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Published in final edited form as: Gene. 2008 Oct 28;430(1-2):123–131. doi: 10.1016/j.gene.2008.10.011

An Allelic Exchange System for Compliant Genetic Manipulation of the Select Agents Burkholderia pseudomallei and Burkholderia mallei

Mohamad A Hamad 1, Sheryl L Zajdowicz 1, Randall K Holmes 1, Martin I Voskuil 1,*
PMCID: PMC2646673  NIHMSID: NIHMS90455  PMID: 19010402

Abstract

Burkholderia pseudomallei and B. mallei are Gram-negative bacterial pathogens that cause melioidosis in humans and glanders in horses, respectively. Both bacteria are classified as category B select agents in the United States. Due to strict select-agent regulations, the number of antibiotic selection markers approved for use in these bacteria is greatly limited. Approved markers for B. pseudomallei include genes encoding resistance to kanamycin (Km), gentamicin (Gm), and zeocin (Zeo); however, wild type B. pseudomallei is intrinsically resistant to these antibiotics. Selection markers for B. mallei are limited to Km and Zeo resistance genes. Additionally, there are few well developed counter-selection markers for use in Burkholderia. The use of SacB as a counter-selection method has been of limited success due to the presence of endogenous sacBC genes in the genomes of B. pseudomallei and B. mallei. These impediments have greatly hampered the genetic manipulation of B. pseudomallei and B. mallei and currently few reliable tools for the genetic manipulation of Burkholderia exist. To expand the repertoire of genetic tools for use in Burkholderia, we developed the suicide plasmid pMo130, which allows for the compliant genetic manipulation of the select agents B. pseudomallei and B. mallei using allelic exchange. pMo130 harbors an aphA gene which allows for Km selection, the reporter gene xylE, which allows for reliable visual detection of Burkholderia transformants, and carries a modified sacB gene that allows for the resolution of co-integrants. We employed this system to generate multiple unmarked and in-frame mutants in B. pseudomallei, and one mutant in B. mallei. This vector significantly expands the number of available tools that are select-agent compliant for the genetic manipulation of B. pseudomallei and B. mallei.

Keywords: Burkholderia pseudomallei, B. mallei, allelic exchange

1. Introduction

The Gram-negative bacteria Burkholderia mallei and Burkholderia pseudomallei are the etiological agents of glanders and melioidosis, respectively (Gilad, 2007). B. mallei is a host adapted pathogen that primarily infects solipeds including horses, donkeys, and mules (Neubauer et al., 2005; Whitlock et al., 2007). Human infections with B. mallei are rare and are associated with close contact with infected animals (Lehavi et al., 2002). B. pseudomallei is a saprophytic free-living bacterium that is endemic to wet soil and stagnant water of Southeast Asia and Northern Australia (Dance, 2000; Cheng and Currie, 2005). Human infections with B. pseudomallei are commonly restricted to endemic areas and are associated with environmental exposure through inhalation, ingestion, or direct inoculation (Cheng and Currie, 2005). Treatment of human melioidosis remains challenging due to the high level of resistance of B. pseudomallei to a wide range of antibiotics (Chaowagul, 2000). Standard antibiotic therapy of human melioidosis requires long treatment regimens and relapse is common (Cheng and Currie, 2005; Limmathurotsakul et al., 2006). Furthermore, melioidosis is increasingly recognized as an emergent infectious disease, in part due to the global travel of both humans and animals (Dance, 2000; Rolim et al., 2005; Inglis et al., 2006; Jones et al., 2008). Both B. mallei and B. pseudomallei can cause fatal infections in humans and currently there is no approved protective vaccine against these pathogens (Cheng and Currie, 2005; Wiersinga et al., 2006). These issues have raised concerns regarding the use of B. pseudomallei and B. mallei in biological warfare and have lead to the classification of these pathogens as category B select agents in the United States (Rotz et al., 2002; Cheng et al., 2005).

Select-agent guidelines greatly restrict the introduction of antibiotic resistance markers into B. pseudomallei and B. mallei. Under these guidelines the use of resistance markers in B. pseudomallei and B. mallei is limited to antibiotics not used in the treatment of humans, animals, or for agricultural purposes. Currently, the only approved antibiotic resistance markers for B. pseudomallei are genes encoding resistance to kanamycin (Km), zeocin (Zeo), and gentamicin (Gm). The approved antibiotic resistance markers for B. mallei are genes encoding resistance to Km and Zeo. Unfortunately the usefulness of these resistance markers is very limited in B. pseudomallei since wild type strains are intrinsically resistant to all three antibiotics (Leelarasamee and Bovornkitti, 1989; Cheng and Currie, 2005). The select-agent guidelines and the paucity of suitable selection markers have impeded the development of genetic tools for the manipulation of B. pseudomallei.

A second obstacle in the development of genetic tools for select-agent compliant manipulation of B. pseudomallei and B. mallei is the lack of an effective counter-selectable marker required for selection of plasmid-free clones. Common counter-selection methods utilizing tetracycline resistance and streptomycin susceptibility cannot be used in the United States under select-agent regulations, which prohibit the creation of tetracycline or streptomycin resistant Burkholderia strains (Reyrat et al., 1998). The non-antibiotic counter-selection marker sacB has been employed in Burkholderia with very limited success due to the frequent presence of an intrinsic sacBC operon in B. pseudomallei and B. mallei genomes (Chan et al., 2004; Holden et al., 2004; Essex-Lopresti et al., 2005). These limitations have greatly hampered the genetic manipulation of B. pseudomallei and B. mallei, and hindered the understanding of the biology and pathogenesis of these deadly pathogens.

