burkholderia 
 pseudomallei ( bp ) is the causative agent 
 for the bacterial septic disease melioidosis . 

melioidosis is endemic throughout the tropical southeast pacific region 
 and is considered a growing global health threat.bp , an environmental saphrophyte 
 by nature , is intrinsically resistant to multiple antibiotics due 
 to factors including drug inactivation ( e.g. , -lactamase activity ) , 
 a multidrug efflux pump system , and an adaptive physiology that can 
 alter drug target sites throughout disease progression . as a result
, mortality rates from melioidosis 
 can approach 50% even with antibiotic treatment . despite increased scrutiny in recent years
, the 
 mechanisms by which bp can establish and propagate 
 an infection have remained unclear .
bioinformatics analyses of bp and its genetic relatives burkholderia mallei ( bm ) and burkholderia thailandensis ( bt ) indicate that substantial parts of their genomes 
 are dedicated to polyketide ( pk)- and non - ribosomal peptide ( nrp)-based 
 secondary metabolism , which we have hypothesized could be a key component 
 of burkholderia virulence .
while many of the gene clusters 
 common to all three species have been studied , clusters that are solely 
 found within the bp genome have remained uncharacterized , 
 and to date , the specific roles that secondary metabolism has in establishing 
 melioidosis have not been evaluated . herein , we begin the characterization 
 of the unique secondary metabolome encoded by bp and 
 show that it is a key factor in melioid virulence .
the importance 
 of these molecules to bp pathogenicity suggests that 
 inhibition of their biosynthesis could present a viable avenue for 
 future therapeutic intervention .
cryptic and/or silent small - molecule 
 biosynthetic gene clusters 
 are routinely found within sequenced bacterial genomes .
while biosynthetic gene clusters encoding molecules 
 required for bacterial pathogenesis must be produced in vivo during an infectious event , many may remain silent in the laboratory 
 setting due the lack of appropriate environmental stimuli needed to 
 induce their activation ( figure 1 ) . due to 

its nih / cdc select agent status , the manipulation of the bp genome for gaining access to silent biosynthesis has been cumbersome . 
 as bt
is generally considered to be avirulent in 
 most mammalian hosts , it has been used as a convenient surrogate model 
 to study biosynthesis in pseudomallei - group burkholderia species . as much of the bp secondary metabolome has been characterized through bt , neither the identity of the molecules encoded by bp - specific gene clusters nor
recently , the schweizer group developed 
 a bp adenine auxotroph ( bp82 ) that 
 has been removed from the select agent list due to its inability to 
 survive outside the laboratory , providing 
 a strain with the genetic background with which to study cryptic secondary 
 metabolism of this important pathogen . examining the influence 
 of secondary metabolism on bacterial pathogenicity . 

( a ) bacterial pathogens typically produce a limited number of metabolites 
 under laboratory fermentation conditions , ( b ) while having the capacity 
 to produce multiple metabolites within the context of an infection , 
 which can function as virulence factors .
( c ) cryptic / silent gene clusters 
 can be identified bioinformatically in sequenced pathogen genomes . 
 through genome engineering ,
individual biosynthetic clusters can be 
 both activated for accessing the encoded molecules and disrupted for 
 determining their influence on bacterial pathogenicity .
( d ) virulence 
 factor biosynthesis can be targeted for the development of new disease - specific 
 therapeutics .
a comparative genome analysis ( figure 2 and table s1 ) of pseudomallei - group burkholderia 
 revealed that , although bp , bm , 
 and bt share many pk / nrp gene clusters , three gene 
 clusters are unique to bp ( figure 2 ; clusters 2 , 14 , and 15 ) , and one cluster ( cluster 11 ) is 
 shared between bp and bm ( but not bt ) . while extensively truncated versions of clusters 14 
 and 15 appear in the bm genome , cluster 2 is completely 
 absent .
gene clusters 2 and 15 are predicted to encode for novel lipopeptides , 
 cluster 11 is also predicted to encode a novel metabolite but has 
 a gene organization and content similar to that found in the bt - specific thailandamide biosynthetic cluster , and cluster 14 is predicted to encode a syrbactin - type 
 structure ( see discussion below ) ( tables s1 and 
 s2 ) .
we hypothesized that bp - specific cryptic 
 gene clusters may play an important , and overlooked , role in melioidosis . 
