Journal of Bacteriology, August 2000, p. 4673-4676, Vol. 182, No. 16
Department of Genetics, The University of
Adelaide, Adelaide, South Australia 5005, Australia
Received 21 January 2000/Accepted 17 May 2000
Three insertion sequences (IS) elements were isolated from the
phytopathogen Ralstonia solanacearum. Southern
hybridization using these IS elements as probes revealed hybridization
profiles that varied greatly between different strains of the pathogen. During a spontaneous phenotype conversion event, the promoter of the
phcA gene was interrupted by one of these IS elements.
Ralstonia solanacearum is
the causative agent of bacterial wilt disease in taxonomically diverse
plant hosts, and the biological heterogeneity of the species is
reflected in its genetic (4, 6), genomic, and biochemical
complexities (12, 13, 16). Genomic variation in many
bacterial species is generated by mobile DNA, which is common among
bacterial genomes (14). Insertion sequence (IS) elements are
the simplest transposable DNA in bacteria and have been major causes of
genomic changes, such as insertion, deletion, and inversion
(9). In pathogenic bacteria, IS elements modify the
expression and cause the transposition of genes controlling interactions of the pathogen with the host organism (8, 10, 15), suggesting an important role for DNA transposition and rearrangement in the evolution of host-pathogen interactions.
During a screening for repetitious DNA that was unique to biovar 2 strain ACH0158 of R. solanacearum (Table
1), we isolated and characterized three
novel IS elements, designated ISRso4, ISRso3, and
ISRso2. Phenotype conversion (PC) (2) was also investigated for potential association with the movement of an IS
element. PC is a phenomenon in which wild-type strains of R. solanacearum spontaneously lose their ability to produce large amounts of extracellular polysaccharide and a range of secreted proteins and become more motile, resulting in mutant strains with greatly reduced virulence (2). In several cases, PC has been reported to result from mutations in phcA (1, 2),
a global regulatory gene controlling the transcription of genes
associated with the virulence of the pathogen (18). In this
study, ACH0158-M81C (Table 1) was identified as a PC mutant resulting
from the insertion of ISRso4 within phcA (1,
2).
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Novel Insertion Sequence Elements Associated with
Genetic Heterogeneity and Phenotype Conversion in Ralstonia
solanacearum
ABSTRACT
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TABLE 1.
Bacterial strains and plasmids
Isolation of IS elements.
Plasmid clones of Sau3AI-
and SalI-digested genomic DNA of the wild-type R. solanacearum biovar 2 strain ACH0158 were prepared with
pBluescript or pUC19 (Table 1), and duplicate DNA blots from colonies
were hybridized with [
-32P]dATP-labeled total genomic
DNA from ACH0158 and ACH0171 (a taxonomically distant biovar 3 strain).
Colonies showing strong hybridization to the ACH0158 probe and little
or no hybridization to the ACH0171 probe were selected for further
characterization. Several such clones were sequenced, and those that
showed similarity to known IS elements were used as probes for a
plasmid library of EcoRI-digested genomic DNA from ACH0158
to yield clones containing full-length IS elements. A 2.7-kb
EcoRI fragment (pSV102; Table 1) was sequenced and found to
contain two adjacent IS elements, designated ISRso4 and
ISRso3. Similarly, sequencing of a 5.2-kb EcoRI
fragment (pISBE; Table 1) revealed a third IS element, designated
ISRso2.
Characterization of IS elements. ISRso4 was readily identified because of its similarity to known IS elements, such as IS1031 (7). It is 855 bp long and has perfectly matched 17-bp inverted repeats (IRs) flanked by 4-bp CTAG direct repeats (DRs). The deduced amino acid sequence for a putative transposase within ISRso4 showed similarity to those of several IS elements belonging to the IS5 family from various bacterial species (17), including IS1031 from Acetobacter xylinum (35.2% identical, 57.2% similar). Amino acid sequences for the putative transposase of ISRso4 and those of the homologous IS elements had two conserved domains (17), N3 with D+I(G/A)(Y/F) and C1 with R+3E as invariant motifs that are typical features of the IS5 family.
The second element, ISRso3, is 1,209 bp long and has imperfect 18-bp IRs with 4 nucleotide mismatches and 4-bp CTAG DRs. The 3' DR of ISRso3 is shared with the 5' DR of ISRso4. For ISRso3, the amino acid sequence of the predicted transposase showed about 50% identity and 70% similarity to those of IS elements from distantly related Pseudomonas species and Xanthomonas campestris pv. vesicatoria. This second group also shared the typical features of the IS5 family, but the core sequences of two domains were further apart than in ISRso4 (compare Fig. 1A and 1C). Therefore, ISRso3 is also a member of the IS5 family but has significant differences from ISRso4 in sequence and size.
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, IRs of each
IS element; 
, ORFs of each IS element; 
, locations of the
signatures for N3 [D+I(G/A)(Y/F)]; 
, C1 (R+3E) motif. The arrow
indicates the direction of transcription.
Distribution of IS elements.
To ascertain the copy numbers and
sequence distribution of the IS elements within the R. solanacearum genome and to look for differences between
genetically different isolates, the three IS elements were used
individually as probes in Southern analyses. ISRso4
hybridized to six EcoRI fragments in three biovar 2 strains (Fig. 2A) and was concluded to be present
in at least six copies because of the lack of internal EcoRI
sites in its sequence. In these biovar 2 strains, ISRso3
hybridized to numerous (>40) EcoRI fragments (Fig. 2B) and
ISRso2 hybridized to approximately 12 fragments (Fig. 2C).
