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Yersinia Research Unit, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France
Correspondence
Elisabeth Carniel
carniel2{at}pasteur.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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yamA mutant of Y. pseudotuberculosis IP32953 was constructed by allelic exchange with a kanamycin cassette. The fact that yamA mutants were obtained indicated that this gene is not essential for Y. pseudotuberculosis viability. The IP32953yamA mutant strain grew as well as the wild-type in a rich medium at both 28 °C and 37 °C. It also grew normally in a chemically defined medium at 28 °C, but exhibited a growth defect at 37 °C. In contrast to the Dam adenine methyltransferase, a mutation in yamA did not impair the functions of DNA repair or resistance to detergents. However, the yamA mutant exhibited a virulence defect in a mouse model of intragastric infection. The in silico analysis indicated that the chromosomal region carrying the Y. pseudotuberculosis yrmI locus has been replaced in Y. pestis by a horizontally acquired region which potentially encodes another methyltransferase. YamA might thus be dispensable for Y. pestis growth and virulence because this species has acquired another gene fulfilling the same functions.
Two supplementary tables and a sequence alignment are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Heusipp et al., 2007; Kobayashi, 2001; Murray, 2002; Noyer-Weidner & Trautner, 1993; Wion & Casadesus, 2006). RM systems are thought to protect bacteria against invading foreign DNA (phages, plasmids, etc.) (Kobayashi, 2001). They are classically classified as type I, II, or III on the basis of their subunit number and organization, cofactor requirements, enzymic mechanism and sequence specificity (Kessler & Manta, 1990; Murray, 2002). Type I systems are the most complex since they are formed of a single protein which is composed of three heterologous subunits responsible for DNA binding (HsdS), methylation (HsdM) and restriction (HsdR). Their activity requires S-adenosyl-L-methionine (AdoMet), ATP and Mg2+ as cofactors (Sistla & Rao, 2004). DNA cleavage can take place at variable sites, several hundred base pairs away from the recognition site. Type II RM systems are the simplest, most common, and best-studied systems. They consist of two separate restriction and modification enzymes, which function independently and require only Mg2+ as a cofactor. Type III RM systems are formed by a complex of the MTase and the restriction enzyme and require AdoMet, ATP and Mg2+ as cofactors. The MTase provides DNA recognition for both restriction and modification and catalyses modification independently of the restriction enzyme. DNA cleavage requires a complex of both subunits and occurs at a specific site approximately 25 bp from the recognition site. A type IV class of RM systems has been recently described. In these systems, the restriction endonuclease only cleaves DNA substrates that have been modified (Tock & Dryden, 2005).
Some DNA MTases are also found in bacteria in the absence of any cognate restriction endonuclease and participate in various processes such as DNA mismatch repair, DNA replication and gene regulation (Heitman, 1993; Kessler & Manta, 1990). One of the best-studied examples of a solitary adenine methylase is Dam, a type II MTase, which places a methyl group on the adenine of the sequence 5'-GATC-3' (Barras & Marinus, 1989; Messer et al., 1988). Dam plays a number of key roles in bacterial processes, including mismatch repair, timing of DNA replication, and transcription of some genes (Noyer-Weidner & Trautner, 1993; Wion & Casadesus, 2006). dam interruption alters the repair system that discriminates between the parental and daughter DNA strands, resulting in an increased rate of spontaneous mutations in Escherichia coli (Barras & Marinus, 1989). Dam also acts as a global regulator of gene expression in E. coli (Barras & Marinus, 1989; Oshima et al., 2002), and in Salmonella it represses the expression of at least 20 in vivo-induced genes (Heithoff et al., 1999).