 to test this hypothesis
, we used homologous recombination to delete 
 the initial biosynthetic domains ( detailed in table s4 ) within the first either pk or nrp megasynth(et)ase 
 gene of clusters 2 , 11 , 14 , and 15 , thereby creating four strains 
 with individually disrupted biosynthetic gene clusters .
this set of 
 deletions was created in a fully sequenced clinical isolate , b. pseudomallei1026b ( bp1026b ) , under strict bsl3 level biocontainment .
these gene cluster disruption 
 strains were then tested for pathogenicity using an intranasal 
 murine infection model ( figure 3 ) .
remarkably , 
 disruption of any of the three gene clusters that are uniquely found 
 in bp ( clusters 2 , 14 , and 15 ) completely abrogated bp1026b murine virulence , even at titers up to 10 times 
 the bp1026b ld50 inoculum .
disruption 
 of cluster 11 , which is shared between bp and bm , led to a strain with virulence that was indistinguishable 
 from the wild - type strain in the murine model .
each of the three nrp / pk 
 metabolites encoded solely within the bp genome is 
 therefore individually essential to the pathogenicity of this bacterium . 
 additionally , the growth rates of these three mutants were identical 
 to that of wild - type bp1026b ( figure s2 ) , indicating that the disruption of biosynthesis 
 has no impact on general bacterial fitness . based on their importance 
 to bp virulence
, we initiated the structural and 
 functional characterization of the small molecules encoded by this 
 collection of bp - specific gene clusters . here
the 
 third cluster ( cluster 2 ) , predicted to encode a five - amino - acid lipopeptide 
 structure , has so far remained recalcitrant to characterization .
future 
 studies will focus on activating this remaining silent gene cluster 
 in the laboratory setting .
comparison of the secondary metabolomes encoded 
 by b. pseudomallei1026b ( bp ) , b. mallei atcc 23344 ( bm ) , and b. thailandensis e264 ( bt ) .
positioning of individual nrp- and pk - encoding 
 gene clusters within each genome is displayed .
clusters shared among 
 the three species are indicated by the connective lines between genomes 
 ( detailed in table s1 ) .
genomes are linearized 
 and arranged for clarity , with the purple arrowhead designating direction 
 from the first gene in each chromosome ( i.e. , nucleotide + 1 ) .
clusters 
 2 , 14 , and 15 ( red ) are unique to bp . cluster 11 
 ( yellow )
genes dedicated to nrp / pk - based secondary 
 metabolism encompass approximately 6% of the bp genome .
21-day murine intranasal infection challenge 
 of wild - type bp and the disruption mutants of biosynthetic 
 clusters 2 , 
 11 , 14 , and 15 .
inoculum : 10 ( 10 for cluster 15 ) colony - forming 
 units . in previous work with bt
, we developed a promoter - replacement 
 strategy for up - regulating or activating secondary metabolite gene 
 clusters in burkholderia . in this approach
, 
 the rhamnose - inducible promoter prhab is inserted directly 
 upstream of a key operon in a gene cluster of interest , providing 
 a conditional switch that activates or represses biosynthesis in the 
 presence or absence of rhamnose , respectively . to investigate cryptic 
 virulence - associated secondary metabolism in bp , 
 we created prhab recombination cassettes targeting the 
 promoters upstream of the most biosynthetic - rich operons found in 
 each gene cluster of interest ( i.e. , clusters 2 , 14 , and 15 ) .
these 
 cassettes were ligated into the allele replacement vector pexkm5 for 
 use in transformation , recombination , selection , and ultimately promoter 
 replacement in bp82 . using this approach
, we were able to induce high - level metabolite 
 production from clusters 14 and 15 ( syr and mpn clusters , respectively ) , 
 and then through a combination of bioinformatics , mass spectroscopy , 
 and nmr , we characterized the molecules encoded by these two clusters , 
 as follows .
on the basis of bioinformatics analysis , we predicted 
 that the 
 mpn gene cluster was composed of a unidirectional five - gene locus 
 ( mpna - e ) .
three of these genes , mpnbcd , encode large modular nrp megasynthetases ( nrpss ) that together 
 are predicted to synthesize a 12-amino - acid peptide ( figure 4a ) .
the mpnb initiation module is predicted to incorporate 
 an acyl group as a starter unit , suggesting that the mpn cluster would 
 encode a lipopeptide .
the two additional 
 genes predicted to reside in the mpn cluster would appear to be responsible 
 for generating unnatural amino acids for use by the nrpss .
the first , mpna , encodes a diaminobutyrate-2-oxoglutarate 
 transaminase that is commonly used in 2,4-diaminobutyric 
 acid ( dab ) biosynthesis , which is supported by the predicted dab substrate 
 binding specificity of one of the nrps adenylation domains , a7 ( figure 4b ) . the final gene , mpne , is predicted to encode for a syrp - like aspartic / glutamic 
 acid hydroxylase .
