Short, interspersed, and repetitive DNA sequences in Spiroplasma species

PLASMID

17,

110-l 16 (1987)

Short, Interspersed, and Repetitive DNA Sequences in Spiroplasma Species ISRAEL NuR,' Mycoplasma Allergy

and Bacterial and Infectious

DONALD

Virulence Diseases,

Sections, Frederick

J. LEBLANC,

AND JOSEPH G. TULLY

Laboratory of Molecular Microbiology, Cancer Research Facility, Frederick,

National Maryland

Institute 21701

of

Received August 6, 1986; revised December 8, 1986 Small fragments of DNA from an 8-kbp plasmid, pRA1, from a plant pathogenic strain of citri were shown previously to be present in the chromosomat DNA of at least two species of Spiroplasma. We describe here the shot-gun cloning of chromosomal DNA from S. citri Maroc and the identification of two distinct sequences exhibiting homology to pRA 1. Further subcloning experiments provided specific molecular probes for the identification of these two sequences in chromosomal DNA from three distinct plant pathogenic species of Spiroplasma. The results of Southern blot hybridization indicated that each of the pRA 1-associated sequences is present as multiple copies in short, dispersed, and repetitive sequences in the chromosomes of these three strains. None of the sequences was detectable in chromosomal DNA from an additional nine Spiroplasma strains examined. 0 1987 Academic h. 1~. Spiroplasma

Spiroplasmas are a group of helical, wallless prokaryotes (class Mollicutes) found in insects, other arthropods, and both in and on a variety of plant hosts (Tully and Whitcomb, 198 1). At present, the group is composed of 7 established species and approximately 20 putative species, with distinctions based primarily upon serological and genome differences (Whitcomb, 198 1; Bove, 1984). Three species (Spiroplasma citri, S. kunkelii, and Spiroplasma strain P40) are documented plant pathogens, while two species (S. melliferum and S. apis) are pathogenic for insects (Bove, 1984; Clark et al., 1985; Whitcomb et al., 1986). Many spiroplasmas possess extrachromosomal DNA, either as cryptic plasmids, ranging in size from 2 to 50 kilobase pairs (kbp), or as replicative forms of viruses (Ranhand et al., 1980; Archer et al., 1981; Cole, 1983; Mouches and Bove, 1983;

MATERIALS

’ To whom correspondence and reprint requests should be addressed. Present address: Genomic Structure and Function Section, Laboratory of Biochemical Pharmacology, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, Bldg. 8, Room 204, Bethesda, MD 20205. 0147-619X/87

$3.00

Copyright 0 1987 by Academic Pres, Inc. All rigbu of wroduction in any form reserved.

Mouches et al., 1984; Dickinson and Townsend, 1984). We have found that an 8-kbp plasmid (pRA 1) isolated from early passages of the pathogenic RSAZ-Maroc strain of S. citri could be used as a sensitive probe to detect this organism in both infected plant material or the hemolymph of infected leafhoppers, the principal vector of the plant disease (Nur et al., 1986a). Subsequent results suggested that the high sensitivity of the probe was due to multiple copies of pRA lrelated sequences integrated into the S. citri chromosome and that the integrated plasmid sequences consisted only of small fragments, and not the entire plasmid (Nur et al., 1986b). In the present work, we cloned from S. citri DNA two repetitive S. citri chromosomal sequences with homology to pRA1 and to chromosomal DNA of several plant pathogenic species of Spiroplasma. AND METHODS

Strains, plasmids, and growth conditions. The Spiroplasma species, their strain designations, and the Spiroplasma and Escherichia coli plasmids used in this work are 110

DNA SEQUENCES TABLE SPIROPLASMAS

I

IN STUDY, AND THEIR SEROLOGIC GROUPINGS AND HOST ORIGINS

EMPLOYED

Common or binomial name

Sero group

Strain (passage history)”

I-l 1-2

Maroc (p. 300+) BC3 (p. 14)

Rabbit tick Green leaf bug Maryland flower Coconut palm Vinca plant

I-3 I-4 I-5 I-6 I-7 I-8

E275 (p. 40) 277F (p. 25) LB-12 (p. 6) M55 (p. 6) N525 (p. 8) P40 (p. 25)

tick Cucumber beetle Potato beetle Flower

VI XII xx XXI

Y-32 DU-1 LD-I w115

Spiroplasma S. mellifeum

citri

S. kunkelii

111

IN Spiroplasma

Host origin (pathogenicity) Citrus (“stubborn disease”) Honeybee (bee spiroplasmosis) Corn (“corn stunt disease”) Haemaphysalis sp. Trigonotylus

sp.

