0.2% MgCl2, 0.5%
glycerol, pH 7.4 prior to sterilization) was used for conjugation
experiments.
Figure 4. Analysis of prlH deletion mutant. (a) HPLC elution peaks
were monitored at 355 nm. (b−d) Mass spectral analysis of
pyralomicin 1a (m/z 455.68 [M + H]+), 1b (m/z 455.68 [M +
H]+), and 2a (m/z 459.81 [M + H]+), respectively.
PCR Amplification of 2-epi-5-epi-Valiolone Synthase Homo-
logues from N. spiralis. Degenerate primers, ValA-1S, ValA-2S, and
ValA-2R (Table S2), were designed from sequence alignments of sugar
phosphate cyclases, including the 2-epi-5-epi-valiolone synthases ValA
from the validamycin pathway and AcbC from the acarbose pathway.
Amplification using ValA-2S and ValA-1R was successful in PCR,
amplifying a homologue sequence from N. spiralis. The Promega
Wizard Kit was used to isolate genomic DNA from N. spiralis
according to the manufacturer’s specifications. Promega GoTaq was
used for PCR amplification reactions according to the manufacturer’s
specifications. A 100 ng amount of template DNA was heated at 70 °C
for 10 min prior to addition to the PCR reaction mixtures. PCR
amplifications were conducted with 1 cycle at 95 °C for 7 min
followed by 5 cycles of 95 °C for 30 s, 42 °C for 30 s, and 72 °C for 1
min, followed by 30 cycles of 95 °C for 30 s, 48 °C for 30 s, and 72 °C
for 1 min, followed by 1 cycle of 72 °C for 5 min. Samples were held at
4 °C until they were analyzed by gel electrophoresis. PCR products of
approximately 400 base pairs were gel purified, subcloned into pBsk
(Stratagene), and submitted for DNA sequencing at the Center for
Genome Research and Biocomputing (CGRB) at Oregon State
University.
Construction and Screening of the N. spiralis Genomic
Library. N. spiralis was grown for 5 days in seed medium. Ten
milliliters of culture was centrifuged at 1400g, and the cell pellet was
utilized to isolate genomic DNA using a modified cetyl trimethy-
lammonium bromide (CTAB) method. The cell pellet was washed
with 10 mL of 10.3% sucrose and repelleted, and the supernatant was
discarded. The pellet was washed twice with 10 mM Tris and 1.0 mM
EDTA buffer, pH 7.4, and repelleted by centrifugation, and the
supernatant was discarded. The cell pellet was resuspended in 6 mL of
lysis buffer (10 mM Tris pH 8.0, 25% sucrose, 0.08 mM EDTA, and
50 mg of lysozyme). The sample was incubated at 37 °C for 90 min. A
100 mL sample of proteinase K (20 mg mL−1) and 3.6 mL of 10%
SDS were added, and the solution was incubated for an additional 90
min at 37 °C. A 1.6 mL portion of CTAB (10%) was added to the
solution, and the solution was incubated at 65 °C for 10 min. An equal
volume of 1:1 phenol−chloroform pH 8.0 was added to the solution
and mixed by inversion until a homogeneous emulsion was created.
The solution was centrifuged, and the aqueous layer removed to a
fresh tube, where it was extracted in phenol−chloroform for a second
time. The aqueous solution was removed to a fresh tube and extracted
using an equal volume of chloroform. The aqueous layer was removed,
1/10 volume of 3 M sodium acetate was added, and the DNA was
precipitated by adding one volume of room-temperature 2-propanol.
The resulting DNA was spooled from the mixture and resuspended in
500 μL of sterile H2O.
This lack of aglycone accumulation may not be due to a polar
effect, as the nucleotidyltransferase gene prlG is the only
downstream gene present within the same operon as prlH.
Rather, it may be due to either negative feedback inhibition of
the intermediate(s) production or the lack of a suitable
transport system for the aglycone. Nevertheless, the results
confirm the critical function of PrlH in pyralomicin biosyn-
thesis.
In conclusion, we have cloned, sequenced, and identified the
pyralomicin biosynthetic gene cluster from N. spiralis IMC A-
0156. The cluster contains 27 ORFs predicted to encode all of
the functions for pyralomicin biosynthesis. Those include the
NRPS and PKS enzymes, which are required for the formation
of the benzopyranopyrrole core unit, a suite of tailoring
enzymes, and regulatory and transport proteins. Targeted
disruption of the gene encoding the N-glycosyltransferase, prlH,
abolished pyralomicin production, and recombinant expression
of PrlA confirms the activity of this enzyme as a sugar
phosphate cyclase involved in the formation of the C7-cyclitol
moiety. Further studies are necessary to dissect the biosynthetic
mechanisms of the unique ring cyclization and proline
rearrangement and the attachment of the cyclitol unit to the
benzopyranopyrrole core moiety.