( a ) biosynthetic 
 gene cluster 
 encoding the malleipeptins ( genes bp1026b_ii1742 - 1746 , table s2 ) , with prhab promoter 
 exchange ( red ) . ( b ) predicted nrps domain architecture and adenylation 
 domain selectivity for mpnbcd .
parentheses indicate amino acids observed 
 in the nmr - determined final malleipeptin structure that differ 
 from the bioinformatics prediction .
abbreviations : c , condensation 
 domain ; cs , starter condensation domain ; cd , 
 dual condensation / epimerization domain ; a , adenylation domain ; t , 
 peptidyl carrier domain ; te , thioesterase ; dhb , 2,3-dehydrobutyric 
 acid ; dab , 2,4-diaminobutyric acid ; hglu , 4-hydroxyglutamic 
 acid .
( c ) hplc traces ( diode array : 254 nm ) of culture broth extracts 
 from ( a ) bp82 , ( b ) bp82:prhab - mpn ( no rhamnose ) , and ( c ) bp82:prhab - mpn ( rhamnose 
 induced ) . ( d ) structure of malleipeptins a ( 1 ) 
 and b ( 2 ) with key hmbc / roesy correlations . predicted 
 tailoring enzyme functionalities ( mpna and mpne ) are highlighted . 

the induction of mpn 
 biosynthesis was achieved through prhab insertion upstream 
 of mpna to give strain bp82:prhab - mpn . upon induction with rhamnose
, 
 two major strain - specific metabolites appeared in culture broth extracts 
 ( figure 4c ; 1 and 2 , esi [ m+h]m / z 1384.3 
 and 1398.2 , respectively ) .
these metabolites were absent in extracts 
 from both wild - type bp82 and uninduced bp82:prhab - mpn cultures , indicating that , while the mpn cluster 
 is critical for virulence in vivo , it remains silent 
 under simple laboratory fermentation conditions .
compounds 1 and 2 were purified by preparative reverse - phase chromatography 
 from the methanol eluent of hp-20 resin - infused rhamnose - induced bp82:prhab - mpn cultures .
cosy / tocsy analyses 
 of major compound 2 showed 14 
 spin systems ( figures s3 and s4 ) . on the 
 basis of h / c chemical shift data and hmqc / hmbc 
 correlations , 12 of these are predicted to be amino acids .
c hmbc correlations between amide protons and 
 adjacent carbonyl groups , with supporting h  h roesy correlations ,
defined the order of the 12 amino acids 
 ( figures 4d and s3 ) .
intramolecular cyclization through the threonine side chain ( position 
 9 ) to form a 13-member macrolactone is supported by an hmbc correlation 
 between the -carbon methine proton ( h 5.37 ) 
 of threonine and the carbonyl carbon of the c - terminal isoleucine 
 residue ( c-1 169.2 ) .
the final two cosy spin 
 systems are predicted to be part of an acyl substituent .
an hmbc correlation 
 between the carbonyl ( c-1 165.8 ) that is 
 connected by hmbc correlations to the olefin - containing spin system 
 and the serine amide proton ( h 8.24 ) confirms attachment 
 of acyl group to the n - terminus .
ultimately , 
 the exact length of the acyl substituent ( 2-(e)-decenoic ) 
 was determined on the basis of the molecular formula predicted by 
 hrms - tof ( m / z [ m+h ] calcd 
 for c62h105n14o22 , 1397.7511 ; 
 found , 1397.7528 ) .
minor product 1 differs from 2 by ch2 , based on the ms - predicted formula ( hrms - tof m / z [ m+h ] calcd for c61h103n14o22 , 1383.7372 ; found , 1383.7365 ) . 

