Institut für Infektiologie, Zentrum für Molekularbiologie der Entzündung (ZMBE), Universitätsklinikum Münster, von-Esmarch-Str. 56, 48149 Münster, Germany
Correspondence
Gerhard Heusipp
heusipp{at}uni-muenster.de
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ABSTRACT |
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INTRODUCTION |
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Mutants of Salmonella typhimurium lacking the Dam enzyme are avirulent in mice, suggesting a role for DNA adenine methylation in virulence of this pathogen. Dam is involved in the regulation of a subset of specifically in vivo induced (ivi) genes. The expression of more than 20 ivi genes was significantly repressed in Salmonella dam mutant strains (Heithoff et al., 1999). Furthermore, the dam mutant strain was unable to disseminate to deeper tissues in mice and could be successfully used as a live vaccine against salmonellosis in different animal models (Dueger et al., 2001
, 2003
; Garcia-Del Portillo et al., 1999
; Heithoff et al., 1999
, 2001
). Recently it was shown that the pathogenicity of Vibrio cholerae, Yersinia pseudotuberculosis, Pasteurella multocida and Haemophilus influenzae is also affected by DNA adenine methylation. As in Salmonella, Dam-overproducing strains of Y. pseudotuberculosis confer protective immunity in mice. Additionally, Dam overproduction leads to expression and secretion of Yop virulence proteins under non-permissive conditions (Chen et al., 2003
; Julio et al., 2001
, 2002
; Watson et al., 2004
).
Although a role for Dam in the regulation of virulence has been described for various pathogens, the underlying molecular mechanisms and regulatory networks have mainly been analysed in E. coli. We cloned the dam gene of the human-pathogenic bacterium Y. enterocolitica in an initial effort to characterize the role of DNA methylation in Y. enterocolitica compared to the E. coli model and in virulence gene expression. We show that in contrast to E. coli, Dam is essential for growth in Y. enterocolitica. This is possibly linked to an altered expression of essential genes, but not to Dam's role in mismatch repair. Initial experiments support the anticipated role of Dam in virulence factor expression in Y. enterocolitica. Our studies contribute to the elucidation of the multifaceted role of DNA methylation in Y. enterocolitica.
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METHODS |
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Construction of mutant strains.
For the construction of a Y. enterocolitica dam mutant strain, a 2 kb EcoRI fragment encoding the (SmR/SpR) cassette of pSmUC was ligated into the unique EcoRI site of the dam gene in pBS-dam1/2, resulting in pBS-dam : : Sm. This plasmid was used as a template in a PCR to amplify a dam : :
(SmR/SpR) fragment using the primers GH-dam1 and GH-dam2, and Pfu polymerase. The PCR product was phosphorylated by polynucleotide kinase and ligated into the SmaI site of pEP185.2 (Kinder et al., 1993
), resulting in pEP-dam : :
(SmR/SpR). This suicide plasmid was transferred to Y. enterocolitica JB580v by conjugation and integrated into the chromosome by homologous recombination after selection for chloramphenicol and streptomycin resistance. The result was a merodiploid dam+-dam : :
(SmR/SpR) strain. This was confirmed by Southern blot analysis. Subsequently, cycloserine enrichment was used in an effort to isolate CamS SmR SpR exconjugants as previously described (Kinder et al., 1993
). To show that the dam gene is essential, plasmid pVLT-dam1/2 was transferred to the merodiploid dam+-dam : :
(SmR/SpR) strain, and CamS exconjugants were enriched by treatment with cycloserine. The presence of the dam : :
(SmR/SpR) genotype on the chromosome was analysed by Southern blotting of CamS SmR SpR KanR exconjugants (data not shown). For the complementation experiment with the E. coli dam, a 950 bp fragment containing the E. coli dam ORF was amplified by PCR using the primers SF-ECdam2 and SF-ECdam5 and plasmid pTP166 as template, digested with EcoRI and cloned into the EcoRI-digested vector pVLT33, resulting in plasmid pVLT-ECdam2/5. Correct orientation of the insert was confirmed by a restriction enzyme digest. The expression of a functional E. coli Dam was confirmed in the E. coli dam mutant strain GM2163 as described above. Plasmid pVLT-ECdam2/5 was transferred to the merodiploid dam+-dam : :
(SmR/SpR) strain by conjugation. Subsequently, cycloserine enrichment was used to generate CamS SmR SpR KanR exconjugants. The presence of the mutant genotype was confirmed by Southern blot analysis and PCR.
Construction of plasmids.
