APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1993, p. 3050-3055 Vol. 59, No. 90099-2240/93/093050-06$02.00/0Copyright © 1993, American Society for Microbiology

Bacteriophage Lambda as a Delivery Vector for TnlO-DerivedTransposons in Xenorhabdus bovienii

MATTHEW S. FRANCIS, ANGELA F. PARKER, RENATO MORONA, AND CONNOR J. THOMAS*Department ofMicrobiology and Immunology, University ofAdelaide,

G.P.O. Box 498, Adelaide, South Australia, Australia, 5001

Received 26 February 1993/Accepted 13 July 1993

Xenorhabdus bovienii wild-type strains lack a functional receptor protein (LamB) in the outer membrane andas a result are unable to adsorb coliphage lambda (X). Introduction of plasmids encoding lamB into X. bovieniiT1228 results in constitutive expression of LamB in the outer membrane of this organism. LamB-expressingstrains of X. bovienii adsorb lambda bacteriophage particles and can be used as hosts for lambda::Tnconstructs. A TnlO-derived transposon, element 9 (J. C. Way, D. Davis, D. Morisato, D. E. Roberts, and N.Kleckner, Gene 32:369-379, 1984) was used to construct a variety of insertion mutants ofX. bovienii. Mutantsthat had altered expression of protease, lipase, DNase, dye-binding capability, and hemolytic activity, inaddition to a series of auxotrophic mutants, were isolated.

Xenorhabdus spp. are entomopathogenic bacteria associ-ated with insect parasitic nematodes of the families Hetero-rhabditidae and Steinemematidae (4, 5, 32). These bacteria,which are currently grouped within the family Enterobacte-riaceae, are carried monoxenically within the foregut ofnonfeeding infective-stage nematodes. Nematodes whichinvade the hemocoel of susceptible insect larvae release thebacteria into the hemolymph. Rapid growth of the bacterialeads to a fatal septicemia and provision of nutritionalconditions necessary for nematode reproduction within thecadaver. Antibiotics secreted by the bacteria restrict thegrowth of other microorganisms (3, 22).Xenorhabdus isolates are unusual in that they produce two

colony forms when cultured in vitro. These colony forms arerepresentative of two phases that differ in some biochemicalcharacteristics. Phase I cells can be readily distinguishedfrom phase II by absorption of dyes and the production ofantibiotic substances (2, 3). Both types are pathogenic forinsect larvae, but phase I forms only associate with infectivenematodes. Phase I cultures are also unstable and readilyconvert to phase II on prolonged in vitro culture or during invivo culture.

Little is known of the molecular events which lead topathogenesis of susceptible insect hosts by these bacteria orof the mechanisms which determine the association of thebacterium with the nematode host. All species ofXenorhab-dus secrete a variety of proteins (e.g., fimbriae, proteinases,lipases, and DNase) which may be implicated in virulenceand colonization of nematodes. One method to determinethe role of these proteins in colonization or virulence is toconstruct isogenic mutants for each of these phenotypes.Transposon mutagenesis can be used as an important tool

for the study of a variety of gram-negative bacteria. This isnormally achieved by introduction of transposons encodedon plasmid or bacteriophage vectors into bacteria which donot normally support replication of the vector DNA. How-ever, most vectors available for introduction of DNA intogram-negative bacteria are limited in versatility, consist ofthe full transposon, and are therefore large and unwieldy and

* Corresponding author. Electronic mail address: cthomas@microb.adelaide.edu.au.

have numerous disadvantages related to presence of thecognate transposase (15). Recently, Xu et al. (34) reportedthe use of TnS transposon mutagenesis as a mechanism forproduction of insertion mutants of Xenorhabdus nemato-philus ATCC 19061. Although a variety of phenotypic mu-tants were obtained, the system used supported only lowfrequencies of transposition and a high level of cointegrationor stable maintenance of the plasmid suicide vector wasobserved. These problems were largely overcome by devel-opment of a negative selection suicide vector for Tn5 (pHX1)based on the Bacillus sacB gene, which mediates productionof levansucrase. Levansucrase is lethal for most gram-negative bacteria, and hence cells maintaining plasmidsencoding sacB do not survive. Nevertheless, introduction ofpHX1 via RP4-based conjugation resulted in fewer than 20mutant colonies per mating.We have developed an alternative approach for transpo-