Despite these impediments, progress towards the development of genetic tools for the manipulation of Burkholderia has been steady (DeShazer et al., 1997; Choi et al., 2006; Cuccui et al., 2007; Flannagan et al., 2008). Most importantly, select-agent compliant genetic tools for B. pseudomallei and B. mallei have recently been developed and described in two separate reports (Barrett et al., 2008; Choi et al., 2008). The first select-agent compliant genetic system was developed by Choi et al. and relies on the natural competency of some B. pseudomallei strains to linear DNA (Choi et al., 2008; Thongdee et al., 2008). This genetic system exploits the natural competency of B. pseudomallei to introduce linear PCR fragments carrying either Km or Zeo markers into B. pseudomallei. These PCR fragments integrate at desired sites in the bacterial genome and selection of transformants is achieved using 700 to 1,000 μg ml−1 Km or 2,000 μg ml−1 Zeo for wild-type B. pseudomallei. The inserted antibiotic markers can then be excised by a site-specific recombinase expressed from a curable plasmid, leaving a scar of 86 nucleotides and creating unmarked, but not necessarily in-frame and non-polar, mutations. The second genetic system was developed by Barrett et al. and relies on a non-antibiotic marker that encodes tellurite resistance for the selection of Burkholderia transformants and a mutant pheS allele for counter-selection of plasmid-free clones (Barrett et al., 2008).

In this report, we add to these genetic tools by describing the suicide vector pMo130 and its utility to generate in-frame and unmarked deletions in B. pseudomallei and B. mallei. As a proof of concept, we describe the deletion and complementation of the flgK gene in B. pseudomallei K96243 and 1026b strains. Additionally, we demonstrate the reliability of our system by constructing twelve unmarked deletion mutants in B. pseudomallei K96243 and one mutant in B. mallei ATCC23344.

2. Materials and methods

2.1 Bacterial strains, growth conditions, and culture media

All bacterial strains and plasmids used in this study are listed in Table 1 and 2, respectively. Manipulations of B. pseudomallei and B. mallei were performed in the BSL3 facility at UCD. Unless otherwise stated, Burkholderia were grown in liquid LB broth or on LB agar plates at 37°C (Fisher scientific, Hampton, NH). E. coli was grown at 30°C in liquid LB broth or LB agar plates, and when appropriate Km was used at 50 μg/ml for plasmid selection. For counter-selection, co-integrants were grown in YT broth, which was made by dissolving 10 grams of tryptone and 10 grams of yeast extract in 1 liter of H2O. Sucrose was added to YT agar at a final concentration of 15% for B. pseudomallei and 5% for B. mallei. Motility was tested by stabbing LB agar plates containing 0.3% agar.

Table 1.

Bacterial strains.

Strains Relevant features Reference
B. pseudomallei K96243 Human Clinical isolate; KmRGmRZeoR PbR (Holden et al.,
B. pseudomallei 1026b Human Clinical isolate; KmR GmR ZeoR PbR (DeShazer et al.,
B. mallei ATCC23344 Human Clinical isolate; KmS GmS ZeoS PbR (Yabuuchi et al.,
B. pseudomallei K96243 flgK∷pMo146 B. pseudomallei K96243 with pMo146 inserted into bpsl0280, xylE + This study
B. pseudomallei 1026b flgK∷pMo146 B. pseudomallei 1026b with pMo146 inserted into bpsl0280, xylE + This study
B. pseudomallei K96243 ΔflgK Unmarked deletion of bpsl0280, nonmotile This study
B. pseudomallei 1026b ΔflgK Unmarked deletion of bpsl0280, nonmotile This study
B. pseudomallei K96243 ΔflgK comp Unmarked deletion of bpsl0280, complemented by an unmarked insertion of bpsl0280 downstream of bpsl3330, motile This study
B. pseudomallei K96243 ΔflgK+ pMo168 Unmarked deletion of bpsl0280 carrying empty replicative vector pMo168, nonmotile This study
B. pseudomallei K96243 ΔflgK+ pMo173 Unmarked deletion of bpsl0280 carrying complementation replicative vector pMo173, motile This study
B. pseudomallei 1026b ΔflgK+ pMo173 Unmarked deletion of bpsl0280 carrying complementation replicative vector pMo173, motile This study
B. pseudomallei K96243 Δbpss1160-1161 Unmarked deletion of bpss1160-1161 This study
B. pseudomallei K96243 Δbpss1161-1162 Unmarked deletion of bpss1161-1162 This study
B. pseudomallei K96243 Δbpsl0074-0075 Unmarked deletion of bpsl0074-0075 This study
B. pseudomallei K96243 Δbpss1359-1360 Unmarked deletion of bpss1359-1360 This study
B. pseudomallei K96243 Δbpss2314 Unmarked deletion of bpss2314 This study
B. pseudomallei K96243 Δbpss2345 Unmarked deletion of bpss2345 This study
B. pseudomallei K96243 Δbpss0031 Unmarked deletion of bpss0031 This study
B. pseudomallei K96243 Δbpss1957 Unmarked deletion of bpss1957 This study
B. pseudomallei K96243 Δbpss1917 Unmarked deletion of bpss1917 This study
B. pseudomallei K96243 Δ bpss0542 Unmarked deletion of bpss0542 This study
B. pseudomallei K96243 Δbpss1803 Unmarked deletion of bpss1803 This study
B. pseudomallei K96243 Δbpss0542-0543 Unmarked deletion of bpss0542-0543 This study
B. mallei ATCC23344 Δbmaa0164 Unmarked deletion of bmaa0164 This study
E. coli DH5α Cloning strain, F- f80lacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rk-, mk+) Laboratory stock
E. coli JM109 Cloning strain, e14−(McrA−) recA1 endA1 gyrA96 thi-1 hsdR17 (rK− mK+) supE44 relA1 Δ(lac- Stratagene
E. coli S17.1 For conjugal transfer, recA thi pro hsdRM+ RP4:2-Tc:Mu:KmTn7 TpR SmR (R. Simon, 1983)
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Pb: polymyxin B, Km: Kanamycin, Gm: Gentamicin, Zeo: zeocin. R: resistance, S: susceptibility.

Table 2.

Plasmids.

