Suicide vectors for antibiotic marker exchange and rapid … · 2007. 3. 8. · Suicide vectors for antibiotic marker exchange and rapid generation of multiple knockout mutants by

7 (2006) 395–407www.elsevier.com/locate/jmicmeth

Journal of Microbiological Methods 6

Suicide vectors for antibiotic marker exchange andrapid generation of multiple knockout mutants by allelic exchange

in Gram-negative bacteria

Inmaculada Ortiz-Martín, Alberto P. Macho, Lotte Lambersten 1,Cayo Ramos, Carmen R. Beuzón ⁎

Área de Genética, Departamento de Biología Celular, Genética y Fisiología, Universidad de Málaga, Campus de Teatinos s/n,Málaga, 29071, Spain

Received 10 January 2006; received in revised form 5 April 2006; accepted 18 April 2006Available online 5 June 2006

Abstract

Allelic exchange is frequently used in bacteria to generate knockout mutants in genes of interest, to carry out phenotypicanalysis and learn about their function. Frequently, understanding of gene function in complex processes such as pathogenesisrequires the generation of multiple mutant strains. In Pseudomonads and other non-Enterobacteriaceae, this is a time-consumingand laborious process based on the use of suicide vectors and allelic exchange of the appropriate mutant version of each gene,disrupted by a different antibiotic marker. This often implies the generation of a series of mutants for each gene, each disrupted by adifferent antibiotic marker, in order to obtain all possible double or multiple mutant combinations. In this work, we have modifiedthis method by developing a set of 3 plasmid derivatives from the previously described suicide vector for allelic exchange,pKAS32, to make antibiotic marker exchange easier and thus accelerate the entire process. Briefly, the construction of each singlegene knockout mutant is carried out by allelic exchange of the chromosomal gene with a mutant allele disrupted by the insertion ofa kanamycin resistance cassette. When a double mutant strain is required, antibiotic marker exchange is performed in either one ofthe single mutants, using any of the three plasmid derivatives that carry the kanamycin resistance gene disrupted by either achloramphenicol, gentamycin, or streptomycin resistance cassette. The single mutant strain, carrying now an antibiotic resistancemarker other than kanamycin, can be used to introduce a second mutation using the original plasmid constructs, to generate adouble mutant. The process can be repeated sequentially to generate multiple mutants. We have validated this method bygenerating strains carrying different combinations of mutations in genes encoding different transcriptional regulators of the Hrptype III secretion system in Pseudomonas syringae. We have also tested the genetic organisation and stability of the resultingmutant strains during growth in laboratory conditions as well as in planta.© 2006 Elsevier B.V. All rights reserved.

Keywords: Allelic exchange; Pseudomonas syringae; Pathogenesis; Type III secretion system; Virulence

⁎ Corresponding author. Tel.: +34 952131959; fax: +34 952132001.E-mail address: [email protected] (C.R. Beuzón).

1 Current address: Department of Bacteriology, Mycology andParasitology, Building 43, Room 405A, Statens Serum Institut,Artillerivej 5, 2300 Copenhagen S, Denmark.

0167-7012/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.mimet.2006.04.011

1. Introduction

The availability of more than 150 complete bacterialgenome sequences (bacterial genome repository atNCBI http://www.ebi.ac.uk/genomes/bacteria.html)

http://www.ebi.ac.uk/genomes/bacteria.htmlmailto:[email protected]://dx.doi.org/10.1016/j.mimet.2006.04.011

396 I. Ortiz-Martín et al. / Journal of Microbiological Methods 67 (2006) 395–407

has provided a great amount of information on themolecular structure and organisation of a multitude ofgenes whose functions need to be determined. This isusually achieved by reverse genetic analysis of thegenes of interest, i.e. knockout of the target gene viaallelic exchange followed by phenotypic characterisa-tion of the resulting mutant strain (Donnenberg andKaper, 1991; Miller and Mekalanos, 1988). Thoroughfunctional characterisation of complex processes oftenrequires the generation of multiple mutant strains. Suchis the case for type III secretion systems (TTSS) andtheir effector proteins, where individual mutationsfrequently display no phenotype (Ruiz-Albert et al.,2002), or multi-factorial regulatory systems (Beuzón etal., 2000, 2001). Foresight of potentially interestingmutant combinations allows antibiotic marker combi-nation to be taken into account when generating thecorresponding single mutants. However, in reversegenetics it is mostly through phenotypic analysis ofsingle mutants how the relevance of a particularcombination of mutations becomes apparent. Thus, denovo construction of mutants with the appropriateselective markers is usually required (Shea et al.,1999).

One of the most common strategies to generateknockout mutants in Gram-negative bacteria aresuicide vectors for allelic exchange, such as thosecarrying the replication origin of R6K that replicateonly in strains producing the π protein from λ phage(Miller and Mekalanos, 1988). Maintenance of R6Kderivatives under selective pressure requires plasmidintegration by homologous recombination. Thesevectors can be used to carry out allelic exchange ofwild-type genes by plasmid-encoded disrupted alleles.This is a two-step procedure with plasmid integrationwithin the target gene by a recombination event,followed by its excision via a second crossover, whichrenders allelic exchange. Integration of the plasmidsinto the chromosome can be selected by means of theantibiotic marker used to disrupt the target gene (firstrecombination event). Excision of the integratedplasmid that results in allelic exchange (secondrecombination event), may be either selected usingcounter-selectable markers (Donnenberg and Kaper,1991; Selbitschka et al., 1993), or screened for on thebasis of antibiotic resistance (Miller and Mekalanos,1988). This method requires cloning and disruption ofthe target gene into the suicide vector prior to allelicexchange, a time-consuming and laborious process,particularly when generating multiple mutants since itfrequently implies de novo disruption. Although one-step mutagenesis methods have recently been devel-

oped that considerably simplify a generation of singleand multiple knockout mutant strains in Enterobacter-iaceae (Datsenko and Wanner, 2000), similar methodsare not available for Pseudomonads, or other Gram-negative bacteria.