son mutagenesis of Xenorhabdus bovienii based on use ofbacteriophage X delivery systems for modified TnlO trans-posons (33). One of these bacteriophage vehicles (X1105)enables isolation of highly stable insertions coupled with acomparatively high frequency of transposition in X. bovienii.This transposon delivery system produces small, stableinsertions by virtue of a transposase gene (ISJOR) locatedoutside of inverted repeats which flank a kanamycin antibi-otic selection marker. Furthermore, unlike suicide plasmidvectors such as pSUP2021 (29), Omegon-Km (12), and pGS9(27), this system is not hampered by formation of cointe-grates or stable maintenance of a plasmid vector. Conse-quently, every infection of a cell by a X::Tn particle has thepotential to initiate transposition and thereby facilitate iso-lation of independent insertion mutants.

MATERIALS AND METHODS

Growth media. All strains of Xenorhabdus and Esche-richia coli were routinely cultivated on Oxoid nutrient agar(NA) and Oxoid nutrient broth (NB). BTB agar (NA plusbromothymol blue, 0.0025% [wt/vol], and tetrazolium chlo-ride, 0.004% [wt/vol]) was used to differentiate phase I andphase II Xenorhabdus variants. Congo red agar is NAsupplemented with filter-sterilized Congo red (1% [wt/vol] toa final concentration of 0.001% [wt/vol]). DNA agar consists

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TABLE 1. Strains, plasmids, and bacteriophages used in this study

Strain, plasmid, Relevant characteristics Source or referenceor bacteriophage

StrainsX. bovienii T228 Apr, KmS, Tcs R. J. AkhurstE. coli S17-1 recA4 thi pro hsdR- M+, (RP4:2-Tc:Mu:Km:Tn7), Tpr, Smr 30E. coli NK5012 leu-6 thi-1 supE44 lacY tonA N. KlecknerE. coli CC118 araD A(ara leu)7697 AlacX74phoA A20 galE galK thi rpsE rpoB argE(Am) recA1 sup0 20

PlasmidspNK862 Kmr (element 9) 33pTROY41613 Tcr, LamB(Con) 16pTROY9 Tcr, malK::IS-3, LamB(Con) 10

BacteriophagesAvir Laboratory collectionX::TnphoA Kmr, phoA 14X940 Cmr (element 4) 33X1104 Kmr (element 2) 33X1105 Kmr (element 9) 33

of 0.2% (wt/vol) herring sperm DNA (Sigma) dissolved inNA by boiling for 10 min. Tween 60 agar was prepared asdescribed by Sierra (28) and skim milk agar was prepared asdescribed previously (13). Blood agar was NA supplementedwith 4.5% (vol/vol) sheep blood. Minimal medium able tosupport growth of X. bovienii T228 consisted of 1.5% (wt/vol) Bacto-Agar, 10% (vol/vol) lOx M9 salts (21), 0.01%(vol/Vol) MgSO4, and 0.5% (vol/voll glucose supplementedwith 1 ,ug of nicotinate (BDH) ml- . Auxotrophic mutantswere identified on media described by Davis et al. (8).All antibiotics were purchased from Sigma and were usedat the following final concentrations: ampicillin, 100 jgml-'; chloramphenicol, 25 ,ug ml-'; kanamycin, 50 ,ug ml-;and tetracycline, 8 ,ug ml-'.

Bacterial strains, plasmids, and bacteriophages. All bacte-rial strains, plasmids, and bacteriophages used are listed inTable 1. Xenorhabdus spp. were incubated at 28°C, and E.coli strains were incubated at 37°C, unless otherwise stated.

Bacteriophage propagation. Bacteriophage stock wasmixed with an equal volume of an overnight culture ofE. coliNK5012 and then incubated overnight at 37°C on NA platesin a soft agar overlay. A single bacteriophage plaque wasremoved and incubated with 0.45 ml of NB and 0.15 ml oflog-phase NK5012 cells at 37°C for 10 min and then placedon NA plates in a soft overlay and incubated at 37°C for 6 to7 h. Overlays were harvested into 10 ml of NB and 0.1 ml ofchloroform and centrifuged, and the supernatant was storedat 4°C. Titers (PFU) of bacteriophage stocks were deter-mined by adding 0.1 ml of overnight culture of E. coliNK5012 to 0.1-ml dilutions of bacteriophage stock andincubating the mixture on NA soft overlay plates.Lambda adhesion assay. The ability of bacteriophage X to

adhere to the surface of X. bovienii (pTROY9) constructswas assessed as follows. Bacteriophage (3 x 10 PFU ml-1)was added to an equal volume of overnight culture of the teststrain (5 x 109 cells ml-') and incubated at 28°C for 60 min.An overnight culture of the X-sensitive indicator strain, E.coli NK5012 (1 x 107 cells ml-'), was added and incubatedat 28°C for 15 min. The mixture was plated in a soft overlayon NA and incubated overnight at 37°C.