Plasmids Relevant features and use Reference
pET-28b Source of spacer DNA Novagen
algD-pVDX18 Source of xylE (Mohr et al., 1990)
pJPS8 Source of RK2 oriT (Sanchez-Romero et al., 1998)
pSCrhaB2 Source of oripBBR1 and mob region (Cardona and Valvano, 2005)
pK19mobsacB Source of sacB and aphA (Schafer et al., 1994)
pUC19 Vector back bone, source of ColE1 ori (Yanisch-Perron et al., 1985)
pMo13 pUC19 with MCS-1 and MCS-2 This study
pMo13X pMo13 plus xylE This study
pMo13Xmob-km pMo13X plus RK2 oriT and aphA, KmR This study
pMo130Xmob-km pMo13Xmob-km plus spacer DNA This study
pMo130 Suicide vector for allelic exchange in Burkholderia; ColE1 ori, RK2 oriT, xylE, sacB, KmR This study
pMo146 Suicide vector used to construct B. pseudomallei K96243 ΔflgK and B. pseudomallei 1026b ΔflgK; ColE1 ori, RK2 oriT, xylE, sacB, KmR This study
pMo168 Replicative vector for Burkholderia; oripBBR1, mob+, xylE, KmR This study
pMo167 Suicide vector used for in-cis complementation of B. pseudomallei K96243 ΔflgK; ColE1 ori, RK2 oriT, xylE, flgK, sacB, KmR This study
pMo173 pMo168 plus flgK used for in-trans complementation of B. pseudomallei ΔflgK; oripBBR1, mob+, flgK, xylE, KmR This study
pMo176 Suicide vector used to construct B. pseudomallei K96243 Δbpss1917 and B. mallei ATCC23344 Δbmaa0164; ColE1 ori, RK2 oriT, xylE, sacB, KmR This study
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2.2 DNA methods, PCR, and Cloning

Cloning methods were performed as described previously (Sambrook, 2001). Plasmid DNA was isolated using QiaPrep Spin kit (Qiagen, Inc., Studio City, CA). Restriction enzymes and T4 DNA ligase were purchased from NEB and used as outlined by the manufacturer (New England Biolabs, Beverly, MA). Chromosomal Burkholderia DNA was isolated using a QIAamp DNA mini kit (Qiagen). PCR products were purified using QIAquick PCR purification kit or QIAquick gel extraction kit (Qiagen). DNA from Burkholderia was amplified using Advantage Taq DNA polymerase (Clontech, Palo Alto, CA) or ExTaq DNA polymerase (TAKARA, Otsu, Japan). PfuTurbo DNA polymerase was used for the amplification of DNA fragments used to construct pMo130 and pMo168 as well as site-directed mutagenesis (Stratagene, La Jolla, CA). Primers were purchased from Integrated DNA Technologies (Coralville, IA). A list of all the primers sequences used in this study is presented in supplementary Table 1S.

Touchdown PCR was performed using ExTaq or Advantage Taq DNA polymerases under the following conditions: 95°C for 5 min; 30 cycles, each consisting of 95°C for 30 sec, 66°C for 30 sec (the temperature of this step was lowered 0.3°C each cycle), and 72°C for 1 min/kb; immediately following this series, a second series of 20 cycles, consisting of 95°C for 30 sec, 56°C for 30 sec, and 72°C for 1 min/kb were performed. A final extension step of 72°C for 10 min was included.

2.3 Promoter prediction and cloning

Promoter prediction was done using the Neural Network Promoter Prediction website (http://www.fruitfly.org/seq_tools/promoter.html). Cloning of promoters, leader, and terminator sequences was done using repetitive PCR. Initial PCR was performed using primers that amplify the gene of interest. The resulting PCR product was then used as a template for a second PCR using a second set of primers that overlaps the first primer set, thus introducing the desired sequence upstream or downstream from the target gene. This step was repeated until the desired promoter, leader, or terminator was introduced into the gene of interest.

The xylE gene was amplified by PCR to include a tacI promoter lacking the operator region and is referred to as tacI* in this study (de Boer et al., 1983). The xylE gene was amplified by PCR using the primers XylE-F1 and XylE-R and an algD-pVDX18 vector as a template (Mohr et al., 1990). To introduce a tacI* promoter to the xylE gene, the xylE PCR product was used as a template for a second PCR using the primers XylE-F2 and XylE-R. The resulting PCR product was then used for a final PCR reaction using the primers XylE-F3 and XylE-R. This final PCR product consisting of tacI* driven xylE contains a BglII, SmaI, HindIII sites upstream the xylE gene and a NotI site downstream of the gene.

The modified sacB gene used in the construction of pMo130 was generated by PCR to include three features: the original Bacillus subtilis sacB leader signal was replaced by the presumed leader signal of the B. mallei sacB gene; the expression of the sacB gene was driven by a predicted groES promoter from B. pseudomallei; and the ribosomal transcriptional terminator rrnB T1 from E. coli was introduced downstream the sacB gene (Sarmientos et al., 1983). All three modifications were introduced into the sacB gene by repetitive PCR using overlapping primers. The coding sequence for the first 22 amino acids of the B. subtilis sacB gene was replaced by the coding sequence for the first 37 amino acids of the B. mallei sacB gene. The sacB gene was amplified from pK19mobsacB (Schafer et al., 1994) by PCR using the primers SacB-L1F and SacB-Tr1. The resulting PCR product was then used a template for a second PCR reaction using the primers SacB-L2F and SacB-Tr2. This procedure was repeated three more times using the primers SacB-L3F, SacB-P1F, and SacB-P2F in that order as forward primers and the SacB-Tr2 as a reverse primer. This repetitive PCR resulted in the amplification of the modified sacB gene and was designed to include a SpeI and an EcoRI site upstream and downstream from the gene, respectively.