Pseudomonas syringae is a plant pathogenicbacteria of major agricultural and economic concern.The ability of P. syringae to cause infection insusceptible hosts is dependent on a large number ofvirulence factors that include many regulatory pro-teins, a type III protein secretion system (Hrp TTSS),and a suite of secreted effector proteins (Buell et al.,2003; Feil et al., 2005; Joardar et al., 2005). Molecularanalyses of the contribution that each factors plays inthe virulence process, as well as the functionalrelationships between them are essential to understandthe pathogenesis of P. syringae, thus requiringphenotypic analysis of multiple mutant strains. Thespecies P. syringae is divided into pathovars largelyon the basis of host specificity (Dye et al., 1980). P.syringae pv. phaseolicola causes halo blight in beanand requires, among other virulence determinants, afunctional Hrp type III secretion system (Lindgren etal., 1986). Expression of hrp genes in P. syringae isactivated by HrpL, an alternative sigma factor of theECF (extracytoplasmic factor) family (Xiao et al.,1994). Transcription of hrpL is in turn activated byHrpR and HrpS, a two component regulatory systemof the NtrC family that work in concert with σ54

(Grimm et al., 1995; Grimm and Panopoulos, 1989).Lon protease has been reported to degrade HrpR inhrp-repressing conditions therefore repressing theexpression of hrp genes (Bretz et al., 2002).Additionally, HrpV has been reported to downregulate expression of hrp genes in hrp-inducingconditions although it is unclear how it acts (Prestonet al., 1998). To date, no analysis of double ormultiple mutant in any combination of these regula-tory factors has been carried out.

In this work, we have developed a set of plasmidderivatives of pKAS32, an oriR6K vector (Skorupskiand Taylor, 1996), to make antibiotic marker exchangeeasier and thus accelerate the process of generatingmultiple mutants. Single gene knockout mutants aregenerated by allelic exchange of the wild-type alleleswith mutant alleles disrupted by an aphA gene (Oka etal., 1981), that confers resistance to kanamycin, andcarries its own promoter but no transcriptional termina-tor. When a double mutant strain is required, antibioticmarker exchange is carried out in any of the singlemutants using one of three pKAS32 derivatives carryingthe aphA gene disrupted by either a chloramphenicol

Table 1Bacterial strains used in this studya

Strain Description Source or reference

DH5α F-endA1 hsdR17supE44 thi-1 recA1gyrA96 relA1ΔlacU189 f80ΔlacZDM15

(Hanahan, 1983)

CC118λpir RifR Δ(ara-leu) araDΔlacX74 galE galKphoA20 thi-1 rpsErpoB argE(Am) recA1,λpir lisogen

(Herrero et al., 1990)

S17–1λpir Thi-Pro-Hsd-recA-zzz::RP4-2 (tet::Mu, kan::Tn7 [TpR, SmR])

(Simon et al., 1983)

HB101 SmR recA thi por leuhsdRM+

(Kessler et al., 1992)

P. syringae pvphaseolicola1448a

Race 6, wild-type strain D. Teversonb

IOM3 ΔhrcC (KmR) This workIOM7 ΔhrpL (KmR) This workIOM9 ΔhrpV (KmR) This workIOM10 Δlon (KmR) This workIOM13 ΔhrpV (GmR) This workIOM16 ΔhrcC (GmR) ΔhrpL

(KmR)This work

IOM28 Δlon (SmR) This workIOM29 ΔhrcC (GmR) ΔhrpL

(SmR)This work

IOM30 ΔhrcC (GmR) ΔhrpL(SmR) Δlon (KmR)

This work

IOM31 Δlon (SmR) ΔhrpL(KmR)

This work

IOM33 Δlon (SmR) ΔhrpV(KmR)

This work

IOM34 ΔhrpL (CmR) This worka Rif R, KmR, GmR, SmR, CmR, and TpR, indicate resistance to

rifampicin, kanamycin, gentamycin, streptomycin, chloramphenicol,and trimetropin, respectively.b Horticulture Research International, Wellesbourne, UK.

397I. Ortiz-Martín et al. / Journal of Microbiological Methods 67 (2006) 395–407

(Cm), gentamycin (Gm), or streptomycin (Sm) resis-tance gene. Thus, a second mutation can be introducedinto the new strain using the original plasmid constructpreviously applied to the generation of the singlemutant. This allows double or multiple mutant strainsto be rapidly generated and tested for potentiallyinteresting phenotypes.

We demonstrate the validity of this approach bydisrupting hrpL, hrpV, and lon genes of P. syringae pv.phaseolicola, as well as hrcC, which encodes astructural component of the type III secretion apparatus(Charkowski et al., 1997), and generating severalcombinations of double and triple mutations. We alsoanalyse the genetic organisation and stability of theresulting mutant strains when growing in laboratoryconditions as well as in planta.

2. Materials and methods

2.1. Bacterial strains and growth conditions

Bacterial strains used in this study are listed inTable 1. Bacteria were grown at 37 °C (Escherichiacoli strains) or 28 °C (P. syringae pv. phaseolicolastrains) with aeration in LB medium supplementedwith ampicillin (100 μg/ml for E. coli strains; 300 μg/mlfor liquid cultures and 500 μg/ml for plates, forP. syringae pv. phaseolicola strains), kanamycin(15 μg/ml), chloramphenicol (30 μg/ml for E. colistrains; 6 μg/ml for P. syringae pv. phaseolicolastrains), streptomycin (50 μg/ml), or gentamycin(10 μg/ml), as appropriate.

2.2. Plasmids

Plasmids used in this work are listed in Table 2.pUC18N-Km is a pUC18Not derivative (Biomedal,Sevilla, Spain; pUC18Not is a pUC18 derivative [GenBank/EMBL L09136]) that carries the SphI fragment ofpMKm (Murillo et al., 1994) containing a kanamycinresistance cassette (aphA). This fragment was treated torender blunt ends, and cloned into the SmaI site ofpUC18Not.