For ISRso3 and ISRso2, these copy numbers may be
underestimated because of the possibility of multiple copies of the
elements in the more strongly hybridizing fragments. The differences in
hybridization intensity may reflect the presence of truncated fragments
or relics that have diverged sufficiently in sequence to reduce the
level of hybridization at high stringency. As we have sequenced only
single examples of ISRso3 and ISRso2, we cannot
distinguish between these possibilities.
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ISRso4 disrupts the phcA gene in a PC mutant. To investigate the possibility of active transposition of these IS elements, the behavior of ISRso4 during PC was examined (2). Three spontaneous PC mutants were isolated from wild-type ACH0158, and their EcoRI-digested genomic DNA was probed with ISRso4 in Southern analyses. The hybridization pattern of ACH0158-M81C showed an additional 2.1-kb band (Fig. 2D, lane 7) compared with the patterns of the wild type (Fig. 2D, lane 6) and the other two PC mutants (Fig. 2D, lanes 8 and 9).
The regions flanking the new insertion in ACH0158-M81C were obtained by inverse PCR and cloned as pM81C (Table 1). Sequence comparisons of this clone and DNA databases revealed greater than 99% amino acid sequence identity to the product of the phcA gene of R. solanacearum strain AW1 (2). The phcA gene has been characterized as a global regulator of virulence genes and as being central to the mechanism of PC (1, 3). Brumbley et al. (1) characterized the PhcA protein as a member of the LysR family of transcriptional regulators that control the expression of genes encoding virulence factors (3, 18). Brumbley and Denny (2) observed hybridization of the sequences flanking phcA to repetitious DNA in the genomes of AW1 and related strains. The new copy of ISRso4, integrated between nucleotide positions 120 and 121 within the phcA gene of ACH0158-M81C, created 5-bp (CTGAG) DRs (results not shown), and the sequence differed from the CTAG DRs in pSV102 and the CTAG DRs in a third, independently isolated copy of ISRso4 (results not shown). Variations in target duplication length and sequence at the point of insertion are not unusual (9) and may be influenced by different helix conformations of the nucleotide sequence of the phcA gene during the initial cleavage by ISRso4. However, additional variations in the length and sequence of the DR motif between different genomic copies of ISRso4 cannot be ruled out, as we have characterized only three of seven possible insertion sites. These results suggest that IS elements contribute to genomic variation and heterogeneity among strains and also to significant phenotypic changes related to virulence in R. solanacearum. The phcA genes were also cloned from wild-type ACH0158 and two additional PC mutants (ACH0158-M3 and ACH0158-M8) in clones pWT, pM3, and pM8, respectively (Table 1). The two PC mutants showed sequence lesions within phcA, confirming the importance of this gene in PC. However, ISRso4 was not involved, and neither were ISRso3 and ISRso2. ACH0158-M3 (Table 1) had between nucleotides 23 and 155 a 132-bp deletion that removed the start codon of phcA (results not shown). ACH0158-M8 (Table 1) contained at nucleotide position 648 in the ORF of phcA a 2-bp (TG) insertion causing a frameshift that resulted in an early stop codon and a truncated protein similar to that previously described for the phcA1 allele from a spontaneous PC mutant, AW1-PC (1). Therefore, the movement of ISRso4 contributes to a range of different mutations that cause PC. However, the transposition of DNA into phcA is not a common cause of PC. Southern analysis indicated that genomic DNA of only 1 in 10 PC mutants probed with phcA showed a band shift consistent with IS element insertion (results not shown). The mutation in ACH0158-M3 is clearly irreversible, whereas the reversibility of PC may be important in vivo. The latter is possible in ACH0158-M81C by perfect IS element excision. However, so far we have been unable to demonstrate the restoration of wild-type ACH0158 from either ACH0158-M81C or ACH0158-M8. It is possible that many insertions are merely "dead ends"
giving rise to cells which
not only are incapable of infection but also have lost the capacity to
regain these functions.
Concluding remarks. Southern hybridization and sequence analyses suggested that the IS elements isolated in R. solanacearum may have been horizontally transferred because of the presence of highly homologous IS elements in very distantly related bacterial taxa and the lack of a clear lineage for the IS elements within diverse strains of R. solanacearum.
Only a single member of each element was fully characterized by sequencing, and evidence for the presence of other genomic copies was obtained from Southern hybridization. We describe one event where the transposition of ISRso4 to a new genomic location causes a specific gene mutation and a consequent change in phenotype. It remains to be fully explored whether these IS elements are all actively involved in genomic rearrangements that contribute to the extensive genetic heterogeneity among different isolates of this species. The availability of mechanisms, in the form of multiple IS elements, that enable fast and extensive genome changes may be essential in R. solanacearum and in other bacterial species that constantly modify the expression of genes responsible for host-pathogen interactions (8), pathogenicity (10), and metabolic pathways (15), either by mutation or regulation.Nucleotide sequence accession numbers. The nucleotide sequences of ISRso4, ISRso3, and ISRso2 have been deposited in GenBank under accession numbers AF079849, AF183890, and AF186082, respectively. The GenBank accession number for the phcA gene from R. solanacearum strain ACH0158 is AF184046.
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ACKNOWLEDGMENTS |
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We thank The University of Adelaide for an international postgraduate research scholarship to E.-L. Jeong. This study was supported in part by grant PN9452 from the Australian Centre for International Agricultural Research.
We thank Tim Denny, Viji Krishnapillai, Chris Hayward, and Mark Fegan for comments on the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Genetics, The University of Adelaide, Adelaide, South Australia 5005, Australia. Phone: (61) 8 8303-3013. Fax: (61) 8 8303-4399. E-mail: eljeong{at}genetics.adelaide.edu.au.
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Journal of Bacteriology, August 2000, p. 4673-4676, Vol. 182, No. 16
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