Infection of LamB-expressing strains of X. bovienii byA.:Tn. Log-phase cultures of X. bovienii or E. coli teststrains grown in selective media (5 x 109 cells ml-') werepelleted, and the cells were resuspended in 1 ml of NB

containing 20 mM MgCl2. The cell suspension (0.25 ml) wasmixed with bacteriophage stock (3 x 109 PFU ml-') beforeincubation at 28°C for 1 h. NB (10 ml) was added beforeshaking for 2 h at 28°C. Bacterial cells were harvested bycentrifugation, then resuspended in 1 ml of NB, and platedon NA containing an appropriate antibiotic selection. Con-trols included test strains incubated in the absence of phageand a suppressor-free E. coli strain (CC118) infected withbacteriophage stock.

Isolation of DNA. Plasmid DNA was prepared by thestandard three-step procedure (18). DNA was extractedfrom suspensions of X: :Tn as described previously (7).Chromosomal DNA was isolated by a modification of amethod described by Manning et al. (19).

Plasmid transformation. Cells competent for transforma-tion by DNA were prepared according to a protocol de-scribed by Brown et al. (6).

Bacterial conjugation. Overnight broth cultures grown inNB were grown to early exponential phase with slow agita-tion to avoid damage to the sex pili of donor cells. The cellswere washed in NB and resuspended in 10 ml of NB. Donorand recipient bacteria were mixed at a ratio of 1:10, and thecells were pelleted by centrifugation (5,000 rpm for 5 min ina bench centrifuge). The pellet was gently resuspended in300 ,ul of broth and spread onto a nitrocellulose membranefilter (0.45-p.m pore size; type HA, Millipore Corp.) on anNA plate. This plate was incubated for 6 to 16 h at 28°C. Thecells were then resuspended in 10 ml of NB, and sampleswere plated onto selective agar and incubated overnight at280C.

Preparation of radiolabelled DNA probes. Denatured probeDNA was labelled by randomly primed incorporation of32P-labelled dATP and dCTP (Amersham International, Am-ersham, England) by the method of Feinberg and Vogelstein(11).

Southern hybridization. Unidirectional transfers of DNAfrom agarose gels to nitrocellulose paper (Schleicher &Schuell) or nylon (Hybond N; Amersham) were performedas described by Southern (31) and modified by Reed (23).Filters were soaked for 6 h at 42°C in prehybridizationsolution and then incubated for 16 to 24 h at 42°C inhybridization solution. Hybrids were detected by using thestandard protocol (18).

Whole-cell membrane preparations. Ten-milliliter over-

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night or log-phase cultures were pelleted in a bench centri-fuge, resuspended in 30 mM Tris-HCl, pH 8.0, and recentri-fuged. The pellet was resuspended in 20% (wt/vol)sucrose-30 mM Tris-HCl, pH 8.1, and transferred to centri-fuge tubes. A 20-,u volume of 1-mg ml-' lysozyme in 0.1 MEDTA was added, and the mixture was incubated on ice for30 min, frozen in an ethanol-dry ice bath for 30 min, andallowed to thaw before addition of 3 ml of 3 mM EDTA, pH7.3. After four sonications of 15 s on ice, the solution wascentrifuged at 5,000 rpm in an SM24 rotor for 5 min in aSorvall RC-5 centrifuge. The supernatant was recovered andcentrifuged at 15,000 rpm in an SM24 rotor for 60 min. Thetubes were drained, and the pellet was resuspended in 0.1 mlof sample buffer (125 mM Tris-HCl [pH 8.0], 2% [wtlvol]sodium dodecyl sulfate [SDS], 10% [vol/vol] glycerol, 5%[vol/vol] ,B-mercaptoethanol, and 0.002% [wt/vol] bromophe-nol blue) and stored at -20°C.SDS-PAGE. SDS-polyacrylamide gel electrophoresis

(PAGE) was a modification of the procedure of Lugtenberget al. (17), as described by Achtman et al. (1). Samples wereheated to 100°C for 3 min and loaded on 12% gels, whichwere electrophoresed at 80 V through the stacking gel and120 V through the running gel. Protein staining was done byincubation overnight at room temperature in 0.06% (wt/vol)Coomassie brilliant blue in 5% (vol/vol) perchloric acid.Destaining was done by incubation in 5% (vol/vol) aceticacid for 24 h, with at least two changes of the destainingsolution.

