Two Systems for Targeted Gene Deletion in Coxiella burnetii

Paul A. Beare, Charles L. Larson, Stacey D. Gilk, and Robert A. Heinzen

Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes ofHealth, Hamilton, Montana, USA

Coxiella burnetii is a ubiquitous zoonotic bacterial pathogen and the cause of human acute Q fever, a disabling influenza-likeillness. C. burnetii’s former obligate intracellular nature significantly impeded the genetic characterization of putative virulencefactors. However, recent host cell-free (axenic) growth of the organism has enabled development of shuttle vector, transposon,and inducible gene expression technologies, with targeted gene inactivation remaining an important challenge. In the presentstudy, we describe two methods for generating targeted gene deletions in C. burnetii that exploit pUC/ColE1 ori-based suicideplasmids encoding sacB for positive selection of mutants. As proof of concept, C. burnetii dotA and dotB, encoding structuralcomponents of the type IVB secretion system (T4BSS), were selected for deletion. The first method exploited Cre-lox-mediatedrecombination. Two suicide plasmids carrying different antibiotic resistance markers and a loxP site were integrated into 5= and3= flanking regions of dotA. Transformation of this strain with a third suicide plasmid encoding Cre recombinase resulted in thedeletion of dotA under sucrose counterselection. The second method utilized a loop-in/loop-out strategy to delete dotA anddotB. A single suicide plasmid was first integrated into 5= or 3= target gene flanking regions. Resolution of the plasmid cointe-grant by a second crossover event under sucrose counterselection resulted in gene deletion that was confirmed by PCR andSouthern blot. �dotA and �dotB mutants failed to secrete T4BSS substrates and to productively infect host cells. The repertoireof C. burnetii genetic tools now allows ready fulfillment of molecular Koch’s postulates for suspected virulence genes.

The intracellular bacterium Coxiella burnetii causes the zoo-notic disease Q fever. Symptomatic infections normally man-

ifest as an acute, debilitating influenza-like illness with rare butserious long-term sequelae, including chronic endocarditis. Theorganism is highly infectious, environmentally stable, and usuallytransmitted to humans via inhalation of contaminated aerosolsgenerated by animal husbandry operations. Sheep, goats, anddairy cattle are important animal reservoirs, with large-scale dairygoat farming in the Netherlands recently associated with the larg-est Q fever outbreak ever recorded (�4,000 cases) (21, 32).

C. burnetii has a unique intracellular lifestyle that involves acid-activated metabolism within a phagolyosome-like vacuole (15,20). The abilities of C. burnetii to replicate to high numbers in thedegradative confines of this compartment and to modulate hostcell functions that promote replication vacuole biogenesis repre-sent important pathogenic strategies (46). Many C. burnetii genesencoding putative virulence factors were revealed by genome se-quencing, including a Dot/Icm type IVB secretion system (T4BSS)(6, 36). Several C. burnetii proteins have been shown to be trans-located into the host cell cytoplasm in a T4BSS-dependent fashionthat are predicted to have important effector functions (9, 10, 23,29, 45, 47). However, the lack of methods for targeted gene inac-tivation has greatly hindered establishment of the functional rolesof T4BSS effector proteins and other putative virulence factors inC. burnetii pathogenesis.

Genetic transformation of C. burnetii was first accomplished in1996 by Suhan et al., who transformed the organism to ampicillinresistance using a shuttle vector containing a 5.8-kb C. burnetiiautonomous replication sequence (40, 41). More than a decadeelapsed before the next report of transformation, achieved usingthe mariner family transposon Himar1 to transform the organismto chloramphenicol resistance (4). These two genetic tools weredeveloped using eucaryotic host cell-based propagation of C. bur-netii, a limitation that imposes significant technical challenges.

However, C. burnetii has recently been rescued from an obligateintracellular lifestyle by robust host cell-free (axenic) growth in amedium termed acidified citrate cysteine medium (ACCM) (27,28). Axenic growth of C. burnetii in liquid medium and as clonalcolonies on agarose plates substantially reduces the time of trans-formant isolation. Indeed, in the last 2 years axenic growth hasenabled development of an improved Himar1 transposon system(5), RSF1010 ori-based shuttle vectors (3, 10, 27), a Tn7 system forsingle-copy, site-specific, in cis complementation (3, 5), and a sys-tem for inducible gene expression using an anhydrotetracycline(aTc)-inducible promoter (3).

A key remaining challenge in C. burnetii genetic manipulationis development of reliable protocols for targeted gene disruption.Conventional allelic-exchange methods generally rely on homol-ogous recombination between an introduced mutant allele and awild-type copy present on the chromosome. An important dis-covery made by Suhan et al. (41) is that homologous recombina-tion occurs between the C. burnetii 5.8-kb autonomous replica-tion sequence present on plasmid transformation DNA and thecorresponding region of the chromosome. Subsequent genomesequencing further supported functional homologous recombi-nation machinery in C. burnetii by revealing chromosomal rear-rangements mediated by recombination between abundant inser-tion sequence elements as small as 1.1 kb in size (6, 36).

Several strategies for targeted bacterial gene inactivation exist

Received 19 March 2012 Accepted 12 April 2012

Published ahead of print 20 April 2012

Address correspondence to Robert A. Heinzen, rheinzen@niaid.nih.gov.

Supplemental material for this article may be found at http://aem.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.00881-12

4580 aem.asm.org Applied and Environmental Microbiology p. 4580–4589 July 2012 Volume 78 Number 13

on Septem

ber 1, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from

that would exploit the homologous recombination ability of C.burnetii. Inactivation could occur via recombination between awild-type gene and a linear DNA fragment containing a mutatedallele (13). However, this strategy requires a low-frequency dou-ble-crossover event. Insertional duplication via single-crossoverrecombination with a suicide plasmid carrying an internal targetgene fragment can result in target gene copies with 5= and 3= de-letions (42, 44). Deficiencies of this method include spontaneousdeletion of the integrated plasmid, resulting in reversion back towild type, and difficultly in inactivating small genes due to subop-timal recombination substrate. A more sophisticated method ofgene deletion exploits both single-crossover plasmid integrationand a counterselectable marker (31). The first step involves chro-mosomal integration of a suicide plasmid carrying upstream anddownstream regions of a target gene, and a counterselectablemarker. In the next step, the “cointegrant” is resolved by a secondrecombination event between the plasmid encoded flanking re-gion and the reciprocal region in the chromosome, resulting inremoval of the wild-type gene. This “loop-in/loop-out” strategy(17, 25) frequently uses the counterselectable sacB, conferring su-crose sensitivity, which allows positive selection of the secondcrossover event, and can be configured to generate unmarked mu-tations (31, 34).