Flowers Cocos palm tree Catharanthus

(periwinkle disease) Ixodes

(p. 6) (p. 12) (p. 10) (p. 14)

Ixodes pac$cus Diabrotica sp. Leptinotarsa sp. Prunis sp.

a Passage history recorded as number of times strain has been passaged on artificial culture media.

listed in Tables 1 and 2. Spiroplasmas were grown in I- to 2-liter volumes of SP-4 or M IA culture media (Whitcomb, 1983) at 30 or 32°C for l-5 days, depending upon the time required to reach late logarithmic phase. The cultures were harvested by centrifugation (12,000g for 30 min) at 4°C. Cell pellets were washed once in cold 0.25 M NaCl containing 0.01 M EDTA, and then stored at -20°C until used. E. coli strains were cultured in LB broth (Maniatis et al.,

1982), containing ampicillin and kanamycin (25 pg/ml), priate.

(100 pg/ml) when appro-

DNA preparation and analysis. Spiroplasma DNA was prepared according to Marmur (196 1). Plasmid DNA was purified from 500 ml cleared lysates of S. citri (R8A2-Maroc, subclone B) by CsCl-ethidium bromide density gradient centrifugation (Radloff et al., 1967). Spiroplasma chromosomal DNA lysates were prepared from

TABLE 2 PLASMIDS

Plasmid or phage

Vector

-

AND PHAGES

Size W-v)

PRAl puc13

-

8.00 2.70

pUCl9

-

2.70

pL8

puc13

5.50

pol7

puc13

6.90

Ml3 mp8 M13/F5 M13/Fl8 Ml3/G15

Ml3 mp8 Ml3 mp8 Ml3 mp8

-

EMPLOYED

7.22 7.47 7.78 7.96

IN THIS STUDY

Insert (kbp) S. citri

Manx plasmid

EcoRI chromosomal frag. of S. citri (2.8) EcoRI chromosomal frag. of S. citri (4.25) DraI DraI DraI

frag. of pO17 (0.25) frag. of pO17 (0.56) ffag. of pL8 (0.74)

Reference Nur et al., Vieira and 1982 Vieira and 1982 Nur et al.,

1986b Messing, Messing, 1986b

Nur et al., 1986b Maniatis et al., 1982 This work This work This work

112

NUR,

LE BLANC,

2-liter volumes of cultures grown in SP-4 medium, using techniques described earlier (Ranhand et al., 1980). Routine screening of E. coli transformants was performed by a rapid alkaline extraction procedure (Birnboim and Doly, 1979). For purification of E. coli plasmids, cleared lysates were obtained from 400-ml broth cultures (Clewell and Helinski, 1969). Plasmid DNAs and M 13 DNA replicative forms (RF) were analyzed by agarose gel electrophoresis in 0.8 and 1.0% horizontal slab gels in Tris-borate-EDTA buffer (Maniatis et al., 1982). Restriction endonucleases were purchased from Bethesda Research Laboratories (BRL, Gaithersburg, MD), and the digestions were carried out as recommended by the supplier. Individual restriction fragments were isolated by electroelution from agarose gels, followed by purification with Elutip-d Systems (Schleicher & Schuel, Inc. Keene, NH). The method of Southern (1975) was used to transfer DNA from agarose to nitrocellulose filters. Hybridization of DNA on the filters was carried out with 32P-labeled DNA probes at 62”C, following the method of Thayer (1974). Radiolabeled probes were prepared by nick translation (Rigby et al., 1977). Singlestranded Ml 3 recombinant DNA was extracted from the phage and radiolabeled by in vitro synthesis of the complementary strand, using DNA polymerase I and 32P-labeled dATP (BRL) and a synthetic primer (Gruenbaum et al., 198 1). The radiolabeled probes were denatured by adding one part formaldehyde and boiling for 5 min. Cloning strategies. EcoRI fragments of pRA 1 and S. citri chromosomal DNAs were cloned into the EcoRI sites of plasmids pUC 19 and pUC 13, respectively. The E. coli strain TBl (BRL) was transformed with DNA by the method of Hanahan ( 1983), and recombinant clones were detected on agar medium containing 5-bromo-4-chloro-3-indolyl &Dgalactoside (X-gal, 40 pg/ml) plus 100 wg/ml of ampicillin. After overnight incubation, transformants containing vector DNA with pRA1 or S. citri chromosomal inserts appeared as white colonies and were chosen for further characterization (Norrander et al., 1983). DraI fragments from