RESULTS

The maltose-inducible LamB protein is an outer mem-brane protein of E. coli associated with the maltose transportsystem and acts as the receptor for bacteriophage A (26). Thehost range of X can be extended to gram-negative bacteriaother than E. coli by introducing plasmids carrying copies oflamB. For example, plasmids such as pTROY9 (10) andpTROY41613 (16) are multicopy plasmids which constitu-tively express LamB in a variety of gram-negative hosts. Itwas anticipated that expression of the LamB protein on thesurface of Xenorhabdus exconjugants would allow attach-ment of bacteriophage X to the cell surface and subsequentintroduction of bacteriophage DNA. Hence, LamB-express-ing strains would allow the use of X::Tn mutagenesis vectorsas the basis for creating Xenorhabdus mutants. LamB-expressing strains could also be used as hosts for cosmidDNA.

Transfer of pTROY9 plasmids to X. bovieni. Plasmid DNAof pTROY9 and pTROY41613 was used to transform E. coliS17-1. This strain provides RP4 transfer functions necessaryfor mobilization of these plasmids into the phase I form ofX.bovienii T228. These plasmids were then transferred to X.bovienii T228 by conjugation through ampicillin and tetracy-cline selection. The frequency of stable tetracycline-resis-tant exconjugates obtained ranfed from 1 x 10- to 2 x 10-7for pTROY9 and from 2 x 10- to 8 x 10-7 for pTROY41613per mating.

Expression of the A receptor by X. bovienii. Expression ofLamB on the outer membrane of X. bovienii exconjugantswas assessed by SDS-PAGE analysis of whole-membranepreparations of wild-type T228, T228(pTROY9), andT228(pTROY41613) (Fig. 1). Whole-membrane preparationsof cultures of E. coli S17-1 grown with and without 0.2%(wtlvol) maltose, E. coli S17-1(pTROY9), and E. coli S17-l(pTROY41613) served as positive controls for the LamBprotein, while T228 grown with or without 0.2% (wt/vol)

FIG. 1. SDS-PAGE analysis of whole-membrane preparationsof E. coli and X. bovienii T228, T228(pTROY9), and T228(pTROY41613) grown with or without maltose medium supplement.The arrow shows the position of migration of the 49-kDa LamBprotein. Lanes: a, molecular mass markers; b, E. coli S17-1(pTROY9); c, E. coli S17-1(pTROY41613); d, T228(pTROY9); e,T228(pTROY41613); f, T228 grown with 0.2% (wt/vol) maltose; g, E.coli S17-1 grown with 0.2% (wt/vol) maltose; h, E. coli S17-1 grownwithout maltose. Molecular mass markers are rabbit muscle phos-phorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), hen eggwhite ovalbumin (42.7 kDa), bovine carbonic anhydrase (31 kDa),soybean trypsin inhibitor (21.5 kDa), and hen egg white lysozyme(14.4 kDa).

maltose was used as a negative control. As expected,maltose-induced E. coli S17-1 produced a more intenselystained protein band of approximately 49 kDa than S17-1grown in the absence of maltose (25). Preparations of S17-1(pTROY9) and S17-1(pTROY41613) grown in the absenceof maltose also produced the 49-kDa LamB protein. Wild-type X. bovienii T228 did not produce a protein of equivalentsize even when grown in the presence of maltose. Of the teststrains, only the T228(pTROY9) constructs produced aprotein comparable in size to LamB. Production of thisprotein was consistently observed in whole-membrane prep-arations of at least 10 independently selected T228(pTROY9)exconjugants (data not shown). None of the T228(pTROY41613) constructs produced a protein of a size equivalent tothat of LamB. The reason for the lack of expression ofLamBfrom pTROY41613 was not apparent.Adsorption of phage A by X. bovienii with pTROY9. A X

adsorption assay was used to demonstrate adsorption of Xvirby T228(pTROY9) constructs. Reduction in the number ofPFU recovered from an inoculum of X after adsorption byLamB-expressing strains of T228 compared with the numberrecovered after adsorption by the T228 wild type was takenas indicative of a functional receptor present on the outermembrane of these constructs. Bacteriophages not adsorbedwere detected by plating on X-sensitive indicator strain E.coli NK5012. Results of adsorption experiments are shownin Table 2. Although some X bacteriophages adsorbed toT228 wild-type cells, at least twice as many bacteriophages