Protocols that exploit both bacterial homologous recombina-tion and the activities of the heterologous, site-specific recombi-nases Flp and Cre have also proven successful in mutating/delet-ing bacterial genes (8, 22, 35, 38). Flp recombinase fromSaccharomyces cerevisiae promotes recombination between two34-bp Flp recombinase target sites (22, 35), whereas Cre recom-binase from P1 bacteriophage promotes recombination betweentwo 34-bp loxP sites (8, 38). The Cre-lox system was recently usedto delete multiple genes of the Borrelia burgdorferi plasmid lp54(8). Suicide plasmids containing kanamycin or streptomycin re-sistance genes, and a loxP site, were integrated into lp54 by singlecrossover events. In one deletion mutant, eight plasmid genes en-compassing approximately 4,000 bp were completely or partiallydeleted following transformation with a third suicide plasmid en-coding Cre recombinase.

Results of the present study expand the repertoire of C. burnetiigenetic tools to include two protocols for targeted gene inactiva-tion. As proof of principle, we describe deletion of dotA usingCre-lox-mediated recombination and deletion of dotA and dotBusing a loop-in/loop-out system. Both strategies used sacB-medi-ated sucrose counterselection to select for mutants that had un-dergone the desired deletion event. Mutant strains did not secretecharacterized T4BSS substrates and showed severe growth defectsin mammalian host cells. Both phenotypes were rescued uponcomplementation.

MATERIALS AND METHODSBacterial strains, plasmids, and mammalian cell lines. The plasmidsused in the present study are listed in Table 1. C. burnetii Nine Mile phaseII (NMII) clone 4 was used in all transformation experiments and waspropagated microaerobically in ACCM-2 or ACCM-2 agarose as previ-ously described (27). Escherichia coli TOP10 (Invitrogen, Carlsbad, CA)was used for recombinant DNA procedures and cultivated in Luria-Ber-tani (LB) broth. E. coli transformants were selected on LB agar platescontaining 50 �g of kanamycin/ml or 10 �g of chloramphenicol/ml.THP-1 cells, a human acute monocytic leukemia cell line (TIB-202;American Type Culture Collection [ATCC]), and African green monkeykidney (Vero) cells (CCL-81; ATCC) were maintained in RPMI 1640 me-

dium (Invitrogen) containing 10% fetal calf serum (Invitrogen) at 37°Cand 5% CO2. C. burnetii replication in host cells or ACCM-2 was mea-sured by quantitative PCR of genome equivalents (GE) as previously de-scribed (20, 28) using a probe specific to CBU1206.

Plasmid construction. The plasmids used here are depicted in Fig. 1and detailed descriptions of their construction are found in Table S1 in thesupplemental material. Restriction enzymes were obtained from NewEngland Biolabs (Ipswich, MA). PCR was performed using Accuprime Pfxor Accuprime Taq (Invitrogen). PCR primers were obtained from Inte-grated DNA Technologies (San Diego, CA), and their sequences are listedin Table S2 in the supplemental material. All cloning procedures wereconducted using an In-Fusion PCR cloning system (BD Clontech, Moun-tain View, CA).

Transformation and sucrose counterselection. Electroporation ofNMII was conducted as previously described (27). Selection of NMII“loop-in” transformants with chromosomal integration of a suicideplasmid was conducted by culture of bacteria in ACCM-2 containingkanamycin (final concentration, 350 �g/ml) and chloramphenicol (fi-nal concentration, 3 �g/ml). Resolution of sacB-encoding plasmidcointegrants was accomplished by subculture of transformants for 3days in ACCM-2 supplemented with 1% sucrose and kanamycin.NMII strains containing gene deletions were subsequently expandedby culture in ACCM-2 containing kanamycin. For Cre-lox mediatedrecombination of transformants harboring cat-loxP and sacB-kan-loxPencoding plasmid cointegrants, transformants were electroporatedwith a cre-encoding suicide plasmid, followed by culture for 4 days inACCM-2 to allow expression of Cre recombinase. The medium wasthen supplemented with sucrose (1% final concentration), and growthof the transformants continued for an additional 3 days to counterse-lect against organisms that had not undergone Cre-lox mediated re-combination. Mutant NMII strains were cloned by picking coloniespropagated on ACCM-2 agarose as previously described (27) or bylimiting dilution in ACCM-2.

Genes conferring resistance to chloramphenicol, kanamycin, or am-picillin are approved for C. burnetii genetic transformation studies by theRocky Mountain Laboratories Institutional Biosafety Committee and theCenters for Disease Control and Prevention, Division of Select Agents andToxins Program.

PCR and Southern blot verification of gene deletions. Deletion ofdotA and dotB was verified using PCR and Southern blotting. Verificationby PCR was accomplished by amplifying an internal gene fragment withspecific primer pairs (see Table S2 in the supplemental material), withwild-type and mutant genomic DNAs as a template. Southern blot verifi-cation was conducted by digesting wild-type and mutant genomic DNAswith BglII and EcoRI (dotA) or PstI and SalI (dotB), followed by separa-tion of fragments by electrophoresis in a 0.8% agarose gel. DNA wastransferred by blotting to Hybond N� membranes (GE Healthcare, Pis-cataway, NJ) as described by Sambrook et al. (33), except that the transfermedium contained 0.4 M NaOH. Probe DNA specific to regions flankingeither dotA or dotB were generated by PCR using specific primer pairs (seeTable S1 in the supplemental material). A probe specific to the 1-kb PlusDNA marker (Invitrogen) was also used. Probe DNA (200 ng) labelingand subsequent blot hybridizations were conducted using instructionsand reagents provided by a Gene Images AlkPhos Direct Labeling andDetection kit (GE Healthcare).

