Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Pflanzenschutz im Obstbau, D-69221 Dossenheim, Germany1
Author for correspondence: Carmine Marcone. Tel: +39 0971 205519. Fax: +39 0971 205503. e-mail: ma513agr{at}unibas.it
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ABSTRACT |
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Keywords: prokaryotes, genome size, yellows diseases, pulsed-field gel electrophoresis, ribosomal genes
Abbreviations: AP, apple proliferation; ESFY, European stone fruit yellows; SPLL, sweet potato little leaf; WX, western X-disease
a Present address: Università degli Studi della Basilicata, Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, Campus Macchia Romana, I-85100 Potenza, Italy.
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INTRODUCTION |
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METHODS |
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Chromosome isolation and restriction endonuclease digestion.
Phytoplasma chromosomes were extracted from leaf midribs, cortical stem tissue or stem phloem preparations. Phloem was prepared as reported by Lauer & Seemüller (2000) , while sample preparation for PFGE and PFGE analysis was carried out as described by Neimark & Kirkpatrick (1993)
, except that the lysis solution was changed several times, SeaPlaque agarose (FMC BioProducts) was used, and the blocks were not gamma-irradiated. PFGE was carried out in a CHEF DRIII apparatus (Bio-Rad Laboratories). Entire linearized ESFY phytoplasma chromosomes were isolated by PFGE, excised from the gel, washed twice for at least 30 min per wash with TE (10 mM Tris/HCl, 1 mM EDTA, pH 8·0) on ice and once with the appropriate restriction buffer and then digested with rare cutting enzymes as reported by Lauer & Seemüller (2000)
. Double digestions were carried out sequentially in the same preparation. After the first digestion, each preparation was first washed twice with TE and then with the appropriate restriction buffer, and then digested with a second restriction endonuclease. In addition, most major restriction fragments were excised from the gel and digested with the second enzyme individually. A concatameric lambda DNA ladder, Saccharomyces cerevisiae chromosomes (Bio-Rad Laboratories) and a Low Range PFG Marker (New England BioLabs) were used as size markers.
Southern blot hybridization.
Southern transfers were carried out as described by Neimark & Kirkpatrick (1993) . The following probes were used for hybridization: (a) linearized chromosomal DNA of strain GSFY1 recovered from the pulsed-field gel; (b) a PCR-amplified 1800 bp rDNA fragment of the ESFY phytoplasma comprising the entire 16S rRNA gene and 16S/23S rDNA spacer region (Schneider et al., 1995
); (c) a cloned fragment containing a gene encoding an immunodominant membrane protein (Berg et al., 1999
); (d) a cloned fragment containing a gene encoding the elongation factor Tu (tuf) (Berg & Seemüller, 1999
); (e) randomly cloned fragment IH184 (Bonnet et al., 1990
); (f) randomly cloned fragment AT67 (Schneider & Seemüller, 1994
); and (g) cloned fragment IH196 containing a putative nitroreductase gene (Jarausch et al., 1994
). For probe preparation from entire GSFY1 chromosomes, DNA was recovered from the pulsed-field gel by agarase (Roche Diagnostics) digestion. PCR products as well as cloned fragments were eluted from conventional agarose gels using the QIAquick gel extraction kit (Qiagen). Probes were labelled with [
-32P]dATP using the Prime-It II kit (Stratagene). Hybridization was carried out according to the protocol of Hoheisel et al. (1991)
.