AND

TULLY

cloned S. citri chromosomal DNA were ligated to liincII-digested M 13mp8 DNA, and recombinant clones were obtained by transfection of E. coli strain JM 103 and the production of colorless plaques on agar medium containing X-gal and isopropyl-Dthiogalactoside. S. citri chromosomal DNA cloned into pUC 13 or Ml 3mp8 was tested for sequences with homology to pRA 1 by dot blot hybridization. Dot blot hybridization. Recombinant M 13 DNA was extracted as described above. Chromosomal DNA from spiroplasma strains for use in dot blots was prepared as follows: 1 ml of a logarithmic phase spiroplasma culture was centrifuged in an Eppendorf centrifuge for 10 min; the packed cells were resuspended in 100 ~1 of a mixture of 0.1 M EDTA and 1% SDS and incubated for 10 min at 60°C. After addition of 12 ~1 of 0.2 N NaOH and heating for 10 min at 60°C, the lysates were cooled to room temperature. Equal volumes each of chloroform-isoamyl alcohol (24: 1) and buffer-saturated phenol were added. The extraction mixture was vortexed for 20 set and the phases were separated by centrifugation in an Eppendorf centrifuge for 5 min. A 1O-p1 portion was used to estimate the concentration and purity of the DNA by electrophoresis, staining with ethidium bromide, and comparison to known amounts of lambda DNA. The remaining DNA was denatured by addition of 300 ~1 of 12% formaldehyde and heating to 60°C for 20 min. The denatured DNA was cooled quickly by the addition of 1.125 ml of cold 10X SSC (1X SSC = 0.15 M NaCl, 0.015 M Na citrate), and 200~~1 samples were loaded onto slots of a vacuum chamber block (Hydriblot manifold, BRL) containing a sheet of nitrocellulose film. Hybridization conditions were as described previously (Nur et al., 1986b). RESULTS

Derivation of cloned DNA sequencesfrom pRA1 and from the S. citri (Maroc) chromosome. Restriction endonuclease maps of pRA 1, and cloned EcoRI fragments used in this study, from pRA1 or from the chromo-

DNA SEQUENCES Hind

s I

H I

NRI II

113

IN Spiroplasma H”‘c III

Hind

RI I

pRAl

1

EcoRI

mcs RI t

puc19

pTDAI

D

Oral digsstlon; HinclI llgatlon

D MlWG15

RIPH 111

S

RVH RI po17 Dral digastion; Hincll ligation

1.0 Kb

FIG. 1. Restriction maps of pRA 1, pTDA 1, pL8, p0 17, M 13/G15, and M 13/F18. The construction of the pUC19/pRAl hybrid plasmid, pTDA1, the pUCl3/S. citri chromosomal DNA hybrids pL8 and p017, and the Ml3 recombinant phage DNAs M13/G15 and M13/F18, derived from pL8 and p017, respectively, is described in the text. The heavy solid bars designate regions of homology to fragment G 15, and the cross-hatch bars designate regions of homology to fragment F18. Abbreviations for restriction endonuclease sites on the maps follow: Hind, HindIII; RI, EcoRI; S, Suu3A 1; C, CkzI; N, NcoI; H, HpuI; D, DraI; RV, EcoRV; P, &I; mcs, multiple cloning site.