2.4 Construction of pMo130 and pMo168

Construction of pMo130 and pMo168 is outlined in supplementary Figure 1S. GenBank accession numbers for pMo130 and pMo168 are EU862243 and EU862244, respectively. pUC19 was used as the backbone for the construction of pMo130 and pMo168. The BamHI site located in lacZα of pUC19 was removed by site-direct mutagenesis using primer pUC19-ΔBamH1-F and pUC19-ΔBamH1-R as described previously (Hamad and Nilles, 2007). The resulting pUC19-ΔBamHI vector was modified by introducing the following restriction sites: EcoRV, ApaI, NheI, BglII upstream of lacZα and NotI, PstI, BamHI, XbaI, SphI downstream of lacZα to create multiple cloning sites 1 and 2, respectively. This was accomplished by PCR amplification of the region spanning lacZα using primers P-MCS1-F and P-MCS2-R while the plasmid backbone was PCR amplified using P-MCS2-F and P-MCS1-R. Both PCR fragments were digested with BamHI and NheI and ligated to create pMo13. Next the xylE amplicon containing the tacI * promoter was digested by BglII and NotI and cloned into linearized pMo13 digested with the same enzymes to generate pMo13X.

To generate pMo13Xmob-km, a Km resistance cassette (aphA) and origin of transfer (oriT) were cloned into pMo13X. This was accomplished by a three-piece ligation of the pMo13X vector backbone, oriT, and aphA. The primers OriT-F and OriT-R were used to amplify the oriT region from pJPS8 (Sanchez-Romero et al., 1998), while the aphA was amplified from pK19mobsacB (Sarmientos et al., 1983) using the primers Kan-F and Kan-R. The vector backbone of pMo13Xmob-km was amplified using the primers pUC19-F and pUC19-R. The resulting oriT PCR product was digested using PacI and SacII, the aphA amplicon was digested with SacII, and pMo13X PCR backbone was digested with PacI and SspI. All three PCR fragments were ligated to generate pMo13Xmob-km. Next a 930 bp DNA fragment was cloned as a spacer DNA between oriT and the aphA gene. This spacer DNA was amplified from pET-28b (Novagen) using the primers Spacer-F and Spacer-R. The PCR fragment was digested with SpeI and SacII and cloned into pMo13Xmob-km to create pMo130Xmob-km. The resulting pMo130Xmob-km vector has unique SpeI, EcoRI and SacII sites, which were used to generate pMo130 and pMo168.

To generate pMo130 the modified sacB gene described in the previous section was cloned into pMo130Xmob-km vector. The modified sacB PCR product was digested with EcoRI and SacII and cloned into pMo130Xmob-km linearized with the same enzymes to generate pMo130.

pMo168 was created as a mobilizable and replicative vector in Burkholderia. The replication origin, replication gene, and moblilization region used in pMo168 were PCR amplified from pSCrhaB2 (Cardona and Valvano, 2005) using the primers Rep-F and Rep-R. The resulting PCR product was digested with NheI and SacII and cloned into pMo130Xmob-km to generate pMo168.

2.5 Conjugative transfer of vectors into Burkholderia

Mobilizable vectors were introduced into B. pseudomallei and B. mallei by biparental mating. Briefly, cultures of E. coli S17-1 harboring the mobilizable vector were grown in flasks in LB broth supplemented with 50 μg/ml Km at 30°C overnight with shaking. Burkholderia were grown in flasks in LB broth overnight at 30°C with shaking. One ml of the donor E. coli and recipient Burkholderia were centrifuged separately and washed with 1 ml of fresh LB containing no antibiotics. The donor and recipient pellets were then suspended with 500 μl fresh LB broth and cultures were combined and filtered onto a 0.45 μM analytical filter (Nalgene, Rochester NY). The filter was placed onto LB agar containing no antibiotics and incubated for 4-8 hours at 37°C to allow for conjugation. Following incubation, the mating mixture was scraped from the filter, suspended in 3 ml of phosphate saline buffer, and 200 μl aliquots were plated by spreading onto LB agar containing Km to select for Burkholderia transformants and Zeo or polymyxin B (Pb) to kill donor E. coli.

For B. pseudomallei, Km was used at a concentration of 200 μg/ml while Zeo was used at 50 μg/ml. For B. mallei, Km was used at a concentration of 50 μg/ml for selection of B. mallei transformants and Pb was used at 30 μg/ml to kill donor E. coli. Plates were incubated for 2 days at 37°C and resulting colonies were exposed to a mist of 0.45 M pyrocatechol (Sigma-Aldrich). To prevent formation of aerosols containing B. mallei or B. pseudomallei, plates were open-faced in the BSL3 biosafety cabinet and pyrocatechol was sprayed indirectly above the plates allowing the mist to fall by gravity onto the colonies. Burkholderia transformants, based on yellow-colored appearance, were streaked for isolation onto LB agar containing 200 μg/ml Km and 50 μg/ml Zeo for B. pseudomallei and 50 μg/ml Km and 30 μg/ml Pb for B. mallei. Mating frequencies were calculated as the ratio of transformants over the number donor cells. The following minor notes are suggested to improve mating efficiency and recovery of transformants. Growth of donor E. coli and recipient Burkholderia should be performed in shaking flasks for increased aeration of cultures. Pb should not be used as counter-selection against donor E. coli in B. pseudomallei mating reactions, since we found that Pb increases the natural resistance of B. pseudomallei to Km (data not shown). Finally we would like to stress the importance of avoiding production of aerosols that contain bacteria during the pyrocatechol spraying, which should be performed indirectly as described above.

2.6 Sucrose resolution of pMo130 derivative vectors from Burkholderia co-integrants

Resolution of co-integrants was achieved by plating onto YT agar containing sucrose. Independently isolated co-integrant colonies were inoculated into 1 ml of YT broth and grown at 37°C while shaking for a minimum of 4 hours. Cultures were then serially diluted using YT broth and dilutions were plated onto YT agar containing 15% sucrose to select for resolved B. pseudomallei co-integrants or YT agar containing 5% sucrose to select for resolved B. mallei co-integrants. Resulting colonies were exposed to pyrocatechol as described above and white colonies (presumptive resolved co-integrants) were analyzed by PCR to confirm the desired deletion.

