Department of Fungal and Bacterial Plant Pathology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK1
Author for correspondence: Ian K. Toth. Tel: +44 1382 562731. Fax: +44 1382 562426. e-mail: itoth{at}scri.sari.ac.uk
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ABSTRACT |
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Keywords: prokaryotic differential gene expression, Erwinia, plant pathogen, cDNA-AFLP
Abbreviations: Eca, Erwinia carotovora subsp. atroseptica; Ecc, Erwinia carotovora subsp. carotovora; AFLP, amplified fragment length polymorphism
The GenBank accession numbers for the EL1, EL2, EL3, EP5, EP22, EP26, EP11 and EP21 sequences determined in this work are AJ274641AJ274648, respectively.
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INTRODUCTION |
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Traditionally, transposon mutagenesis has been used as a method to identify genes involved in bacterial processes such as pathogenicity (reviewed by Mills, 1985 ). This technique has yielded important information about a number of genes involved in disease development in Er. carotovora (e.g. genes encoding pectolytic enzymes or involved in secretory and regulatory processes; Pérombelon & Salmond, 1995
). However, hot-spots for transposon insertion, secondary transposition with possible genome rearrangements, low throughput and the generation of lethal mutations limit the use of this method (Mills, 1985
). In essence, many subtle interactions between the pathogen and its host may be lost when the pathogen is disabled by mutagenesis. An alternative approach to studying plantpathogen interactions is to investigate changes in gene expression during the interaction.
Methods for profiling gene expression, such as differential display (DD) RT-PCR (Liang & Pardee, 1992 ) and cDNA-amplified fragment length polymorphism (cDNA-AFLP; Bachem et al., 1996
) have yielded important differentially expressed genes from eukaryotes. Both the purification of mRNA and synthesis of cDNA for these techniques involves an oligo(dT) primer that anneals to the poly(A) tail at the 3' end of the transcript. The low levels of polyadenylation in prokaryotic mRNAs mean that efficient methods for cDNA synthesis that exclude rRNA are lacking. Nevertheless, DD RT-PCR techniques have been reported for prokaryotes (Wong & McClelland, 1994
; Abu Kwaik & Pederson 1996
; Akins et al., 1998
), although insufficient amplification products were generated to allow an entire genome to be screened for differentially expressed genes. Recently, a method has been developed for profiling gene expression in members of the Enterobacteriaceae that provides a broad genome coverage (Fislage et al., 1997
). This method uses combinations of short (10-mer and 11-mer) oligonucleotide primers that anneal, respectively, to conserved sequences in the 5' and 3' regions of Escherichia coli genes. We report the use of these primers for representative cDNA synthesis from Eca and Ecc and the subsequent use of cDNA-AFLP to profile gene expression for the first time in bacteria.
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METHODS |
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RNA extraction and cDNA synthesis.
Total RNA was prepared from bacterial cells as described by Aiba et al. (1981) . Prior to cDNA synthesis, RNA was treated with RNase-free DNase I (Pharmacia Biotech) (1 U DNase I:20 µg RNA) and incubated for 20 min at 37 °C. The RNA was then heat-denatured at 65 °C for 10 min, simultaneously inactivating DNase I. A mixture of all 11-mer oligonucleotide primers (Ea1Ea10) at 100 ng µl-1 (Fislage et al., 1997
) was used to generate first-strand cDNA from 10 µg DNase-I-treated RNA following the procedure described in the First-strand cDNA Synthesis Kit (Pharmacia Biotech). The second strand of cDNA was synthesized using the Universal RiboClone cDNA Synthesis System (Promega).
PCR amplification.
Approximately 200 ng genomic DNA and 1 µl of a 100-fold dilution of first-strand cDNA were subjected to 35 cycles of amplification by PCR. In addition to template DNA, the amplification mixture contained, in a final volume of 50 µl, 2·5 U Taq polymerase (Gibco-BRL), 10 mM Tris/HCl (pH 8·4), 50 mM KCl, optimized amounts of MgCl2 within the range 1·52·5 mM, 100 µmol of each deoxynucleotide triphosphate and 50 ng of each primer for a given gene (Table 1).
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Northern hybridization.
Total RNA (10 µg) was denatured, separated by gel electrophoresis and blotted onto Amersham Hybond-N+ (Pharmacia Biotech) membranes following the procedure of Fourney et al. (1988) . Hybridization was performed with DNA probes in 5xSSPE, 0·2% SDS, 500 µg denatured herring sperm DNA (Boehringer Mannheim) ml-1 and 5xDenhardts solution (Sambrook et al., 1989
). After hybridization, filters were washed twice with 5xSSC, 0·5% SDS at 65 °C for 20 min and twice with 1xSSC, 0·5% SDS at 65 °C for 20 min. Probes were generated using, as template, PCR products purified through Wizard columns (Promega) by a random-primed reaction with High Prime (Boehringer Mannheim). Unincorporated nucleotides were eliminated from the probe by purification through Sephadex Nick-Columns (Pharmacia Biotech).