To obtain pIOM14, pIOM15, and pIOM17, threeintermediary plasmids, pUC18N–Km–Gm, pUC18N–Km–Sm, and pUC18N–Km–Cm, were constructed. A2 Kb SalI fragment from each pMGm and pSmUC(Murillo et al., 1994) containing a gentamycinresistance and a streptomycin resistance cassette,respectively, was cloned into the XhoI site ofpUC18N–Km, rendering pUC18N–Km–Gm andpUC18N–Km–Sm. The XhoI site cleaves the kanamy-

cin resistance cassette in two bands of approximately1 Kb and 0.8 Kb. To generate pUC18N–Km–Cm, afragment containing a chloramphenicol resistancecartridge was amplified by PCR from pRK600 (Kessleret al., 1992) using the appropriate primers (Table 3),which introduced a XhoI site at both 5′ and 3′ ends ofthe fragment. The resulting PCR product was digestedwith XhoI and cloned into the XhoI site of pUC18N–Km (Fig. 1). pIOM14, pIOM15, and pIOM17 wereobtained digesting pUC18N–Km–Gm, pUC18N–Km–Sm, and pUC18N–Km–Cm, with NotI and cloning thefragments corresponding to the antibiotic resistancecartridges into NotI-digested pKAS32 (Skorupski andTaylor, 1996).

Table 2Plasmids used in this studya

Plasmids Description Antibiotic resistance Source or reference

pBluescript SK (+) Cloning vector Ap Stratagene (La Jolla, CA)pUC18Not Cloning vector Ap Genbank accession no. L09136pKAS32 rpsL, oriRK6, mob Ap (Skorupski and Taylor, 1996)pMKm aphA gene Km (Murillo et al., 1994)pMGm aacC1 gene Gm (Murillo et al., 1994)pSmUC aadA gene Sm (Murillo et al., 1994)pRK600 cat gene Cm (Kessler et al., 1992)pUC18N–Km pUC18 derivative, aphA gene Ap, Km This workpKAS32–hrcC Contains hrcC ORF plus 0,5 Kb on each side Ap This workpKAS32–hrpL Contains hrpL ORF plus 0,5 Kb on each side Ap This workpKAS32–hrpV Contains hrpV ORF plus 0,5 Kb on each side Ap This workpBSK–lon Contains lon ORF plus 0,5 Kb on each side Ap This workpBSKlonKm Contains Δlon::aphA Ap, Km This workpUC18N–Km–Gm Contains aphA::aacC1 Ap, Gm This workpUC18N–Km–Sm Contains aphA::aadA Ap, Sm This workpUC18N–Km–Cm Contains aphA::cat Ap, Cm This workpIOM8 Contains ΔhrcC::aphA Ap, Km This workpIOM10 Contains ΔhrpL::aphA Ap, Km This workpIOM11 Contains ΔhrpV::aphA Ap, Km This workpIOM14 Contains aphA::aacC1 Ap, Gm This workpIOM15 Contains aphA::aadA Ap, Sm This workpIOM16 Contains lon::aphA Ap, Km This workpIOM17 Contains aphA::cat Ap, Cm This workpIOM18b Contains aphA::catb Ap, Cm This work

a ApR, KmR, GmR, SmR and CmR indicate resistance to ampicillin, kanamycin, gentamycin, streptomycin and chloramphenicol, respectively.b Orientation of cat in respect to aphA is opposite to that in pIOM17.


The ORFs from hrcC, hrpL, hrpV and lon, togetherwith their flanking regions were amplified by a PCRusing genomic DNA from P. syringae pv phaseolicola(Pph) 1448a as template, and the appropriate primers(Table 3). These primers introduced a KpnI restrictionsite at the 5′ and a SacII site at the 3′ ends of theamplified fragments. PCR products corresponding tohrpL and lon were digested with KpnI and SacII, andcloned into the KpnI–SacII sites of pKAS32, generatingpKAS32–hrpL, and pBluescript SK (+) (Stratagene; LaJolla, California, USA), generating pBSK–lon. Frag-ments corresponding to hrcC and hrpV were treated togenerate blunt ends, and cloned into the EcoRV site ofpKAS32, rendering pKAS32–hrcC and pKAS32–hrpV(Fig. 2A). pKAS32–hrcC, pKAS32–hrpV, pKAS32–hrpL and pBSK–lon, were amplified by PCR usingExpand Long Template Polymerase (Roche; Mannheim,Germany) and the appropriate primers (Table 3). ThesePCR reactions amplify the entire length of the plasmidexcept the target gene, since primers are designed toamplify outwards from the ORF. Primers used for PCRof pKAS32–hrcC, pKAS32–hrpV and pKAS32–hrpL,introduced XhoI sites at both 5′ and 3′ ends of theamplified products. Primers used for PCR of pBSK–lonintroduced SphI sites instead. PCR products were

digested with XhoI or SphI as appropriate, and ligatedto a 1.7 Kb SalI or SphI fragment from pMKmcontaining the aphA gene, generating pIOM8,pIOM11, pIOM10 and pBSKlonKm. pIOM16 wasgenerated by cloning a 2.7 Kb KpnI–SacI fragmentfrom pBSKlonKm into pKAS32 digested with KpnI andSacI (Fig. 2B).