2.7 Cloning of deletion vectors and generation of Burkholderia deletion mutants

The plasmid pMo146 was used to generate an unmarked deletion in the B. pseudomallei flgK gene, bpsl0280, by allelic exchange in both K96243 and 1026b strains. pMo146 was constructed by cloning two PCR fragments, approximately 1 kb in length, spanning the upstream and downstream regions of the flgK of B. pseudomallei K96243. The upstream fragment was amplified using primers ΔflgK-US-NheI and ΔflgK-US-BgLII. The resulting PCR product was digested with NheI and BglII. The downstream fragment was amplified using ΔflgK-DS-BgLII and ΔflgK-DS-HindIII and the resulting PCR product was digested with BglII and HindIII. Digested upstream and downstream fragments were ligated into vector pMo130 linearized with NheI and HindIII to generate pMo146.

pMo146 was introduced into B. pseudomallei K96243 and 1026b strains by biparental mating as described above. Following sucrose counter-selection, plasmid-free colonies were assayed for loss of motility. Confirmation of the B. pseudomallei K96243 ΔflgK mutant was done by PCR using the primers FlgK-screen-F and FlgK-screen-R.

pMo176 was used to generate an unmarked deletion of the bpss1917 gene of B. pseudomallei K96243 and the bmaa0164 gene of B. mallei ATCC23344. The upstream region of bpss1917 was amplified using primers ΔBpss1917-US-NheI and ΔBpss1917-US-BgLII and resulting PCR was digested with NheI and BglII. The downstream fragment was amplified using ΔBpss1917-DS -BgLII and ΔBpss1917-DS-HindIII and the resulting PCR product was digested with BglII and HindIII. Digested upstream and downstream fragments were ligated into pMo130 linearized with NheI and HindIII to generate pMo176. Deletion mutants were confirmed by PCR using the primers ΔBpss1917-US-NheI and ΔBpss1917-DS-HindIII.

The generation of the deletion vectors used to create the B. pseudomallei K96243 mutants shown in figure 5 will be described elsewhere and is available upon request.

Figure 5.

Figure 5

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PCR confirmation of 12 unmarked deletions in B. pseudomallei K96243 (lanes 2-23), and one unmarked deletion in B. mallei ATCC23344 (lanes 24-25). L: DNA ladder. Wt and M are PCR performed on wild type and mutant strains, respectively. Lane 1-2: PCR confirmation of Δbpss1917, wild type allele is 2666 bp and mutant allele is 2000 bp. Lane 3-4: PCR confirmation of Δbpss1161-1162, wild type allele is 1257 bp and mutant allele is 303 bp. Lane 5-6: PCR confirmation of Δbpss1803, wild type allele is 2570 bp and mutant allele is 2000 bp. Lane 7-8: PCR confirmation of Δbpss2345, wild type allele is 2175 bp and mutant allele is 229 bp. Lane 9-10: PCR confirmation of Δbpss1160-1161, wild type allele is 3102 bp and mutant allele is 800 bp. Lane 11-13: PCR confirmation of Δbpss0542 and Δbpss0542-0543 wild type allele is 5393 bp, Δbpss0542, M1, is 3758 bp, and Δbpss0542-0543, M2, is 2090 bp. Lane 14-15: PCR confirmation of Δbpss0031, wild type allele is 2582 bp and mutant allele is 2000 bp. Lane 16-17: PCR confirmation of Δbpss1957, wild type allele is 1394 bp and mutant allele is 641 bp. Lane 18-19: PCR confirmation of Δbpsl0074-0075, wild type allele is 3151 bp and mutant allele is 1165 bp. Lane 20-21: PCR confirmation of Δbpss1359-1360, wild type allele is 2071 bp and mutant allele is 284 bp. Lane 22-23: PCR confirmation of Δbpss2314, wild type allele is 2017 bp and mutant allele is 284 bp. Lane 24-25: PCR confirmation of bmaa0164 deletion in B. mallei ATCC23344, wild type allele is 2666 bp and mutant allele is 2000 bp.

2.8 Complementation of ΔflgK mutants of B. pseudomallei

pMo167 was used for the in-cis complementation of B. pseudomallei ΔflgK mutant. pMo167 was designed to insert the flgK gene into the large chromosome of B. pseudomallei K96243 downstream of the bpsl3330 gene. pMo167 was constructed by cloning three PCR products consisting of the flgK gene including 210 bp upstream from the gene to retain possible ribosomal binding site, and two fragments of 1 kb in length each spanning upstream and downstream regions from the insertion site. The upstream and downstream regions were used to flank the flgK gene in the same orientation as the bpsl3330 gene. A 1 kb DNA fragment downstream from the insertion region was amplified using the primers flgK-Comp-DS-SmaI and flgK-Comp-DS-Hind III. The PCR product was digested with SmaI and HindIII and cloned into pMo130 to generate pMo165. The flgK gene was amplified by PCR using the primers flgK-BglII and flgK-EcoRV. A 1 kb DNA fragment upstream from the insertion site was amplified by PCR using flgK-Comp-US-NheI and flgK-Comp-US-BglII. The flgK gene PCR product was digested with EcoRV and BglII while the upstream fragment was digested with BglII and NheI and both fragments were ligated into pMo165 linearized with SmaI and NheI to create pMo167. pMo167 was introduced into the B. pseudomallei K96243 ΔflgK mutant strain by biparental mating as described above. Following sucrose resolution, plasmid-free colonies were assayed for restoration of motility. Confirmation of the in-cis complementation in the B. pseudomallei K96243 ΔflgK mutant was done by PCR using the primers flgK-comp-screen F and flgK-comp-screen R.

pMo173 was constructed as a replicative vector in Burkholderia for in-trans complementation of the ΔflgK B. pseudomallei mutants. The flgK gene was amplified by PCR to include a predicted native promoter of the bpsl0270 gene, which is part of the flagellar operon. The flgK gene and the predicted promoter were generated by two repetitive PCR reactions using flgK-rep-comp-F1 and flgK-rep-comp-F2 as forward primers and flgK-comp-R as the reverse primer. The PCR product was then digested with NheI and HindIII and ligated with pMO168 to generate pMo173. pMo173 was then introduced into the ΔflgK mutants of B. pseudomallei K96243 and 1026b strains by biparental mating.