MATERIALS AND METHODS
■
General Experimental Procedures. Low-resolution electrospray
ionization (ESI) mass spectra were recorded on a ThermoFinnigan
liquid chromatograph-ion trap mass spectrometer. Gas chromatog-
raphy−mass spectrometry was performed on a Hewlett-Packard 5890
Series II gas chromatograph and a Hewlett-Packard 5971 Series mass
selective detector. High-performance liquid chromatography was
carried out on a C18 column (C18 Symmetry 10 × 150 mm, Waters)
with the detector set at 355 nm. Thin-layer chromatography was
performed on aluminum sheets with silica gel 60 F254 (EMD
Chemicals Inc.).
Bacterial Strains and Culture Conditions. E. coli strains
DH10B, EPI300, ET12567, and BL21(DE3)pLysS were grown in
LB Miller broth with appropriate supplements as indicated. Gene
expression was conducted using LBBS (LB Miller broth containing 2.5
mM betaine and 1 M sorbitol) supplemented with appropriate
antibiotics.
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The CopyControl Fosmid library kit from Epicentre was utilized to
prepare a genomic library using the N. spiralis DNA. Briefly, 20 μg of
isolated DNA was end-filled and gel purified to recover 40 kb DNA
fragments. N. spiralis DNA fragments were ligated into the fosmid
vector, pCC1Fos, overnight at 16 °C. The ligation extract was
packaged into lambda phage using the Gigapack III gold packaging
extract from Stratagene according to the manufacturer’s specifications.
Phage extracts were used to infect competent E. coli EPI300 cells
grown in LB Miller containing 10 mM MgCl2.
culture temperature was reduced to 25 °C and the expression of prlA
was induced by the addition of 0.3 mM IPTG. Cultures were
incubated at 25 °C for 15 h, and bacterial cell pellets collected by
centrifugation at 1400g. Bacterial cell pellets were lysed in K2HPO4−
KH2PO4 (25 mM) and NaCl (300 mM), pH 7.4, containing 0.2 mM
CoCl2 and the protease inhibitor 4-(2-aminoethyl)benzenesulfonyl
fluoride (200 μg mL−1). The cell lysate was sonicated for two 30 s
bursts on a Microson Ultrasonic Cell Disruptor XL on setting 6 and
clarified by centrifugation to yield the cell-free extract. This mixture
was used directly in the cell-free protein assay, or the enzyme was
further purified using Ni-NTA agarose beads according to the
manufacturer’s specifications (Qiagen). The purified protein was
dialyzed in 2 L of dialysis buffer [K2HPO4−KH2PO4, pH 7.5 (25
mM), CoCl2 (0.2 mM)] and visualized using 10% SDS-PAGE
followed by Coomassie staining.
Enzyme activity was analyzed by the addition of cell-free extract or
purified protein to the reaction mixture, as described below. The
enzyme assay (100 μL volume) was performed at 30 °C for 2 h in 25
mM phosphate buffer (pH 7.5) containing 50 μL of protein (0.3 mg
mL−1), 5 mM substrate, 0.05 mM CoCl2, 1 mM NAD+, and 2 mM KF.
The substrate, aminoDAHP, was a gift from Prof. Heinz G. Floss. The
remaining substrates, including sedoheptulose 7-phosphate, DAHP,
glucose 6-phosphate, fructose 6-phosphate, mannose 6-phosphate, and
ribose 5-phosphate, were purchased from Sigma. After incubation,
samples were visualized using silica gel thin-layer chromatography
(TLC) (butanol−ethanol−water, 9:7:4), and the substrate and
reaction product were detected as blue spots with a cerium sulfate−
phosphomolybdate-containing reagent. The reaction mixture was
lyophilized, and the reaction products were extracted with methanol.
After drying, the extracts were mixed with drops of Sigma SIL-A and
dried under argon. The products were reextracted with n-hexane and
analyzed by GC-MS.
Construction of the prlH Deletion Mutant. Construction of a
prlH suicide vector to use in single crossover recombination
experiments was designed using the pBsk cloning vector (Stratagene).
A 697 base pair BamHI-ApaI fragment of the prlH gene was cloned
into the pBsk vector for single crossover recombination into N. spiralis.