The high-copy-number plasmid pTP166 carries the E. coli dam gene under the control of the Ptac promoter (Marinus et al., 1984). For use in Y. enterocolitica, the bla gene of pTP166 was removed by digestion with DraI and AatII. A blunt-ended 2·2 kb EcoRI fragment encoding the kanamycin-resistance cassette of pCNB5 (De Lorenzo et al., 1993
) was ligated to the remaining part of pTP166 treated with T4 DNA polymerase, resulting in plasmid pTP166Kan. Plasmid pTP166Kan-dam
was generated by digestion of pTP166Kan with MluI and EcoRI, treatment with T4 DNA polymerase and religation, thereby deleting a 1·2 kb fragment containing the Ptac promoter and the 5' part of the dam gene. Plasmids were transferred to Y. enterocolitica by electroporation.
For the construction of a Y. enterocolitica Dam-overproducing strain, the E. coli dam gene from pTP166 was removed as a 1·2 kb XbaI/PvuII fragment. The 930 bp KpnI/XbaI fragment containing Y. enterocolitica dam from pBS-dam1/2 was treated with T4 DNA polymerase to produce blunt ends and cloned into the remaining pTP166 fragment, which had been treated with T4 DNA polymerase, resulting in pTP166-YEdam. For use in Y. enterocolitica, the kanamycin-resistance cassette from pCNB5 (De Lorenzo et al., 1993) was excised as a 2·2 kb EcoRI fragment, treated with T4 DNA polymerase and cloned into pTP166-YEdam, which had been digested with PstI and treated with T4 DNA polymerase, generating pTP166Kan-YEdam. For use as a control plasmid, pTP166Kan-YEdamrev was constructed based on pTP166Kan-YEdam by insertion of the dam fragment in the opposite orientation. Plasmids were electroporated into Y. enterocolitica.
Analysis of frequencies of spontaneous mutation and of resistance to 2-aminopurine (2-AP).
All assays were performed in triplicate as previously described (Ostendorf et al., 1999). For the determination of spontaneous mutation frequencies, appropriate dilutions of overnight cultures in LB were plated on selective (LB containing 30 µg streptomycin ml1 or 710 µg chloramphenicol ml1) and nonselective (viable count) media and incubated at 26 °C for 23 days in the presence of 1 mM IPTG to induce dam expression from Ptac. The mutation frequency is expressed as the ratio of the number of resistant cells to the number of viable cells.
The influence of 2-AP on survival (based on the frequency of lethal mutations) was determined as described by Ostendorf et al. (1999) with the following modifications. Overnight cultures of Y. enterocolitica strains grown in LB at 26 °C were diluted to 2x107 cells ml1. 2-AP was added to 10 ml of culture at final concentrations of 0, 10, 50, 100 and 200 µg ml1. The cultures were then incubated with aeration for 3 h at 26 °C before the bacterial cells were collected, washed twice in LB and resuspended in 1 ml LB. Appropriate dilutions were plated on LB agar, cultivated for 2 days at 26 °C and viable counts were determined.
Construction of transcriptional fusions.
All transcriptional fusions to the lacZ gene were constructed in pFUSE or in pKN8, a pFUSE derivative containing a BglII restriction site instead of the SmaI site (Bäumler et al., 1996). Both vectors contain an R6K origin of replication, which is not functional in Y. enterocolitica. Therefore, constructs integrate into the chromosome by homologous recombination after conjugation to Y. enterocolitica and selection for CamR. Integration of the plasmids was confirmed by Southern blot analysis (data not shown).
For the construction of (fyuA : : lacZ),
(rscR : : lacZ) and
(mdoH : : lacZ) fusion strains, approximately 650 bp fragments containing the 5' end of the respective gene including the promoter region were amplified by PCR using the primer pairs SF-fyu1/SF-fyu2, SF-rsc1/SF-rsc2 and SF-mdo1/SF-mdo2, respectively (Table 2
), with genomic DNA of Y. enterocolitica JB580v as template. The PCR products were digested with BglII and XbaI, and ligated into BglII/XbaI-restricted pKN8, resulting in pKN8-fyuA, pKN8-rscR and pKN8-mdoH, respectively, which were subsequently conjugated into Y. enterocolitica JB580v.
For the construction of the (hreP : : lacZ) strain, a 1 kb EcoRV fragment of pWSK-4J7opp was ligated into SmaI-digested pFUSE, resulting in pFUSE-hreP. The correct orientation of the insert was analysed by restriction enzyme digestion. Subsequently, pFUSE-hreP was conjugated into Y. enterocolitica JB580v.
