Vol. 57, No. 9APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1991, p. 2597-26010099-2240/91/092597-05$02.00/0Copyright © 1991, American Society for Microbiology

Plasmids and Phase Variation in Xenorhabdus spp.

M.-C. LECLERCt AND N. E. BOEMARE*

Universite' Montpellier II, Sciences et Techniques du Languedoc, Laboratoire de Pathologie compar~e,Institut National de la Recherche Agronomique-Centre National de la Recherche Scientifique

(URA no. 1184), 34095 Montpellier Cedex 05, France

Received 26 December 1990/Accepted 14 June 1991

Three strains of Xenorhabdus nematophilus (A24, Fl, NC 116) and strain Dan of Xenorhabdus bovienii weretested to evaluate whether the phase variation observed in these bacteria was in any way connected withplasmids. The plasmid patterns of both phases of A24 and Fl strains were the same, whereas the two NC116phases had only one band each. No difference was observed between the undigested or digested plasmidpatterns of the two phases from the three strains. No plasmid was detected in either phase of strain Dan. Theplasmid probes were prepared from the six bands of A24 phase 1. By hybridization studies, three plasmids intwo forms (open circular and supercoiled) were detected in the strain A24. Two were estimated at 12 kb, andthe smallest was about 4 kb. Attempts to hybridize plasmid probes with either undigested or digestedchromosomal DNA of the two phases of strain A24 were unsuccessful. The results suggest that neither a

difference in plasmid content nor a plasmid recombination with the chromosome is involved in phase variation.The hybridizations revealed homologous DNA sequences among the three plasmids of strain A24 and among

the plasmids of strains such as A24 and NC116, which were isolated from geographically distant countries,suggesting that plasmids may encode similar proteins.

Xenorhabdus spp. (family Enterobacteriaceae) are bacte-rial symbionts of entomopathogenic nematodes of the fami-lies Steinernematidae and Heterorhabditidae (27) used in thebiological control of insect pests. These bacteria are partic-ularly located in an intestinal vesicle of the L3 juvenilestages of the Steinernematidae (7). After release in the insecthemocoel by the parasitic nematodes, they induce a septi-cemia and provide suitable nutrient conditions for nematodedevelopment in the insect cadavers (24).Every strain of Xenorhabdus spp. occurs in two colony

form variants, named phase 1 and phase 2 (2, 5). Phase 2appears spontaneously during in vitro culture or duringrearing of the nematode hosts on artificial diets. Phase 2 doesnot provide the suitable conditions for the nematode repro-duction, whereas phase 1 does. Phase 1 colonies of Xe-norhabdus spp. are convex, adsorb dyes, and produceantimicrobial substances (1). Phase 2 colonies are flattenedand are negative for the above-mentioned properties. De-pending on the species, other variable characters have beenrelated either to phase 1 (protoplasmic paracrystalline inclu-sions [6, 10] or lecithinase [1]) or to phase 2 (lipases [5]).What is the genetic mechanism involved in this variable

phenotypic expression? In Salmonella typhimurium the twoflagellar types are produced alternatively by two differentexpression genes controlled by the inversion of a chromo-somal fragment, which switches one of these two genes off(8, 20). Phenotypic variants observed in Streptomyces spp.are caused by gene deletions or transposon insertions, andregions of amplified DNA could account for certain deletionsafter recombination events (11, 18). Rhizobium spp. havesome morphological variants that change their symbioticproperties with respect to nitrogen fixation; homologousrecombination between different groups of amplified DNAhas been proposed to be the cause of these variations (12,

* Corresponding author.t Present address: Institut Jacques Monod, 2 Place Jussieu,

F-75251 Paris Cedex 5, France.

13). In archaebacteria, DNA rearrangements are frequentand have been related to A+T-rich regions (22). Conse-quently, some DNA rearrangements could be the mecha-nism of phase variation in Xenorhabdus spp.

Plasmids, which are extrachromosomal DNA, confer pro-visional characteristics such as resistance to antibiotics andto other chemical compounds (4, 15, 16). These differentfeatures can be carried by the plasmid DNA itself or bytransposable elements, which can be situated on the plasmidor on the chromosome. Plasmids and transposons can fit intothe chromosome without any disturbance of the expressionof neighboring genes, but recombination and transpositioncan change certain bacterial physiological functions. InXenorhabdus spp., plasmids have been reported (10). Aplasmid of 50 to 56 kb was found in phase 1 of Xenorhabdusluminescens (23), and another plasmid of 7.1 kb was found inphase 1 of X. luminescens Hm (14). The latter plasmid wasused as a probe against phase 2 genomic DNA, and Frack-man and Nealson detected a homologous sequence and werestudying its significance. We investigate here possible differ-ences in plasmid patterns ofXenorhabdus phase 1 and phase2 and the possibility of integral or partial plasmid insertioninto the chromosome.