A Dam homologue has been identified in Yersinia pestis, the causative agent of plague, and in the two enteropathogens Yersinia pseudotuberculosis and Yersinia enterocolitica. Dam was found to be essential for the viability of Y. pseudotuberculosis strain YPIII and its overproduction led to an attenuation in mouse virulence (Julio et al., 2001). Dam overproduction also resulted in a dysregulation of Yop expression and secretion in Y. pseudotuberculosis and Y. enterocolitica (Falker et al., 2006; Julio et al., 2001, 2002). However, dam inactivation was not lethal in another strain of Y. pseudotuberculosis (IP32953) (Taylor et al., 2005) or in Y. pestis (Robinson et al., 2005), but it resulted in a dramatic increase in the 50 % lethal dose (LD50) of both species in a mouse model of infection. The fact that, at least in Y. pseudotuberculosis, the dam mutation was accompanied by the concomitant loss of the pYV virulence plasmid (Taylor et al., 2005) may explain the virulence defect observed.
With the exception of Dam, no other DNA MTase has been identified and characterized in Y. pseudotuberculosis. An analysis of the genome sequence of Y. pseudotuberculosis IP32953 allowed the identification of one RM locus composed of three genes (YPTB0535–YPTB0537) which was previously shown to be absent from the genome of 13 strains of Y. pestis tested (Chain et al., 2004). The aim of this study was to further investigate the distribution of this locus in the species Y. pestis and Y. pseudotuberculosis, to determine whether the newly identified MTase is important for Y. pseudotuberculosis survival and growth in vitro, and to estimate its influence on the virulence of this species.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. The Y. pseudotuberculosis strains used for PCR screening are listed in Supplementary Table S1, available with the online version of this paper. Bacteria were grown in Luria–Bertani (LB), or M63 [KH2PO4, 0.1 M, (NH4)2SO4, 0.2 %, MgSO4, 0.02 %, FeSO4, 0.00005 %] with 0.2 % glucose and 0.2 % Casamino acids broth (M63S) and on LB or LB-Sac (without NaCl and supplemented with 10 % sucrose) agar plates. Yersinia strains were grown at 28 °C or 37 °C and E. coli strains at 37 °C. Chloramphenicol (Cm: 25 µg ml–1), kanamycin (Kan: 30 µg ml–1) or spectinomycin (Spe: 50 µg ml–1) were added to the media when necessary. To measure bacterial growth, bacteria were cultivated overnight in LB or M63S at 28 °C or 37 °C and a bacterial inoculum of OD600 0.03 was seeded in the same medium and grown at the same temperature as used for precultivation.
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) and Y. pseudotuberculosis IP32953 (Chain et al., 2004) were compared pairwise using the Artemis Comparison Tool (ACT) (http://www.sanger.ac.uk/Software/ACT). DNA and protein sequences were analysed with BLAST programs (Altschul et al., 1997) and on http://www.genome.jp/kegg-bin/show_genomemap_top?org_id=yps. RM systems were studied using REBASE (http://rebase.neb.com).
DNA and gene manipulation.
Genomic and plasmid DNA were extracted with the Isoquick nucleic acid extraction Kit (Isoquick) and the Qiagen Plasmid Maxi Kit (Qiagen), respectively. Mutagenesis of the chromosomal target gene was performed by the 3LFHR-PCR procedure described by Derbise et al. (2003). Recombinant plasmid DNA and PCR fragments were introduced by transformation into E. coli or by electroporation into Yersinia as described by Conchas & Carniel (1990).
PCR.
The primers used in this study are listed in Supplementary Table S2, available with the online version of this paper. PCRs were performed with 1 unit of Taq polymerase (Roche) or 1 unit of a 1 : 2 mixture of Taq and Isis (QBiogen) polymerases in the supplier's buffer. PCR amplification reaction mixtures contained 10 µM of each primer and 1 mM of dNTPs. The PCR programme involved one step at 95 °C for 5 min, followed by three steps of 30 cycles of amplification at (i) 95 °C for 30 s, (ii) 55 °C for 30 s, and (iii) 72 °C for 1–3 min, depending on the fragment length. PCR products were maintained at 72 °C for 5 min, subjected to electrophoresis in 1 % agarose gels, and stained with ethidium bromide. Amplification of the kan cassette with long flanking homologous regions of the Yersinia target DNA was done with the LFHR-PCR programme described by Derbise et al. (2003).
