Laboratoire de Biologie Cellulaire et Moléculaire, Institut de Biologie Végétale Moléculaire, Institut National de la Recherche Agronomique and Université Victor Segalen Bordeaux 2, Domaine de la Grande Ferrade, BP 81, 33883 Villenave dOrnon cedex, France1
Author for correspondence: Frédéric Laigret. Tel: +33 5 56 84 31 50. Fax: +33 5 56 84 31 59. e-mail: laigret{at}bordeaux.inra.fr
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ABSTRACT |
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Keywords: Spiroplasma citri, fructose operon, xylitol, gene disruption
Abbreviations: 1-PFK, 1-phosphofructokinase; 6-PFK, 6-phosphofructokinase; fructose-PTS, phosphoenolpyruvate:fructose phosphotransferase system; XylR, xylitol resistant; wt, wild-type
The GenBank accession number for the sequence reported in this paper is AF202665.
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INTRODUCTION |
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Mutant GMT 553 has drawn our attention to the possible involvement of fructose metabolism in the phytopathogenicity of S. citri and also the possible involvement of other sugars such as glucose and trehalose in its virulence and insect transmission. However, no method has been described to specifically obtain sugar-auxotrophic mutants of S. citri because the culture media contain animal serum and have complex compositions. To produce such targeted fructose operon mutants, we have explored two different strategies. The first is based on the fact that xylitol, a fructose analogue, is toxic for many bacteria, including S. citri (Labarère & Barroso, 1989 ). Therefore, xylitol-resistant mutants might be mutated in the fructose operon. The second strategy involves disruption of the fructose operon by homologous recombination involving one crossing-over.
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METHODS |
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DNA and RNA methods.
Total DNA from the S. citri wt strain GII-3 and mutants was isolated as follows. Thirty millilitres of spiroplasma culture were collected by centrifugation and resuspended in 270 µl PBS buffer. Cells were lysed by adding 30 µl 10% (w/v) SDS and incubated for 30 min at 37 °C. The DNA was further purified by phenol/chloroform deproteinization and ethanol/acetate precipitation. Restricted DNA fragments were separated by agarose gel electrophoresis, and blotted onto charged nylon membranes by the alkali transfer procedure. Total RNA from S. citri cells, extracted by the guanidinium thiocyanate/caesium chloride method (Chirgwin et al., 1979 ), was separated by electrophoresis on 1% (w/v) agarose formaldehyde 16·6% (v/v), MOPS 1xgel, and blotted onto C-extra membrane (Amersham). Hybridization with [
-32P]dATP-labelled probes was performed using standard stringency conditions (Sambrook et al., 1989
). The fruR probe was the 0·4 kbp HincII fragment obtained from plasmid pRAK (Gaurivaud et al., 2000
). Expression of fructose operon genes was monitored using a fruAfruK-specific probe (the 2·4 kbp HpaI fragment from plasmid pRAK). DNA sequencing was performed with the T7 DNA sequencing kit (Pharmacia), [35S]dATP
S and appropriate primers. Topology prediction of membrane proteins was done by the HMM method (Sonnhammer et al., 1998
). The sequence of the fructose operon has been deposited in GeneBank (accession number AF202665). The ProDom database was used to study protein-domain arrangements (Corpet et al., 1999
). Multiple sequence alignment was done with the Multalin program (Corpet, 1988
). Primers FruA1 (AATTCGCTTCTCGTTCGG) and FruK (CCTTTTCCACCAATCACC), corresponding to nucleotides 20542071 and 43504368, respectively, in the sequence of the fructose operon, and primers FruA1 and Rev (GGAAACAGCTATGACCATG), corresponding to nucleotides 979997 in the sequence of the pBS plasmid, were used in PCR amplification with 100 ng genomic DNA of S. citri in a 50 µl mixture containing 20 mM Tris/HCl pH 8·5, 10 mM (NH4)2SO4, 2 mM MgCl2, 100 µg BSA ml-1, 0·1% (v/v) Triton X-100, 200 µM (each) deoxynucleoside triphosphate, 0·5 µM (each) of primers and 2·5 U Taq DNA polymerase (Gibco-BRL). Thirty-five cycles were performed using three steps (1 min/92 °C, 30 s/52 °C and 4 min/72 °C) in a thermojet thermocycler (Eurogenetec).