MATERIALS AND METHODS

Bacteria, plasmids, and growth conditions. Three strains ofXenorhabdus nematophilus phase 1 and phase 2, A24 (Aus-tralia), Fl (France), NC116 (United States), and strain Danof Xenorhabdus bovienii (Denmark) were used (Table 1).Phase 1 and phase 2 of Xenorhabdus strains are indicated assuffixes /1 and /2 added to strain designations.

Five strains of Escherichia coli containing the followingsized reference plasmids were supplied by Institut Pasteur(Paris): R-sa (39.5 kb), RP4 (54 kb), piP 135/1 (70 kb), piP 112(100.5 kb), -and piP 55 (130 kb). We also used E. coli 517,which contains eight plasmids (2.1, 2.7, 3, 3.9, 5.1, 5.55, 7.2,and 53.7 kb).The Xenorhabdus strains were grown in Luria broth (LB)

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TABLE 1. Numbers and size of plasmid bands ofXenorhabdus strains

Strain No. of plasmid bands Size' of each band (kb)

A24/1 5b 3.3, 5.5, 12, 15, 396c 3.3, 5.5, 12, 15, 38-39, 39

A24/2 5b 3.3, 5.5, 12, 15, 396c 3.3, 5.5, 12, 15, 38-39, 39

Fl/l 5b 3.3, 5.5, 12, 15, 39Fl/2 5b 3.3, 5.5, 12, 15, 39NC116/1 lb 2.5

1 2.5NC116/2 lb 2.5

1V 2.5Dan/2 ob

0c

a Sizes were estimated by comparison with reference plasmids of E. coliextracted and run in the same conditions.

b Determined as described by Kado and Liu (17).Determined as described by Clewell and Helinski (9).

at 28°C. Phase identification was examined on nutrient agarsupplemented with 25 mg of bromothymol blue and 40 mg oftriphenyltetrazolium chloride per liter at each subculture;phase 1 colonies are blue, and phase 2 colonies are maroon-red (1). Phase 1 organisms were checked more accurately bydetermining antibiotic production against Micrococcus lu-teus (1). To prepare plasmid and genomic DNA, bacterialstrains were grown in LB to the stationary phase (Xenorhab-dus spp. for 48 h at 28°C and E. coli for 24 h at 37°C).

Extraction, purification, and isolation of plasipid DNA.Small-scale preparations of plasmid DNA were performedby the rapid alkaline lysis procedure of Kado and Liu (17).Large-scale preparations were performed by the clear lysateprocedure of Clewell and Helinski (9). Isolation of E. colireference plasmids was conducted in parallel with isolationof the Xenorhabdus plasmids to estimate the Xenorhabdusplasmid sizes and to control the efficiency of the prepara-tions. The plasmid DNA was electrophoresed on a 0.7%agarose gel in Tris-borate-EDTA or Tris-acetate-EDTAbuffer at 35 V for 18 h at room temperature.

Plasmids to be used as probes were isolated by theprocedure of Clewell and Helinski (9) followed by purifica-tion on a cesium chloride gradient (21). The purified plasmidswere separated by electrophoresis on 0.7% low-melting-temperature agarose in Tris-acetate-EDTA buffer at 35 V for18 h at 4°C. Each visible plasmid was collected by cutting theband from the gel and heating for 5 min at 65°C, and DNAwas further purified as described by Maniatis et al. (21).

Plasmid DNA was digested with the restriction enzymeDraI, SspI, or AsnI as recommended by the enzyme manu-facturer.

Extraction of genomic DNA. Genomic DNA was preparedfrom 3 ml of bacterial culture; the bacterial pellet was lysedby the addition of 400 ,ul of the lysis solution (50 mMglucose, 10 mM EDTA, 25 mM Tris-base [pH 8], 4 mg oflysozyme per liter) and then 10% (vol/vol) sodium dodecylsulfate. The mixture was heated for 2 min at 65°C, and DNAwas further purified by one phenol and one chloroformextraction. The upper phase (400 ,ul) was collected andincubated for 2 h at 37°C in the presence of proteinase K (50,ug ml-'). The DNA was further purified by one phenol andone chloroform extraction and ethanol precipitation. Ge-nomic DNA was digested with restriction enzyme DraI asrecommended by the enzyme manufacturer.