Cre-lox mediated deletion of Tn7 sequences bounded by loxP sites wasverified by PCR using primers specific to the chloramphenicol acetyl-transferase (CAT) gene and CBU1788 (see Table S2 in the supplementalmaterial).

Immunoblotting. Expression of adenylate cyclase (CyaA) fusion pro-teins or DotB was assessed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis and immunoblotting. Membranes were incubated with arabbit polyclonal antibody directed against L. pneumophila (Philadel-phia-1 strain) DotB or a mouse monoclonal antibody directed againstBordetella pertussis CyaA (clone 3D1; Santa Cruz Biotechnology, Santa

Coxiella Gene Inactivation

July 2012 Volume 78 Number 13 aem.asm.org 4581

on Septem

ber 1, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from

Cruz, CA). C. burnetii and L. pneumophila DotB proteins display 63%amino acid identity. Reacting proteins were detected using anti-rabbit oranti-mouse IgG secondary antibodies conjugated to horseradish peroxi-dase (Pierce, Rockford, IL) and chemiluminescence using ECL Pico re-agent (Pierce).

CyaA translocation assay. CyaA translocation assays were performedas previously described using a cyclic AMP (cAMP) enzyme immunoassay(GE Healthcare) (45, 47). Construction of the CyaA reporter plasmidspJB-CAT-CyaA, pJB-CAT-CyaA-A15, and pJB-CAT-CyaA-A16 has beenreported elsewhere (45).

Indirect immunofluorescence. Vero cells infected with mutant orwild-type NMII were fixed for 20 min in 4% paraformaldehyde plus phos-phate-buffered saline (PBS; 1 mM KH2PO4, 155 mM NaCl, 3 mMNa2HPO4 [pH 7.4]), followed by permeabilization for 5 min in 0.1%saponin in PBS. Cells were stained for indirect immunofluorescence as

previously described (4, 19). Guinea pig anti-C. burnetii serum and amouse monoclonal antibody directed against LAMP-3 (CD63) (cloneH5C6; BD Biosciences) were used as primary antibodies. Alexa Fluor 488and Alexa Fluor 594 IgG (Invitrogen) were used as secondary antibodies.Coverslips were mounted using ProLong Gold containing DAPI (4=,6=-diamidino-2-phenylindole; Invitrogen) to visualize nuclei. Epifluores-cence microscopy images were acquired with a TE-2000 microscopeequipped with a CoolSNAP HQ digital camera (Roper Scientific, Tucson,AZ). Images were obtained using Metamorph software (Molecular De-vices, Inc., Downingtown, PA) and processed with ImageJ software (writ-ten by W. S. Rasband at the U.S. National Institutes of Health, Bethesda,MD [http://rsb.info.nih.gov/ij/]).

Statistical analysis. Statistical analyses were performed using a one-way analysis of variance and Prism software (GraphPad Software, Inc., LaJolla, CA).

TABLE 1 Strains and plasmids used in this study

Strain or plasmid Genotype and/or phenotypea Source or reference

StrainsC. burnetii

Nine Mile phase II (NMII) Phase II, clone 4 Beare et al. (6)NMII/Tn7-CAT-311P-MC-sacB NMII containing a Tn7 transposon inserted between CBU1787 and CBU1788; Cmr This study����/�dotA-loxP Cre-lox-mediated dotA deletion mutant; Kanr This study����/�dotA dotA deletion mutant; Kanr This study����/�dotB dotB deletion mutant; Kanr This study

E. coliTOP10 F� mcrA �(mrr-hsdRMS-mcrBC) 80dlacZ�M15 �lacX74 recA1 araD139

�(ara-leu)7697 galU galK rpsL (Strr) endA1 nupGInvitrogen

PlasmidspJC84 ColE1 ori; Kanr Wehrly et al. (48)pUC19 pUC ori; Ampr InvitrogenpJB-Kan pJB2581 containing kan driven by 1169P; Kanr Omsland et al. (27)pJB-CAT pJB2581 containing cat driven by 1169P; Cmr Omsland et al. (27)pJC-CAT pJC84 containing cat driven by 1169P; Cmr This studypJC-CAT:DotB5=3= pJC-CAT containing 2 kb of dotB 5= and 3= flanking sequences; Cmr This studypJC-CAT::DotB5=3=-Kan pJC-CAT::DotB5=3= containing a 1169P-Kan cassette cloned into a unique AgeI

restriction site; Cmr Kanr

This study

pJC-CAT::DotA5=3= pJC-CAT containing 2 kb of dotA 5= and 3= flanking sequences; Cmr This studypJC-CAT::DotA5=3=-Kan pJC-CAT::DotA5=3= containing a 1169P-Kan cassette cloned into a unique AgeI

restriction site; Cmr Kanr

This study

pMiniTn7T-CAT 1169P-CAT cloned into pUC18R6K-mini-Tn7T; Cmr Beare et al. (3)p1898-Tn Himar1 transposon vector; Cmr Beare et al. (4)pJB2581 cyaA fusion vector; Ampr Cmr Bardill et al. (2)pJB2581:: 311P-MC 311P-MC-CAT-1169P cassette cloned into pJB2581; Ampr Cmr This studypMiniTn7T-CAT::311P-MC loxP-311P-MC cassette cloned into pMiniTn7T-CAT; Cmr This studypMiniTn7T-CAT::311P-MC-sacB sacB-loxP fragment cloned into pMiniTn7T-CAT::311P-MC; Cmr This studypMiniTn7T-Kan-loxP-SacB 1169P-Kan-loxP-sacB cassette cloned into EcoRI/SalI-digested pMiniTn7T-CAT;