-Galactosidase assay experiments.
-Galactosidase assays were performed as previously described (Miller, 1972
). Overnight cultures grown in LB at 26 °C were diluted 1 : 20 in fresh medium and subcultured for 3 h at 26 °C or at 37 °C. The bacterial cells were collected by centrifugation and washed in 0·85 % (w/v) NaCl before enzyme activity assays. Enzyme activities are expressed as arbitrary Miller units and were averaged from at least three independent experiments, each performed in triplicate.
Tissue culture invasion assays.
Invasion assays were performed using CHO cells as previously described (Miller & Falkow, 1988). Y. enterocolitica strain JB580c, which is cured of the pYV virulence plasmid, carrying either pTP166Kan (GHY151) or pTP166Kan-dam
(GHY148) was grown for 1518 h at 26 °C before infection. Assays were repeated six times, each in triplicate, and mean results are reported as percentage invasion [=100x(number of bacteria resistant to gentamicin/initial number of bacteria)] with GHY148 referred to as the wild-type.
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RESULTS |
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Complementation of an E. coli dam mutant strain with the Y. enterocolitica dam gene
To confirm functionality of the cloned dam gene, we subcloned a 930 bp fragment of pBS-dam1/2 containing the Y. enterocolitica dam ORF into pVLT33 under the control of an inducible Ptac promoter, resulting in pVLT-dam1/2, and introduced it into the dam mutant strain E. coli GM2163. Plasmid DNA isolated from E. coli GM2163 carrying either pVLT33 or pVLT-dam1/2 and grown in the presence of IPTG was analysed for DNA methylation by a restriction digest with Sau3AI or MboI. Both enzymes recognize the sequence GATC. While Sau3AI cleaves this sequence independent of its methylation status, MboI only cleaves unmethylated DNA. DNA isolated from E. coli GM2163 carrying pVLT-dam1/2 could only be digested with Sau3AI but not with MboI, indicating methylation of GATC sequences, whereas DNA isolated from E. coli GM2163 carrying pVLT33 could be digested with Sau3AI and with MboI, confirming that the cloned DNA fragment from Y. enterocolitica encodes a protein with DNA adenine methylase activity similar to the Dam enzyme of E. coli.
Dam is essential for viability of Y. enterocolitica
Dam regulates a variety of physiological processes in the bacterial cell and in addition plays a role in virulence of several enteric bacteria. To analyse the function of Dam in Y. enterocolitica, we attempted to generate a Y. enterocolitica dam mutant strain. Strains lacking a functional dam gene have been described for E. coli, Salmonella typhimurium, Serratia marcescens and H. influenzae (Bale et al., 1979; Bayliss et al., 2002
; Ostendorf et al., 1999
; Torreblanca & Casadesus, 1996
). However, Dam is essential for viability in Y. pseudotuberculosis and Vibrio cholerae (Julio et al., 2001
). To construct a Y. enterocolitica dam mutant strain, an
(SmR/SpR) cassette was inserted into the unique EcoRI site of dam, cloned into pEP185.2 and mated into Y. enterocolitica JB580v. This led to several merodiploid conjugants with an integrated plasmid. However, despite intense and repeated efforts, a CamS SmR SpR exconjugant could never be obtained. This suggested that dam is essential and required for growth in Y. enterocolitica, as found for Y. pseudotuberculosis and V. cholerae. To confirm the essentiality of dam, pVLT-dam1/2 was introduced into a merodiploid dam+ dam : : pEP-dam : :
(SmR/SpR) strain. Subsequently, we screened for CamS SmR SpR exconjugants in which the second cross-over had occurred, leaving behind dam : :
(SmR/SpR) on the chromosome. When a wild-type copy of the dam gene was provided in trans, exconjugants with a
(SmR/SpR) insertion in the chromosomal dam gene could be generated; 57 % of all chloramphenicol-sensitive exconjugants were dam : :
(SmR/SpR) as confirmed by the SmR SpR phenotype, PCR and Southern blot analysis (data not shown). From these data we conclude that dam is an essential gene in Y. enterocolitica as in Y. pseudotuberculosis and V. cholerae.