Deletion of yamA from the chromosome of Y. pseudotuberculosis IP32953.
Primer pairs 336G/393A and 393B/336H were used to amplify the 5' and 3' extremities of yamA and flanking regions, respectively (Fig. 1), and primer pair 136A/136B to amplify the kan cassette (Supplementary Table S2). Following the LFHR-PCR procedure (Derbise et al., 2003), a linear PCR fragment composed of the kan gene with its promoter region and flanked by 
500 bp homologous to the regions located on each side of yamA was generated. This fragment was introduced by electroporation into strain IP32953(pKOBEG-sacB) and correct allelic exchange between the PCR fragment and the chromosomal gene was checked by PCR with primer pairs 400A/167, 400B/166 and 400A/400B (Fig. 2, Supplementary Table S2). IP32953 clones with the appropriate mutation were grown on LB-Sac agar plates to select for colonies cured of pKOBEG-sacB.
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yamA and selection of recombinant strains was done on Spe plates. The presence of the plasmid with the appropriate insert was checked after plasmid extraction and digestion with BamHI and HindIII.
Extraction of RNA and reverse transcription PCR (RT-PCR).
Bacteria grown in M63S at 37 °C were harvested at the early exponential phase (OD600 0.2). Ten millilitres of bacterial culture was centrifuged for 5 min at 6000 g at 4 °C. Pellets were suspended in 1 ml RNAwiz (Ambion), treated with 200 µl chloroform, vigorously shaken for 20 s, and incubated at room temperature for 10 min. The aqueous phase was recovered after centrifugation at 4 °C (12 000 g for 10 min) and diluted with 450 µl H2O. RNA was precipitated by the addition of 900 µl 2-propanol and pelleted by centrifugation at 12 000 g for 30 min at 4 °C. Pellets were rinsed with cold 70 % ethanol, dried for 10 min and suspended in 50 µl H2O. DNA was removed from the RNA samples by adding 4 U DNase following the procedure of the DNA-free Kit (Ambion). The quality of the RNA was checked by PCR amplification with primer pair 18/19 to ensure that the preparation did not contain any trace of contaminating DNA. RT-PCR was performed using the SuperScript One Step RT-PCR with the Platinum Taq Kit (Invitrogen). The reactions contained the supplier buffer (1x) mixed with 1 µl RT/Platinum Taq mix, 50 ng RNA template and 0.1 µM of primer pairs 0634F/0634R, 0536F/0536R or 0535F/0535R, amplifying an internal portion of yamA, yreA or yrsA, respectively. Each amplification reaction involved one step for 30 min at 50 °C, one step for 2 min at 94 °C and 30 cycles of three steps as follows: 94 °C for 15 s, 55 °C for 30 s, and 72 °C for 1 min.
Stability of the pYV plasmid.
The number of pYV-cured colonies of Y. pseudotuberculosis IP32953 and IP32953yamA was estimated after growth on MOX agar (0.25 M sodium oxalate, 0.25 M magnesium chloride, 1 M D-glucose, Brain-Heart Infusion) at 28 °C and 37 °C.
Determination of spontaneous mutation frequencies.
Three individual colonies from each strain were grown in LB at 28 °C for 18 h and the bacterial suspensions were diluted in phosphate-buffered saline (PBS) to obtain a concentration of 109 bacteria ml–1. Then 200 µl of each suspension was streaked on LB plates containing either rifampicin (100 µg ml–1) or nalidixic acid (50 µg ml–1). The mutation frequency was calculated as the ratio of the number of antibiotic-resistant colonies to the number of bacteria plated.
Sensitivity to bile salts and 2-aminopurine.
Y. pseudotuberculosis IP32953 and IP32953yamA were grown overnight at 28 °C in LB broth. The cultures were adjusted to 103 c.f.u. ml–1, and 100 µl of the bacterial suspension was plated in duplicate on LB plates containing either 1 % bile salts (Sigma), 400 µg ml–1 of the base analogue 2-aminopurine (2-AP) (Sigma), or no supplement. The plates were incubated at 28 and 37 °C for 48 h and colonies were counted.