To identify mutant colonies, an apramycin resistance cassette from
pOJ446 was cloned downstream of the partial prlH gene into the pBsk
vector backbone at the KpnI restriction site. In addition, the 800 base
pair oriT loci from pOJ446 was also cloned into the knockout vector at
the NotI restriction site to enable conjugal transfer of the vector using
the helper plasmid pUB307. The completed prlH knockout vector is
referred to as pKO3A4.
Single crossover recombination of the prlH deletion plasmid
pKO3A4 was achieved using conjugation of N. spiralis with E. coli
ET12567 transformed with pKO3A4 and the pUB307 conjugation
helper plasmid. N. spiralis was inoculated and grown in RARE3
medium at 30 °C for 5−7 days. The night before conjugation, N.
spiralis cultures were reinoculated at a 1:10 dilution into RARE3
medium and grown overnight at 30 °C. An overnight culture of E. coli
ET12567 transformed with pKO3A4 and pUB307 was inoculated into
LB Miller broth supplemented with thiamine hydrochloride (25 μg
mL−1), 0.1% D-glucose, 25 mM MOPS, chloramphenicol (25 μg
mL−1), kanamycin (50 μg mL−1), and apramycin (50 μg mL−1) and
incubated at 37 °C. On the day of conjugation, cultures of N. spiralis
and E. coli were centrifuged at 1400g and the supernatant was
removed. The N. spiralis cells were resuspended directly in 1/10
volume of RARE3. The E. coli cells were washed three times in RARE3
medium to remove antibiotics and traces of LB Miller broth and were
then resuspended in 1/10 volume of RARE3 medium. Aliquots of 500
μL of E. coli and 500 μL of N. spiralis were mixed and incubated
without shaking for 16, 24, 48, or 72 h at 30 °C. Following incubation,
200 μL aliquots of 1-fold, 10-fold, and 100-fold dilutions were plated
onto BTT agar containing apramycin (50 μg mL−1) and nalidixic acid
(25 μg mL−1). Growth of mutant strains was evaluated after 4−6
weeks incubation at 30 °C. Mutant colonies were restreaked onto BTT
agar containing apramycin (50 μg mL−1) and nalidixic acid (25 μg
mL−1). Growth of mutant strains in RARE3 liquid broth was used to
make 10% glycerol stocks that were stored at −80 °C.
The library of approximately 5000 colonies was replica plated into
liquid culture using the Genetix QPix2 colony picker into sets
containing two 96-well plate replicates. All of the plates were grown
overnight at 37 °C. One set of plates was used to make glycerol stock
cultures (10% glycerol) that were stored at −80 °C. The second set of
liquid plates was used to replica plate the library onto 150 mm agar
plates containing 100 μL of CopyControl Induction Solution
(Epicentre). Agar plates were grown at 37 °C overnight and were
lifted onto Hybond-N nitrocellulose membrane (Amersham). Briefly,
the colony lifts were performed on prechilled agar plates. The
nitrocellulose membrane was placed on the agar for 1 min and marked
using a needle to retain orientation. The membrane was then floated in
succession in three solutions for 15 min each: denaturation solution
(0.5 M NaOH, 1.5 M NaCl); neutralization solution (1.5 M NaCl, 1.0
M Tris-HCl, pH 7.4); and 2XSSC (0.3 M NaCl, 0.03 M Na-citrate, pH
7.0). After the last incubation, the blots were cross-linked using UV
light in a UV cross-linker at 1200 W. After cross-linking the blots were
incubated for 1 h at 37 °C with proteinase K solution (2.5 mg mL−1).
The blots were then washed liberally with water, dried on Whatman
paper overnight, and stored at 4 °C until used for hybridization.
The lifts were hybridized in succession with the 2-epi-5-epi-valiolone
homologue prlA and the pyoluteorin halogenase gene pltA (kindly
provided by Dr. Joyce Loper) in DIG-Easy Hyb Solution (Roche)
containing DIG-labeled DNA probe (15 ng mL−1) overnight at 50 °C.
After each round of hybridization the blots were washed twice with
2XSSC−0.1% SDS at room temperature for 5 min each and then
subsequently washed twice with 0.5XSSC−0.1% SDS at 50 °C for 15
min each wash. The blots were then incubated in blocking solution
[0.1 M maleic acid, 0.15 M NaCl, pH 7.5, 1× blocking reagent
(Roche), and 5% nonfat dry milk] for 1 h at room temperature. The
blots were incubated in blocking solution containing 1:10 000 dilution
antidig monoclonal Ab for 30−45 min at room temperature. The blots
were then washed three times for 15 min in wash buffer (0.1 M maleic
acid, 0.15 M NaCl, and 0.3% Tween 20, pH 7.5). The blots were
briefly rinsed in detection buffer (0.1 M Tris, 0.1 M NaCl, pH 9.5) and
incubated for 10 min in detection buffer containing 0.025 mM CSPD
(Roche) at 37 °C. The blots were then put down to film for 30 min.