Gene disruption of the fructose operon.
Plasmids H and E used for gene disruption of the fructose operon of wt strain GII-3 were constructed as follows. Plasmid pRAK (Gaurivaud et al., 2000 ) was partially digested with HpaI. The 2·9 kbp HpaI fragment was cloned into plasmid pBS. The recombinant plasmid (pHinc2.9.1) was digested with BamHI and SphI and the 2·9 kbp fragment was cloned in plasmid pBOT digested with the same enzymes. The plasmid was named plasmid H. Plasmid pHinc2.9.1 was digested with BstXI and treated with exonuclease III for different times, extremities were blunt-ended with S1 nuclease, and the plasmid was recircularized with T4 DNA ligase, yielding plasmid pEXO35. This plasmid is characterized by a deletion of the intergenic region between fruA and fruK, and a deletion of the 3' extremity of fruA and the 5' extremity of fruK. This plasmid was digested with BamHI and SphI, and the resulting 2·5 kbp fragment was cloned in pBOT, yielding plasmid E. The wt strain GII-3 was transformed with plasmids E and H. After selection on SP-4 plates supplemented with 2 µg tetracycline ml-1 and multiplication of colonies in SP-4 liquid medium during 15 passages, integration of the plasmids into the chromosome was followed by Southern blots.
Carbohydrate fermentation.
Catabolism of carbohydrate in S. citri results in lactic acid production. Acidification of the growth medium was used as an indicator of carbohydrate utilization. S. citri growth requires complex media supplemented with calf fetal or horse serum, the latter bringing carbohydrates and enzymes such as invertase which may interfere with fermentation of sugar added to the medium. For these reasons we used HSI medium [mycoplasma broth base 15 g l-1, PPLO serum fraction 1% (v/v), sorbitol 7% (w/v), phenol red 30 mg l-1, penicillin 2x105 U ml-1] (Whitcomb, 1983 ) for monitoring carbohydrate fermentation. In HSI, a sugar-free serum fraction (PPLO serum fraction) replaces the normal serum; thus, without sugar addition no fermentation is observed. S. citri cells growing exponentially in SP-4 medium were harvested by centrifugation (20 min, 12000 g, 20 °C), washed twice and resuspended with 8 mM HEPES pH 7·4, 10% (w/v) sorbitol. HSI medium supplemented with glucose, fructose or sorbitol (0·5% w/v) was inoculated with the washed cells. Decrease of pH was followed by the colour change of phenol red from red to yellow.
Determination of the minimal inhibitory concentration of xylitol.
The MIC of xylitol was determined in HSI medium with glucose as carbon source. S. citri cells grown in SP-4 medium were harvested by centrifugation (20 min, 12000 g, 20 °C), washed twice and resuspended in 8 mM HEPES pH 7·4, 10% sorbitol. HSI medium supplemented with 0·5% glucose and xylitol at concentrations ranging from 0 to 8% (w/v) was inoculated with the washed cells and incubated at 32 °C for 1 week. The MIC of xylitol was the lowest concentration which prevented acidification of the medium.
Enzyme assays.
Protein concentration was determined by the procedure of Bradford (1976) , using the Bio-Rad protein assay kit. The activity of PTS was measured as described by Navas-Castillo et al. (1993)
. The 6-phosphofructokinase (6-PFK) and 1-phosphofructokinase (1-PFK) activities were determined as described by Pollack (1995)
.
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RESULTS AND DISCUSSION |
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In order to select spontaneous xylitol-resistant mutants, wt strain GII-3 was inoculated into HSI medium containing 0·5% glucose and 2% xylitol, and incubated at 32 °C for 3 weeks, at the end of which time acidification of the medium eventually occurred. Spiroplasma cells were plated on solid SP-4 medium, and five colonies (xyl1 to xyl5) were tested for their resistance to xylitol. They showed a xylitol MIC higher than 8%. Fermentation of glucose or fructose, in HSI medium, showed that mutants xyl1, xyl2, xyl4 and xyl5 used glucose and fructose, whereas mutant xyl3 used glucose but not fructose. Fructose-PTS activity was not detected in mutant xyl3 whereas 1-PFK activity remained unchanged (Table 1.) In these experiments Fru- mutants could be obtained in HSI medium in the presence of glucose and xylitol because the fructose operon of S. citri is constitutively expressed and is not catabolically repressed by glucose (F. Laigret and others, unpublished results). This is also evidenced by the fact that in SP-4 medium, which contains 0·5% added glucose, spiroplasma cells can be shown to possess an active fructose PTS (Table 1
).