2.3. Conjugations

S17–1λpir (Simon et al., 1983) was used as donorstrain for bi-parental conjugations, whereas CC118λpir(Herrero et al., 1990) and HB101 carrying pRK600(Kessler et al., 1992), were used for tri-parentalconjugations, as donor and auxiliary strains, respec-tively. Donor (D), receptor (R) and auxiliary (A)strains were grown overnight in LB at 28 °C or 37 °Cwith aeration, as appropriate. Cultures were thenadjusted to an OD600 nm of approximately 4, combinedin 1:4 or 1:1:4 volume ratios of D:R or A:D:R,respectively, rinsed in fresh LB medium three times,spotted onto sterile filters set on LB plates, andincubated at 28 °C. Following overnight incubation,filters were removed from the plates, placed into NaCl0.9% and vortexed. The resulting bacterial suspensions

Table 3Primers used in this study

Name Sequence 5′-3′ Locationa Product (bp)

hrpL-F AAT GGT ACC TCG CCT TCA GCT CCATCT TCC 1504993-5014 1690 (ORF: 555)hrpL-R AAT CCG CGG AAG AGT ATT CGG AGT TGT CG 1506661-81hrpV-F AAT GGT ACC CTG ATG GTC GAT GGA ACT GC 1493858-77 1335 (ORF: 351)hrpV-R AAT CCG CGG ATT CAT TGC AAG GGT GAG GAC G 1495168-90lon-F AAT GGT ACC AAG CTG TTC GAG ATG GAA GG 1963969-89 3225 (ORF: 2397)lon-R AAT CCG CGG TCT TAC CAG TCT GAG GGT TGC 1967171-92hrcC-F AAT GGT ACC ATG GAT TTC AGT GAG TTC GC 1491681-701 2985 (ORF: 2100)hrcC-R AAT CCG CGG CAA AAT CAC GCT GTA CAT CC 1494644-63hrpLinv-F AAT CCT CGA GAT TGA AGC AGC AGATTG ACC 1506186-205 5430hrpLinv-R AAT CCT CGA GTA CAG CTT TTG GCA CAA ACG 1505496-515hrpVinv-F AAT CCT CGA GAC CAA CCT GGATGA CAT ACG 1494702-21 5450hrpVinv-R AAT CCT CGA GAC TTC ACG ACT GTG TAATCC 1494384-403loninv-F ATC GGC ATG CTA AAT GGATTG ACG AAG TCC 1966711-30 5400loninv-R ATC GGC ATG CGA GAG GCA ATT CAATAG TGG 1964454-73hrcCinv-F AAT CCT CGA GAA ATG GCG AAA GAG AGT CGG 1494165-84 5440hrcCinv-R AAT CCT CGA GGT AAG CCG TGT GTT TCC AGG 1492191-210Cm600-F TCA CTC GAG TCT GGATTT GTT CAG AAC GC From pRK600b 800Cm600-R TCA CTC GAG TCT TTC AC TGA GCC TTT CGKm–aphA–F ATG AGC CAT ATT CAA CGG From pUC18NKmc 820Km–aphA–R AGC ATC AAATGA AAC TGCa Position in the annotated genome.b These primers amplify the cat gene with its own promoter but without the transcriptional terminator.c These primers amplify the aphA gene with its own promoter but without the transcriptional terminator.


were rinsed three times in NaCl 0.9% and plated ontoselective media. Replica plates of the resultingcolonies were carried out in plates supplementedwith ampicillin (500 μg/ml) to determine whethereach transconjugant was the result of plasmidintegration or allelic exchange.

2.4. Plant experiments

Seeds of Phaseolus vulgaris cultivar CanadianWonder (Thomas Etty Esquire [Kent, UK]) weregerminated and grown with 16 h light–8 h dark cycles.Bacterial lawns were grown for 48 h in LB plates andresuspended in 10 mM MgCl2. Cell densities wereadjusted to an OD600 nm of 0.1 (corresponding toapproximately 5×10−7 colony forming units [cfu] perml) and serial dilutions were carried out to reach theinoculation dose (104 cfu/ml). Eight days-old beanplants were inoculated by infiltrating 200 μl ofbacterial suspension into the intracellular spaces ofthe primary leaves. Infiltration was achieved bypressing the bacterial suspension against the leafunderside with a 2 ml syringe without needle.Symptoms were analyzed 14 days post-inoculation(dpi). Leaf disks were taken with a 7 mm–diametercork borer from infected tissue at 14 dpi. Ten leaf diskswere taken per plant, placed into 1 ml of sterile distilled

water, and homogenized by mechanical disruption.Serial dilutions of the resulting bacterial suspensionwere plated onto selective plates.

2.5. DNA manipulations

Basic DNA and molecular techniques were per-formed following standard methods (Sambrook et al.,2001). Genomic DNA was extracted using the Jet FlexExtraction Kit (Genomed; Löhne, Germany), andplasmid DNA was extracted using either NucleobondAX, or NucleoSpin Plasmid Quick Pure, depending onthe scale of the extraction (Macherery-Nagel; Düren,Germany). Routine clone analysis was carried out byboiling plasmid extraction (Holmes and Quigley, 1981).DNA gel-purification was carried out using the GFXPCR DNA kit (Amersham; Little Chalfont, Buckin-ghamshire, England). Taq Polymerase or Expand LongTemplate Polymerase (Roche; Mannheim, Germany)was used as appropriate.

DNA hybridization was performed following stan-dard methods (Sambrook et al., 2001), using a DIG-Nucleic Acid Detection Kit (Roche; Mannheim, Ger-many), following the instructions provided by themanufacturer. DNA was transferred onto a nylonmembrane by upward capillary transfer, and cross-linked by UV irradiation. Prehybridization and

NotI digestion andcloning into pKAS32 NotI

OriR6K

mob

BglII/ EcoRV/ KpnI/ NotI/ SacII/ SacI/ EcoRI

MCS

pKAS32 4619 bp

pUC18N-Km-AntR 6386 bp

NotI

NotI

XhoI/SalI

XhoI/SalI

Ligation

NotI

NotI

pUC18N-Km 4386 bp

XhoI

SalI

SalI

XhoI

SalI

SalI

XhoI

aacC1

aadA

cat (CmR)

(SmR)

(GmR)

OriR6K

mob

NotINotI

pIOM14 7450 bp

a

OriR6K

mob

NotINotI

pIOM15 7450 bp

mob

OriR6KNotI

NotI

pIOM17 6630 bp

(ApR, CmR)

(ApR, SmR)

(ApR, GmR)

Fig. 1. Construction of pKAS32 derivatives for antibiotic marker exchange. pUC18N–Km is a pUC18Not derivative into which the aphA gene frompMKm, conferring kanamycin resistance, has been cloned. Three mutant alleles of aphA were obtained by cloning in its XhoI site either a SalIfragment from pMGm containing aacC1 (which confers resistance to gentamycin), a SalI fragment from pSmUC containing aadA (which confersresistance to streptomycin), or a XhoI fragment obtained by PCR from pRK600 containing cat (which confers resistance to chloramphenicol). TheseSalI fragments are collectively referred to as AntR in the figure. NotI fragments containing aphA::aacC1, aphA::aadA, and aphA::cat were obtainedfrom the resulting plasmids and cloned into NotI-digested pKAS32, rendering pIOM14, pIOM15, and pIOM17, respectively.