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RESULTS AND DISCUSSION |
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To investigate whether the cDNA generated from Eca and Ecc was representative of genes expressed under the conditions used, we designed pairs of oligonucleotide primers that anneal to 14 previously reported Er. carotovora genes (Table 1) to test for the presence of these sequences in each cDNA population by PCR amplification. PCR was performed using the cDNAs as templates with each of the primer pairs in Table 1
. As controls, the DNase-I-treated RNA populations used for cDNA synthesis, and Eca, Ecc and Es. coli genomic DNAs were also used as PCR templates. The results of the PCRs are given in Table 2
. Fig. 2
shows a typical PCR amplification result for primers that anneal to the mopB gene. In all cases, PCR was performed on duplicate samples of independently synthesized cDNAs and gave reproducible results. All primer pairs derived from Eca gene sequences amplified DNA fragments of expected sizes (Table 1
) from genomic DNA and cDNA templates of Eca (Table 2
). Similarly, Ecc-derived primer pairs amplified DNA fragments of expected sizes from genomic DNA and cDNA templates of Ecc. No amplification products were generated from Es. coli cDNA or genomic DNA templates. In addition, no amplification products were obtained from DNase-treated RNA samples, demonstrating the absence of contaminating genomic DNA (results not shown). In some cases, primer pairs (Y1/Y2, Mop F/R, Ogl F/R, CelN F/R; Table 1
) amplified DNA fragments of expected sizes from both Eca and Ecc cDNA templates, indicating that they annealed to conserved regions of analogous genes within each subspecies (Table 2
). Interestingly, the profile of PCR amplification for the ogl gene suggested that it is differentially expressed in Ecc but not in Eca under the conditions used in this study. In addition, the rffDG operon, which was not detectable in Ecc, appeared to be differentially expressed in Eca. Successful amplification from cDNA templates with all primer pairs indicated that the combination of all ten 11-mers had resulted in the synthesis of representative first-strand cDNA. In addition, amplification of a product of over 1000 bp in size (rffDG) implied that primer extension is extremely efficient during the process of reverse transcription.
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cDNA-AFLP from Eca after growth in LB or MM-pectin media
To test whether the synthesized cDNA, as described above, could be used for the isolation of differentially expressed genes, we prepared templates from Eca grown on LB and MM-pectin media for cDNA-AFLP (see Fig. 1 for procedure). Until the present work, the cDNA-AFLP technique, derived from the DNA fingerprinting method of Vos et al. (1995)
, had only been used for profiling differential gene expression in eukaryotes (Bachem et al., 1996
).
First-strand cDNAs from Eca were converted to double-stranded cDNA and used to prepare cDNA-AFLP templates. An AFLP template from Eca genomic DNA was prepared for comparison. Following PCR amplifications with eight independent MseI/EcoRI primer combinations, radiolabelled PCR products were visualized by PAGE. Amplification products specific to a template, as well as many common to all templates, were observed (Fig. 3). A mean of 34±9 amplification products were detected from the genomic DNA template. In contrast, means of 26±10 and 17±3 amplification products were obtained from cDNA templates derived from LB and MM-pectin, respectively (Fig. 3
). The larger number of amplification products generated from genomic DNA may have been due to either of two factors: (i) additional DNA fragments present in this AFLP template were derived from non-transcribed portions of the genome, or (ii) gene sequences were detected that were not expressed on LB or MM-pectin media. Similarly, the larger number of amplification products generated from LB-derived template than from MM-pectin-derived template (Fig. 3
) may be representative of more complex gene expression on a complete medium than on a minimal medium. On three separate occasions, fresh cDNA-AFLP templates generated from independent RNA extractions, after growth of Eca on LB and MM-pectin media, yielded reproducible amplification profiles when PCR amplified with the same primer combinations (results not shown). In all cases, PCR amplification was performed using MseI and EcoRI primers with two nucleotide extensions. A total of 256 combinations of such primers can be made, suggesting that as many as 6656 amplification products may be generated from LB-derived template and 4352 from MM-pectin-derived template. Given that there are approximately 4300 expressed genes in the closely related enterobacterium Es. coli, cDNA-AFLP appears to provide a broad coverage of the Eca transcriptome. Nevertheless, it is unlikely that all amplification products are derived from independent genes or that all genes contain an EcoRI site. It is therefore advisable to employ more than one restriction enzyme combination, and to include 6-bp-cutting enzymes such as PstI, which recognizes sites with a greater G+C content and may thus, in some cases, increase the chances of digesting G+C-rich cDNA sequences.
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Adaptation of bacteria to their environment can be highly efficient, involving many metabolic and physiological changes. This work shows that it is possible to reproducibly profile gene expression in a bacterial plant pathogen under different environmental conditions, and to isolate differentially regulated sequences using a modification of the cDNA-AFLP protocol of Bachem et al. (1996) .
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ACKNOWLEDGEMENTS |
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Received 26 July 1999;
revised 20 September 1999;
accepted 5 October 1999.