Cmr Kanr

This study

pUC19-Kan-loxP-sacB pUC19 containing a 1169P-Kan-loxP-sacB cassette; Kanr This studypUC19-Kan-loxP-sacB::DotA5=flank pUC19-Kan-loxP-sacB containing 2 kb of dotA 5= flanking DNA; Kanr This studypJC-CAT-loxP pJC-CAT containing a loxP site; Cmr This studypJC-CAT-loxP::DotA3=flank pJC-CAT-loxP containing 2 kb of dotA 3= flanking DNA; Kanr This studypBBR1MCS-2::TetR-pTetA pBBR1 ori; Kanr Starr et al. (39)pBSV25-flgBp-cre Cre expression vector; Kanr Bestor et al. (8)pUC19::1169P-cre pUC19 containing cre driven by 1169P; Ampr This studypUC19::tetRAP-cre pUC19 containing cre driven by tetAP; Ampr This studypTnS2::1169P-tnsABCD pTnS2 containing tnsABCD driven by 1169P; Ampr Beare et al. (3)pMiniTn7T-CAT::icmVP-icmV-dotA icmVP-icmV-dotA cloned into pMiniTn7T-CAT; Cmr This studypMiniTn7T-CAT::dotDP-dotDCB dotDP-dotDCB cloned into pMiniTn7T-CAT; Cmr This studypJB-CAT-CyaA C. burnetii cyaA fusion vector; Cmr Voth et al. (45)pJB-CAT-CyaA-A15 cbua0015 in pJB-CAT-CyaA; Cmr Voth et al. (45)pJB-CAT-CyaA-A16 cbua0016 in pJB-CAT-CyaA; Cmr Voth et al. (45)

a Cmr, chloramphenicol resistance; Ampr, ampicillin resistance; Strr, streptomycin resistance; Kanr, kanamycin resistance.

Beare et al.

4582 aem.asm.org Applied and Environmental Microbiology

on Septem

ber 1, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from

RESULTS AND DISCUSSIONCre-lox-mediated gene excision using sacB counterselection. Asan initial step toward our goal to develop efficient methods oftargeted gene deletion in C. burnetii, we tested the Cre-lox recom-bination system. This method relies on Cre recombinase catalysisof recombination between directly repeated 34-bp loxP recogni-tion sites that flank a region targeted for deletion (1, 18).

To test whether the Cre-lox system functions in C. burnetii, weconstructed a Tn7 derivative (pMiniTn7T-CAT::311P-MC-sacB) thatcontains a CAT gene (Cmr), and loxP sites flanking the mCherry redfluorescent protein-encoding gene (MC) and sacB driven as a singletranscriptional unit by the P1 porin (CBU0311) promoter (311P) (5)(Fig. 1). We previously demonstrated that, consistent with otherGram-negative bacteria, Tn7 inserts as a single copy into an inter-

FIG 1 Suicide plasmids used in the present study.

Coxiella Gene Inactivation

July 2012 Volume 78 Number 13 aem.asm.org 4583

on Septem

ber 1, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from

genic region of the C. burnetii chromosome immediately down-stream from glmS (CBU1787) (5). The Bacillus subtilis sacB gene wasincluded in the transposon as a counterselectable marker (31). ThesacB gene product is the secreted enzyme levansucrase (sucrose: 2,6,-�-D-fructan 6-�-D-fructosyltransferase; EC 2.4.1.10) that convertssucrose to levans (high-molecular-weight-fructose polymers). Pro-duction of levans is toxic to most Gram-negative bacteria, includingLegionella pneumophila, a close relative of C. burnetii (11, 14).

NMII was cotransformed with pMiniTn7T-CAT::311P-MC-sacB and pTnS2::1169P-tnsABCD (3), encoding the Tn7transposase, and chloramphenicol-resistant transformants clonedfrom ACCM-2 agarose plates (Fig. 2a). Sequence analysis con-firmed correct insertion of the Tn7 transposon. This strain wasthen transformed with a suicide plasmid encoding constitutivelyexpressed cre (pUC19::1169P-cre) or cre under the control of anaTc-inducible promoter (pUC19::tetRAP-cre) (3) (Fig. 1). Organ-

FIG 2 Recovery of C. burnetii Cre-lox-mediated recombinants is enhanced by sacB-based sucrose counterselection. (A) Schematic showing Cre-lox mediateddeletion of the 311P-MC-sacB gene cassette carried by NMII transformed with pMiniTn7T-CAT::311P-MC-sacB. (B) Agarose gel showing the efficiency ofcassette deletion by the NMII-Tn7 transformant in the absence of Cre recombinase, or when cotransformed with a suicide plasmid encoding constitutivelyexpressed cre (pUC19::1169P-cre), or cre under the control of an aTc-inducible promoter (pUC19::tetRAP-cre), and grown in the presence or absence of 1%sucrose. An intact Tn7 sequence is indicated by a 3,356-bp PCR product. Deletion of the Tn7-encoded 311P-MC-sacB gene cassette is indicated by a 639-bp PCRproduct.

FIG 3 Cre-lox-mediated deletion of dotA. (A) Schematic showing Cre-lox mediated replacement of dotA with a Kanr gene cassette. The remaining loxP site, BglII(B) and EcoRI (E) restriction sites, and two regions corresponding to Southern blot probe DNA, are shown. (B) Southern blot of BglII/EcoRI-digested genomicDNA from NMII and the NMII/�dotA-loxP mutant strain hybridized with two DNA probes specific to regions flanking dotA. Disruption of the dotA-containing3.9-kb BglII/EcoRI fragment of NMII in the NMII/�dotA-loxP mutant strain is indicated by hybridizing 0.7-kb BglII/EcoRI and 1.85-kb EcoRI fragments. (Aunique EcoRI site was introduced by the Kanr gene cassette.) (C) PCR using primers specific to dotA showing the absence of target sequence in the NMII/�dotA-loxP mutant strain.

Beare et al.

4584 aem.asm.org Applied and Environmental Microbiology

on Septem

ber 1, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from

isms were cultivated for 4 days in ACCM-2, with aTc present incultures of pUC19::tetRAP-cre transformants. PCR was conductedon Cre-expressing and -nonexpressing control organisms usingprimers specific to the CAT gene and CBU1788. Without Crerecombinase, a single 3,356-bp PCR product was amplified, indi-cating the Tn7T-CAT::311P-MC-sacB sequence was intact in theseorganisms (Fig. 2). With Cre recombinase, the 3,356-bp PCRproduct was the primary amplicon. However, a modest amount ofa 639-bp PCR product was also produced, indicating some Cre-lox mediated excision of the mCherry-sacB gene cassette had oc-curred (Fig. 2).