Animal infections.
Prior to infections, the presence of known unstable elements such as the pYV plasmid and the high-pathogenicity island was systematically verified by PCR with primer pairs located on these elements: 18/19 (irp2) and 160A/160B (yopM) (Supplementary Table S2). To follow the kinetics of lethality, groups of ten 4-week-old C57BL/6 female mice (Janvier) were infected intragastrically (i.g.) with suspensions containing 104 Y. pseudotuberculosis cells. The LD50 (Reed & Muench, 1935) of the various strains tested was determined on groups of five 5-week-old C57BL/6 female mice infected i.g. with tenfold serial dilutions of the bacterial suspensions. Lethality was recorded daily for 3 weeks.
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RESULTS |
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) suggested that this locus is restricted to the Y. pseudotuberculosis group (i.e. lost by Y. pestis after its emergence), we decided to analyse this region further. A search for this locus on the Y. enterocolitica 8081 genome sequence (www.sanger.ac.uk/Projects/Y_enterocolitica/) did not reveal any similar locus. A detailed in silico analysis of the IP32953 genome indicated that this region is composed of three adjacent genes oriented in the same direction (Fig. 1a
). The first gene is YPTB0537, a coding sequence (cds) of 2592 nt which has the potential to encode an 863 aa product (97.3 kDa). This putative protein is highly similar (93 % aa identity, 96 % aa similarity) to the type I RM system M subunit of Serratia proteamaculans 568 (EAV32736) and possesses the signature sequence (VVSNPPY) of N6-adenine-specific DNA methylases (EC 2 . 1 . 1 . 72) as well as other domains characteristics of this family (see Supplementary Fig. S1, available with the online version of this paper). Based on this homology we designated this cds yamA (for Yersinia adenine methyltransferase A). The second cds (YPTB0536) is 1284 nt long and could encode a 427 aa protein (48.3 kDa). BLAST searches indicated that it shares the highest homology (43 % aa identity, 58 % aa similarity) with the putative type I restriction enzyme S subunit of Desulfitobacterium hafniense (AAAW04000001.1). This cds was thus called yrsA (for Yersinia recognition site A). The last gene, YPTB0535, is a 3249 nt cds which could encode a 1082 aa product (121.9 kDa). This gene is highly homologous (90 % aa identity and 94 % aa similarity) to the type I site-specific DNase HsdR subunit of S. proteamaculans 568 (EAV32734.1). We consequently designated it yreA (for Yersinia restriction enzyme A). Since the YPTB0535–YPTB0537 locus has the potential to encode the three subunits characteristic of type I RM systems, we called this system yrmI (for Yersinia restriction modification system, type I).
The yamA, yrsA and yreA genes have G+C contents of 45.6, 37.8 and 44.5 mol%, respectively, which are slightly lower than that of the core Y. pseudotuberculosis genome (48.9 %), suggesting that the system might have been acquired by lateral gene transfer. The regions flanking the yrmI locus are highly conserved in both Y. pseudotuberculosis IP32953 and Y. pestis CO92 despite a large inversion of the region downstream of yrmI in CO92. Interestingly, the yrmI locus of IP32953 has been replaced in Y. pestis by a specific region composed of 10 genes (YPO0387 to YPO0397), most of them of unknown functions except one (YPO0391) encoding a putative MTase (Chain et al., 2004; Hinchliffe et al., 2003).