Plasmid probes, DNA transfers, and hybridizations. Non-

1 r) ) A R i 7 R 9 1 11 1 9 1R

FIG. 1. Plasmid patterns with the Kado and Liu procedure (17):reference E. coli plasmids piP 135 (lane 1), RP.4 (lane 2), R-sa (lane3), piP 55 (lane 4), piP 112 (lane 5); plasmid pattern of E. coli 517(lane 6); plasmid patterns of X. bovienii pap/2 (lane 7) and X.nematophilus NC116/2 (lane 8), NC116/1 (lane 9), F1/2 (lane 10),Fi/l (lane 11), A24/2 (lane 12), and A24/1 (lane 13). chr, chromo-somal DNA.

radioactive probes were prepared according to the technicalinstructions of the digoxigenin kit from Boehringer Mann-heim. DNA was blotted on Schleicher and Schuell nitrocel-lulose filters as described by Maniatis et al. (21). Hybridiza-tion was carried out as described in the digoxigenin kit ofBoehringer.

RESULTS

Plasmid contents of Xenorhabdus phase 1 and phase 2DNAs. To evaluate some differences between the two phasesof each strain, we isolated and electrophoresed plasmidDNAs.With the Kado and Liu method (17), no plasmid was

observed in Dan/2 (Fig. 1, lane 7), whereas NC116/1 andNC116/2 (lanes 9 and 8, respectively) exhibited bands at ca.2.5 kb; compare these with the plasmids of E. coli 517 (lane6). Strains Fl/i and F1/2 (lanes 11 and 10, respectively) andA24/1 and A24/2 (lanes 13 and 12, respectively) showed threebands (3.3, 12, and 15 kb), but in some preparations fivebands were observed for these strains (Table 1).When the method of Clewell and Helinski (9) was used,

the patterns of A24/1 and A24/2 (Table 1) were the same andconsisted in six bands (Fig. 2, lane 1 for A24/1); four bandswere located under the chromosomal DNA (3.3, 5, 12, and15 kb), and two bands just above the chromosomal DNAwere smaller than 39 kb (compare, with the E. coli Rsa39.5-kb band in Fig. 2, lane 2).Fl and A24 strains have similar plasmids, whereas one

plasmid in strain NC116 is different from those of Fl andA24. We detected no plasmid in strain Dan. In no strain didwe observe any difference between phase 1 and phase 2DNAs.The DraI, SspI, and AsnI digestion patterns of the plasmid

DNAs of both phases of A24 were identical. The sum of thedigested fragments was approximatively 16 kb with Dral and

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1 2 3 4 5 6

FIG. 2. Plasmid patterns with.the Clewell and Helinski proce-dure (9): plasmids of X. nematophilus A24/1 (lane 1), E. coli R-saplasmid (lane 2), plasmids of E. coli 517 (lane 3), and E. coli piP 135plasmid (lane 4). Plasmid bands la, lb, 2, 3, 4, and 5 were used toprepare cold probes. chr, chromosomal DNA.

20 kb with SspI and AsnI. Since the sum of the sizes ofundigested A24 plasmids, calibrated with the reference plas-mids of E. coli strains, was arpund 115 kb (Table 1 and Fig.2), we suspected that several forms of the same plasmidswere observed on the gels. Hybridization experiments witheach A24 plasmid band (Fig. 2) used as a probe will allow usto confirm these results.

Hybridization patterns with genomic DNA and phase vari-ation. In a second step, we hybridized the probes preparedfrom the six plasmid bands of strain A24/1 (Fig. 2) with thegenomic -DNAs of A24/1 and A24/2 to detect differenthybridization patterns resulting from plasmid recombina-tion.Three different hybridization patterns were obtained (Fig.

3, 4, and 5): bands la and 2, bands lb and 3, and bands 4 and5 (Fig. 2) have to be considered in pairs as two forms, opencircular and supercoiled, of the same plasmids, since thesebands as probes gave the same hybridization patterns.Therefore, three plasmids have been demonstrated here.The plasmid contents of E. coli reference strains are wellknown, and the two extraction methods used gave correctpatterns (Fig. 1 and 2). The extraction conditions were moredrastic for Xenorhabdus plasmids than for E. coli plasmids,and with the Xenorhabdus plasmids we obtained both theopen circular and supercoiled forms.The three hybridization probes did not show any differ-

ence in pattern between the genomic DNAs of A24/1 andA24/2, whether digested or undigested (Fig. 3, 4, and 5).Moreover, the hybridization patterns of digested and undi-gested genornic DNA were the same as those of digested andundigested plasmid DNA of A24/1 (control) (lanes 3 and 6 ofFig. 3 and 4; lane 3 of Fig. 5). Since extracted genomic DNAis the total DNA, which contains both plasmid and chromo-somal DNA, it is concluded that the plasmid probes did nothybridize with the chromosomal DNA but only with theplasmid part of the genomic DNA. There is no homologybetween chromosomal DNA and extrachromosomal DNA atthis level of sensitivity, which is less than 0.1 pg of homol-ogous DNA. This experiment allows us to conclude thatphase variation cannot be explained by a difference inplasmid content between the two phases and by plasmidinsertion into chromosomal DNA.