3. Results

3.1 Features of the select-agent compliant vectors pMo130 and pMo168

The maps of the select-agent compliant plasmids pMo130 and pMo168 are presented in Figure 1A and their construction is depicted in supplementary Figure 1S. pMo130 carries a ColE1 origin of replication derived from pUC19 plasmid and is a suicide vector in Burkholderia. This suicide vector can be used for allelic exchange to generate in-frame deletions or insertions into the genomes of Burkholderia. pMo168 carries the pBBR1 origin of replication and can be maintained extra-chromosomally in Burkholderia. The replicative vector pMo168 can be used for in-trans complementation or gene expression in Burkholderia. pMo130 and pMo168 carry an origin of transfer, which allows for their mobilization into Burkholderia through conjugation. Both pMo130 and pMo168 carry the reporter gene xylE which encodes a catechol-2,3-dioxygenase, an enzyme that turns colorless catechol substrate into bright yellow-colored 2-hydroxymuconic semialdehyde (Zukowski et al., 1983; Lee et al., 1996). xylE is expressed from a tacI promoter lacking the operator region which allows for constitutive and high level of expression in Burkholderia. Flanking xylE are two multiple cloning sites, MCS1 and MCS2, which contain unique restriction sites that can be used for cloning. Selection for pMo130 and pMo168 is achieved by the activity of an aphA gene, which confers Km resistance and is an approved antibiotic selection marker for B. pseudomallei and B. mallei. Finally, counter-selection against pMo130 is achieved through a modified sacB gene optimized for Burkholderia. The modified B. subtilis sacB gene has the leader sequence of the B. mallei sacB and is expressed from a predicted promoter of the groES genes of B. pseudomallei.

Figure 1.

Figure 1

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Plasmid maps and allelic exchange. A. Plasmid maps of pMo130 and pMo168. pMo130 and pMo168 carry a kanamycin resistance cassette (KmR) and the reporter gene xylE. pMo130 carries a ColE1 origin of replication (ori) and an RK2 origin of transfer (oriT) and is a mobilizable and suicide vector in Burhkolderia. Multiple cloning sites for pMo130 are MCS-1: ApaI, NheI, BglII, SmaI, HindIII and MCS-2 NotI, PstI, BamHI, XbaI, SphI. pMo168 is a mobilizable vector derived from the vector pSCrhaB2 and is a replicative vector in Burkholderia. pMo168 carries the mobilization region (mob) and replication features of pBBR1. Multiple cloning sites for pMo168 are MCS-1: ApaI, NheI, BglII, SmaI, HindIII and MCS-2: PstI, BamHI, XbaI, SphI. B. The use of allelic exchange to construct a flgK deletion in B. pseudomallei. DNA fragments upstream and downstream the flgK gene are cloned into pMo130 to generate pMo146. pMo146 is introduced into B. pseudomallei through conjugation and co-integrant are recovered by kanamycin selection and screening for xylE positive yellow colonies. Resolved co-integrant are recovered by sucrose counter-selection and screening for xylE negative white colonies. Colonies are analyzed by PCR or phenotypic analysis to distinguish deletion mutants from wild type isolates.

3.2 Use of pMo130 to create an unmarked flagellar B. pseudomallei mutant

To demonstrate the utility of pMo130 for allelic exchange, the bpsl0280 gene of B. pseudomallei encoding the flagellar gene flgK was targeted for deletion. One kb upstream and downstream fragments flanking the bpsl0280 gene of B. pseudomallei K96243 were cloned into pMo130. The resulting plasmid, pMo146, was conjugated into B. pseudomallei K96243 and 1026b to isolate co-integrants representing the first crossover event required for allelic exchange (Fig. 1B). Selection and identification of B. pseudomallei co-integrants was achieved using a combination of Km selection and visual detection of XylE activity following catechol treatment (Fig. 2A). Wild-type B. pseudomallei is resistant to Km mainly through the activity of efflux pumps (Moore et al., 1999; Choi et al., 2008). However we found that plating of wild type B. pseudomallei on LB agar plates supplemented with 200 μg/ml Km results in a reduction of 7.3 log10 and 6.2 log10 in colony counts for the K96243 and 1026b strains, respectively. To overcome this leaky selection capacity of Km, colonies that grew on Km agar plates were sprayed with catechol to detect XylE activity. The activity of XylE in Burkholderia turns co-integrant colonies into bright yellow color while spontaneously Km resistant colonies remain white in color (Fig. 2A-C). Typically 30-50% of B. pseudomallei colonies recovered on Km agar plates were true transformants (Fig. 2A), and the efficiency of co-integrants formation was 2.3×10-7 and 5.6×10-7 per donor cell for the K96243 and 1026b strains, respectively.

Figure 2.

Figure 2

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Utility of a pMo130 derivative vector in Burkholderia. A. Use of xylE of pMo130 for detection of Burkholderia transformants. A 200 μl of conjugation reaction between wild type B. pseudomallei K96243 and donor E. coli to introduce the pMo130 derivative, pMo146, were plated on Km-Zeo LB agar. Following 2 days incubation, plates were indirectly sprayed with catechol to visualize yellow-colored transformants and to distinguish them from white-colored wild type nontransformant colonies. B. Pure culture of B. pseudomallei K96243 and B. pseudomallei K96243 carrying a single copy of pMo146 integrated in the large chromosome prior to catechol treatment. C. Same as B after catechol treatment. D. Use of the modified sacB of pMo130 for killing of B. pseudomallei K96243 carrying a single copy of pMo146 integrated in the large chromosome. Wild type B. pseudomallei K96243 and pMo146 co-integrant were serially diluted at 1:10 and plated on LB agar or YT agar plus 15% sucrose, and colonies were scored 2 days post incubation. E. Use of the modified sacB for counter-selection of plasmid-free resolvants of B. pseudomallei K96243. pMo146 co-integrant culture were plated onto LB agar and YT agar plus 15% sucrose and incubated for 2 days. Resulting colonies were sprayed with catechol to determine resolution.