some of the host strain, S. citri (Maroc), are presented in Fig. 1. Preliminary results indicated that the region of homology between plasmid pRA1 and the host chromosome was within the 3.7-kbp EcoRI fragment of pRA1. This fragment was cloned into pUC19, yielding the hybrid plasmid, pTDA 1. EcoRI-digested chromosomal DNA from a late passage culture of S. citri Maroc, containing no detectable plasmid DNA, was cloned into pUC 13. Dot blots were prepared with DNA from recombinant clones and hybridized with 32P-labeled pRA 1. Two hybrid plasmids, pL8 and p0 17, containing chromosomal fragments exhibiting homology to pRA1 were chosen for further study.

The regions of homology to pRA1 in the two cloned chromosomal fragments were delineated further by subcloning short DruI fragments into Ml 3mp8 vector DNA. Recombinant plaques containing DraI fragments with homology to the 3.7-kbp EC&I fragment from pTDA1 were identified by dot blotting. One such fragment from pL8 (Gl5) and one from p0 17 (F18) were identified (Fig. 1). Localization of specijc homologous sequences within pTDA1, pL8, and ~017. DNA from the M 13 recombinant clone containing fragment F18 was used as a hybridization probe in Southern blots of p0 17, pL8, and pTDA 1 DNAs digested with EcoRI plus

114

NUR, LE BLANC, AND TULLY

HpaI, and DraI plus HpaI (Fig. 2). Several DNA bands in each lane exhibited homology with the probe, and those indicated by arrows in Fig. 2 contain the homology with fragment F18. Similar experiments were also performed with the M 13 recombinant clone containing fragment G 15 (data not shown). The results are summarized in Fig. 1. Sequences with homology to fragment F18, cross-hatched bar, were located in specific DruI fragments of ~017 and pL8, and in a DruI/ClaI fragment, as well as the adjacent CluI/EcoRV fragment of pTDA 1. On the other hand, fragment G 15, solid bar, hybridized to a different DruI fragment in each of the hybrid plasmids. However, in each case, this fragment was adjacent to the region of homology to fragment F18.

gl5 peDNA

1

2

3

4

5

6

7

6

0

10

11

12

50,000

5

FIG. 3. Dot blot hybridizations showing homology between chromosomal DNA from 3 of 12 serologically distinct spiroplasmas and subcloned fragments G15 (top panel) and F 18 (bottom panel). Numbers at the left of the figure refer to picograms of chromosomal DNA on blot. Numbers across the top of each panel refer to chromosomal DNA from S. citri Maroc (1); S. melliferum BC3 (2); S. kunkelii E275 (3); and Spiroplasma strains 277F (4); LB-12 (5); M55 (6); N525 (7); P40 (8); Wl 15 (9); Y-32 (10); LD-1 (11); and DU-1 (12).

FIG. 2. Hybridization of M 13/Fl8 DNA to restriction endonuclease fragments from pGl7, pL8, pTDA 1, and pUCl9. Hybrid plasmids pO17 (lanes 1 and 2), pL8 (lanes 3 and 4), pTDA1 (lanes 5 and 6), and pUC19 (lane 7) were digested with EcoRI plus DruI (lanes 1,3, and 5) or with DraI plus HpuI (lanes 2,4, and 6); the resulting fragments were separated by agarose gel electrophoresis and then transferred to a nitrocellulose filter by the method of Southern (1975). The blot was then hybridized with a 32P-labeled Ml3/Fl8 probe under conditions described under Materials and Methods. After obtaining an autoradiograph of the hybridized filter, the probe was removed by denaturation and washing, and the filter again hybridized with ‘*P-labeled M 13 mp8, to determine which of the hybridizing bands were due to homologous fragments in the respective vector portions of the hybrids (results not shown). Arrows designate hybridizing bands due to homology between the F18 fkagment of Ml3/Fl8 and the pRA1 (pTDA1) or S. citri chromosomal (p0 17 and pL8) DNA in the hybrids.