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TABLE 2. Adsorption of Xvir by the suppressor-free E. coliCC118, X. bovienii T228, T228(pTROY9),

and T228(pTROY41613)a

No. of % ReductionAdsorbing strain plaques, 102 in plaques

NB control 244 0E. coli CC118 54 79X. bovienii T228 208 15X. bovienii T228(pTROY9) 166 32X. bovienii T228(pTROY41613) 191 21

a Data shown are percent reductions in phage titers following adsorption byLamB-expressing strains. The X-sensitive indicator bacterium E. coli NK5012was used to determine titers of X before and after adsorption.

were adsorbed by T228(pTROY9) constructs. By compari-son, most T228(pTROY41613) constructs adsorbed X atlevels equivalent to that of the wild-type strain.Transposon mutagenesis of X. bovienii with different Tn

elements. On the basis of SDS-PAGE and X adsorptionstudies, a single T228[pTROY9] construct was selected as asuitable host for A: :Tn mutagenesis vectors such asK::TnphoA (a TnS derivative [14]) and several TnlO deriva-tives described by Way et al. (33) (A940 [Cmr], A1104 [Kmr]and K1105 [KmrI). The protocol for infection consisted of a1-h adsorption phase in the presence of either 20 or 200 mMMgCl2 followed by either a 2- or 24-h incubation for expres-sion of the antibiotic resistance marker. The multiplicity ofinfection used was 10. Suppressor-free E. coli CC118, whichis nonpermissive for K replication, was used as the positivecontrol, and X. bovienii T228 incubated in NB served as anegative control. All positive controls for each separateadsorption event produced confluent growth on selectionplates, demonstrating that each of the transposable elementsefficiently transposed in E. coli. Comparison of all the K::Tnvectors tested with X. bovienii T228(pTROY9) demonstratedthat infections with K1105 resulted in the highest frequency(5 x 10-8) of kanamycin-resistant transductants (20 mMMgCl2 in 2 h of incubation). Other K::Tn vectors producedappropriate antibiotic-resistant transductants at frequenciesat least 1 order of magnitude lower.To ensure that kanamycin-resistant colonies were indeed

due to element 9 insertions, HindIll-digested, chromosomalDNA preparations of 10 randomly selected kanamycin-resistant T228(pTROY9) clones arising from K1105 infectionwere probed with a 32P-labelled 1.7-kb BamHI fragmentencoding the kanamycin resistance determinant of pNK862(Fig. 2A). pNK862 contains the transposable element ofK1105 (element 9). All preparations of DNA contained twofragments which hybridized to probe DNA. Eight of the tenmutants examined contained unique insertions. On the basisof these data, we were able to estimate that the operationalfrequency of mutation due to transposon insertion was about0.8%. This suggested that the transposable element of K1105(element 9) must have been inserted randomly in targetDNA. Two fragments were obtained, because the kanamy-cin resistance gene within element 9 contains a single Hin-dlll restriction endonuclease site. HindIII-digested pNK862DNAwas used as a positive control. E. coli S17-1, T228, andT228(pTROY9) DNA preparations did not hybridize withprobe DNA under the stringency conditions used.

Phenotypic characterization ofX. bovienii::Tn mutants. Theefficiency of the K1105 system as a means of creatinginsertional mutants was determined from the number ofauxotrophic mutants obtained from screening 2,000 colonies