Effect of Dam overproduction on spontaneous mutation frequency and resistance to 2-AP
In E. coli, strains which lack or overproduce the Dam enzyme show an increased spontaneous mutation frequency and are sensitive to base analogues like 2-AP. As dam mutant strains of Y. enterocolitica are not viable, we investigated the influence of Dam overproduction on mutation avoidance. As an initial control, we investigated whether overproduction of Dam might be detrimental for growth of Y. enterocolitica. No obvious growth defect could be observed in a strain overexpressing dam from a Ptac promoter during growth from lag through stationary phase. We then analysed the effect of the Dam enzyme on mutability in Y. enterocolitica. Overproduction of the Y. enterocolitica Dam enzyme from plasmid pTP166Kan-YEdam increased the spontaneous resistance to chloramphenicol and streptomycin by a factor of 4·4 and 1·6, respectively, in comparison to control cells carrying plasmid pTP166Kan-YEdamrev (Table 3). In addition to the increased spontaneous mutability, Dam-overproducing strains of Y. enterocolitica also showed an increased sensitivity towards 2-AP at concentrations above 150 µg ml1 (Fig. 1
). However, in comparison to the effects reported in E. coli and Serratia marcescens (Glickman, 1979
; Ostendorf et al., 1999
), the increase in sensitivity towards 2-AP related to Dam overproduction appears to be only marginal.
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Functional complementation of a Y. enterocolitica dam strain by the E. coli dam gene in trans
As dam is an essential gene in Y. enterocolitica but not in E. coli, and as overproduction of the E. coli Dam enzyme has a strong effect on spontaneous mutability compared to the Y. enterocolitica Dam enzyme, we were interested in determining if the E. coli dam gene is able to complement a dam mutant of Y. enterocolitica. Plasmid pVLT-ECdam2/5 carrying the E. coli dam gene under the control of the Ptac promoter was introduced into the merodiploid dam+-dam : : (SmR/SpR) Y. enterocolitica strain GHY121. As already shown for the complementation with the Y. enterocolitica dam gene, CamS SmR SpR exconjugants could be obtained. All chloramphenicol-sensitive exconjugants were dam : :
(SmR/SpR) as confirmed by the SmR SpR phenotype, PCR and Southern blot analysis (data not shown). From these data we conclude that the essentiality of Dam in Y. enterocolitica is not due to different activities in mismatch repair in comparison to Dam of E. coli. Instead, Dam is essential as an altered methylation pattern of GATC sequences in promoters interferes with the proper expression of essential genes in Y. enterocolitica, while in E. coli different subsets of (non-essential) genes are expressed in a Dam-dependent fashion.
The expression of hre loci is not influenced by Dam overproduction
Multiple aspects of virulence of various enteric pathogens are influenced by the expression of Dam (Chen et al., 2003; Low et al., 2001
; Watson et al., 2004
). As it was shown by Heithoff et al. (1999)
that the expression of at least 20 genes identified as induced during an infection (the so-called in vivo-induced or ivi genes) is altered in a dam mutant strain of Salmonella typhimurium, we were particularly interested in the effect of Dam overproduction on expression of hre loci of Y. enterocolitica specifically expressed during an infection in the mouse model of yersiniosis (Young & Miller, 1997
). We used transcriptional fusions of the lacZ gene to the hre genes rpoE, hreP, rscR, mdoH and fyuA, and analysed their expression after overproduction of Dam. These fusions were chosen as they represent genes involved in various aspects of virulence of Yersinia and other pathogens (RpoE is an extracytoplasmic function sigma factor; HreP is a protease; RscR is a regulator involved in systemic dissemination of Y. enterocolitica; MdoH is involved in biosynthesis of membrane-derived oligosaccharides; FyuA is an iron siderophore receptor). As shown in Table 4
, overproduction of Dam had no significant effect on the expression of the hre genes investigated. We conclude from these data that Dam overproduction has no general regulatory effect on the expression of the in vivo-expressed genes of Y. enterocolitica analysed here.
