Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, 35043 Marburg, Germany1
Author for correspondence: Matthias S. Ullrich. Tel: +49 6421 178 101; Fax: +49 6421 178 109. e-mail: ullrichm{at}mailer.uni-marburg.de
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ABSTRACT |
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Keywords: temperature, differential gene expression, thermoadaptation, phytotoxin, plant pathogen
Abbreviations: COR, coronatine; GUS, ß-glucuronidase; HR, hypersensitive reaction; X-Gluc, 5-bromo-4-chloro-3-indolyl glucuronide
The GenBank accession numbers for the nucleotide sequences of mutants 560, 561, 562, 563, 564, 568, 570, 574, 590, 591, 593, 596, 599, 601, 605, 608, 613, 617, 618, 626 and 632 determined in this work are AF274322AF274342, respectively.
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INTRODUCTION |
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The bacterial blight pathogen Pseudomonas syringae pv. glycinea PG4180 infects soybean plants (Glycine max), inducing typical leaf spot symptoms characterized by water-soaked regions which soon develop into necrotic lesions surrounded by chlorotic haloes. Like most representatives of P. syringae, the pathovar glycinea is an opportunistic pathogen that requires water films and aerosol formation for infection of plant tissues via the plants stomata or open wounds. The symptoms of bacterial blight are most severe during or following periods of cold, humid weather conditions (Dunleavy, 1988 ).
P. syringae pv. glycinea produces the chlorosis-inducing polyketide phytotoxin coronatine (COR) (Bender et al., 1991 ). COR-deficient mutants of P. syringae pvs tomato, glycinea and maculicola were shown to be severely impaired in virulence on tomato, soybean and Chinese cabbage plants, respectively (Bender et al., 1987
; Mittal & Davis, 1995
; Budde & Ullrich, 2000
; Tamura et al., 1998
). PG4180 synthesizes high levels of COR at 18 °C, whereas no detectable toxin is produced at 14 or 28 °C (Palmer & Bender, 1993
; Budde et al., 1998
). Previously, we reported that the temperature-sensitive transcriptional activation of three promoters within the 32·8 kb plasmid-borne COR biosynthetic gene cluster might explain thermoregulation of COR biosynthesis (Ullrich & Bender, 1994
; Liyanage et al., 1995
; Ullrich et al., 1995
). Two of these promoters control the expression of biosynthetic operons whilst the third is located upstream of corS, a gene encoding the histidine protein kinase CorS which, together with the two response regulators CorR and CorP, forms a temperature-dependent two-component regulatory system (Ullrich et al., 1995
; Budde et al., 1998
).
The higher infection rate and stronger symptom development at lower temperatures prompted us to investigate whether COR biosynthesis represented the only temperature-controlled factor of P. syringae pv. glycinea. A comprehensive search for additional thermoregulated genetic loci was conducted to better understand which factors might potentially be involved in the response of P. syringae to minor temperature shifts possibly taking place during micro-climatic changes on the leaf surface. Such temperature fluctuations are often associated with alterations in humidity. Since P. syringae pv. glycinea requires high humidity for plant infection, low temperatures might be a signal for the epiphytically growing pathogen to start an effective invasion process.
In this study, P. syringae pv. glycinea PG4180 was randomly mutagenized using the transposon miniTn5 harbouring a promoterless ß-glucuronidase (GUS) gene as the reporter system (Wilson et al., 1995 ). Mutants were screened for differential expression of GUS at 18 and 28 °C. Following a three-step screening procedure, interesting mutants were differentiated with respect to insertions inside or outside the COR biosynthetic gene cluster. Insertion loci of PG4180 mutants not affected in phytotoxin synthesis were subcloned and sequenced, allowing the identification of additional thermoresponsive genetic loci of P. syringae pv. glycinea PG4180.
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METHODS |
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DNA procedures.