Distribution of yrmI among Y. pestis and Y. pseudotuberculosis strains
A previous study based on the analysis of 12 strains of Y. pestis suggested that this species does not possess the yrmI region (Chain et al., 2004). In this work, the screening by PCR of a panel of 46 additional strains of Y. pestis of various geographical origins and biovars with primers internal to each of the three genes (Supplementary Table S2, Fig. 1a) confirmed that the RM system is absent from this species (data not shown). The analysis of 39 strains of Y. pseudotuberculosis of diverse serotypes and geographical origins, including the nine strains initially used for a preliminary screening, indicated that the three genes are not equally distributed among Y. pseudotuberculosis isolates (Supplementary Table S1). Four different patterns of gene distribution were observed (Fig. 1b). Only approximately one-third (39 %) of the Y. pseudotuberculosis isolates tested had the three genes (Fig. 1b, Supplementary Table S1). The yreA and yrsA genes were found in only 58 % and 52 % of the strains, respectively, while yamA was the only cds which was systematically present in all strains tested (Fig. 1b). No strict correlation between the geographical origin or serotype of the isolates and their pattern of gene distribution was noted (Supplementary Table S1).
The presence of yamA in all Y. pseudotuberculosis strains tested while either one or the two other components of the yrmI system are missing suggests that YamA is a solitary MTase which may have some specific functions, independently of YreA and YrsA. The maintenance of yamA despite the loss of the other two genes further suggests that this gene has been positively selected because it confers some advantages to Y. pseudotuberculosis during its epidemiological cycle.
Expression of the three genes composing the yrmI system
To determine whether yamA and the two other genes composing the yrmI system are transcribed in strain IP32953 (which possesses a complete gene cluster), RT-PCR experiments were performed after RNA extraction and amplification with primers internal to each gene (Fig. 1a). As shown in Fig. 2, a transcript was clearly seen for yamA, a detectable but weak band was visible for yrsA, and no signal was detected for yreA. These results thus indicate that yamA is effectively expressed in IP32953. In contrast, the transcription of the other two genes is either low or absent, at least under the experimental conditions used.
Construction of a Y. pseudotuberculosis yamA mutant
To investigate the role of YamA in Y. pseudotuberculosis, the yamA gene was replaced by allelic exchange with a non-polar kan cassette (Fig. 1a), yielding IP32953yamA. Correct allelic exchange was checked with primer pairs 400A/167, 400B/166 and 400A/400B (Supplementary Table S2, Fig. 1a). Absence of yamA transcript in the IP32953yamA mutant strain was confirmed by RT-PCR (Fig. 2). Since yamA is the first gene in the yrmI locus (Fig. 1), RT-PCR analyses were also performed to ensure that its interruption had no polar effect on the transcription of the yrsA and yreA downstream genes. As shown in Fig. 2, although yamA transcription was abolished in the IP32953yamA mutant, the intensity of the yrsA fragment was similar to that observed in the wild-type strain, confirming that mutagenesis of yamA did not affect the transcription of the downstream gene.
Effect of YamA on Y. pseudotuberculosis multiplication in vitro
In order to determine whether deletion of yamA could impair the ability of Y. pseudotuberculosis to multiply in vitro, both IP32953 and the yamA derivative were cultured in LB at 28 °C (optimal temperature for Yersinia growth) or 37 °C (in vivo temperature). In this medium, the two strains grew equally well at the two temperatures (data not shown). Similarly, no difference was observed when these strains were grown in a chemically defined medium (M63S) at 28 °C (data not shown). However, the yamA mutant exhibited a growth defect in M63S at 37 °C (Fig. 3a). To confirm that the observed growth defect of IP32953yamA was the consequence of yamA disruption, the functional yamA gene of IP32953 was cloned into the low-copy-number plasmid pAM239 (yielding pAM-yamA) and the recombinant plasmid was introduced into the deletant strain IP32953yamA. As shown in Fig. 3(b), introduction of pAM239 alone into the deletant strain IP32953yamA did not restore the ability to optimally grow in M63S at 37 °C, while trans-complementation with a functional yamA allele abolished the growth defect. This result confirms that the observed growth impairment was due to yamA inactivation and not to a polar effect of the mutation on the downstream genes.
