APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1991, p. 2597-2601

Vol. 57, No. 9

0099-2240/91/092597-05$02.00/0 Copyright © 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 were tested to evaluate whether the phase variation observed in these bacteria was in any way connected with plasmids. The plasmid patterns of both phases of A24 and Fl strains were the same, whereas the two NC116 phases had only one band each. No difference was observed between the undigested or digested plasmid patterns of the two phases from the three strains. No plasmid was detected in either phase of strain Dan. The plasmid probes were prepared from the six bands of A24 phase 1. By hybridization studies, three plasmids in two forms (open circular and supercoiled) were detected in the strain A24. Two were estimated at 12 kb, and the smallest was about 4 kb. Attempts to hybridize plasmid probes with either undigested or digested chromosomal 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 bacterial symbionts of entomopathogenic nematodes of the families Steinernematidae and Heterorhabditidae (27) used in the biological control of insect pests. These bacteria are particularly located in an intestinal vesicle of the L3 juvenile stages of the Steinernematidae (7). After release in the insect hemocoel by the parasitic nematodes, they induce a septicemia and provide suitable nutrient conditions for nematode development 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 2 appears spontaneously during in vitro culture or during rearing of the nematode hosts on artificial diets. Phase 2 does not provide the suitable conditions for the nematode reproduction, whereas phase 1 does. Phase 1 colonies of Xenorhabdus spp. are convex, adsorb dyes, and produce antimicrobial substances (1). Phase 2 colonies are flattened and are negative for the above-mentioned properties. Depending on the species, other variable characters have been related either to phase 1 (protoplasmic paracrystalline inclusions [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 two flagellar types are produced alternatively by two different expression genes controlled by the inversion of a chromosomal 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, and regions of amplified DNA could account for certain deletions after recombination events (11, 18). Rhizobium spp. have some morphological variants that change their symbiotic properties with respect to nitrogen fixation; homologous recombination between different groups of amplified DNA has been proposed to be the cause of these variations (12,

13). In archaebacteria, DNA rearrangements are frequent and have been related to A+T-rich regions (22). Consequently, some DNA rearrangements could be the mechanism of phase variation in Xenorhabdus spp. Plasmids, which are extrachromosomal DNA, confer provisional characteristics such as resistance to antibiotics and to other chemical compounds (4, 15, 16). These different features can be carried by the plasmid DNA itself or by transposable elements, which can be situated on the plasmid or on the chromosome. Plasmids and transposons can fit into the chromosome without any disturbance of the expression of neighboring genes, but recombination and transposition can change certain bacterial physiological functions. In Xenorhabdus spp., plasmids have been reported (10). A plasmid of 50 to 56 kb was found in phase 1 of Xenorhabdus luminescens (23), and another plasmid of 7.1 kb was found in phase 1 of X. luminescens Hm (14). The latter plasmid was used as a probe against phase 2 genomic DNA, and Frackman and Nealson detected a homologous sequence and were studying its significance. We investigate here possible differences in plasmid patterns of Xenorhabdus phase 1 and phase 2 and the possibility of integral or partial plasmid insertion into the chromosome. MATERIALS AND METHODS Bacteria, plasmids, and growth conditions. Three strains of Xenorhabdus nematophilus phase 1 and phase 2, A24 (Australia), Fl (France), NC116 (United States), and strain Dan of Xenorhabdus bovienii (Denmark) were used (Table 1). Phase 1 and phase 2 of Xenorhabdus strains are indicated as suffixes /1 and /2 added to strain designations. Five strains of Escherichia coli containing the following sized 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)

* Corresponding author. t Present address: Institut Jacques Monod, 2 Place Jussieu, F-75251 Paris Cedex 5, France. 2597

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LECLERC AND BOEMARE

2598

TABLE 1. Numbers and size of plasmid bands of Xenorhabdus strains Strain

A24/1

No. of plasmid bands

5b 6c

A24/2

Fl/l Fl/2 NC116/1

5b

6c 5b 5b lb

1

NC116/2

lb

Dan/2

ob

1V

1

r)

)

A

R

i

7

R

9 1

11

19

1R

Size' of each band (kb)