Presence and distribution of sequences exhibiting homology to fragment F18 and GIS in spiroplasma chromosomul DNA. Fragments F 18 and G 15 were used as hybridization probes with dot blots containing DNA extracted from 12 different Spiroplasma strains (Fig. 3). The DNAs from three of the strains (S. citri Maroc, S. kunkelii E275, and S. sp. P40) hybridized to both of the probes. The intensities of hybridization observed with lo-fold dilutions of the extracted DNAs suggested that there were more copies of the Fl g-associated sequence than of the G 15-a+ sociated sequence in the three Spiroplasma strains. This was contkmed by Southern blot hybridization of chromosomal DNAs from the three strains (Fig. 4). More EcoRI fragments hybridized to fragment F 18 (Fig. 4B)

115

DNA SEQUENCES IN Spiroplasma C

B

A 123

12

3

123

FIG. 4. Southern blot hybridizations between EcoRIdigested chromosomal DNA from three plant pathogenic strains of Spiroplasma and subcloned DraI fragments from S. c&n’ Maroc chromosomal DNA exhibiting homology to pRA1. In each panel, one lane contained EcoRI-digested DNA from S. citri Maroc (lane l), S. kunkelii E275 (lane 2), or strain P40 (lane 3). 32P-labeled probes were fragments G 15 (A), F18 (B) and F5 (C), the derivations ofwhich are described in the text.

than to fragment Gl5 (Fig. 4A). These results established the repetitive nature of the two homologous sequences in the chromosomes of the three plant pathogenic Spiroplasma species. The M 13 subcloning experiment that provided fragments F 18 and G 15 also generated a third DraI fragment (F5) that exhibited homology to pTDA 1. However, this fragment only hybridized to four chromosomal EcoRI fragments from S. citri Maroc and one from S. kunkelii E275, and did not hybridize to the DNA from Spiroplasma species P40 (Fig. 4C). DISCUSSION

We have shown here that DNA sequences from two regions of the S. citri plasmid, pRA 1, are present as short, dispersed, repetitive sequences in the chromosomal DNA of three distinct plant pathogenic species of

Spiroplasma. We estimate that there are 30-40 copies of these sequences per genomic equivalent (Figs. 3 and 4 and data not shown). These plasmid sequences were not detectable in chromosomal DNA from nine other strains of Spiroplasma representing nonpathogenic plant isolates, nor in nonpathogenic and pathogenic strains from a number of insect hosts (Table 1). Our results do not directly implicate the pRAl-associated sequences in virulence, particularly since the strains used here had been passaged repeatedly in the laboratory, and such latepassage strains would be unable to complete a normal cycle via leafhopper vectors of plant infections (Whitcomb and Williamson, 1975). Nevertheless, the presence of pRAlrelated sequences in three distinct Spiroplasma species suggest that they can serve as broad generic indicators of species which produce plant disease. Earlier studies (Nur et al., 1986b) indicated that pRA 1 DNA is a very sensitive molecular probe for the detection of S. citri infections in plant material or insect hemolymph. The present results showing the highly repetitive nature of two short sequences from pRA1 helps explain this high sensitivity. However, since only about onethird of the known spiroplasmas have been probed thus far, further study of the distribution of repetitive DNA in these organisms and their ultimate utility as diagnostic probes is desirable. ACKNOWLEDGMENTS We thank David Rose for his assistance in spiroplasma cultivation and for other technical support to the study, Kevin Hackett for supplying the cell pellet of the LD-1 strain, and Jon Ranhand for his helpful and constructive discussions to the study. I.N. was supported during this study by a fellowship from the Rothschild Foundation (Israel).

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CLARK, T. B., WHITCOMB, R. F., TULLY, J. G., MOUCHES, C., SAILLARD, C., BOVE, J. M., WROBLEWSKI, H., CARLE, P., ROSE, D. L., HENEGAR, R. B., AND WILLIAMSON, D. L. (1985). Spiroplasma melliSerum, a new species from the honeybee (Apis melli.@a).