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RESULTS |
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Restriction endonuclease digestion
Strain GSFY1 chromosomal DNA was digested individually with 11 different restriction enzymes possessing GC-rich recognition sequences. Restriction enzymes NotI, BglI and Cfr42I failed to cleave the chromosomal DNA whereas digestions with SalI or HpaII yielded more than 10 fragments and were not used for mapping. Restriction enzymes SmaI, ApaI, BssHII, BamHI and XhoI produced two, three, five, six and ten fragments, respectively, and were chosen to construct the map (Table 1; see Figs 1
, 3
, 4
, 6
and 7
). PFGE-purified chromosomal DNA of strain GSFY1 was used to probe Southern blots of PFGE-separated SmaI, BssHII, ApaI, BamHI and XhoI restriction fragments. The banding pattern revealed two, five, three, six and ten bands for SmaI, BssHII, ApaI, BamHI and XhoI, respectively, as seen in ethidium-bromide-stained gels. Reciprocal double digestions resulted in restriction fragments with sizes between 9 and 438 kb (Table 1
; see Figs 1a
, 3a
, 4a
, 6a
and 7a
). Analysis of the restriction data indicated that for each double digestion there must be, in addition to the clearly identified fragments, one or two fragments between 1 and 5 kb that were too small to be detected due to insufficient amounts of phytoplasma DNA in the gel (see Table 1
). Different pulse parameters and running times were used so that the size of each restriction fragment could be determined under optimal electrophoretic conditions. Pulse times were varied between 0·7 and 120 s, with different extents of ramping depending on the size range of molecules to be resolved. The mean size of restriction fragments, which was established from a minimum of three different gels, is shown in Table 1
. From the sums of restriction fragments generated from all double digests, the size of the strain GSFY1 chromosome was calculated to be 635 kb (see Fig. 8
). This value is slightly larger than the mean calculated from single digests (633 kb) as well as that estimated from the migration of entire chromosomes in the PFGE gels (630 kb) (Marcone et al., 1999b
).
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Map construction
Data obtained on fragment sizes from single and reciprocal double digestions as well as from hybridization analyses were used to locate the restriction sites on the map shown in Fig. 8. Construction of the map started with double digestion of the strain GSFY1 chromosome with SmaI and BssHII, which showed that one of the two SmaI sites was located in the large BssHII fragment (Fig. 1a
) whereas the second was very close to one of the BssHII sites. This BssHII site was chosen as the starting point of the map. From summation of resulting restriction fragments as well as from staining intensity, there was evidence that digestion of the strain GSFY1 chromosome with BssHII resulted in two fragments of 50 kb which could not be resolved by PFGE. In order to arrange these two fragments on the map, the 350 and 284 kb SmaI fragments, designated S1 and S2, respectively, were separated under appropriate PFGE conditions, excised from the gel and digested separately with BssHII. From each of the SmaI fragments, one 50 kb BssHII fragment was obtained (Fig. 1a
). Southern blot hybridization with probe IH184 revealed that the two 50 kb BssHII fragments were linked (Fig. 2a
). The linking restriction site was chosen as the starting point of the map.
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As there was evidence that XhoI digestion resulted in four doublets 80, 25, 12 and 9 kb in size, their presence was verified by separate digestion of the two SmaI fragments with XhoI (Fig. 4a). Each of the resulting restriction patterns contained fragments of these four sizes. Southern blot hybridization with probe IH184 revealed that the 12 kb XhoI fragments were linked and were located near the BssHII site at position 0 (Figs 4b
and 8
). This region also contained ApaI, SmaI and BamHI restriction sites (Figs 2a
, 3b
, 4b
, 6d
and 7d
). Southern blot hybridization with the other gene probes allowed positioning of the other XhoI sites within the S1 and S2 fragments and confirmed the position of other sites on the map. However, it was impossible to unambiguously allocate the restriction sites XhoI, SmaI, BamHI and ApaI clustering with the BssHII site at position 0 because these sites were too close together and the fragments too small to be observed in pulsed-field gels. A similar situation was observed for the XhoI sites adjacent to the SmaI site at position 283.