The resolution of pMo146 co-integrants was achieved through sucrose counter-selection which is required for the second crossover event and vector loss (Fig. 1B). The plating efficiency of B. pseudomallei containing a single copy of pMo146 on YT agar containing 15% sucrose vs. LB agar was approximately 1% (Fig. 2D). In contrast, plasmid-free B. pseudomallei grew equally well on YT agar containing 15% sucrose and on LB agar (Fig. 2D). Because counter-selection using 15% sucrose does not inhibit growth of plasmid-free B. pseudomallei, it is unlikely to cause selection for unintended secondary mutations. To determine the efficiency of sucrose resolution, pMo146 co-integrants were plated onto LB plates or 15% sucrose YT agar and resolution was determined visually through XylE activity. None of the colonies from the LB plates had lost the integrated vector; however, all of the colonies that grew on the sucrose agar plate were plasmid-free as determined by the loss of XylE activity (Fig. 2E). Experiments utilizing pMo146 and other pMo130 derivatives show that during sucrose resolution, greater than 99% of resulting colonies are plasmid free for both K96243 and 1026b strains of B. pseudomallei.

Screening for deletion mutants is typically performed by PCR of resolved colonies. However, since this flagellar deletion has a known phenotype, the flgK deletion mutant was detected by its loss-of-motility phenotype. Of the twenty resolved colonies screened from each strain, thirteen colonies of the K96243 strain and eight colonies of the 1026b strain were found to be non motile. The motility of the wild type and the ΔflgK mutants in the 1026b and K96243 strains are shown in Figure 3.

Figure 3.

Figure 3

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Motility plate assays. A. Motility of wild type B. pseudomallei K96243 (Wt), its ΔflgK derivative mutant (ΔflgK), and the in-cis ΔflgK complemented mutant (ΔflgK comp). B. Motility of wild type B. pseudomallei K96243, its ΔflgK derivative mutant carrying empty replicative vector pMo168, and its ΔflgK derivative mutant carrying the complementation vector pMo173. C. Motility of wild type B. pseudomallei 1026b, its ΔflgK derivative mutant, and the ΔflgK mutant complemented in-trans with pMo173.

3.3 Complementation of the B. pseudomallei flagellar mutants

Complementation of the flagellar B. pseudomallei mutants was done both in-cis and in-trans to demonstrate that the mutation was non-polar, and that pMo130 can be reused for allelic exchange in the same mutant strain. For in-cis complementation, pMo130 was used to construct an unmarked insertion of flgK into the large chromosome of the ΔflgK mutant of B. pseudomallei K96243. The flgK gene, bpsl0280, was inserted by allelic exchange downstream and in the same orientation as the bpsl3330 gene. flgK was cloned between the desired insertion site into pMo130 and the resulting vector was introduced into the ΔflgK mutant of B. pseudomallei K96243 by conjugation. Following sucrose counter-selection, resolved colonies were screened for complementation by the restoration of motility. Nine out of the twenty resolved colonies tested were found to be motile. Motility of the B. pseudomallei K96243 ΔflgK mutant complemented through the insertion of flgK downstream from the bpsl3330 is shown in figure 3A. PCR was then used to confirm that the flgK gene “bpsl0280” was inserted downstream of the bpsl3330 gene and not at the homologous DNA sequence of the bpsl0280 deletion allele. PCR screening using primers flanking of the flgK gene confirmed the deletion of flgK (Fig. 4, lanes 1- 2) and demonstrated that the flgK gene did not reinsert at the homologous deletion allele in the complemented flgK mutant (Fig. 4, lanes 1-3). PCR screening of the complemented mutant using primers flanking the insertion region confirmed that the flgK gene was indeed inserted at the desired region downstream of bpsl3330 (Fig. 4, lanes 4-6). Taken together, these results demonstrate that pMo130 is suitable for genomic complementation in B. pseudomallei and that this vector can be reused to introduce multiple mutations or insertions into the same strain.

Figure 4.

Figure 4

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PCR confirmation of B. pseudomallei K96243 ΔflgK mutant and the in-cis complemented derivative (ΔflgK comp). PCR confirmation of the flgK deletion was performed using the outside primers flgK-screen-F and flgK-screen-R. PCR confirmation of the insertion of flgK downstream bpsl3330 was performed using the primers flgK-comp-screen-F and flgK-comp-screen-R which are outside the complementation region. The ΔflgK mutant had a 2 kb deletion in comparison to wild-type B. pseudomallei K96243. The complemented strain had a 2 kb insertion in comparison to wild-type or un-complemented ΔflgK mutant.

In-trans complementation was achieved by cloning the flgK gene into the replicative vector pMo168 to generate pMo173. pMo168 and pMo173 were then introduced into the ΔflgK mutant strains of B. pseudomallei by conjugation and transformants were recovered using Km selection and detection of XylE activity. Complementation of the non-motile ΔflgK mutants through the introduction of pMo173 restored motility in both mutant strains, while the un-complemented mutant or the mutant carrying empty vector remained non-motile (Fig. 3B-C). These results demonstrate the utility of pMo168 for in-trans complementation and gene expression in Burkholderia.