Genomic DNA was isolated from P. syringae by established procedures (Staskawicz et al., 1984 ). Agarose gel electrophoresis, restriction digests, purification of DNA fragments from agarose gels, electroporations, Southern blot hybridizations and small-scale plasmid DNA preparations were performed by standard techniques (Sambrook et al., 1989
). For the detection of RFLPs in COR- mutants of PG4180 by Southern blot hybridization, plasmids pMU234 and pMU567 (Ullrich et al., 1994
) were used as probes. Subclones were generated in pBluescript SK II(+) (Stratagene). For large-scale preparations, plasmid DNA from E. coli was isolated by alkaline lysis and purified with Qiagen columns. The oligonucleotide primer used for sequencing of the DNA adjacent to the miniTn5-uidA insertions was 5'-AGATCTGATCAAGAGACAG-3' and was derived from the I-end sequence of IS50 (Phadnis & Berg, 1987
). Plasmid DNA from P. syringae cultures was isolated by the procedure described by Kado & Liu (1981)
.
DNA sequencing and analysis.
Nucleotide sequencing reactions were performed by the dideoxynucleotide method (Sambrook et al., 1989 ) with the Thermo Sequenase fluorescent labelled primer cycle sequencing kit (Amersham-Buchler) and Cy5-labelled oligonucleotide primers (Pharmacia). Automated DNA sequencing was accomplished using an ALF Express sequencing apparatus (Pharmacia). Additional nucleotide sequencing was carried out commercially (MWG Biotech, Ebersberg, Germany). The single-strand nucleotide sequence data obtained (approximately 300650 bp) were aligned and processed with the DNASTAR version 4·1 software package (Lasergene, Madison, WI). DNA and protein sequence homology searches of the GenBank, EMBL, PIR and SWISS-PROT databases were performed using the University of Wisconsin Genetics Computer Group (UWGCG) programs BLASTN, BLASTX, FASTEMBL, GAP and BESTFIT.
Assays for GUS activity.
GUS activities of transposon mutants were initially screened on MG plates containing 20 µg of the GUS substrate X-Gluc (5-bromo-4-chloro-3-indolyl glucuronide) ml-1 and incubated at 18 or 28 °C for 57 d. To standardize the procedure, single mutant colonies were transferred into 96-well microtitre plates filled with MG medium and incubated overnight on a rotary shaker at 100 r.p.m. and 28 °C. A 96-needle metal stamp was used to transfer aliquots of the mutant suspensions onto MG plates containing X-Gluc. Intensities of blue colour formation were visually estimated by three individual researchers in independent screenings. Photometric GUS quantification using p-nitrophenyl glucuronide as substrate was carried out as described by Wilson et al. (1992) . Fluorometric analysis of GUS activity (Xiao et al., 1992
) was carried out using a Fluorolite-1000 micro-plate reader (Dynatech) and 96-well microtitre plates.
Detection and quantification of COR synthesis.
Organic acids were extracted from cell-free bacterial supernatants (1·5 ml) and analysed for the presence of COR using the HPLC method described elsewhere (Palmer & Bender, 1993 ).
UV sensitivity assay.
Strains of P. syringae were grown to mid-exponential phase in 5 ml KB medium broth. The cultures were pelleted by centrifugation and resuspended in 5 ml 0·9% PBS (58 mM Na2HPO4, 17mM NaH2PO4, 68 mM NaCl, pH 7·4). UV sensitivity assays were performed using the method of Simonson et al. (1990) , and survivors were enumerated after plating cells on MG agar plates at 28 °C. At least three replicate UV sensitivity assays were performed for each strain.
Plant inoculation experiments.
Soybean plants (Glycine max cv. Maple Arrow) were grown in a greenhouse at 2025 °C and 60% humidity, and with a 12 h photoperiod (15000 lux; cold white fluorescent light). Four-week-old soybean plants were sprayed with bacterial cultures grown at 23 °C to an OD600 of 1·0 (approx. 1 x 109 c.f.u. ml-1). The soybean plants were then transferred to a growth chamber (Controlled Environments) at 23 °C and 80% humidity, and with a 12 h photoperiod (15000 lux; cold white fluorescent light). For determination of the in planta growth of P. syringae, three leaf disks (each with an area of 0·5 cm2) from different plants were removed 7 d post-inoculation, surface-sterilized with 70% ethanol, pooled and homogenized in sterile 0·9% NaCl. Serial dilutions were subsequently plated onto KB agar plates for determination of bacterial numbers (c.f.u. ml-1).