The overall poor recovery of the desired deletion strainprompted us to test whether sucrose counterselection would im-prove recovery of bacteria having undergone Cre-lox-mediatedrecombination. NMII displayed no obvious growth defect inACCM-2 containing up to 5% sucrose (data not shown), aconcentration commonly used in sacB-based sucrose counterse-lection schemes (14, 16, 34). After transformation of NMII/Tn7T-CAT::311P-MC-sacB with pUC19::1169P-cre or pUC19::tetRAP-cre, organisms were cultivated in ACCM-2 for 4 days, and thensucrose was added to a final concentration of 0.5, 1, 2, 3, 4, or 5%,and the cultures were allowed to grow for 3 days. Growth wasobvious only in medium containing 0.5 or 1% sucrose. Organismssubjected to 1% sucrose counterselection were then PCR geno-

typed. Complete excision of mCherry-sacB cassette, originallybounded by loxP sites, was observed with organisms transformedwith pUC19::1169P-cre or pUC19::tetRAP-cre (Fig. 2). Thus, sacB-based sucrose counterselection substantially increased recovery oforganisms having undergone Cre-lox mediated recombination byeliminating transformants that retained the intact Tn7 trans-poson.

Targeted gene deletion using Cre-lox. Our optimized sacB-based Cre-lox system was then tested for efficacy in generating atargeted gene deletion. As proof of concept, dotA (CBU1648),which encodes a structural component of the T4BSS of C. burnetii,was targeted for deletion. The homologous protein in L. pneumo-phila is essential for type IVB secretion of effector proteins andproductive infection of macrophages (7, 26).

NMII was sequentially transformed with the suicide plas-mids pUC19-Kan-loxP-sacB::DotA5=flank and pJC-CAT-loxP::DotA3=flank (Fig. 1) that contain approximately 2,000 bp ofchromosomal DNA immediately flanking the 5= and 3= regionsof dotA, respectively. Correct chromosomal integration of bothplasmids was confirmed by PCR (data not shown). This strainwas subsequently transformed with pUC19::1169P-cre and cul-tured in ACCM-2 using sucrose counterselection as describedabove. Southern blotting of a mutant clone revealed 0.7-kbBglII/EcoRI and 1.85-kb EcoRI fragments as opposed to a

FIG 4 Deletion of dotA and dotB using a single suicide plasmid and a loop-in/loop-out strategy. (A) Schematic showing the replacement of dotA with a Kanr genecassette. BglII (B) and EcoRI (E) restriction sites are shown, as are two regions corresponding to probe DNA used in Southern blots. (B) Southern blot ofBglII/EcoRI-digested genomic DNA from NMII and the �dotA mutant hybridized with two DNA probes specific to regions flanking dotA. Disruption of thedotA-containing 3.9-kb BglII/EcoRI fragment of NMII in the �dotA mutant is indicated by a hybridizing 2.5-kb BglII/EcoRI fragment. (C) PCR using primersspecific to dotA show the absence of target sequence in the �dotA mutant. (D) Schematic showing the replacement of dotB with a Kanr gene cassette. PstI (P) andSalI (S) restriction sites are shown, as are two regions corresponding to probe DNA used in Southern blots. (E) Southern blot of PstI/SalI-digested genomic DNAfrom NMII and the �dotB mutant hybridized with two DNA probes specific to regions flanking dotB. Disruption of dotB-containing 3.4-kb SalI and 2.0-kbPstI/SalI fragments of NMII in the �dotB mutant is indicated by hybridizing 4.1-kb SalI and 1.2-kb PstI/SalI fragments (F) PCR using primers specific to dotBshowing the absence of target sequence in the �dotB mutant. (G) Immunoblot of �dotB mutant lysate showing the absence of the 41.3-kDa DotB protein.

Coxiella Gene Inactivation

July 2012 Volume 78 Number 13 aem.asm.org 4585

on Septem

ber 1, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from

3.9-kb BglII/EcoRI fragment present in wild-type genomicDNA, indicating the deletion of dotA via Cre-mediated recom-bination between loxP sites (Fig. 3A and B). PCR analysis alsoconfirmed the absence of dotA (Fig. 3C). A schematic represen-tation of Cre-lox mediated deletion of dotA is shown in Fig. S1in the supplemental material.

To our knowledge, the NMII/�dotA-loxP mutant strain is thefirst example of a C. burnetii mutant generated by targeted genedeletion. In addition to deleting individual genes, the Cre-lox sys-tem has proven effective in deleting bacterial chromosomal re-gions as large as 67.3 kb (43). Such a procedure may prove usefulin constructing a new generation of attenuated C. burnetii strainsas vaccine candidates.

Loop-in/loop-out deletion of dotA and dotB. Gene deletionsin C. burnetii can be generated using Cre-lox-based recombina-tion. However, the procedure is somewhat cumbersome in requir-ing sequential transformation with three different suicide plas-mids. Therefore, we developed a simplified loop-in/loop-outsystem that uses a single suicide plasmid and sacB counterselec-tion. Using this approach, dotA and dotB (CBU1645) genes weretargeted for deletion. The DotB homolog in L. pneumophila is acytoplasmic ATPase that is essential for secretion of T4BSS sub-strates and productive infection of macrophages (37). Suicideplasmids were constructed containing a kanamycin resistancegene bounded by approximately 2,000 bp of 5= and 3= regionsflanking the dotA gene (pJC-CAT::DotA5=3=-Kan) or the dotBgene (pJC-CAT::DotB5=3=-Kan) (Fig. 1). Plasmids also containeda cat-sacB gene cassette driven by the CBU1169 (hsp20) promoter(4). Resistance to chloramphenicol and kanamycin was used toselect for cointegrant transformants (loop-in), and plasmid inte-gration into the chromosome confirmed by PCR (data notshown). Cointegrant transformants were subcultured for 3 days inACCM-2 containing 1% sucrose and kanamycin to select fortransformants with resolved (loop-out) plasmid sequences. Selec-tion for both sucrose and kanamycin resistance was conducted topromote recovery of a Kanr-marked deletion of dotA or dotB.Without selection for kanamycin resistance, there was roughlyequal probability of recovering the wild-type chromosome by

simple in toto excision of introduced plasmid DNA (17) (see Fig.S2 in the supplemental material).