3.4 Construction of twelve B. pseudomallei mutants and a B. mallei mutant

To further demonstrate the reliability of pMo130 in generating unmarked deletions in B. pseudomallei, we generated numerous other deletions in the K96243 strain. The K96243 strain, rather than strain 1026b, was used for constructing these mutations because the genomic sequence of the K96243 is available. Twelve unmarked mutants were constructed by cloning the appropriate flanking DNA fragments into pMo130 and using the resulting vectors for allelic exchange as described above for the flagellar deletion. PCR confirmation of the twelve mutants is shown in figure 5 (Fig. 5 lanes 2-23). These mutants included deletions of bpss1161-1162, bpss2345, bpss1160-1161, bpsl0074-0075, bpss1359-1360, and bpss2314 which encode putative response regulators and/or sensor kinases; bpss0031, bpss1917, bpss1803 which encode putative transcriptional regulators; bpss1957 which encodes a putative 6-phosphofructokinase; and bpss0542 and bpss0542-0543 which encode putative sacC and sacBC genes, respectively. The transformation and resolution efficiencies obtained during the creation of these mutants were similar to those observed for the flgK deletion. The rate of mutant recovery differed for each deletion, and generally 20-50% of the screened resolved colonies contained the deleted form as confirmed by PCR.

The use of pMo130 for generating deletions in B. mallei ATCC23344 strain was also evaluated. The bmaa00164 gene of B. mallei is homologous to bpss1917 of B. pseudomallei K96243. pMo176, which was used to delete bpss1917 in B. pseudomallei K96243, was used to delete bmaa00164 in B. mallei through allelic exchange. pMo176 was introduced into B. mallei by conjugation and co-integrants were selected using 50 μg/ml Km since B. mallei is sensitive to Km. The efficiency of co-integrants formation was 2.3×10-7 per donor cell, and all of the recovered Km resistant colonies were true co-integrants as determined by XylE activity. B. mallei co-integrants were plated onto YT agar plus 5% sucrose to allow for the resolution of the integrated pMo176. The resolution rate of B. mallei co-integrants was lower than that of B. pseudomallei and only 10-20% of the colonies that grew on sucrose plates were plasmid-free as determined by loss of XylE activity. Resolved colonies were screened by PCR and five out of the sixteen analyzed colonies were found to contain the mutation. PCR of the wild type B. mallei gene bmaa00164 as well as its deletion allele are shown in figure 5 (Fig. 5 lanes 24-25).

4. Discussion

Genetic manipulation of B. pseudomallei and B. mallei has been difficult and their classification as category B select agents has greatly limited the development of new and useful genetic tools. The vectors presented in this study add to the repertoire of available tools that allow for the select-agent compliant manipulation of B. pseudomallei and B. mallei. Our system overcomes some of the limitations of other systems, including the recovery of false positive transformants despite the use of high antibiotic concentrations for selection, lack of natural competence of specific Burkholderia strains for DNA uptake, and generation of DNA scars following marker excision (Choi et al., 2008). Additionally, our system uses homologous recombination to exchange mutant alleles for wild type alleles (or vice-versa) in the genome of Burkholderia. Our genetic system is reliable for generating deletion mutants in B. pseudomallei, and we have successfully utilized this system to generate numerous in-frame and unmarked deletions both in B. pseudomallei K96243 and 1026b (this work; M. Hamad and S. Zajdowicz unpublished). We have also demonstrated the usefulness of this system to produce mutations in B. mallei ATCC23344 (this work; and S. Zajdowicz). A minor drawback in the manipulation of B. mallei is that sucrose counter-selection generates a lower proportion number of resolved colonies than in B. pseudomallei and can lead to recovery of spontaneous deletions of the endogenous sacB region of B. mallei (DeShazer et al., 2001). However, these drawbacks can be overcome by using our allelic exchange vector in a ΔsacB B. mallei background. A ΔsacB B. mallei background strain has been created and is fully virulent in the mouse model of glanders infection (DeShazer et al., 2001; Schell et al., 2008).

In conclusion, the genetic tools described herein are a valuable addition to the repertoire of Burkholderia genetic systems and will help to advance our understanding of the biology and pathogenesis of these dangerous pathogens.

Supplementary-material

01. Figure 1S.

Construction of pMo130 and pMo168. Only restriction sites and primers relevant for the vector construction are shown, primers are depicted as arrows. Multiple cloning sites are MCS-1 and MCS-2. ApR and KmR are bla and aphA genes encoding ampicillin and Km resistance, respectively. The ribosomal transcriptional terminator rrnB T is shown as the letter t. p-bla, p-groES, and p-tacI* are promoters of the original pUC19 bla and groES genes of B. pseudomallei, and the tacI prompter lacking operator sequence, respectively. ori and oriT represent a ColE1 origin of replication and an RK2 origin of transfer, respectively. rep, mob, and oripBBR1 are replication and mobilization features derived from pSCrhaB2. Bma leader is the predicted secretion signal of the B. mallei sacB gene.

02

Acknowledgments

We would like to thank Dr. Mike Schurr for providing algD-pVDX18 vector, Dr. Miguel Valvano for providing pSCrhaB2 vector, and Dr. Victor De Lorenzo for providing pJPS8 vector. This research was supported Rocky Mountain Regional Center of Excellence grant AI-065357 awarded to MIV and NIH R21 grant AI074606-01 awarded to RKH.

Abbreviations

Pb

Polymyxin B

Gm

gentamicin

Km

kanamycin

Zeo

zeocin

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01. Figure 1S.

Construction of pMo130 and pMo168. Only restriction sites and primers relevant for the vector construction are shown, primers are depicted as arrows. Multiple cloning sites are MCS-1 and MCS-2. ApR and KmR are bla and aphA genes encoding ampicillin and Km resistance, respectively. The ribosomal transcriptional terminator rrnB T is shown as the letter t. p-bla, p-groES, and p-tacI* are promoters of the original pUC19 bla and groES genes of B. pseudomallei, and the tacI prompter lacking operator sequence, respectively. ori and oriT represent a ColE1 origin of replication and an RK2 origin of transfer, respectively. rep, mob, and oripBBR1 are replication and mobilization features derived from pSCrhaB2. Bma leader is the predicted secretion signal of the B. mallei sacB gene.

NIHMS90455-supplement-01.pdf (156.2KB, pdf)
02
NIHMS90455-supplement-02.doc (28.5KB, doc)

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