KpnI

pBSK-lon

SphI SphI

SacIISacI

OriR6KhrcC hrpVor

pKAS32-hrcC or pKAS32-hrpV

XhoI XhoI

mob

BglII/ EcoRV/ KpnI/ NotI/ SacII/ SacI/ EcoRI

MCS

OriR6K

mobpKAS32 4619 bp

gene

KpnI SacII

Amplification oftarget gene by PCR

A

B

KpnI SacIIOriR6K

hrpL

pKAS32-hrpL

XhoI XhoI

mob

KpnI-SacI digestion andcloning into KpnI-SacI sitesof pKAS32

KpnI

SacIISacI

pBluescript SK (+) 2958 bp

mob

OriR6KKpnI

XhoI/SalISacII

XhoI/SalI

pIOM8pIOM10pIOM11

mob

OriR6KSacI

SphISphISacI

pIOM16 pBSKlonKm

KpnISphI SphISacII

SacI

XhoI orSphI

aphA

SalI or SphI

+ (ligation)XhoI orSphI

SalI or SphI

Fig. 2. Generation of pKAS32 derivatives for deletion of hrpV, hrcC, hrpL, and lon genes. (A) hrpV, hrcC, and hrpL ORFs plus 0.5 kb flankingregions from each side were amplified and cloned into pKAS32, whereas lon ORF plus its l.5 Kb flanking regions were amplified and cloned intopBluescript SK (+). PCR reactions were carried out using the resulting plasmids as DNA template and outward facing primers that amplify the entireplasmid, except the ORFs of each gene, and introduce a restriction site. Dotted lines represent the amplified sequences (B) The aphA gene carrying itsown promoter but no transcriptional terminator, was ligated to the inverse PCR products obtained, rendering pIOM8, pIOM10, pIOM11, and pBSK–lon::aphA. The fragment containing Δlon::aphA from the former was cloned into pKAS32 460 rendering pIOM16.


hybridization stages were carried out at 65 °C. DNAprobe was labelled by PCR reaction with chemilumi-niscent digoxigenin-dNTPs using a DIG Labelling Mix

(Roche; Mannheim, Germany), primers Km–aphA–Fand Km–aphA–R (Table 3) and pUC18N–Km as DNAtemplate.


3. Results

3.1. Construction of pKAS32 derivatives for antibioticmarker exchange

To facilitate generation of multiple knockoutmutants, we constructed a set of suicide vectors forantibiotic marker exchange on mutant strains. Wecloned the aphA gene from pMKm (Murillo et al.,1994; Oka et al., 1981), carrying its own promoter butno transcriptional terminator, into pUC18Not. Then,we disrupted aphA with three different selectionmarkers, aacC1, aadA, and cat, which confergentamycin, streptomycin, and chloramphenicol resis-tance, respectively (Kessler et al., 1992; Murillo et al.,1994). All three markers carry their own promoter, butonly aadA carries a transcriptional terminator. Theresulting vectors, pIOM14 (aphA::aacC1), pIOM15(aphA::aadA), and pIOM17 (aphA::cat), contained theselection marker genes flanked by fragments of aphA(Fig. 1). These flanking regions provide homology toallow the allelic exchange of aphA wild-type copies.These vectors can be introduced into the receptorstrain by transformation or conjugation. To beintroduced by conjugation, tra functions have to beprovided in trans either by the donor or a helper strainsince the pKAS32 plasmid backbone carries a moblocus.

pKAS32 also carries the rpsL gene from E. coli, thatencodes ribosomal protein S12. Mutations in rpsLconferring resistance to streptomycin are recessive in astrain expressing the wild-type protein (Lederberg,1951), allowing rpsL to be used as a counter-selectablemarker for plasmid loss. Resistance to streptomycin dueto expression of aadA is not affected by expression ofrpsL.

3.2. Construction and genetic characterisation of hrpL,hrpV, hrcC, and lon mutants

P. syringae pv. phaseolicola (Pph) is divided intoraces according to cultivar specificity (Taylor et al.,1996). Race 6 groups a number of strains capable ofcausing infection in all bean cultivars (Tsiamis et al.,2000). Strain 1448a is the representative strain of race 6and its genome has been fully sequenced (Joardar et al.,2005). Using the genome sequence, primers weredesigned to amplify the ORFs plus their flankingregions from hrpL, hrpV, hrcC, and lon. Amplifiedfragments were cloned and the complete ORFs deleted,and disrupted with the aphA gene (Fig. 2). The resultingplasmids, pIOM8 (ΔhrcC::aphA), pIOM10 (ΔhrpL::

aphA), pIOM11 (ΔhrpV::aphA), and pIOM16 (Δlon::aphA) were introduced by conjugation into 1448a.

Transconjugants were selected for resistance tokanamycin. In these experiments, as in all conjugationexperiments carried out throughout this work, isolatedtransconjugant colonies were screened for sensitivity toampicillin (pKAS32 derivatives selective marker), inorder to identify which transconjugants have undergoneallelic exchange, and therefore did not carry the plasmidstill integrated (merodiploids for the target gene).Frequencies of both transconjugants and allelic ex-change are shown in Table 4.