The putative dotA and dotB deletion mutants were cloned us-ing ACCM-2 agarose, expanded in ACCM-2, and then theirgenomic DNA was examined by Southern blotting. Replacementof dotA with the Kan cassette resulted in a predicted 2.5-kb BglII/EcoRI fragment as opposed to a 3.9-kb fragment present in wild-type DNA (Fig. 4A and B). Replacement of dotB with the Kancassette resulted in predicted 1.2- and 4.1-kb PstI/SalI fragmentsas opposed to the 2.0- and 3.4-kb fragments present in wild-typeDNA (Fig. 4D and E). The availability of DotB antibody allowedconfirmation by immunoblotting of defective protein productionby the �dotB mutant (Fig. 4G). PCR analysis also showed a com-plete absence of dotA (Fig. 4C) or dotB (Fig. 4F) in mutant strains.

dotA and dotB are required for cytosolic delivery of T4BSSsubstrates by NMII and productive infection of mammaliancells. L. pneumophila dotA and dotB mutants are defective in se-cretion of Dot/Icm T4BSS substrates, a deficiency that correlateswith an inability to productively infect mammalian host cells. Thesame phenotypes are associated with C. burnetii strains harboringtransposon insertions in icmD (3) and icmL (9). To test whether

FIG 5 The �dotA and �dotB mutants are deficient in secretion of T4BSSsubstrates. Cytosolic levels of cAMP after infection of THP-1 macrophages for2 days with NMII or �dot mutants expressing CyaA fused to the previouslydefined Dot/Icm substrates CpeD and CpeE. Control infections were con-ducted with NMII expressing CyaA alone. Elevated levels of cAMP, indicatingsecretion, were observed only with NMII expressing CyaA-CpeD or -CpeEfusion proteins. The results shown are from one experiment conducted induplicate and representative of two independent experiments.

FIG 6 �dotA and �dotB mutants are defective in intracellular growth. Six-daygenome equivalent (GE) increases of NMII, �dot mutants, or complemented(comp) �dot mutants grown in ACCM-2 (A) or Vero cells (B). ACCM-2results are expressed as the means of three independent experiments. Errorbars indicate the standard deviations from the means. Vero cell results areexpressed as the means of two biological replicates from three independentexperiments. Error bars indicate the standard deviations from the means, andasterisks indicate a statistically significant difference (P � 0.0001) from cellsinfected with NMII.

Beare et al.

4586 aem.asm.org Applied and Environmental Microbiology

on Septem

ber 1, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from

deletion of dotA or dotB is associated with defects in T4BSS secre-tion and intracellular growth, CyaA translocation (3) and Verocell growth assays (12), respectively, were conducted.

THP-1 macrophages were infected with NMII or �dot mutantstransformed with the plasmids pJB-CAT-CyaA-A15 or pJB-CAT-CyaA-A16 that encode CyaA fusions to the C. burnetii T4BSS ef-fector proteins CpeD and CpeE, respectively (45). CpeD and CpeEfusion proteins were secreted by NMII as indicated by a �100-foldincrease in cAMP levels relative to organisms expressing CyaAalone (Fig. 5). Conversely, the AMP levels generated by the �dotmutants did not exceed the CyaA-alone negative control level,indicating no secretion (Fig. 5). Negative secretion was not due toa lack of CyaA fusion protein since immunoblotting revealedequal amounts of fusion protein produced by NMII and the �dotmutants (data not shown).

To test whether the lack of secretion of two defined C. burnetiiDot/Icm substrates correlated with an inability to productivelyinfect mammalian cells, growth measurements of the �dot mu-tants were made at 6 days postinfection of Vero cells. Axenicgrowth of NMII and the �dot mutants was indistinguishable, withall strains attaining roughly 1,000-fold increases in GE after 6 days

of incubation (Fig. 6A). Conversely, �dotA and �dotB mutantsexhibited only 3.3- and 4.6-fold increases, respectively, in GE at 6days postinfection of Vero cells compared to a 1,000-fold increaseby NMII (Fig. 6B). This modest amount of replication was previ-ously observed for a NMII icmD mutant in THP-1 macrophagesand was attributed to the mutant’s ability to undergo a few roundsof genomic replication in the acidic but nutritionally deficientphagolysosome (3).

To confirm that deficient intracellular replication of the �dot mu-tants was due to their respective gene deletions, complementationstudies were performed. C. burnetii dotB is the third gene in a pre-dicted operon with dotD and dotC, while dotA is the second gene in apredicted operon with icmV (24). Thus, rescue of the intracellulargrowth defects of �dot mutants was attempted by complementationwith Tn7 constructs encoding dotD, dotC, and dotB (pMiniTn7-CAT::dotDP-dotDCB) or icmV-dotA (pMiniTn7-CAT::icmVP-icmV-dotA) (Fig. 1). Constructs contained approximately 200 bp upstreamof their respective operon predicted to encode an endogenous pro-moter. Transformation of the �dotA and �dotB mutants with Tn7constructs resulted in functional complementation as scored by GEincreases similar to NMII at 6 days postinfection (Fig. 6B).

FIG 7 �dotA and �dotB mutants are defective in replication vacuole development. Fluorescence micrographs of Vero cells infected for 4 days with NMII, �dotmutants, or complemented (comp) �dot mutants. C. burnetii (red) and LAMP-3 (green) are stained by indirect immunofluorescence. Nuclei (blue) are stainedwith DAPI. Micrograph insets of cells infected with the �dotA or �dotB mutant show LAMP-3 (upper inset), C. burnetii (middle inset), and the merged image(lower inset). Mutants were harbored as single organisms in dispersed, tight-fitting LAMP-3-positive vacuoles, while NMII and the complemented mutants wereobserved in single large and spacious replication vacuoles. Bars, 10 �m.