the same general nmr / ms arguments used to define the structure of 2 were used to establish the structure of 1 , 
 with the exception that h , c , and both the 
 cosy and tocsy spectra indicate the c - terminal amino acid is a valine 
 instead of an isoleucine .
we have assigned these new lipopeptides 
 the names malleipeptin a ( 1 ) and malleipeptin 
 b ( 2 ) .
malleipeptins a and b are 12-amino - acid 
 lipopeptides with 
 a novel peptide sequence and a rarely seen 13-membered terminal lactone .
the primary malleipeptin amino acid sequence is in very good 
 agreement with the bioinformatics prediction from the primary mpnbcd 
 nrps ( figures 4b ) .
one exception is that 2,3-dehydro-2-aminobutanoic 
 acid ( dhb ) , a dehydrated threonine residue , is incorporated at the 
 position predicted to contain a threonine .
the biosynthesis of the 
 hydroxyglutamic acid ( hglu ) moiety seen at position 10 is supported 
 by the presence mpne , which is a predicted syrp - like aspartic / glutamic 
 acid hydroxylase.h and cosy 
 spectra with supporting h 
c hmqc correlations 
 indicate that the oxidation occurs at the c-4 methine ( c-4 66.3 ) , instead of the more common c-3 position ( figure s5 ) .
the presence of dual condensation / epimerization 
 domains in five of the nrps modules indicates 
 the incorporation of d - amino acids directly upstream of these 
 domains at positions-1 , 3 , 5 , 7 , and 10 
 ( figure 4b ) , as is drawn in figure 4d .
the tandem thioesterases ( te1te2 ) found at the n - terminus of mpnd are commonly seen in large 
 lipopeptide biosynthetic clusters and are predicted to encode 
 for both intramolecular cyclization and proofreading to ensure the 
 fidelity of the biosynthetic assembly line .
the malleipeptins are the first lipopeptides characterized 
 from pseudomallei - group burkholderia and are distinct from lipopeptides 
 produced by unrelated burkholderia species ( e.g. , burkholdine , occifungin ) .
the nrpss responsible for the assembly of the malleipeptins 
 do not align well to any deposited nrps sequences outside of those 
 found in other sequenced bp strains , indicating that 
 the mpn cluster encodes for a novel structural family of lipopeptides 
 that is so far solely associated with bp virulence .
the syr cluster is comprised of nine genes ( syra  i ) that are unidirectionally oriented ( figure 5a ) .
bioinformatics analysis predicted this gene cluster was likely 
 to encode a syrbactin - type proteasome inhibitor , as indicated by homologues 
 of genes known to encode the hybrid pk / nrp syrbactin  warhead  
 that binds to the 20s proteasome active site ( syregfhi ) .
in addition , this cluster is predicted 
 to contain a set of genes ( syrabcd ) putatively responsible 
 for synthesis and transfer of an acyl group ( figure 
 s23 ) . while syrbactins have been characterized from bacteria 
 that are pathogenic to plant and insect hosts , this is the first syrbactin - type 
 cluster identified within a mammalian pathogen . to study this cluster
, 
 we exchanged the promoter upstream of syra with prhab , yielding strain bp82:prhab - syr .
hplc analysis of ethyl acetate extracts from rhamnose - infused 
 cultures of bp82 and bp82:prhab - syr showed a dramatic increase in two metabolites in the 
 induced bp82:prhab - syr cultures ( figure 5b ; 3 and 4 ) .
these metabolites 
 are absent in extracts from uninduced bp82:prhab - syr cultures , confirming the activation / repression phenotype 
 provided by the prhab promoter and coupling compound 3 and 4 production to syr gene cluster induction . 

compounds 3 and 4 were purified from ethyl 
 acetate extracts using a modified kupchan scheme followed by silica 
 gel flash chromatography and preparative reverse - phase hplc .
ms and 
 nmr data indicate that compound 3 is identical to the 
 syrbactin glidobactin c ( hrms - tof m / z [ m+na ] calcd for c29h48n4o6na , 571.3514 ; found , 571.3483 ) , which is characterized 
 by the acylation of the pk / nrp warhead with a 2(e),4(e)-diene derivative of myristic acid ( figure 5c ) .
the hrms - predicted molecular formula for 4 differs from that of 3 by one oxygen atom ( hrms - tof m / z [ m + na ] calcd for c29h48n4o5na , 555.3522 ; found , 
 555.3520 ) .
h and c chemical shift data as 
 well as cosy , hmqc , and hmbc correlation data indicate that compound 4 contains lysine , instead of 4-hydroxyllysine , within the 
 warhead substructure . to our knowledge , this syrbactin variant , which 
 we assign the name deoxyglidobactin c ( 4 ) , has 
 not been reported previously .