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FIG. 2. Southern hybridization analysis of chromosomal DNApreparations of kanamycin-resistant isolates of X. bovieniiT228(pTROY9) obtained by infection with X1105. DNA was probedwith a 32P-labelled BamHI fragment encoding the kanamycin resis-tance determinant of pNK862 (containing element 9). (A) All DNApreparations digested with HindlIl. Lanes: a, pNK862; b, E. coliS17-1; c, X bovienii T228; d, X. bovienii T228(pTROY9); e throughn, randomly selected kanamycin-resistant isolates obtained by in-fection of T228(pTROY9) with X1105. Note that the DNA fragmentswhich hybridized to the probe show that element 9 has undergone asingle random insertion into the T228(pTROY9) chromosome. ProbeDNA hybridized with two fragments in each lane because a HindIllrestriction site exists within the kanamycin resistance gene of theelement 9. Arrows indicate 8.9-, 3.6-, and 0.9-kb fragments (top tobottom) obtained by HindlIl digestion of X1105 DNA. (B) All DNApreparations digested with EcoRI. Lanes: a, pNK862; b, T228; c,T228(pTROY9); d, XM147 (Leu-); e, XM148; f, XM304; g, XM636;h, XM707 (Leu-); i, XM736 (Cys- and Met-); j, XM740 (Cys- andMet-); k, XM801 (Cys- and Met-); 1, XM806 (Cys- and Met-); m,XM812 (Cys- and Met-); n, XM816 (Cys- and Met-); o, XM847; p,XM1019 (Thr-); q, XM1051; r, XM1519; s, XM1522; t, XM1530.Arrows indicate 14.3-, 5.2-, and 3.5-kb fragments (top to bottom)obtained by EcoRI digestion of K1105 DNA.

derived from a series of independent infections. Preliminaryexperiments demonstrated that wild-type T228 was able togrow on M9 minimal medium containing only nicotinate (1,ug m-). By using this medium, auxotrophic mutants wereidentified on the basis of the procedures used by Davis et al.(8). This protocol allowed isolation of mutants requiringleucine (XM147 and XM707), threonine (XM1019), andcysteine and methionine (XM736, XM740, XM801, XM806,XM812, and XM816).

Southern analysis of auxotrophic mutants was used toconfirm the presence of transposon insertions. This ap-proach also allowed grouping of mutants which have inser-tions in the same region of the chromosome and which giverise to similar mutant phenotypes. Chromosomal DNA prep-

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TABLE 3. Mutants of X. bovienii T228 created by X1105-mediated transposon mutagenesis of X bovienii T228(pTROY9)

Differential No. of Mutantmedium Phenotype isolates' frequency

Minimal media Auxotrophic 18 0.9Blood agar Hemolysin negative 10 0.5Congo red agar Congo red binding negative 2 0.1DNase agar DNase negative 1 0.05DNase agar DNase overproducers 5 0.25Skim milk agar Protease negative 6 0.3Tween 60 agar Lipase overproducers 4 0.2

a Out of 2,000 colonies screened.

arations digested with EcoRI were probed with a 32P-labelled1.7-kb BamHI fragment encoding the kanamycin resistancedeterminant of pNK862 (Fig. 2B). EcoRI-digested pNK862served as the positive control, which hybridized with theprobe, while the T228 and T228(pTROY9) chromosomalDNA negative controls did not. Probe DNA hybridized tosingle chromosomal DNA fragments for all the auxotrophicmutants examined. The hybridization banding patterns ofthe auxotrophic mutants generated by Southern analysiscorrelated with their respective auxotrophies.Phenotypes of X. bovienii::Tn mutants were screened by

plating kanamycin-resistant colonies on appropriate differ-ential media. Protease mutants were selected on skim milkagar, lipase overproducers were selected on Tween 60 agar,

Congo red binding mutants were selected on Congo red agar,and DNase mutants were selected on DNA agar. Nonhe-molytic mutants were selected on NA containing sheepblood erythrocytes. A list of putative mutants isolated isshown in Table 3. None of these mutations were analyzed bySouthern blot hybridization.

DISCUSSION

In this study, we have extended the host range of thebacteriophage X to Xenorhabdus spp. X represents a power-

ful tool for molecular genetic analysis but is essentiallylimited for use in E. coli. In E. coli, the outer membraneprotein LamB facilitates adsorption of X (26). Extension ofthe X host range, however, has been achieved for a numberof diverse bacteria, such as Salmonella typhimurium, Kleb-siella pneumoniae (10), Agrobacterium, Rhizobium, andPseudomonas aeruginosa strains (16), and Erwinia strains(24), by conjugal transfer of plasmids like pTROY9,pTROY41613, and pHCP2, all of which encode expressionofLamB in these hosts. Surface expression ofLamB rendersthese bacteria susceptible to X infection, although the phageis unable to replicate. However, these LamB-expressingstrains are capable of infection by X recombinant DNAlibraries, A::Tn insertion mutagenesis vectors, and in vivoX-packaged cosmids. Given the versatility of this system fortransfer and manipulation of DNA, we investigated exten-sion of the host range of X to Xenorhabdus spp. as a

mechanism for introduction of transposon insertion muta-tions.