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), we wondered whether yamA inactivation may have the same effect. As expected, after growth of the control pYV-cured IP32953 strain on MOX agar at 28 °C or 37 °C, the number of colonies was similar. In contrast, both IP32953 and IP32953yamA harbouring the pYV exhibited a temperature-dependent growth inhibition. While approximately 100 colonies of each strain were observed on MOX agar after growth at 28 °C, no colonies grew at 37 °C (data not shown). This indicates that, in contrast to dam, the yamA mutation does not impair pYV stability.
Impact of YamA on DNA repair in Y. pseudotuberculosis
Dam has been shown to play a role in mismatch repair and dam mutants are susceptible to agents that cause mismatches. To determine whether YamA may play the same role in Y. pseudotuberculosis, both IP32953 and its yamA derivative were grown in the presence of the base analogue 2-AP at both 28 °C and 37 °C. As shown in Table 2, the two strains grew equally well despite the presence of high concentrations of the base analogue (400 µg ml–1). To determine whether yamA participates in the control of the mutation rate of Y. pseudotuberculosis, the same inoculum of IP32953 or its yamA derivative was plated on nalidixic acid and rifampicin agar plates and the number of spontaneous antibiotic-resistant colonies was counted. In all instances, the mutation rate was in the region of 10–9 (Table 3), indicating that YamA has no major impact on the mutation rate of Y. pseudotuberculosis. Altogether, these results suggest that YamA is not involved in the methyl-directed mismatch repair mechanisms of Y. pseudotuberculosis.
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; Pucciarelli et al., 2002) and Y. pseudotuberculosis IP32953 (Taylor et al., 2005), a mutation in dam confers an increased sensitivity to detergents such as bile salts. To determine whether inactivation of yamA may also alter the resistance of IP32953 to detergents, the wild-type and the yamA mutant were grown at 28 or 37 °C in the presence of 1 % bile salts. In contrast to dam, inactivation of yamA did not impair the capacity of IP32953 to grow at both temperatures in the presence of this detergent (Table 2).
Effect of YamA on Y. pseudotuberculosis pathogenicity
In order to determine whether YamA plays a role in Y. pseudotuberculosis virulence, 104 c.f.u. of the wild-type strain IP32953 and the deletant IP32953yamA were injected i.g. to groups of 10 C57BL/6 female mice and mortality was recorded daily for 21 days. As shown in Fig. 4, all ten mice infected with the wild-type strain died, while only two animals infected with IP32953yamA did so (P<0.0003 with the 
2 test). These results demonstrate that YamA is necessary for the full virulence of Y. pseudotuberculosis by the oral route. To quantify the virulence defect, serial dilutions of the two strains were injected i.g. into groups of five mice and the LD50 values were determined. A 31-fold increase in LD50 was observed in the yamA mutant (8.8x104 c.f.u.) as compared to the wild-type strain (2.8x103 c.f.u.). To ensure that this defect was due to yamA inactivation and not to a polar effect on the downstream genes yrsA and yreA, mice were infected i.g. with 104 c.f.u. of the trans-complemented strain IP32953yamA(pAM-yamA) and their mortality was compared to that of the wild-type. The number of dead mice infected with IP32953yamA(pAM-yamA) (7/10) was slightly lower than that of IP32953 (10/10) but the difference was not statistically significant (P>0.05 with the 2 test), confirming that the virulence defect observed was the consequence of yamA inactivation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Murray, 2002; Noyer-Weidner & Trautner, 1993; Roberts & Macelis, 1998; Tock & Dryden, 2005). In type I systems, genes specifying the specificity (S) and modification (M) subunits of the complex are always adjacent, whereas that encoding the restriction (R) subunit can be separated from them by a few hundred base pairs. The restriction activity of an RM system cannot usually exist in a cell in the absence of its cognate MTase activity, since this would cause suicidal degradation of the cell DNA. However, the reciprocal situation – solitary existence of a MTase – can be encountered (Heusipp et al., 2007; Noyer-Weidner & Trautner, 1993; Wion & Casadesus, 2006). Remarkably, all Y. pseudotuberculosis strains tested have kept the MTase-encoding yamA gene, while more than 60 % of them have lost (or rearranged) either one or the two other genes (yrsA and yreA) forming the yrmI RM system. Similarly to Dam in E. coli, YamA may thus be considered as a solitary MTase in most Y. pseudotuberculosis strains. Since solitary MTases from only type II RM have been described and characterized until now, YamA may represent the first type I solitary MTase described.