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Production of fructose-negative mutants by homologous recombination.
Two pBOT-derived plasmids carrying mutated versions of fruA+fruK were constructed (Fig. 2.) In plasmid H, fruA carries a deletion of the first 99 bases, and fruK, a deletion of the last 105 bases. In plasmid E, fruA and fruK carry deletions of both 5' and 3' ends, and the intergenic region is deleted. After transformation of the wt strain GII-3 with plasmid E or H, 60 colonies from each transformation experiment were propagated during 15 passages in SP-4 medium supplemented with 2 µg tetracycline ml-1. The culture from each colony was screened for fructose fermentation and glucose fermentation in HSI medium, and for resistance to 2% xylitol. Fifty-seven colonies transformed with plasmid H were able to use glucose (Glc+) but only 5 of these could not use fructose (Fru-), and they were resistant to 2% xylitol (XylR). In the case of plasmid E, 8 of 59 cultures were Glc+, Fru- and XylR. Cultures H31 and H45, obtained with plasmid H, and cultures E38 and E53, obtained with plasmid E, were randomly selected and triply cloned, as previously described for mollicutes (Tully, 1983
). The expected phenotype (Glc+ Fru- XylR) was verified at the end of the cloning procedure. For control purposes, culture H11, from plasmid H, was selected and cloned because it was able to use glucose and fructose and was sensitive to xylitol. Fructose-PTS activity and 1-PFK activity were not detected in clones H31, H45, E38 and E53, whereas 6-PFK activity was present (Table 1
). Clone H11 exhibited activities of fructose-PTS, 1-PFK and 6-PFK (Table 1
). These results confirm inactivation of the fructose operon in strains H31, H45, E38 and E53, but not strain H11.
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The second approach consisted in fructose operon gene disruption by homologous recombination between fruAfruK of the chromosomal fructose operon and partially deleted fruAfruK carried by replicative plasmid pBOT. Recombinational mutants were obtained in which two copies of disrupted fruAfruK genes were present in the spiroplasmal chromosome in the following order: fructose operon promoter/copy 1/pBOT plasmid/copy 2 (Fig. 2). This is characteristic of homologous recombination involving one crossing-over. Only copy 1 is transcribed. This copy may contain a complete fruA gene (see Fig. 2
) which can thus be transcribed. A complete fruK gene can be present in copy 2, but copy 2, and thus fruK, is not transcribed. Therefore, in certain mutants, where fruA, but not fruK, can be transcribed and translated, fructose-PTS activity could occur in the absence of 1-PFK activity. This activity would result in accumulation and toxicity of fructose 1-phosphate in the spiroplasmal cell as no 1-PFK activity is present to remove fructose 1-phosphate. Toxicity of fructose 1-phosphate has been described for E. coli (Ferenci & Kornberg, 1973
) and Bacillus subtilis (Gay & Rapoport, 1970
), precisely in strains lacking 1-PFK activity. In fact with our recombinational mutants, we have not observed such toxicity. In the case of mutant H31, which has been studied in more detail, a complete fruA gene was transcribed from copy 1 but in spite of this no active fructose permease occurred as judged from the absence of fructose-PTS activity. Interestingly, it could be shown that a mutation affected the permease gene, rendering the enzyme inactive. Probably, mutation of the fruA gene is a way for the spiroplasma cell to prevent fructose 1-phosphate toxicity. We believe that such fruA mutations are not restricted to strain H31, but probably occur in other mutants with a complete fruA gene in copy 1 in the absence of a complete fruK gene.
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ACKNOWLEDGEMENTS |
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Received 5 May 2000;
accepted 30 May 2000.