Wild-type X. bovienii T228 does not express an outermembrane protein capable of acting as a functional receptorfor X bacteriophage. However, when the gene encodingLamB was supplied on a plasmid (pTROY9), wild-typestrains were able to express this protein as an outer mem-brane protein capable of acting as a receptor for X bacterio-

phage. On the basis of these data, T228(pTROY9) constructswere selected for use as host strains for A::Tn vectors.Preliminary experiments showed that of the tested A::Tnderivatives which confer either kanamycin or chloramphen-icol resistance, only X1105 (element 9) reliably producedinsertion mutants at a useful frequency. X::TnphoA, a Tn5derivative (14), was used with only limited success, probablybecause TnS does not transpose at a high frequency (9).Similarly, neitherA940 (element 4) norA1104 (element 2) wasable to create transposon insertions in T228(pTROY9).These observations are, perhaps, not surprising for thefollowing reasons. Efficiencies of phage adsorption to X.bovienii are less than that for E. coli, the natural host.Secondly, transposition frequencies of these transposonelements in their natural hosts are low (33). Thirdly, differ-ences in transpositional frequencies obtained for E. coli (33)and X. bovienii(pTROY9) are to be expected, since transpo-sition may not be as efficient in X. bovienii. Nevertheless,X1105, which provides a comparatively high frequency oftransposition in E. coli, can transpose in X. bovieniiT228(pTROY9) at a frequency sufficient to justify use of thissystem as a mechanism for production of insertion mutants.In our study, five independent infections allowed isolation ofseveral thousand kanamycin-resistant colonies for subse-quent screening of phenotypic traits.

Southern analysis of representative strains from kanamy-cin-resistant colonies obtained by infection of T228(pTROY9) with X1105 demonstrated that this antibioticresistance was due to single transposon insertion events and,consequently, that this system could be used as a means forisolating phenotypic mutants of X. bovienii. A variety ofmutants were isolated with the use of differential tests. Inparticular, by identifying a suitable minimal medium whichsupported the growth of X. bovienii T228, auxotrophicmutants could be isolated at a frequency of at least 0.8%.

Preliminary characterization of the nutritional require-ments of the auxotrophic mutants identified several pheno-types such as Leu- and Thr-. Other mutants, which re-quired either cysteine or methionine for growth, reflecting acommon need for the thiol groups, were isolated. As trans-poson insertions conferred by element 9 are strongly polar, itis possible that a number of mutants with the same pheno-type may result from insertions in different locations withinthe same operon. Indeed, Southern analysis of DNA ex-tracted from the auxotrophic mutants with the same pheno-type showed that several of these contained element 9insertions in the same 14-kb EcoRI fragment. Given the largesize of this fragment and the unlikelihood that an insertionoccurred in the same site within that fragment, it seemslikely that the resultant phenotypes are caused by polareffects on downstream genes within the same operon. Nev-ertheless, the contribution of siblings to this result cannot beexcluded.

In summary, this study has clearly established that X.bovienii strains carrying pTROY9, which encodes the Xreceptor, can adsorb wild-type X particles and can be in-fected by X::Tn insertion mutagenesis vectors. In particular,the A::TnlO derivative, X1105, can be used as a suitablesystem for mutagenesis of Xenorhabdus spp. However, inthis study, no attempt was made to optimize transposition byimproving transcription of the transposase encoded withinthe IS10 of X1105. Since transcription of this gene is underthe control of the ptac promoter, the frequency of transpo-sition should be improved by induction of this system withisopropyl-3-D-thiogalactopyranoside (IPTG). One other ap-proach might be to isolate strains of Xenorhabdus which

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have a rough lipopolysaccharide phenotype. These strains,when expressing LamB, may adsorb X more efficiently thancorresponding smooth strains because the LamB receptorwould be more likely to be exposed. For similar reasons,strains which do not express capsular polysaccharide mayalso be expected to facilitate more efficient adsorption of X.

ACKNOWLEDGMENT

This work was supported in part by the University of Adelaide.

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