In order to verify aphA disruption of the correct targetgene, genomic DNA from the mutant strains wasextracted, digested with the appropriate restrictionenzyme, and subjected to Southern blot analysis usingaphA ORF as specific probe. As expected, a singlehybridization band was obtained for each sample sincerestriction enzymes used in each sample cuts neitherwithin aphA ORF, nor within the target gene (Fig. 3).Band sizes depend on the location of the restriction sitesnearest to each target gene, and corresponded to thosepredicted from the genome sequence.

Genomic DNA of these strains was also digestedusing XhoI, which cuts once inside aphA ORF. UsingaphA ORF as a probe, Southern blot analysis showedtwo hybridization bands for each strain, with sizescorresponding to those predicted for each gene disrup-tion (data not shown).

3.3. Antibiotic marker exchange and generation ofmultiple knockout mutant strains

Strains IOM7 (ΔhrpL; KmR), IOM9 (ΔhrpV; KmR),and IOM10 (Δlon; KmR) were selected for antibioticmarker exchange experiments, and used as receptorstrains for conjugational transfer of plasmids pIOM17(aphA::cat), pIOM14 (aphA::aacC1), and pIOM15(aphA::aadA), respectively. Transconjugants were se-lected as CmR, GmR, and SmR, respectively, andchecked for ampicillin sensitivity (Table 4). Theresulting strains, IOM34 (ΔhrpL; CmR), IOM13(ΔhrpV; GmR), and IOM28 (Δlon; SmR), respectively,were checked for sensitivity to kanamycin (KmS) toconfirm disruption of the aphA gene. Disruption of theaphA gene with cat, aacC1, and aadA in these threestrains was also confirmed using PCR, and Southernblot analyses (Figs. 4, 5 and data not shown).Additionally, we tested the resulting strains for HRinduction in N. tabacum.

Strains IOM13 (ΔhrpV; GmR) and IOM28 (Δlon;SmR), were used as receptor strains for the conjugative

Table 4Frequency of conjugation and allelic exchange

Receptors Transconjugants Allelic exchanged strain

Strain Description cfu/ml a Selectionused

Frequencyb

T /RcFrequencyd

K /T eDescription Strain

Generation of single mutants1448a Wild-type 5×108 Km 1.36×10−6 10−2 ΔhrcC (KmR) IOM31448a Wild-type 5×108 Km 6.96×10−7 10−2 ΔhrpL (KmR) IOM71448a Wild-type 5×108 Km 6.44×10−7 10−2 ΔhrpV (KmR) IOM91448a Wild-type 5×108 Km 4.2×10−7 1.5×10−2 Δlon (KmR) IOM10

Antibiotic marker exchange in single mutantsIOM7 ΔhrpL (KmR) N.d. Cm N.d. 2.5×10−1 ΔhrpL (CmR) IOM34IOM9 ΔhrpV (KmR) 2×108 Gm 1.5×10−7 3×10−2 ΔhrpV (GmR) IOM13IOM10 Δlon(KmR) 1.6×108 Sm 3.12×10−7 4×10−2 Δlon (SmR) IOM28

Generation of double mutantsIOM13 ΔhrpV (GmR) 2×108 Gm, Km 1.35×10−2 6×10−3 ΔhrpV (GmR) ΔhrpL (KmR) IOM16IOM28 Δlon (SmR) 8×106 Sm, Km 5.5×10−5 6×10−3 Δlon (SmR) ΔhrpL (KmR) IOM31IOM28 Δlon (SmR) 1.2×108 Sm, Km 4.7×10−6 3×10−3 Δlon (SmR) ΔhrpV (KmR) IOM33

Antibiotic marker exchange in a double mutantIOM16 ΔhrpV (GmR) ΔhrpL (KmR) 2×108 Gm, Sm 1.03×10−6 5×10−3 ΔhrpV (GmR) ΔhrpL (SmR) IOM29

Generation of a triple mutantIOM29 ΔhrpV (GmR) ΔhrpL (SmR) 3.2×108 Gm, Sm, Km 2×10−6 2×10−2 ΔhrpV (GmR) ΔhrpL (SmR)

Δlon (KmR)IOM30

a Numbers are the results of one conjugation experiment per strain.b Frequency T /R reflects both frequency of conjugation and plasmid integration.c Cfu ml−1 of transconjugants (T) divided by cfu/ml of receptors (R).d Frequency K /T reflects frequency of plasmid excision.e Frequency of knockout mutant (K) (ApS) among transconjugants (T) obtained (KmR).


transfer of plasmid pIOM10 (ΔhrpL::aphA), to generatedouble mutant strains. Transconjugants were selected asGmR KmR, and SmR KmR, respectively, and checkedfor ampicillin sensitivity (Table 4). Disruption of thehrpL gene in both resulting strains (IOM16 and IOM31,respectively) was confirmed using PCR and Southernblot analyses (Figs. 4, 5 and data not shown).

Strains IOM28 (Δlon; SmR) was used as receptorstrain for the conjugative transfer of plasmids pIOM11(ΔhrpV::aphA), to generate a double mutant strain.Transconjugants were selected as SmR KmR, andchecked for ampicillin sensitivity (Table 4). Disruptionof the target genes in the resulting strains (IOM33) wasconfirmed by Southern blot analysis (Fig. 4).

Strain IOM16 (ΔhrpV [GmR]; ΔhrpL [KmR]) wasused as receptor strain for the conjugative transfer ofplasmid pIOM15 (aphA::aadA) to exchange kanamycinresistance for streptomycin resistance. Transconjugantswere selected as GmR SmR, and checked for sensitivityto kanamycin and ampicillin to confirm antibioticmarker exchange (Table 4). Antibiotic marker exchange

in the resulting strain (IOM29) was further confirmedusing PCR (Fig. 5).

Strain IOM29 (ΔhrpV [GmR]; ΔhrpL [SmR]) wasused as receptor strain for the conjugative transfer ofplasmid pIOM16 (Δlon::aphA) to generate a triplemutant strain. Transconjugants were selected as GmR

SmR KmR and checked for ampicillin sensitivity (Table4). Disruption of the lon gene in the resulting strain(IOM30) was confirmed using PCR (Fig. 5).