( a ) biosynthetic gene cluster 
 encoding for the syrbactins ( genes bp1026b_ii1345 - 1353 ; table s2 ) with prhab promoter 
 exchange ( red ) .
( b ) hplc traces ( diode array : 254 nm ) of culture broth 
 extracts from ( a ) bp82 , ( b ) bp82:prhab - syr ( no rhamnose ) , 
 and ( c ) bp82:prhab - syr 
 ( rhamnose induced ) .
( c ) structures of glidobactin c ( 3 ) and deoxyglidobactin c ( 4 ) .
while syrbactins are known proteasome inhibitors , the biological 
 activity of the malleipeptins was unknown . at the highest levels 
 tested , the malleipeptins showed no general toxicity against 
 bacterial ( 100 g / disk ) or human cells lines ( 100 g ml ) .
a common function among non - cytotoxic lipopeptides 
 is surfactant activity . in bacteria ,
biosurfactant production is thought 
 to reduce the surface tension at water / hydrophobic interfaces , thereby 
 helping to increase growth on surfaces ; in pathogens , they have been 
 shown to aid in bacterial invasion mechanisms . using a toluene emulsion assay , we tested the emulsive potential 
 of bp82 and bp82:prhab - mpn cultures both in the presence and in the absence of rhamnose . 

only the induced bp82:prhab - mpn supernatant 
 formed any emulsion when mixed 1:1 with organic solvent , indicating 
 that a potent surfactant property is , in fact , associated with malleipeptin 
 production ( figure 6a ) .
as biosurfactants have 
 also been shown to possess a role in modulating biofilms , we examined the effect of malleipeptin on bp82 biofilm production . at concentrations as low as 1 m , malleipeptin 
 disrupted bp82 biofilm formation ( figures 6b and s32 ) . on the basis 
 of these observations , we believe malleipeptins are biosurfactants 
 that are required at some stage during bp infections .
equal parts toluene and supernatant were combined , vortexed , 
 and then let stand for 2 h. ( a ) bp82 , no rhamnose ; ( b ) bp82 , plus rhamnose ; 
 ( c ) bp82:prhab - mpn , no 
 rhamnose ; ( d ) bp82:prhab - mpn , plus rhamnose ; ( e ) lb medium ( blank ) .
( b ) 
 microtiter plate assay showing increasing disruption of bp82 top biofilm with increasing concentrations of 2 ( 200 
 l cultures , 30 c , 48 h ; 20 magnification ) .
comparative genome analyses 
 led us to three small - molecule biosynthetic 
 gene clusters that are required for bp pathogenicity . 
 in a previous study involving bt , we showed that 
 the lipophilic siderophore malleilactone , which is encoded by 
 a gene cluster that is shared among bp , bm , and bt ( figure 2 , cluster 
 8) , strongly influences the virulence of bt in non - mammalian 
 models .
taken together , these works illustrate 
 that small molecules play critical , yet largely overlooked , roles 
 in burkholderia pathogenesis ( figure 7 ) .
additionally , 
 the identification and characterization of small molecules encoded 
 by cryptic gene clusters associated with virulence , especially when 
 studying highly controlled select agent pathogens like bp , provide a means to potentially more safely dissect the detailed 
 mechanisms of melioidosis .
small - molecule 
 biosynthesis is predicted to influence bp virulence 
 through diverse mechanisms : ( a ) bacterial invasion , ( b ) systemic toxicity , 
 and ( c ) acquisition of nutrients .
the inhibition of these overlooked 
 components of bp virulence should provide novel avenues 
 for the development of bp - specific anti - infective 
 agents . as the pathogenicity of bp
is known to involve 
 intracellular replication following invasion of both epithelial and 
 macrophage cells , potential roles for secondary metabolites within bp pathogenicity can be proposed .