FIG. 3. Hybridizations with probes la and 2. Lanes containedDral-digested genomic DNA of A24/1 (lane 1) and A24/2 (lane 2),Dral-digested plasmid DNA of A24/1 (lane 3), undigested genomicDNA of A24/1 (lane 4) and A24/2 (lane 5), and undigested plasmidDNA of A24/1 (lane 6).

Hybridization patterns and homologies among the plasmids.In addition to the stated purpose, the hybridization experi-ments led us to detect homologies among the plasmids. Thehybridization patterns with the probes la-2 and lb-3 dis-played an inversion of band intensities (Fig. 3 and 4),meaning that there is strong homology between these twoprobes. Consequently, these two plasmids must have manycommon sequences in addition to their similar sizes (around12 kb).

Hybridization patterns with probe 4-5 (Fig. 5) were dis-tinct from the two previous patterns (Fig. 3 and 4). Weshould point out that the bands (Arrows in Fig. 5) are alsorevealed in Fig. 3 and 4 (arrows). So this 4-kb plasmid,smaller than the previous probes, shared some homologoussequences with plasmids la-2 and lb-3. Finally we noticedthat probe 4-5 hybridized with the single undigested plasmidof NC116, indicating a homology between the plasmidscarried by geographically distant strains (A24 and NC116).All of these plasmid DNA homologies within a strain andamong strains suggest similar proteins encoded by plasmidDNA.

DISCUSSION

Among 10 Xenorhabdus strains, 7 strains carried plasmids(10). Both phases of three strains of X. nematophilus had thesame plasmid patterns; the plasmid sizes were between 3.6and 12 kb. Our results are in agreement with these previousdata (10); we found that the A24 plasmid sizes were 12 kb for

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FIG. 4. Hybridizations with probes lb and 3. Lanes containedDral-digested genomic DNA of A24/1 (lane 1) and A24/2 (lane 2),Dral-digested plasmid DNA of A24/1 (lane 3), undigested genomicDNA of A24/1 (lane 4) and A24/2 (lane 5), and undigested plasmidDNA of A24/1 (lane 6).

the two biggest plasmids and 4 kb for the smaller one andthat the NC116 strain had only one plasmid, evaluated at ca.

2.5 kb. We confirmed the results of Couche et al. (10), whoshowed that strain Dan had no plasmid.Using a plasmid extraction in the wells before electropho-

resis, Smigielski (26) detected megaplasmids of 70 and 120kb in strains A24/1-2 and F1/1-2 but found the same patternsas we reported here concerning the small plasmids. InBacillus thuringiensis, two plasmid groups were reported(19), one bigger and one smaller than 22.5 kb, with nohomologous sequences between them. However, the biggestplasmids had some homologies with chromosomal DNA.The megaplasmids detected by Smiglielski (26) should beprobed to characterize some possible homology with thechromosomal DNA.DNA homologies among the three plasmids of A24 and the

small plasmid of NC116 were observed, suggesting thatthese extrachromosomal elements may encode similar pro-teins in X. nematophilus strains isolated from geographicallydistant regions. This kind of result was observed, for in-stance, in Edwardsiella irtaluri, an enteropathogenic agentof fish (25), for which hybridization revealed relationshipsamong plasmids within a strain and between geographicallydistant strains.

Apparently, phenotypic differences of phase variation inthe X. nematophilus species studied are due neither todifferent plasmid contents nor to plasmid DNA insertion inthe chromosome. There are no homologous sequences be-tween plasmid and chromosomal DNAs of strain A24. Thepresence of homologous sequences between one plasmidand the chromosome was reported in X. luminescens (14).This difference from other Xenorhabdus species providesanother argument that X. luminescens should be placed in a

FIG. 5. Hybridizations with probes 4 and 5. Lanes containedDral-digested genomic DNA of A24/1 (lane 1) and A24/2 (lane 2) andDral-digested plasmid DNA of A24/1 (lane 3).

separate genus, as suggested previously (2) and supportedrecently by DNA hybridization data (3).

ACKNOWLEDGMENTS

We thank M.-H. Boyer-Giglio, M. Brehdlin, and G. Devauchellefor their advice in the revision of the manuscript and J. Luciani fortechnical assistance.

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