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RESULTS AND DISCUSSION |
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Transposon mutagenesis of P. syringae pv. glycinea PG4180
P. syringae pv. glycinea PG4180 was mutagenized by random insertion of the miniTn5-uidA transposon (Wilson et al., 1995 ) following its delivery on suicide plasmid pCAM140 by conjugation. Upon miniTn5-uidA insertion, transcriptional fusions could be formed between the promoterless uidA gene and P. syringae promoters and the uidA gene could be expressed, resulting in production of GUS. In total, 7500 individual mutants were obtained, which were subsequently screened for GUS expression on MG agar plates at 18 and 28 °C (see below). The random character of the transposon insertions was verified with respect to the location of the miniTn5-uidA by analysing genomic DNA of 72 randomly picked mutants using Southern blot hybridization with pCAM140 as DNA probe (data not shown). Only three mutants showed signals with similar migration patterns, suggesting that the majority of the mutants contained the miniTn5-uidA insertion at different genomic locations.
Qualitative determination of temperature-dependent reporter gene expression
To analyse all 7500 transposon mutants for temperature-dependent reporter gene expression, a qualitative GUS assay on X-Gluc-containing MG minimal medium plates was carried out at 18 and 28 °C. Three individual researchers visually estimated development of blue coloration after 57 d (Fig. 1). As controls, we included three PG4180 transconjugants on each microtitre plate (marked with open arrows in Fig. 1
). One transconjugant harboured plasmid pRG960sd (Van den Eede et al., 1992
), which carries a promoterless uidA gene and served as negative control. The other two transconjugants carried plasmids pRGMU1 and pRGMU3 (Ullrich & Bender, 1994
), which contained transcriptional fusions of uidA to the cmaABT upstream region and the cmaU promoter region, respectively, from within the COR biosynthetic gene cluster. cmaABT is transcribed in a temperature-dependent fashion, whereas the cmaU promoter functioned constitutively (Ullrich & Bender, 1994
; Budde et al., 1998
). All three transconjugants exhibited the expected phenotype (Fig. 1
). Blue colour formation varied widely among the 7500 mutants tested, with approximately 50% of the transposon mutants expressing GUS (blue colour) and approximately 50% exhibiting a white colony phenotype, indicating that GUS was not expressed. Only in those mutants that were repeatedly selected by all three researchers was GUS activity considered to be influenced by temperature. Three hundred and forty-six transposon mutants (4·7% of the total number of mutants) were selected for re-evaluation by the subsequent quantitative GUS assay.
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Characterization of individual transposon mutants of PG4180
Southern blot experiments with a 2·0 kb NotI fragment of plasmid pCAM140 carrying the uidA gene as the DNA probe and genomic DNA of all 87 mutants treated with restriction enzyme SalI were carried out (data not shown). This experiment aimed to identify duplicates among the selected mutants since the signals obtained should migrate in a manner characteristic for each insertion locus. Interestingly, signals in genomic DNA of 26 mutants occurred simultaneously in two individually picked mutants, suggesting that the transposon insertion loci of these mutants were identical. Therefore, we limited all further experiments to the remaining 61 mutants with individual genotypes. From those, 39 transposon mutants showed an increased GUS expression at 18 °C and 22 exhibited a stronger uidA transcription at 28 °C. As depicted in Fig. 2 the level of temperature induction expressed as the ratio of GUS activities at 18 versus 28 °C or vice versa varied between 2- and 40-fold. The actual GUS expression values ranged between 15 and 2500 U GUS (mg protein)-1 (data not shown). On one hand, the strong variability in total GUS values suggested a high diversity in promoter strengths. On the other hand, this result might indicate that the transposon inserted in variable proximity to the respective promoters, giving rise to higher or lower levels of reporter gene expression.
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Transposon mutants with increased reporter gene expression at 28 °C
Following SalI digestion of genomic DNA, the genetic loci affected by the miniTn5-uidA insertion in the 22 mutants with induced uidA expression at 28 °C were shotgun-cloned into SalI-treated pBluescript SK II(+). Transformants were screened for resistance to ampicillin, streptomycin and spectinomycin, and their plasmid DNA was subsequently digested with SalI to verify the cloning results. The subcloned miniTn5-uidA insertions were given designations between p560 and p626 according to the corresponding mutant numbers (Table 2). Subsequently, the DNA adjacent to the I-end of the insertion sites was sequenced. The nucleotide sequences were compared with database entries and the results are summarized in Table 2
. Unfortunately, the insert DNA derived from eight of these mutants showed no significant sequence similarities to any database entries.