Coxiella Gene Inactivation

July 2012 Volume 78 Number 13 aem.asm.org 4587

on Septem

ber 1, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from

The lack of intracellular replication by �dotA and �dotB mu-tants correlated with failed production of the large and spaciousreplication vacuole typical of C. burnetii infection (Fig. 7). Rather,mutant bacteria were randomly dispersed in LAMP-3-postive,tight-fitting vacuoles. The normal vacuole phenotype was rescuedwhen mutants were transformed with the complementing Tn7constructs (Fig. 7).

In summary, C. burnetii’s historic obligate intracellular lifestylehas thwarted the development of systems for pathogen geneticmanipulation. The present study details two efficient methods fortargeted gene disruption, the development of which was facilitatedby new axenic culture techniques (27). Although this work gener-ated marked gene deletions, the Cre-lox and loop-in/loop-out ap-proaches described here are adaptable to generation of markerlessdeletions or mutations. A markerless approach is desirable whenmaking several mutations in a single genome and eliminates po-tential polar effects of marker genes. The loop-in/loop-out strat-egy also has the added advantage of generating “scarless” muta-tions completely devoid of cointegrant plasmid sequences (31),although the 34-bp loxP site scar remaining after Cre-mediatedrecombination is reported to not exert polar effects on down-stream genes (30). Markerless approaches would necessitate addi-tional screening of the 0.5-mm C. burnetii colonies that form onACCM-2 agarose, a procedure that is still technically challenging.Nonetheless, the targeted gene inactivation techniques describedhere now permit routine mutation and complementation strate-gies for C. burnetii virulence factor discovery.

ACKNOWLEDGMENTS

We thank Philip Stewart for critical review of the manuscript, Aaron Be-stor for pBSV25-flgBp-cre, and Joseph Vogel for anti-DotB antibody.

This study was supported by the Intramural Research Program of theNational Institutes of Health, National Institute of Allergy and InfectiousDiseases

REFERENCES1. Ayres EK, Thomson VJ, Merino G, Balderes D, Figurski DH. 1993.

Precise deletions in large bacterial genomes by vector-mediated excision(VEX): the trfA gene of promiscuous plasmid RK2 is essential for replica-tion in several gram-negative hosts. J. Mol. Biol. 230:174 –185.

2. Bardill JP, Miller JL, Vogel JP. 2005. IcmS-dependent translocation ofSdeA into macrophages by the Legionella pneumophila type IV secretionsystem. Mol. Microbiol. 56:90 –103.

3. Beare PA, et al. 2011. Dot/Icm type IVB secretion system requirementsfor Coxiella burnetii growth in human macrophages. mBio 2:e00175–00111. doi:10.1128/mBio.00175-11.

4. Beare PA, et al. 2009. Characterization of a Coxiella burnetii ftsZ mutantgenerated by Himar1 transposon mutagenesis. J. Bacteriol. 191:1369 –1381.

5. Beare PA, Sandoz KM, Omsland A, Rockey DD, Heinzen RA. 2011.Advances in genetic manipulation of obligate intracellular bacterialpathogens. Front. Microbiol. 2:97.

6. Beare PA, et al. 2009. Comparative genomics reveal extensive trans-poson-mediated genomic plasticity and diversity among potential effectorproteins within the genus Coxiella. Infect. Immun. 77:642– 656.

7. Berger KH, Merriam JJ, Isberg RR. 1994. Altered intracellular targetingproperties associated with mutations in the Legionella pneumophila dotAgene. Mol. Microbiol. 14:809 – 822.

8. Bestor A, et al. 2010. Use of the Cre-lox recombination system to inves-tigate the lp54 gene requirement in the infectious cycle of Borrelia burg-dorferi. Infect. Immun. 78:2397–2407.

9. Carey KL, Newton HJ, Luhrmann A, Roy CR. 2011. The Coxiellaburnetii Dot/Icm system delivers a unique repertoire of type IV effectorsinto host cells and is required for intracellular replication. PLoS Pathog.7:e1002056. doi:10.1371/journal.ppat.1002056.

10. Chen C, et al. 2010. Large-scale identification and translocation of type IVsecretion substrates by Coxiella burnetii. Proc. Natl. Acad. Sci. U. S. A.107:21755–21760.

11. Cianciotto NP, Long R, Einsenstein BI, Engleberg NC. 1988. Site-specific mutagenesis of Legionella pneumophila by allelic exchange usingcounterselectable ColE1 vectors. FEMS Microbiol. Lett. 56:203–208.

12. Coleman SA, Fischer ER, Howe D, Mead DJ, Heinzen RA. 2004.Temporal analysis of Coxiella burnetii morphological differentiation. J.Bacteriol. 186:7344 –7352.

13. Driskell LO, et al. 2009. Directed mutagenesis of the Rickettsia prowazekiipld gene encoding phospholipase D. Infect. Immun. 77:3244 –3248.

14. Gay P, Le Coq D, Steinmetz M, Berkelman T, Kado CI. 1985. Positiveselection procedure for entrapment of insertion sequence elements ingram-negative bacteria. J. Bacteriol. 164:918 –921.

15. Hackstadt T, Williams JC. 1981. Biochemical stratagem for obligateparasitism of eukaryotic cells by Coxiella burnetii. Proc. Natl. Acad. Sci.U. S. A. 78:3240 –3244.

16. Hamad MA, Zajdowicz SL, Holmes RK, Voskuil MI. 2009. An allelicexchange system for compliant genetic manipulation of the select agentsBurkholderia pseudomallei and Burkholderia mallei. Gene 430:123–131.

17. Hamilton CM, Aldea M, Washburn BK, Babitzke P, Kushner SR. 1989.New method for generating deletions and gene replacements in Esche-richia coli. J. Bacteriol. 171:4617– 4622.

18. Hoess RH, Abremski K. 1984. Interaction of the bacteriophage P1 re-combinase Cre with the recombining site loxP. Proc. Natl. Acad. Sci.U. S. A. 81:1026 –1029.

19. Howe D, Melnicakova J, Barak I, Heinzen RA. 2003. Maturation of theCoxiella burnetii parasitophorous vacuole requires bacterial protein syn-thesis but not replication. Cell. Microbiol. 5:469 – 480.