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35, 296-308.

CLEWELL, D. B., AND HELINSKI, D. R. (1969). Supercoiled circular DNA-protein complex in Escherichia coli purification and induced conversion to an open circular DNA form. Proc. Natl. Acad. Sci. USA 62, 1159-l 162. COLE, R. M. (1983). Virus detection by electron microscopy. In “Methods in Mycoplasmology” (J. G. Tully and S. Razin, Eds.), Vol. II, pp. 407-412. Academic Press, New York. DICKINSON, M. J., AND TOWNSEND, R. (1984). Integration of a temperate phage infecting Spiroplasma citri. Isr. J. Med.

Sci. 20, 785-787.

GRUENBAUM, Y., CEDAR, H., AND RAZIN, A. (198 1). Substrate and sequence specificity of a eukaryotic DNA methylase. Nature (London) 295, 620-622. HANAHAN, D. (1983). Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557-580. MANIATIS, T., FRITSH, F., AND SAMBROOK, J. (1982). “Molecular Cloning,” p. 68. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. MARMUR, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3, 208-2 18. MOUCHES, C., AND BOVE, J. M. (1983). A plasmid from S. citri strain Ml4 hybridizes with extrachromosomal DNAs from other spiroplasmas, including corn stunt spiroplasma E275, tick spiroplasma 277F, and coca spiroplasma N525. Yale J. Biol. Med. 56, 723-727. MOUCHES, C., BARROSO, G., GADEAU, A., AND BOVE, J. M. (1984). Characterization of two cryptic plasmids from Spiroplasma citri and occurrence of their DNA sequences among various spiroplasmas. Ann. Microbiol. (Inst. Pasteur A) 135, 17-24. NORRANDER, J., KEMPE, T., AND MESSING, J. M. ( 1983). Construction of improved M 13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26, 101-106. NUR, I., BOVE, J. M., SAILLARD, C., ROTTEM, S., WHITCOMB, R. F., AND RAZIN, S. (1986a). DNA probes in detection of spiroplasmas and mycoplasmalike organisms in plants and insects. FEMS Lett. 35, 157-162. NUR, I., GLASER, G., AND RAZIN, S. (1986b). Free and

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integrated plasmid DNA in spiroplasmas. Curr. Microbiol. 14, 169-176. RADLOFF, R., BAUER, W., AND VINOGRAD, J. (1967). A dye buoyant-density method for the detection and isolation of closed circular duplex DNA: The closed circular DNA in HeLa cells. Proc. Natl. Acad. Sci. VSA51, 1514-1521. RANHAND, J. M., MITCHELL, W. D., POPKIN, T. J., AND COLE, R. M. (1980). Covalently closed circular deoxyribonucleic acids in spiroplasmas. J. Bacterial. 143, 1194-l 199. RIGBY, P. W. J., DUCKMAN, M., RHODES, C., AND BERG, P. (1977). Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113, 237-25 1. SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-5 17. THAYER, R. E. (1974). An improved method for detecting foreign DNA in plasmids of Escherichia coli. Anal. Biochem.

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TULLY, J. G., AND WHITCOMB, R. F. (198 1). The genus Spiroplasma. In “The Prokaryotes” (M. P. Starr, H. Stolp, H. G. Truper, A. Balows, and H. G. Schlegel, Eds.), Vol. II, pp. 2271-2284. Springer-Verlag, New York. VIEIRA, J., AND MESSING, J. M. (1982). The pUC plasmids on M 13 mp7 derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19,259-268. WHITCOMB, R. F. (198 1). The biology of spiroplasmas. Annu.

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WHITCOMB, R. F. (1983). Culture media for spiroplasmas. In “Methods in Mycoplasmology” (S. Razin and J. G. Tully, Eds.), Vol. I, pp. 147-158. Academic Press, New York. WHITCOMB, R. F., AND WILLIAMSON, D. L. (1979). Pathogenicity of mycoplasmas for arthropods. Zentralbl.

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by Douglas

E. Berg