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Regulatory genes might have been affected in mutants 563, 591 and 599. The insertion loci of these three mutants showed similarities, respectively, to a HU-like DNA-binding protein of Rhizobium leguminosarum (Khanaka et al., 1985 ); to VacC, involved in virulence regulation of Shigella flexneri (Durand et al., 1994
); and to MucD, a regulatory periplasmic serine protease involved in alginate biosynthesis of Azotobacter vinelandii and Pseudomonas aeruginosa (Martinez-Salvazar et al., 1996
; Ertesvag et al., 1995
). With respect to the latter mutant, P. syringae is also known to produce this exopolysaccharide and alginate synthesis was shown to be controlled by temperature, with increased algD transcription at 28 °C (Peñaloza-Vázquez et al., 1997
).
Three mutants, 605, 596 and 626, apparently harboured miniTn5-uidA insertions in genes homologous to those, respectively, of a small multi-drug transporter (Sundstroem et al., 1988 ); of the TraL/KorB locus, required for T-DNA and plasmid DNA transfer in Agrobacterium tumefaciens and E. coli (More et al., 1996
; Winans & Walker, 1985
); and of a high-affinity branched-chain amino acid permease from P. aeruginosa (Hoshino & Kose, 1990
). All three loci may be involved in transport or secretion processes. To find out whether the traL/korB locus in mutant 596 was plasmid-borne, its plasmid profile was analysed for an alteration in electrophoretic mobility due to the miniTn5-uidA insertion (Fig. 3
, lane 1). Mutant 596 showed a shift in the band representing the 45 kb indigenous plasmid p4180E, raising the interesting hypothesis that this plasmid might harbour a conjugal traL locus. Subsequently, Southern blot analysis was carried out using the insert DNA of p596 as DNA probe and undigested plasmid DNA of mutant 596 and the PG4180 wild-type (data not shown), indicating that a traL/korB-like locus is only present on the 45 kb plasmid p4180E but not on any of the other indigenous plasmids of PG4180. In a subsequent study, we have identified a total of 12 tra-like genes in close proximity to the traL-like locus of p4180E, suggesting that this plasmid might harbour a tra operon (K. Leykauf & M. S. Ullrich, unpublished observation). Conjugation experiments are currently being conducted in our laboratory to test whether PG4180 plasmids are transferred in a temperature-dependent manner. Conjugal plasmid transfer and plasmid replication in different organisms has previously been shown to undergo temperature regulation (Fullner & Nester, 1996
; Fernandez-Tresguerres et al., 1995
; Tietze & Tschape, 1994
; Banta et al., 1998
).
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Interestingly, the insertion loci of mutants 613 and 618 showed sequence homologies, respectively, to the UV resistance locus rulAB of P. syringae (Sundin et al., 1996 ) and to the uvrA region of Micrococcus luteus (Shiota & Nakayama, 1989
), which both may represent stress-inducible genetic loci. To test this, both mutants were screened for impaired resistance to UV irradiation (Fig. 4
). Mutant 613 was indeed more susceptible to UV damage than the PG4180 parental strain. In contrast, the other mutant was not affected in its ability to withstand UV irradiation under the tested conditions. It could be speculated that expression of UV tolerance genes is more important when the bacteria grow epiphytically on the UV-exposed, warmer surface of plant leaves (Beattie & Lindow, 1999
) than when the bacteria enter the plant apoplast to become virulent. To determine if strain PG4180 shows a temperature-dependent UV tolerance, cells were incubated in KB liquid medium at 18 and 28 °C and dilution series of each culture were plated onto MG agar plates, which were subsequently exposed to UV irradiation of 30 J m-2. Bacterial survival rates were 12% for cells pre-incubated at 28 °C and less than 0·2% for the samples pre-incubated at 18 °C, suggesting that PG4180 is more sensitive to UV damage at the lower temperature. This result is in line with an increased expression of the rulAB locus at 28 °C.