The alignment of the restriction fragments revealed that the chromosome of strain GSFY1 was circular, like that of all other mycoplasmas previously examined. Several genetic loci were mapped by Southern hybridization of restriction fragments with various probes (Figs 1, 2
, 3
, 4
, 5
, 6
, 7
and data not shown). It could be shown that the two rRNA operons that seem to be present in all phytoplasmas were not linked (Fig. 5
). The locus of the gene encoding the elongation factor Tu (tuf) was located on the same fragment as one of the rRNA operons while the randomly cloned fragment AT67 was located on the same fragment as the other rRNA operon (Figs 1b
, 2b
, 5
, 6b
, 7b
, 8
and data not shown). However, the position of both tuf and fragment AT67 relative to the positions of rRNA operons was not determined. The locus of the gene encoding an immunodominant protein as well as that of a putative nitroreductase gene were identified on the 65 kb BssHII and 56 kb BamHIXhoI fragments, respectively (Figs 6c
, 7c
and 8
). The randomly cloned fragment IH184 was identified within the 12 kb XhoI fragment containing the BssHII restriction site at position 0 (Figs 4b
and 8
).
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DISCUSSION |
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A total of 26 sites of five different restriction endonucleases were placed on the map of the GSFY1 chromosome of the ESFY phytoplasma. The mapped restriction sites were not uniformly distributed on the genome but clustered in certain areas. In particular, half of the restriction sites are located in the 100 kb region between positions 615 and 80, flanking the start point. A similar pronounced clustering of restriction sites is known for the chromosome of the phylogenetically closely related AP phytoplasma (Lauer & Seemüller, 2000 ) and for several other mollicute genomes (Pyle & Finch, 1988
; Cocks et al., 1989
; Colman et al., 1990
; Ye et al., 1992
). In contrast, the restriction sites mapped on the WX and SPLL phytoplasma chromosomes are evenly distributed (Firrao et al., 1996
; Padovan et al., 2000
). Clustering in the mapped restriction sites may indicate differences in the overall base composition of the regions involved. It is possible that the 100 kb region flanking the arbitrary starting point is richer in G+C than other parts of the genome.
Most of the restriction enzymes we used were the same as those employed in the AP phytoplasma chromosome mapping. Comparisons of the maps showed that numbers and relative positions of restriction sites are conserved in only some cases. The three ApaI sites and the two SmaI sites are in the same or similar positions as those reported for the AP phytoplasma chromosome. In contrast, strain GSFY1 has more BssHII sites than strain AT (five versus two) of which only one is at the same position (Lauer & Seemüller, 2000 ). Also, there are only six BamHI sites in the GSFY1 chromosome while there are about 12 in the AT phytoplasma chromosome (E. Seemüller, unpublished). Different restriction patterns of closely related organisms are known for the SPLL and tomato big bud phytoplasmas (Padovan et al., 2000
). The same applies for five Mycoplasma hominis strains (Ladefoged & Christiansen, 1992
). Although only some of the restriction sites are conserved in strains GSFY1 and AT, the order of identified genes in the GSFY1 chromosome is similar to that of the AT phytoplasma chromosome. In both cases, the two rRNA operons are unlinked. Such a chromosomal arrangement is also known from the WX and SPLL phytoplasmas and some culturable mollicutes (Pyle & Finch, 1988
; Cocks et al., 1989
; Pyle et al., 1990
; Firrao et al., 1996
; Padovan et al., 2000
). As there is an indication that the gene order is conserved in several mollicutes (Pyle et al., 1990
; Ladefoged & Christiansen, 1992
; Peterson et al., 1995
; Himmelreich et al., 1997
), this marker could be more appropriate than restriction sites in studying genomic diversity of mollicutes.
The presence of different MluI restriction patterns, the fragments of which proved to be of phytoplasma nature, could be explained by the fact that strain GSFY1 consists of a phytoplasma population showing a sequence heterogeneity with respect to MluI restriction sites. Similar observations have recently been reported for the SPLL and tomato big bud phytoplasma chromosomes (Padovan et al., 2000 ).
The physical map of the ESFY phytoplasma chromosome will further enhance genomic studies on this phytopathologically important phytoplasma, and will aid genome comparisons within the Candidatus Phytoplasma genus.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 29 September 2000;
revised 2 February 2001;
accepted 12 February 2001.
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