Genomic analysis of a variety of bacterial systems suggests that many RM homologues are inoperative because of insertions, deletions or point mutations (Kong et al., 2000; Nobusato et al., 2000b). In some cases, the R gene is inactive while the neighbouring M gene seems to be functional (Ibanez et al., 1997). Along the same lines, our RT-PCR analysis of the yrmI system of IP32953 showed that yamA was much more expressed than yrsA, and that yreA was not transcribed (at least under the conditions used). The poor expression or silence of yrsA and yreA thus suggests that they are dispensable for the bacteria and may explain their loss in many strains. In contrast, the presence of yamA in all Y. pseudotuberculosis isolates tested and its high level of transcription argue for a role of yamA in the physiology and/or the pathogenicity of this species.
The N6-adenine MTase activity of YamA could not be confirmed because its methylation site is not known. Furthermore, it was not possible to substantiate the YamA-mediated protection of the restriction site from the cleavage by its cognate restriction endonuclease YreA since this subunit is either absent or probably inactive (as deduced from the absence of detectable transcript) in Y. pseudotuberculosis. Nonetheless, the high similarity of YamA with various bacterial type I RM system M subunits, as well as the presence of DNA methylase motifs and the signature sequence of N6-adenine-specific DNA methylases (EC 2 . 1 . 1 . 72) argue for such an activity.
In an attempt to determine whether YamA is important during some bacterial processes, yamA was deleted by allelic exchange from the chromosome of strain IP32953. The fact that yamA mutants were obtained indicates that this gene is not essential for Y. pseudotuberculosis viability. The yamA mutant was able to grow normally in vitro in a rich medium at 28 °C and 37 °C. Interestingly, a growth defect was observed in a chemically defined medium, but only when the bacteria were grown at 37 °C. This effect under these conditions, which could at least partly mimic the in vivo environment (nutrient limitation, host temperature), suggested that the mutant might be altered in its capacity to multiply in a host. Indeed, while most animals (80 %) infected i.g. with the yamA mutant survived the infection, all mice infected with the wild-type strain died. Our results thus demonstrate that YamA is important both for the growth under specific conditions and for the pathogenicity of Y. pseudotuberculosis.
Dam-deficient mutants of Salmonella and Y. pseudotuberculosis were shown to exhibit an increased sensitivity to detergents such as bile salts (Heithoff et al., 2001; Pucciarelli et al., 2002; Taylor et al., 2005). It has been suggested that this sensitivity might be due to a defect in the cell envelope and that it may be responsible in part for the reduced virulence of the mutants by the oral route of infection (Heithoff et al., 2001; Pucciarelli et al., 2002; Taylor et al., 2005). YamA does not appear to have such a role since the IP32953yamA mutant and the wild-type strain grew equally well in the presence of bile salts.
Dam also plays a role in mismatch repair (Wion & Casadesus, 2006). The growth of Dam-deficient mutants of E. coli (Heithoff et al., 1999), Serratia marcescens (Ostendorf et al., 1999) and Y. pestis (Robinson et al., 2005), but not Y. pseudotuberculosis IP32953 (Taylor et al., 2005), was inhibited due to the accumulation of lethal mutations induced by the presence of the base analogue 2-AP. Inactivation of YamA led neither to an increase nor to a reduction in the mutation rate of IP32953, suggesting that YamA does not participate in methyl-directed mismatch repair mechanisms in Y. pseudotuberculosis.