3.3, 3.3, 3.3, 3.3, 3.3, 3.3, 2.5 2.5 2.5 2.5

5.5, 5.5, 5.5, 5.5, 5.5, 5.5,

12, 12, 12, 12, 12, 12,

15, 15, 15, 15, 15, 15,

39 38-39, 39 39 38-39, 39 39 39

0c a Sizes were estimated by comparison with reference plasmids of E. coli extracted 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 agar supplemented with 25 mg of bromothymol blue and 40 mg of triphenyltetrazolium chloride per liter at each subculture; phase 1 colonies are blue, and phase 2 colonies are maroonred (1). Phase 1 organisms were checked more accurately by determining antibiotic production against Micrococcus luteus (1). To prepare plasmid and genomic DNA, bacterial strains were grown in LB to the stationary phase (Xenorhabdus 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 performed by the rapid alkaline lysis procedure of Kado and Liu (17). Large-scale preparations were performed by the clear lysate procedure of Clewell and Helinski (9). Isolation of E. coli reference plasmids was conducted in parallel with isolation of the Xenorhabdus plasmids to estimate the Xenorhabdus plasmid sizes and to control the efficiency of the preparations. The plasmid DNA was electrophoresed on a 0.7% agarose gel in Tris-borate-EDTA or Tris-acetate-EDTA buffer at 35 V for 18 h at room temperature. Plasmids to be used as probes were isolated by the procedure of Clewell and Helinski (9) followed by purification on a cesium chloride gradient (21). The purified plasmids were separated by electrophoresis on 0.7% low-meltingtemperature agarose in Tris-acetate-EDTA buffer at 35 V for 18 h at 4°C. Each visible plasmid was collected by cutting the band from the gel and heating for 5 min at 65°C, and DNA was further purified as described by Maniatis et al. (21). Plasmid DNA was digested with the restriction enzyme DraI, SspI, or AsnI as recommended by the enzyme manu-

facturer. Extraction of genomic DNA. Genomic DNA was prepared from 3 ml of bacterial culture; the bacterial pellet was lysed by the addition of 400 ,ul of the lysis solution (50 mM glucose, 10 mM EDTA, 25 mM Tris-base [pH 8], 4 mg of lysozyme per liter) and then 10% (vol/vol) sodium dodecyl sulfate. The mixture was heated for 2 min at 65°C, and DNA was further purified by one phenol and one chloroform extraction. The upper phase (400 ,ul) was collected and incubated for 2 h at 37°C in the presence of proteinase K (50 ,ug ml-'). The DNA was further purified by one phenol and one chloroform extraction and ethanol precipitation. Genomic DNA was digested with restriction enzyme DraI as recommended by the enzyme manufacturer. Plasmid probes, DNA transfers, and hybridizations. Non-

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 (lane 3), 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, chromosomal DNA.

radioactive probes were prepared according to the technical instructions of the digoxigenin kit from Boehringer Mannheim. DNA was blotted on Schleicher and Schuell nitrocellulose filters as described by Maniatis et al. (21). Hybridization was carried out as described in the digoxigenin kit of Boehringer. RESULTS

Plasmid contents of Xenorhabdus phase 1 and phase 2 DNAs. To evaluate some differences between the two phases of each strain, we isolated and electrophoresed plasmid DNAs. With the Kado and Liu method (17), no plasmid was observed in Dan/2 (Fig. 1, lane 7), whereas NC116/1 and NC116/2 (lanes 9 and 8, respectively) exhibited bands at ca. 2.5 kb; compare these with the plasmids of E. coli 517 (lane 6). Strains Fl/i and F1/2 (lanes 11 and 10, respectively) and A24/1 and A24/2 (lanes 13 and 12, respectively) showed three bands (3.3, 12, and 15 kb), but in some preparations five bands 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 and consisted in six bands (Fig. 2, lane 1 for A24/1); four bands were located under the chromosomal DNA (3.3, 5, 12, and 15 kb), and two bands just above the chromosomal DNA were smaller than 39 kb (compare, with the E. coli Rsa 39.5-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 and A24. We detected no plasmid in strain Dan. In no strain did we observe any difference between phase 1 and phase 2 DNAs. The DraI, SspI, and AsnI digestion patterns of the plasmid DNAs of both phases of A24 were identical. The sum of the digested fragments was approximatively 16 kb with Dral and

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PLASMIDS IN XENORHABDUS SPP. 1