biosurfactants have been 
 shown to disrupt epithelial integrity , facilitate paracellular infiltration 
 by bacterial pathogens , and support intercellular communication among 
 bacteria by improving the solubility of lipophilic quorum sensing 
 molecules .
the malleipeptins may play 
 similar roles in bp pathogenicity , potentially aiding 
 in bacterial infiltration and disease progression .
proteasome inhibition 
 is known to directly activate programmed cell death through induction 
 of apoptosis and autophagy across multiple cell lines , including macrophages . in the context of bp infections
, autophagy has 
 been shown to be critical for diverting host nutrients to the pathogen .
proteasome inhibition by the glidobactins 
 could therefore provide the mechanistic explanation for both promoting 
 intracellular replication or , in the case of macrophages , immunosuppression , 
 which has long been suspected within bp pathogenicity . in light of the importance of these small molecules 
 to bp pathogenesis , the inhibition of their biosyntheses 
 could prove to be a productive avenue for the development of next - generation 
 therapeutics for combating melioidosis .
deletion of biosynthetic clusters was performed via a two - stage 
 pcr strategy , wherein a key region of genomic dna was excised by homologous 
 recombination of dna designed from genetic regions both upstream and 
 downstream of the targeted excision region .
upstream and downstream regions ( 1 kb in length ) were amplified 
 from bp1026b genomic dna in a volume of 50 l 
 with 50 pmol of primer pair ( primers 2f/2r and primers 3f/3r ; listed 
 in table s3 ) , 1x failsafe premix e ( epicentre ) , 
 and 5 u phusion polymerase ( neb ) .
pcr cycling conditions were as follows : 
 98 c for 30 s , 30 cycles of 98 c for 30 s ; annealing at 
 62.5 c for 30 s ; 60 s extension at 72 c ; 72 c , 1 
 min .
primers were designed to ligate into the sacb - based vector pexkm5 as a smai / xhoi cassette using the in - fusion cloning kit 
 ( clontech ) .
each fragment was gel purified with qiagen pcr purification 
 kit , and the fragments were ligated together into 2 kb fragments 
 by a second round of pcr with the above protocol using primers 2f 
 and 3r and 1 ng of each product from the initial pcr reaction . 
 the extension time
was increased to 2 min , with final 72 c incubation 
 at 2 min .
recombinant 
 derivatives of pexkm5 were electroporated into e. coli s17 - 1 and conjugated with b. pseudomallei for 8 
 h , as described elsewhere.bp1026b transconjugants were selected with kanamycin ( 1000 g 
 ml ) , and polymyxin b ( 25 g ml ) was used to counterselect e. coli s17 - 1 .
optimal conditions for resolution of the sacb constructs were found to be lb agar lacking nacl and containing 
 10% sucrose , with incubation at 25 c for 34 days .
colony pcr was performed by resuspending an isolated 
 sucrose resistant colony in 50 l of water and using 5 l 
 of the suspension in a pcr reaction .
pcr amplifications were performed 
 in a final reaction volume of 50 l containing 1x failsafe premix 
 d ( epicentre ) , 1.25 u of failsafe pcr enzyme mix ( epicentre ) , and 
 1 m pcr primers .
pcr cycling conditions were as follows : 97 
 c for 5 min , 30 cycles of a three - temperature cycling protocol 
 ( 97 c for 30 s , 55 c for 30 s , and 72 c for 1 min ) ; 
 72 c , 10 min .
confirmation primers were designed to show a 500 
 bp region from the fusion of the upstream and downstream regions of 
 the excised target locus , as verified by agarose gel ( figure s1 ) .
b. pseudomallei animal work was 
 conducted under animal biosafety level 3 ( absl3 ) 
 conditions in accordance with institutional animal care and use committee 
 regulations at the u.s .
six- to eight - week - old female balb / c mice ( national cancer 
 institute , frederick , md ) were anesthetized with a 0.10.2 
 ml intraperitoneal injection of ketamine hcl , acepromazine , 
 and xylazine ( k - a - x ) .
the k - a - x solution was prepared by combining 
 5 ml of ketamine hcl ( 100 g ml ) and 0.5 
 ml of acepromazine ( 10 g ml ) with 2.75 
 ml of xylazine ( 20 g ml ) and mixing 1 ml 
 of the resulting solution with 9 ml of saline .