20. Howe D, Shannon JG, Winfree S, Dorward DW, Heinzen RA. 2010.Coxiella burnetii phase I and II variants replicate with similar kinetics indegradative phagolysosome-like compartments of human macrophages.Infect. Immun. 78:3465–3474.

21. Huijsmans CJ, et al. 2011. Single-nucleotide-polymorphism genotypingof Coxiella burnetii during a Q fever outbreak in the Netherlands. Appl.Environ. Microbiol. 77:2051–2057.

22. Kato J, Hashimoto M. 2008. Construction of long chromosomal deletionmutants of Escherichia coli and minimization of the genome. MethodsMol. Biol. 416:279 –293.

23. Luhrmann A, Nogueira CV, Carey KL, Roy CR. 2010. Inhibition ofpathogen-induced apoptosis by a Coxiella burnetii type IV effector pro-tein. Proc. Natl. Acad. Sci. U. S. A. 107:18997–19001.

24. Mao F, Dam P, Chou J, Olman V, Xu Y. 2009. DOOR: a database forprokaryotic operons. Nucleic Acids Res. 37:D459 –D463.

25. Marx CJ. 2008. Development of a broad-host-range sacB-based vector forunmarked allelic exchange. BMC Res. Notes 1:1.

26. Nagai H, Kagan JC, Zhu X, Kahn RA, Roy CR. 2002. A bacterial guaninenucleotide exchange factor activates ARF on Legionella phagosomes. Sci-ence 295:679 – 682.

27. Omsland A, et al. 2011. Isolation from animal tissue and genetic trans-formation of Coxiella burnetii are facilitated by an improved axenicgrowth medium. Appl. Environ. Microbiol. 77:3720 –3725.

28. Omsland A, et al. 2009. Host cell-free growth of the Q fever bacteriumCoxiella burnetii. Proc. Natl. Acad. Sci. U. S. A. 106:4430 – 4434.

29. Pan X, Luhrmann A, Satoh A, Laskowski-Arce MA, Roy CR. 2008.Ankyrin repeat proteins comprise a diverse family of bacterial type IVeffectors. Science 320:1651–1654.

30. Pomerantsev AP, Camp A, Leppla SH. 2009. A new minimal replicon ofBacillus anthracis plasmid pXO1. J. Bacteriol. 191:5134 –5146.

31. Reyrat JM, Pelicic V, Gicquel B, Rappuoli R. 1998. Counterselectablemarkers: untapped tools for bacterial genetics and pathogenesis. Infect.Immun. 66:4011– 4017.

32. Roest HI, et al. 2011. The Q fever epidemic in the Netherlands: history,onset, response and reflection. Epidemiol. Infect. 139:1–12.

33. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a labora-tory manual, 2nd ed. Cold Spring Harbor laboratory Press, Cold SpringHarbor, NY.

34. Schweizer HP. 1992. Allelic exchange in Pseudomonas aeruginosa usingnovel ColE1-type vectors and a family of cassettes containing a portableoriT and the counter-selectable Bacillus subtilis sacB marker. Mol. Micro-biol. 6:1195–1204.

35. Schweizer HP. 2003. Applications of the Saccharomyces cerevisiae Flp-FRTsystem in bacterial genetics. J. Mol. Microbiol. Biotechnol. 5:67–77.

Beare et al.

4588 aem.asm.org Applied and Environmental Microbiology

on Septem

ber 1, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from

36. Seshadri R, et al. 2003. Complete genome sequence of the Q-fever patho-gen Coxiella burnetii. Proc. Natl. Acad. Sci. U. S. A. 100:5455–5460.

37. Sexton JA, Yeo HJ, Vogel JP. 2005. Genetic analysis of the Legionellapneumophila DotB ATPase reveals a role in type IV secretion system pro-tein export. Mol. Microbiol. 57:70 – 84.

38. Song H, Wolschendorf F, Niederweis M. 2009. Construction of un-marked deletion mutants in mycobacteria. Methods Mol. Biol. 465:279 –295.

39. Starr T, et al. 2012. Selective subversion of autophagy complexes facili-tates completion of the Brucella intracellular cycle. Cell Host Microbe11:33– 45.

40. Suhan M, et al. 1994. Cloning and characterization of an autonomousreplication sequence from Coxiella burnetii. J. Bacteriol. 176:5233–5243.

41. Suhan ML, Chen SY, Thompson HA. 1996. Transformation of Coxiellaburnetii to ampicillin resistance. J. Bacteriol. 178:2701–2708.

42. Tao L, LeBlanc DJ, Ferretti JJ. 1992. Novel streptococcal-integrationshuttle vectors for gene cloning and inactivation. Gene 120:105–110.

43. Ullrich S, Schuler D. 2010. Cre-lox-based method for generation of largedeletions within the genomic magnetosome island of Magnetospirillumgryphiswaldense. Appl. Environ. Microbiol. 76:2439 –2444.

44. Vagner V, Dervyn E, Ehrlich SD. 1998. A vector for systematic geneinactivation in Bacillus subtilis. Microbiology 144:3097–3104.

45. Voth DE, et al. 2011. The Coxiella burnetii cryptic plasmid is enriched ingenes encoding type IV secretion system substrates. J. Bacteriol. 193:1493–1503.

46. Voth DE, Heinzen RA. 2007. Lounging in a lysosome: the intracellularlifestyle of Coxiella burnetii. Cell. Microbiol. 9:829 – 840.

47. Voth DE, et al. 2009. The Coxiella burnetii ankyrin repeat domain-containing protein family is heterogeneous, with C-terminal trunca-tions that influence Dot/Icm-mediated secretion. J. Bacteriol. 191:4232– 4242.

48. Wehrly TD, et al. 2009. Intracellular biology and virulence determinantsof Francisella tularensis revealed by transcriptional profiling inside mac-rophages. Cell. Microbiol. 11:1128 –1150.

Coxiella Gene Inactivation

July 2012 Volume 78 Number 13 aem.asm.org 4589

on Septem

ber 1, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from