Every time an antibiotic marker exchange isperformed a “scar” of aphA sequences is left behind.Thus, upon entry of a plasmid carrying a gene disruptedby the aphA gene, integration of the plasmid can takeplace either by homologous recombination into thetarget gene, or by homologous recombination into thedisrupted aphA gene. After excision of the integratedplasmid, the former would generate the double mutantstrain intended. This strain would be resistant tokanamycin as well as the antibiotic corresponding tothe gene used to disrupt aphA. However, excision of theplasmid in the second case would result in the allelic

1448a

IOM7ΔhrpLKmR

IOM34ΔhrpLCmR

IOM10ΔlonKmR

IOM28ΔlonSmR

IOM31ΔlonSmR

ΔhrpLKmR

IOM33ΔlonSmR

ΔhrpVKmR

8.0 Kb

4.3 Kb

3.0 Kb

Fig. 4. Southern blot analysis of antibiotic-marker exchanged anddouble mutant strains. Southern blot analysis of genomic DNA fromstrains 1448a (wild-type), IOM7 (ΔhrpL; KmR), IOM34 (ΔhrpL;CmR), IOM10 (Δlon; KmR), IOM28 (Δlon; SmR), IOM31 (Δlon,SmR; ΔhrpL, KmR), and IOM33 (Δlon, SmR; ΔhrpV, KmR), digestedwith EcoRV, using aphA ORF as a probe. No hybridization signal wasdetected in 1448a. Single bands of 7.5 kb (corresponding to EcoRVfragment containing hrpL::aphA) and 8.4 kb (corresponding to thesame fragment plus the size of the cat gene used to disrupt aphA) weredetected in IOM7 and IOM34, respectively. Single bands of 3.6 kb(corresponding to EcoRV fragment containing lon::aphA), and 5.7 kb(EcoRV fragment containing lon::aphA plus the size of the aadA genedisrupting aphA), were obtained in IOM10 and IOM28, respectively.Bands of 5.7 kb (EcoRV fragment containing lon::aphA plus the sizeof the aadA gene disrupting aphA) and 7.5 kb (EcoRV fragmentcontaining hrpL::aphA), were detected in IOM31, whereas the sameband of 5.7 kb plus a 5.6 kb band (EcoRV fragment containing hrpV::aphA), were detected in IOM33.

1448aIOM3

hrcCIOM9

hrpVIOM7ΔΔΔ hrpL

IOM10Δlon

23.1 Kb

8.0 Kb

4.3 Kb

3.0 Kb

Fig. 3. Southern blot analysis and hypersensitive response in N.tabacum of single mutant strains. Southern blot analysis of genomicDNA from strains 1448a (wild-type), IOM3 (ΔhrcC), IOM9 (ΔhrpV),IOM7 (ΔhrpL), and IOM10 (Δlon), using aphA ORF as a probe,indicate gene disruption of target genes. Genomic DNA from strainsIOM3, IOM7, IOM9 (digested with SacII), and IOM10 (digested withEcoRV) render a single hybridization band in each case, with sizescorresponding to those predicted from the genome sequence.


exchange of a disrupted aphA allele by a wild-type one,and the resulting strains would therefore revert to be asingle mutant strain, only resistant to kanamycin. Byapplying double or triple antibiotic selection, asappropriate in each case, to obtain the transconjugants,those transconjugants resulting from the allelic ex-change of aphAwere eliminated. Only a small decreasein the frequencies of transconjugants were detected inthese experiments (6×10−3 to 2×10−2) when comparedto similar ones carried out to generate the correspondingsingle mutants (10−2 to 1.5×10−2), indicating that ofaphA “scars” do not act as hotspots for allelic exchange(Table 4).

3.4. Genetic stability of multiple mutant strains in vitroand in planta

Double and triple mutant strains generated using thismethod carry fragments of aphA in different loci, thusproviding fragments of homology that can constitutetargets for recombination events. Recombination eventsbetween these fragments would give rise to chromo-somal reorganisations, either inversions if recombina-tion took place between fragments in invertedorientation, or deletions if recombination took placebetween fragments in direct orientation. Thus, recom-bination events between aphA fragments in directorientation would result in the deletion of one of the

copies of aphA, either the wild-type or the disruptedversion, as well as the deletion of the chromosomalregion between them, altering the hybridization, PCRprofile, and antibiotic resistance of the strain. PCRanalyses carried out after either growth in laboratoryconditions or growth in planta of the triple mutant strain(IOM30) did not detect any evidence of a profile thatcould correspond to such reorganisations in the bacterialpopulation of any of the strains generated (Fig. 4).Furthermore, analysis of antibiotics resistance of thisstrain after either growth in laboratory conditions orgrowth in planta, carried out to detect reorganisationsthat could occur at a frequency below the level ofdetection of Southern blot analysis, showed thatreorganisations occur at the same rate as any otherrecombination event in these loci (data not shown).These results further support the conclusion that aphA“scars” do not constitute a “hotspot” for recombinationevents.

1448a

IOM9ΔhrpVKmR

3.0 Kb

1.9 Kb

1.4 Kb

1.0 Kb

0.7 Kb

a b

IOM13ΔhrpVGmR

IOM16ΔhrpVGmR

ΔhrpLKmR

IOM29ΔhrpVGmR

ΔhrpLSmR

IOM30ΔhrpL GmR

ΔhrpV SmR

Δlon KmR

Fig. 5. PCR analysis of antibiotic-marker exchanged, double and triplemutant strains. PCR analysis of strains IOM9 (ΔhrpV; KmR), IOM13(ΔhrpV; GmR), IOM16 (ΔhrpV, GmR; ΔhrpL, KmR), IOM29 (ΔhrpV,GmR; ΔhrpL, SmR), and IOM30 (ΔhrpV, GmR; ΔhrpL, SmR; Δlon,KmR), using primers to amplify aphA ORF. No PCR product wasobtained for strain 1448a; a 0.8 kb amplification product,corresponding to the size of aphA ORF was obtained from strainIOM9; a 2.8 kb band corresponding to the size of aphA::aacC1, wasobtained from strain IOM13; two bands of 0.8 kb and 2.8 kb,corresponding to the sizes of the aphA ORF and aphA::aacC1,respectively, were obtained from strain IOM16; a band of 2.8 kbcorresponding to the sizes of aphA::aadA and aphA::aacC1, wasobtained from strain IOM29; and the same band plus a 0.8 kb bandcorresponding to the size of aphAwas obtained from strain IOM30 (a).The same bands were obtained from IOM30 after 14 days of growth inplanta (b).