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DISCUSSION |
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The dam gene was identified in the unfinished sequence of Y. enterocolitica by identity to the E. coli dam gene. The genes share an identity of 67 %, which is even more pronounced at the protein level (70·6 %). Conserved motifs of aminomethyltransferases are present in the correct sequential order, placing Dam of Y. enterocolitica in the group of N6-adenine aminomethyltransferases (Malone et al., 1995
). The complementation of an E. coli dam mutant with dam of Y. enterocolitica implies a functional homology between the two Dam proteins, as the dam strain retained its ability to methylate adenine in GATC sequences. Moreover, overproduction of the E. coli Dam protein in Y. enterocolitica affects the spontaneous mutation frequency and the resistance to the base analogue 2-AP, comparable to the effect of Dam overproduction in E. coli. However, the effect is less pronounced when the Y. enterocolitica Dam is overproduced, indicating similarities as well as differences in methyl-directed mismatch repair between E. coli and Y. enterocolitica. This is in agreement with previous data obtained from H. influenzae, where a dam mutant strain showed no significant difference in mutation frequency from the wild-type (Bayliss et al., 2002
; Watson et al., 2004
). Data from studies on the methyl-directed mismatch repair in E. coli as a model organism cannot be transferred in every detail to other species. Even in Shigella flexneri, a phylogenetically very close relative of E. coli, a dam mutant strain has a 1000-fold increased spontaneous mutation frequency compared to 20- to 80-fold in E. coli (Honma et al., 2004
). Obviously, although bacteria use similar mechanisms of methyl-directed mismatch repair, the exact function or activity of Dam on the molecular level in this process differs between bacterial species. This needs to be analysed in more detail to gain a better understanding of the role of DNA methylation in mutation avoidance.
In Y. pseudotuberculosis and V. cholerae, Dam is essential for growth (Julio et al., 2001). However, overproduction of Dam does not lead to a detectable growth defect. This is also true for Dam of Y. enterocolitica. Interestingly, a dam mutant strain could be constructed in E. coli, Salmonella typhimurium, Serratia marcescens and H. influenzae (Bayliss et al., 2002
; Marinus & Morris, 1973
; Marinus et al., 1983
; Ostendorf et al., 1999
; Torreblanca & Casadesus, 1996
). It remains to be determined why Dam is essential for viability in some
-proteobacteria, but not in others. The phenotype of an E. coli dam strain is pleiotropic (Marinus, 1996
; Oshima et al., 2002
). Although this has not been studied in detail, it probably also holds true for other bacteria, and therefore it cannot be easily determined which function affected by Dam is important for viability in one bacterial species but not in others. Our studies imply that the role of Dam in methyl-directed mismatch repair is not the reason for essentiality in Y. enterocolitica, although its activity or function seems to be different in this process compared to E. coli. Rather, our data imply that GATC methylation affects the expression of different subsets of genes in different species, and that some or at least one gene(s) affected in Y. enterocolitica, and putatively also in Y. pseudotuberculosis and V. cholerae, are essential for growth.
Besides physiological functions in mutation avoidance and regulation of replication, Dam is also involved in the regulation of virulence-associated genes of diverse human, animal and plant pathogens (Chen et al., 2003; Heithoff et al., 1999
; Julio et al., 2001
; Low et al., 2001
; Watson et al., 2004
). In an initial approach to study the effect of Dam on virulence gene expression in Y. enterocolitica, the specifically in vivo-expressed genes (hre genes) fyuA, hreP, mdoH, rpoE, and rscR (Young & Miller, 1997
) were analysed in wild-type and Dam-overproducing strains for differences in expression, as expression of in vivo-induced genes (ivi) of Salmonella typhimurium was influenced by DNA adenine methylation (Heithoff et al., 1999
). Interestingly, in contrast to Salmonella, disturbance of the DNA methylation pattern had no effect on expression of the hre genes examined, indicating involvement of different regulatory mechanisms in expression of in vivo-induced genes in Y. enterocolitica and Salmonella typhimurium. Alternatively, the lack of an effect of Dam overproduction on hre expression in Y. enterocolitica might also be due to the relatively small number of hre genes in comparison to the ivi genes analysed in the Salmonella study (Heithoff et al., 1999
).
The effect of Dam overproduction on invasion of epithelial cells shows that in Y. enterocolitica not only is Dam involved in physiological processes like mismatch repair and mutation avoidance, but DNA methylation also influences virulence factor expression. In previous studies with Y. pseudotuberculosis, a Dam-overproducing strain did not colonize mucosal tissues of mice differently from the wild-type (Julio et al., 2002). This might imply that invasion is not changed in Y. pseudotuberculosis or that this phenotype is not detectable under in vivo conditions. Therefore it will be interesting to analyse tissue colonization and virulence of a Dam-overproducing strain of Y. enterocolitica in the mouse model of infection.
As we used pYV-cured strains for the invasion assay, we can exclude that the effect of Dam overproduction on invasion depends on the virulence plasmid encoded invasin YadA, implying that Dam acts on a chromosomally encoded invasin like Inv or Ail. This will be addressed in future experiments together with a molecular analysis of regulatory mechanisms underlying the phenotype.
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ACKNOWLEDGEMENTS |
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Received 4 February 2005;
revised 21 March 2005;
accepted 24 March 2005.
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