b. pseudomallei strains were grown overnight in lennox lb broth and then serially 
 diluted in phosphate - buffered saline , and 1010 colony - forming units were used to inoculate groups of 10 
 mice by intranasal instillation .
briefly , 50 l of the 
 bacterial inoculum was gradually released into the nostrils of anaesthetized 
 mice using a gilson pipetman p200 .
the infected animals were monitored 
 daily for a period of 21 days , at which time the survivors were euthanized 
 with co2 , and the 50% lethal dose ( ld50 ) was 
 calculated .
avirulent bp strain , bp82 , was 
 derived directly from bp1026b through deletion of 
 the purm gene responsible for de novo adenine synthesis , and all primers 
 were based on the bp1026b genome sequence .
all media 
 formulated for adenine auxotroph bp82 strain culturing 
 were supplemented with adenine ( 80 g ml ) and thymidine ( 5 g ml ) . for dual activation / suppression 
 of individual biosynthetic gene clusters in bp82 , 
 we previously designed a strategy for replacing native promoter regions 
 upstream of an individual biosynthetic cluster with an inducible promoter 
 imposing tight repression of transcription in burkholderia ( i.e. , 
 promoter exchange ) , detailed by biggins et al .
promoter exchange cassettes containing the rhamnose - inducible promoter 
 prhab and the dhfr trimethoprim resistance 
 gene ( from pscrhab2 vector ) , flanked 
 on each side by 1 kb of sequence homologous to the specific 
 recombination sites in the burkholderia genome , were constructed by 
 successive rounds of pcr / restriction digest / ligation .
each 4.8 
 kb promoter exchange cassette was pcr amplified from its puc - rha parent 
 clone with primer pair 2f/3r , gel purified , treated to afford blunt - ended 
 phosphorylation ( end - it dna repair kit ; epicenter ) , and ligated into 
 vector pexkm5 at the cip - treated smai site within 
 the multicloning region .
each pexkm5-cloned variant was transported 
 into e. coli s17 - 1 and conjugated with bp82 , as described.bp82/e .
coli co - cultures were resuspended in 250 l 
 of lb medium and selected over 23 days on selection media 
 lb agar / kanamycin ( 1000 g ml)/trimethoprim 
 ( 100 g ml ) .
merodiploid bp82 colonies were 
 picked and restreaked on a fresh selection plate for 48 h. a single 
 colony was then grown in 1 ml of yt medium overnight and plated on sacb - curing media yt agar / trimethoprim 
 ( 100 g ml)/x - gluc ( 50 g ml)/15% sucrose for 23 days at 30 c .
excision of the 
 plasmid backbone was verified by white colony phenotype and the inability 
 to survive against kanamycin ( 1000 g ml ) selection .
proper recombinant transformants were verified for proper 
 insertion / recombination by pcr using primer pair 4f/5r for proper 
 4.8 kb fragment containing prhab and flanking hybridization 
 domains .
seamless insertion of promoter prhab was verified 
 by amplifying the region within the prhab promoter and 
 downstream of the insertion site with primer pairs 5f/5r , gel purifying 
 each 1 kb fragment , and sanger sequencing to verify that no 
 mutations occurred during the cloning process .
strain bp82:prhab - mpn was grown overnight 
 to confluence in lb / trimethoprim ( 100 g ml ) , and then 500 l was inoculated into 50 ml of lb / trimethoprim 
 ( 100 g ml)/0.2% l - rhamnose with 
 1.5 g of hp-20 resin added and incubated at 30 c , 200 rpm , 72 
 h. an identical control culture lacked the addition of l - rhamnose 
 to the culture medium .
after 72 h , resin was filtered from the broths , 
 washed twice in equal amounts of deionized water , and dried overnight 
 in a laboratory fume hood .
dried resins were resuspended in 10 ml 
 of methanol , sonicated , decanted , dried in vacuo , 
 and monitored by reverse - phase hplc - ms ( linear gradient from 10:90 
 ch3cn / h2o with 0.1% formic acid to 100% ch3cn with 0.1% formic acid over 50 min ; 0.7 ml min ; waters xbridge c18 , 5 m , 4.6  150 mm ) . rhamnose - induced 
 culture ( illustrated in figure 4c ) displayed 
 two major peaks ( retention times : 22.6 min , 1 ; 23.4 min , 2 ) that are not present in the control cultures .