As expected, frequencies were lower after growth inplanta, probably reflecting that large chromosomalreorganisation would probably be deleterious underthe strong selective pressure to be found within theplant.

4. Discussion

In this work, we have developed a set of plasmidvectors that allows antibiotic marker exchange in asingle conjugation step. We have validated the efficacyand calculated the efficiency of the method in single anddouble mutant strains, as well as established itsapplication to the rapid generation of multiple mutantstrains. Once plasmids for gene knockout have beenconstructed, this set of vectors allows generation ofmultiple mutant combinations in a number of conjuga-tion steps equal to the number of mutations to becombined, eliminating any cloning step and decreasingconsiderably the time usually required for this purpose.

pKAS32 carries the rpsL gene from E. coli. Vectorscarrying rpsL, which encodes ribosomal protein S12,allow the use of streptomycin as counter-selection of the

presence of the vector, since mutations in rpsLconferring resistance to streptomycin are recessive in astrain expressing the wild-type protein (Lederberg,1951). Thus, if streptomycin-resistant strains, due tomutation in rpsL, are used as receptors of these vectorsin conjugation or transformation experiments, plasmidintegration (first recombination event) will renderbacteria sensitive to the antibiotic. Subsequent selectionof SmR permits identification of transconjugants thathave excised the plasmid (second recombination event).However, given the high frequencies of allelic exchangeobtained in our system (4×10−2 to 5×10−3; Table 4),and the fact that streptomycin-resistant 1448a growsconsiderably slower than 1448a, an undesirable charac-teristic for a strain to be tested for growth in planta(Zumaquero A. and Beuzón, C.R. unpublished results),we decided against using rpsL as counter-selectablemarker for the generation of the mutant strains,screening for them instead on the basis of antibioticresistance. When knockout mutants were not detectedamong the transconjugants, they could be found after around of growth in non-selective medium.

Interestingly, if further rounds of growth in non-selective medium were carried out, the frequency ofknockout mutants dropped (data not shown), presum-ably due to a growth advantage of isolates with a higherrpsL gene dosage. Nevertheless, streptomycin ascounter-selection maker can be useful when generatingmutants in strains with lower recombination frequen-cies, or natural resistance to high concentrations ofampicillin.

Antibiotic marker exchange can also be helpful inphenotypic characterisation of single mutants sinceswapping the antibiotic resistance cassette used in aparticular mutation might also imply swapping from apolar to a non-polar mutation or vice versa. Since thismethod uses two types of antibiotic resistance cassettes,with and without transcriptional terminators, allelicexchange of one type of resistance gene (aphA, notranscriptional terminator) for the other (aadA, tran-scriptional terminator), would allow determination ofphenotypic differences due to polarity on downstreamgenes. Furthermore, if aphA disruption of the target geneis designed in order to prevent expression of down-stream genes from the aphA promoter, further disruptionof aphA using pIOM18, that carries the cat gene inopposite orientation to aphA transcription, would causeexpression of downstream genes from the cat promoter,allowing determination of phenotypic differences due toconstitutive overexpression of downstream genes. Thisis illustrated by the results described by Preston et al.(1998), where a hrpT::ntpII mutation caused a reduction


in HrpZ expression through overexpression of hrpT-downstream hrpV gene from the nptII promoter,constituting one of the evidences that indicated thatHrpV was a negative regulator of Hrp genes.

We also show that the “scars” of homology leftbehind in multiple mutants do not constitute a “hotspot”for recombination events, since genetic reorganisationsmediated by them occur at the same rate as otherrecombination events in these loci (Table 4). Thusgenetic stability of the resulting strains is sufficient formost phenotypic characterisations, allowing identifica-tion of mutant combinations of particular interest.Further characterisation of such a mutant combinationcan then be carried out in a reconstructed mutant strain,reducing the number of de novo disruptions to begenerated to those worthy of detailed analysis. Howev-er, although genetic stability of multiple mutants issufficient for phenotypic analysis, it would not suffice ifthe intended use of the resulting strain were the isolationof variants, or its use for mutagenesis and screening,since chromosomal reorganisations do occur and couldbe selected during the screening process.

The method and vectors described in this work havebeen developed and tested in P. syringae, however theycan be potentially used in any genetically amenableGram-negative bacteria in which gene replacement ispossible.

Acknowledgements

This work was supported by a project grant from theMinisterio de Ciencia y Tecnología (Spain) to CarmenR. Beuzón. Carmen R. Beuzón was supported by the“Ramón y Cajal” Programme from the Ministerio deCiencia y Tecnología. Inmaculada Ortiz-Martín wassupported by a Fellowship from Junta de Andalucía.We are grateful to L. Cruzado, J. Guillamet, and T.Duarte for their practical assistance. We also want tothank Javier Ruiz-Albert and Eduardo R. Bejarano fortheir helpful discussion and critical reading of themanuscript.

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Suicide vectors for antibiotic marker exchange and �rapid generation of multiple knockout mutan.....IntroductionMaterials and methodsBacterial strains and growth conditionsPlasmidsConjugationsPlant experimentsDNA manipulations

ResultsConstruction of pKAS32 derivatives for antibiotic marker exchangeConstruction and genetic characterisation of hrpL, hrpV, hrcC, and lon mutantsAntibiotic marker exchange and generation of multiple knockout mutant strainsGenetic stability of multiple mutant strains in vitro and in planta

DiscussionAcknowledgementsReferences

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