Positive colonies were inoculated into 20 mL LB cultures with
chloramphenicol (12.5 μg mL−1) and grown overnight at 37 °C.
Following overnight incubation, 20 μL of copy control induction
solution was added to the remaining culture. The cultures were then
incubated for 5 h at 37 °C before they were harvested using the Bio-
Rad miniprep kit. The resulting DNA was digested with BamHI and
used to run a Southern analysis. Briefly, digested DNA samples were
separated using agarose gel electrophoresis. DNA was transferred to
Hybond-N nitrocellulose membrane using capillary transfer. Southern
membranes were hybridized and analyzed in the same manner as
colony lifts from the library. Both DNA strands from three overlapping
library clones housing either one or both of the screening probes were
sequenced by a combination of chromosome walking and shotgun
pyrosequencing. The 41 kb putative pyralomicin biosynthetic gene
cluster was reconstituted using BioEdit software.
Expression of prlA and Chraacterization of the Recombinant
Enzyme. PCR was used to clone prlA into the pRSET B expression
plasmid (Invitrogen) in frame with the 6XHis-tag at the BamHI/KpnI
restriction sites. Primers PrlA-F2 and PrlA-R2 (Table S2) were used
during the PCR amplification step. Resulting clones were confirmed by
DNA sequencing and were subsequently transferred into E. coli
BL21(DE3)pLysS. Transformed bacteria were grown in LBBS
containing ampicillin (100 μg mL−1) and chloramphenicol (25 μg
mL−1) at 37 °C until the OD600 reached 1.0 to 1.2. At this point the
G
dx.doi.org/10.1021/np400159a | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Analysis of prlH Deletion Mutant. Wild-type and prlH deletion
strains of N. spiralis were cultured for 5−7 days in seed medium at 28
°C. Seed cultures were subsequently diluted 1:10 into production
medium and grown for an additional 5−7 days. Upon harvesting,
cultures of wild-type and prlH deletion strains of N. spiralis were
acidified to pH 3 using HCl. Bacterial mycelia and other precipitates
were removed from the culture medium via centrifugation at 1400g.
The acidified culture medium was extracted three times with an equal
volume of butyl acetate. Butyl acetate fractions were dried by the
addition of sodium sulfate, clarified by filtration, and reduced using
rotary evaporation. Resulting extracts were analyzed using LC-MS on a
Waters C18 Symmetry Prep column with a 25% to 100% MeOH
gradient. Peaks were monitored at 355 nm. Peaks corresponding to
pyralomicin metabolites were identified using electrospray ionization
mass spectrometry.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Tables S1 and S2, Figure S1, and Scheme S1. This material is
available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
The GenBank accession number for the sequence of the
pyralomicin gene cluster is JX424761.
(18) Wehmeier, U. F.; Piepersberg, W. Appl. Microbiol. Biotechnol.
2004, 63, 613−625.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 1-541-737-9679. Fax: 1-541-737-3999. E-mail: Taifo.
Mahmud@oregonstate.edu.
(19) Sanchez, C.; Butovich, I. A.; Brana, A. F.; Rohr, J.; Mendez, C.;
Salas, J. A. Chem. Biol. 2002, 9, 519−531.
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Zhang, G.; Gilbert, E. J.; Wang, P. G.; Van Vranken, D. L.; Thorson, J.
S. ChemBioChem 2006, 7, 795−804.
Present Address
†(P. M. Flatt) Department of Chemistry, Western Oregon
University, Monmouth, Oregon 97361, United States.
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Kalantar-Zadeh, K. ChemPhysChem 2007, 8, 2036−2050.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Drs. H. Naganawa and Y. Takahashi at the
Institute of Microbial Chemistry for providing N. spiralis IMC
A-0156 and M. Walters for technical assistance. The project
described was supported by grant R01AI061528 from the
National Institute of Allergy and Infectious Diseases and by the
Herman-Frasch Foundation. The content is solely the
responsibility of the authors and does not necessarily represent
the official views of the National Institute of Allergy and
Infectious Diseases or the National Institutes of Health (NIH).
(25) Thomas, M. G.; Burkart, M. D.; Walsh, C. T. Chem. Biol. 2002,
9, 171−184.
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