2

3

4

5

2599

6

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

20 kb with SspI and AsnI. Since the sum of the sizes of undigested A24 plasmids, calibrated with the reference plasmids of E. coli strains, was arpund 115 kb (Table 1 and Fig. 2), we suspected that several forms of the same plasmids were observed on the gels. Hybridization experiments with each A24 plasmid band (Fig. 2) used as a probe will allow us to confirm these results. Hybridization patterns with genomic DNA and phase variation. In a second step, we hybridized the probes prepared from the six plasmid bands of strain A24/1 (Fig. 2) with the genomic -DNAs of A24/1 and A24/2 to detect different hybridization patterns resulting from plasmid recombination. Three different hybridization patterns were obtained (Fig. 3, 4, and 5): bands la and 2, bands lb and 3, and bands 4 and 5 (Fig. 2) have to be considered in pairs as two forms, open circular and supercoiled, of the same plasmids, since these bands as probes gave the same hybridization patterns. Therefore, three plasmids have been demonstrated here. The plasmid contents of E. coli reference strains are well known, and the two extraction methods used gave correct patterns (Fig. 1 and 2). The extraction conditions were more drastic for Xenorhabdus plasmids than for E. coli plasmids, and with the Xenorhabdus plasmids we obtained both the open circular and supercoiled forms. The three hybridization probes did not show any difference in pattern between the genomic DNAs of A24/1 and A24/2, whether digested or undigested (Fig. 3, 4, and 5). Moreover, the hybridization patterns of digested and undigested genornic DNA were the same as those of digested and undigested plasmid DNA of A24/1 (control) (lanes 3 and 6 of Fig. 3 and 4; lane 3 of Fig. 5). Since extracted genomic DNA is the total DNA, which contains both plasmid and chromosomal DNA, it is concluded that the plasmid probes did not hybridize with the chromosomal DNA but only with the plasmid part of the genomic DNA. There is no homology between chromosomal DNA and extrachromosomal DNA at this level of sensitivity, which is less than 0.1 pg of homologous DNA. This experiment allows us to conclude that phase variation cannot be explained by a difference in plasmid content between the two phases and by plasmid insertion into chromosomal DNA.

FIG. 3. Hybridizations with probes la and 2. Lanes contained

Dral-digested genomic DNA of A24/1 (lane 1) and A24/2 (lane 2), Dral-digested plasmid DNA of A24/1 (lane 3), undigested genomic DNA of A24/1 (lane 4) and A24/2 (lane 5), and undigested plasmid DNA of A24/1 (lane 6).

Hybridization patterns and homologies among the plasmids. In addition to the stated purpose, the hybridization experiments led us to detect homologies among the plasmids. The hybridization patterns with the probes la-2 and lb-3 displayed an inversion of band intensities (Fig. 3 and 4), meaning that there is strong homology between these two probes. Consequently, these two plasmids must have many common sequences in addition to their similar sizes (around 12 kb). Hybridization patterns with probe 4-5 (Fig. 5) were distinct from the two previous patterns (Fig. 3 and 4). We should point out that the bands (Arrows in Fig. 5) are also revealed in Fig. 3 and 4 (arrows). So this 4-kb plasmid, smaller than the previous probes, shared some homologous sequences with plasmids la-2 and lb-3. Finally we noticed that probe 4-5 hybridized with the single undigested plasmid of NC116, indicating a homology between the plasmids carried by geographically distant strains (A24 and NC116). All of these plasmid DNA homologies within a strain and among strains suggest similar proteins encoded by plasmid DNA. DISCUSSION

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

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LECLERC AND BOEMARE

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

APPL. ENVIRON. MICROBIOL.

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

separate genus, as suggested previously (2) and supported recently by DNA hybridization data (3). the two biggest plasmids and 4 kb for the smaller one and that the NC116 strain had only one plasmid, evaluated at ca. 2.5 kb. We confirmed the results of Couche et al. (10), who showed that strain Dan had no plasmid. Using a plasmid extraction in the wells before electrophoresis, Smigielski (26) detected megaplasmids of 70 and 120 kb in strains A24/1-2 and F1/1-2 but found the same patterns as we reported here concerning the small plasmids. In Bacillus thuringiensis, two plasmid groups were reported (19), one bigger and one smaller than 22.5 kb, with no homologous sequences between them. However, the biggest plasmids had some homologies with chromosomal DNA. The megaplasmids detected by Smiglielski (26) should be probed to characterize some possible homology with the chromosomal DNA. DNA homologies among the three plasmids of A24 and the small plasmid of NC116 were observed, suggesting that these extrachromosomal elements may encode similar proteins in X. nematophilus strains isolated from geographically distant regions. This kind of result was observed, for instance, in Edwardsiella irtaluri, an enteropathogenic agent of fish (25), for which hybridization revealed relationships among plasmids within a strain and between geographically distant strains. Apparently, phenotypic differences of phase variation in the X. nematophilus species studied are due neither to different plasmid contents nor to plasmid DNA insertion in the chromosome. There are no homologous sequences between plasmid and chromosomal DNAs of strain A24. The presence of homologous sequences between one plasmid and the chromosome was reported in X. luminescens (14). This difference from other Xenorhabdus species provides another argument that X. luminescens should be placed in a

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