strain bp82:prhab - syr was grown overnight to confluence 
 in lb / trimethoprim ( 100 g ml ) , and then 
 500 l was inoculated into 50 ml of lb / trimethoprim ( 100 g 
 ml)/0.2% l - rhamnose and incubated at 
 30 c , 200 rpm , 72 h. an identical control culture lacked the 
 addition of l - rhamnose to the culture medium . after 72 h , 
 20 ml of culture broth was extracted with an equal amount of ethyl 
 acetate , dried in vacuo , and monitored by reverse - phase 
 hplc - ms ( linear gradient from 10:90 ch3cn / h2o with 0.2% trifluoroacetic acid to 100% ch3cn with 0.2% 
 trifluoroacetic acid over 50 min ; 0.7 ml min ; 
 waters xbridge c18 , 5 m , 4.6  150 mm ) . rhamnose - induced 
 culture ( illustrated in figure 5c ) displayed 
 two major peaks ( retention times : 31.6 min , 3 ; 34.0 min , 4 ) that are repressed in non - induced cultures .
the emulsive potential of malleipeptin 
 producing extracts of bp82:prhab - mpn cultures 
 was tested against wild - type bp82 , both with ( induction ) 
 and without 0.2% l - rhamnose added to media , using toluene 
 as an organic solvent .
confluent cultures 
 of each strain were grown overnight from a freshly restreaked colony 
 in lb medium .
aliquots of 50 l of lb media , both with and without 
 added 0.2% l - rhamnose , were inoculated with 500 l 
 of each strain and incubated ( 48 h , 200 rpm ; 30 c ) .
one milliliter 
 of each culture was pelleted ( 60 s ; 15000 g ) , from 
 which 500 l was mixed with an equal volume of toluene in a 
 glass vial , vortexed vigorously , and rested for 2 h. lb media alone 
 was used as a blank control .
a confluent 
 culture of bp82 was grown overnight from a freshly 
 restreaked colony 
 in lb medium , inoculated into 50 ml
lb medium , and grown to od600 0.5 ( 200 rpm ; 37 c ) , after which the culture was 
 diluted 1:100 and seeded in 96-well plates ( 200 l / well ) .
the 
 concentration of purified malleipeptin b ranged from 100 g 
 ml ( 70 m ) to 0.34 g ml ( 0.24 m ) ( 2.25-fold dilution increments ) .
blank controls 
 ( methanol solvent vector ) were co - incubated in adjacent wells .
cultures were incubated ( 48 h ; 80 rpm ; 30 
 c ) , and top biofilm formations were visualized through a dissecting 
 stereoscope .
disruption in biofilm is evident in cultures with ligand 
 concentration above 1 m malleipeptin b , wherein 
 biofilms become increasingly porous and fragmented .
still pictures 
 were taken with a leica m60 stereoscope affixed with an ic80hd camera 
 ( 20 magnification ) .
 increasing evidence has shown that 
 small - molecule chemistry in 
 microbes ( i.e. , secondary metabolism ) can modulate the microbe  
 host 
 response in infection and pathogenicity . 
 the bacterial disease melioidosis 
 is conferred by the highly virulent , antibiotic - resistant pathogen burkholderia pseudomallei ( bp ) . whereas 
 some macromolecular structures have been shown to influence bp virulence ( e.g. , secretion systems , cellular capsule , 
 pili ) , the role of the large cryptic secondary metabolome encoded 
 within its genome has been largely unexplored for its importance to 
 virulence . 
 herein we demonstrate that bp - encoded 
 small - molecule biosynthesis is indispensible for in vivo bp pathogenicity . 
 promoter exchange experiments were used to induce 
 high - level molecule production from two gene clusters ( mpn and syr ) 
 found to be essential for in vivo virulence . 
 nmr 
 structural characterization of these metabolites identified a new 
 class of lipopeptide biosurfactants / biofilm modulators ( the 
 malleipeptins ) and syrbactin - type proteasome inhibitors , both 
 of which represent overlooked small - molecule virulence factors for bp . 
 disruption of burkholderia virulence by inhibiting the 
 biosynthesis of these small - molecule biosynthetic pathways may prove 
 to be an effective strategy for developing novel melioidosis - specific 
 therapeutics . 
Introduction
Results and Discussion
Conclusions
Experimental
Procedures