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The genetic loci p561, p568 and p570 showed similarity in their predicted amino acid sequences to the biosynthetic proteins mannuronan C-5-epimerase (AlgE2) of Azotobacter vinelandii (Ertesvag et al., 1995 ), to formaldehyde dehydrogenase of E. coli (Gutheil et al., 1992
) and to a putative short-chain dehydrogenase from Rhizobium strain NGR234 (Freiberg et al., 1997
). The result for mutant 561, which carries the reporter gene insertion in an algE-like locus (Table 2
) is in stark contrast to that of mutant 599, which has an insertion in a mucD-like locus and shows increased GUS expression at 28 °C. If mucD of PG4180 were part of the algT operon, one would expect that the algE-like locus would be expressed preferentially at 28 °C. Transcription of algE in P. aeruginosa and Azotobacter vinelandii was previously shown to be under the control of AlgT (Ertesvag et al., 1995
; Martinez-Salvazar et al., 1996
). Since copper ions induce alginate biosynthesis in P. syringae (Kidambi et al., 1995
), we subsequently tested the alginate production of mutants 561 and 599 on MG agar plates supplemented with 250 µg cupric sulfate ml-1 and compared it to the phenotype of the wild-type. No obvious differences in exopolysaccharide production were observed (data not shown). This result suggested that the algE-like locus affected in mutant 561 might not be involved in alginate biosynthesis.
Mutant 574 carried a transposon insertion in hrpT, a gene involved in assembly of the type III hrp (hypersensitive reaction and pathogenicity) protein secretion apparatus. This secretion system is required for pathogenicity and induction of the plant-borne hypersensitive response (HR) caused by P. syringae (Deng et al., 1998 ). When tested for its HR phenotype, this mutant still elicited a HR on tobacco plants (data not shown). This result might be due to the fact that the transposon had inserted at the 3' end of hrpT, thus not completely blocking its expression. Since hrpT is located at the 3' end of the respective hrpFGCTV operon (Deng et al., 1998
) the miniTn5-uidA insertion might not have caused any polar effects on the transcription of the other hrp genes. Regardless, this finding is interesting since hrp gene expression in Erwinia amylovora has previously been shown to be thermoresponsive (Wei et al., 1992
) and the hrp-mediated protein secretion mechanism in P. syringae had unambiguously been demonstrated to be temperature-dependent with maximal activity at 1820 °C (Van Dijk et al., 1999
).
Two additional loci, designated p601 and p632 (Table 2), showed relatedness, respectively, to the plasmid replication protein RepA of P. syringae pv. phaseolicola (Gibbon et al., 1999
) and to the replication region of the P. aeruginosa plasmid R91-5 (Moore & Krishnapillai, 1982
), suggesting plasmid-borne locations. Agarose gel electrophoresis of undigested plasmid preparations from mutants 632, 601 and PG4180(pRGMU3) (Fig. 3
, lanes 2, 3 and 4) demonstrated that mutant 601 showed a shifted band representing the 45 kb plasmid p4180E. This indicated that the miniTn5-uidA had inserted in this plasmid. In contrast, the plasmid profile of mutant 632 did not differ from that of PG4180(pRGMU3), suggesting that the sequence similarity to the replication region of plasmid R91-5 was misleading (Fig. 3
).
The cloned DNA of mutant 562 showed strong sequence similarity to IS870, an insertion element frequently found in P. syringae and other plant pathogens (Fournier et al., 1993 ). Turner et al. (1990)
reported on the temperature sensitivity of transposition of class II transposons with optimal levels of transposition at room temperature or 30 °C versus 3742 °C. Our result might reflect that transposition of IS870could also be more pronounced at lower temperatures.
Effects of transposon mutations on growth rates in vitro and in planta
To determine whether mutations in the identified genetic loci affected the in vitro growth of P. syringae, we compared the generation times of the PG4180 wild-type and 34 distinct mutants representing all identified genetic loci (12 with increased GUS expression 18 °C and 22 with increased GUS expression at 28 °C) in HSC medium at 18 and 28 °C. The bacterial growth of duplicate cultures was measured photometrically. No significant differences between the wild-type and any of the mutants were observed (data not shown), suggesting that the transposon insertions had no effect on the in vitro growth of the bacteria.
Furthermore, plant inoculation experiments were carried out with all 34 PG4180 mutants, one of the COR- mutants (designated 615) and the wild-type strain. Following spray-inoculation of bacteria grown at 23 °C onto soybean plants (subsequently kept at 23 °C), we monitored disease development and enumerated the bacterial populations inside the plant tissue after 7 d. Most of the mutants tested were as virulent as the wild-type and reached population densities of approximately 37 x 107 c.f.u. cm-2. In contrast, three mutants (615, 564 and 608) showed a decreased development of bacterial blight symptoms on the host plant and lowered bacterial multiplication (1·54 x 106 c.f.u. cm-2) inside the plant tissue, indicating that their transposon insertions interfered with the virulence of the bacteria. Whilst 615 was the COR- mutant tested, thus confirming previous findings on the role of COR as a virulence factor (Bender et al., 1987 ; Mittal & Davis, 1995
; Tamura et al., 1998
; Budde & Ullrich, 2000
), the insertion locus of 564 showed similarities to bacterial adhesins and that of 608 was similar to LPS biosynthetic loci. This result hints at a potential role of both loci in virulence associated processes. The reason why the latter two mutants although impaired in virulence showed an increased GUS expression at 28 °C, a temperature non-conducive for the initial establishment of a successful invasion process for PG4180 (Dunleavy, 1988
; Budde & Ullrich, 2000
) remains obscure. It could be speculated that the two affected genetic loci might be required for later steps of the disease development during which low temperature might not be a triggering key factor anymore. Future experiments in our laboratory will aim at unravelling their respective functions in the plantmicrobe interaction.
Time-dependence of temperature induction among P. syringae mutants
We were interested in determining the time span that is necessary to induce reporter gene expression in the identified PG4180 mutants. Cultures of the 34 thermoresponsive mutants were incubated at the respective non-conducive temperature until they reached an OD600 of 1·0. Subsequently, the bacterial cultures were shifted to the growth temperature that induced GUS expression and were incubated for an additional 24 h. Samples were taken at intervals of 12 h for determination of GUS expression levels and results for all mutants were plotted against time. Results for some representative mutants are depicted in Fig. 5. In general, there was a considerable lag phase for GUS induction (26 h), indicating that extended time spans were required for the bacteria to adjust to the new temperature regime before GUS expression was initiated. As a control, we included incubation of PG4180(pRGMU3), which harbours a constitutively expressed cmaU:uidA fusion (Ullrich & Bender, 1994
). This transconjugant did not show a temperature-dependent induction of GUS synthesis. Its initial GUS values at the time of temperature shift from 18 to 28 °C or vice versa were 154±20 and 210±14 U GUS (mg protein)-1, respectively. Upon temperature shifts, GUS values for this transconjugant reached 243±31 and 279±25 U GUS (mg protein)-1, respectively, after 15 h incubation. This is in line with previously reported results (Ullrich & Bender, 1994
). All identified thermoresponsive fusions showed a slow and steady increase in reporter gene expression once the bacteria were shifted to the inducing temperature. This indicates that the initial screening procedure had biased temperature-shock-dependent promoters. Moreover, these results suggested that the general type of thermoregulation for most of these genetic loci might be similar. If at least some of the identified genetic loci are part of a regulon, subsequent mutagenesis could be used to identify regulatory components that function at higher hierarchical levels of thermoregulation in P. syringae.
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An increase in reporter gene expression at a given temperature could also be interpreted as a repression of transcription at the other temperature. The data and the methods used herein did not allow us to discriminate unambiguously between these possibilities. Use of unstable and hence temperature-independent reporter genes fused to the respective loci will answer this question in future studies. The results of this work have initiated a number of subsequent studies that ultimately will deepen our understanding of the molecular basis of thermoregulation in plant-pathogenic bacteria.
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ACKNOWLEDGEMENTS |
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Received 7 June 2000;
revised 9 July 2000;
accepted 24 July 2000.