Although the yamA mutation had no detectable effects on bacterial resistance to detergent, DNA repair or pYV replication, it nonetheless resulted in impaired growth and pathogenicity of Y. pseudotuberculosis. YamA may either have a direct role on virulence, or may indirectly interfere with gene regulation of virulence factors, as shown for Dam in diverse gamma proteobacteria (Heusipp et al., 2007). Although the latter hypothesis appears the most likely, we do not yet have any evidence to support it.
Some of the sequenced bacterial genomes are impressively rich in RM genes (or their homologues) (Kobayashi, 2001; Roberts & Macelis, 1998). While eleven RM systems are found on the Y. pseudotuberculosis IP32953 genome, only five are present on the Y. pestis CO92 genome, suggesting a loss of these systems during Y. pestis evolution. RM gene complexes can cause genome rearrangements when their presence is threatened (Tock & Dryden, 2005). The interaction between selfish attack by RM systems and defensive homologous recombination system of bacteria is likely responsible for these genome rearrangements (Handa et al., 2001). It may thus be hypothesized that loss by Y. pestis of several RM systems has participated in the high plasticity of its genome. However, despite their function in bacterial processes, there is increasing evidence that some RM systems operate on a primarily ‘selfish’ level (Handa & Kobayashi, 1999; Handa et al., 2000; Jeltsch, 2003; Kobayashi, 2001; Kusano et al., 1995; Naito et al., 1995). That is, the measures taken to maintain themselves in a cell population can lead to adverse consequences for their host cell. This might also be a reason why Y. pestis has eliminated several RM systems.
RM loci are subject to extensive lateral gene transfer (Bujnicki & Radlinska, 1999; Jeltsch et al., 1995; Kobayashi, 2001; Nobusato et al., 2000a). Their GC content and/or codon usage are often different from those of the core genome (Alm et al., 1999; Jeltsch & Pingoud, 1996; Nobusato et al., 2000a). The yrmI system of IP32953 indeed has a G+C content (42.6 mol%) lower than that of the chromosome backbone (48.9 mol%). Although no obvious mobility machinery could be identified in its vicinity, this system seems to be localized in a region prone to lateral gene transfer since it has been replaced in Y. pestis by another horizontally acquired region, designated region 1 (Chain et al., 2004). Interestingly, the replacing chromosomal locus acquired by Y. pestis carries one gene which potentially encodes another DNA MTase. Although this MTase is predicted to be a type II N4-cytosine MTase and not a type I N6-adenine MTase, this observation may nonetheless suggest that YamA is dispensable in Y. pestis, despite its role in Y. pseudotuberculosis multiplication and virulence, because the plague bacillus has acquired another gene fulfilling similar functions.
Altogether, this study demonstrates that, among the three genes composing the type I RM system probably acquired horizontally by Y. pseudotuberculosis, only the yamA gene encoding the N6-adenine MTase subunit has been systematically retained. This gene is not required for Y. pseudotuberculosis viability and is not involved in DNA replication or repair mechanisms, but it plays a role in pathogenicity, possibly explaining its stabilization in this species. Deciphering the mode of action of YamA in vitro and in vivo should bring valuable information for a better understanding of Y. pseudotuberculosis pathogenesis.
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ACKNOWLEDGEMENTS |
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Edited by: M. Schweizer
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.
Barras, F. & Marinus, M. G. (1989). The great GATC: DNA methylation in E. coli. Trends Genet 5, 139–143.[CrossRef][Medline]
Bujnicki, J. M. & Radlinska, M. (1999). Molecular phylogenetics of DNA 5mC-methyltransferases. Acta Microbiol Pol 48, 19–30.[Medline]
Chain, P. S., Carniel, E., Larimer, F. W., Lamerdin, J., Stoutland, P. O., Regala, W. M., Georgescu, A. M., Vergez, L. M., Land, M. L. & other authors (2004). Insights into the evolution of Yersinia pestis through whole-genome comparison with Yersinia pseudotuberculosis. Proc Natl Acad Sci U S A 101, 13826–13831.
Conchas, R. F. & Carniel, E. (1990). A highly efficient electroporation system for transformation of Yersinia. Gene 87, 133–137.[CrossRef][Medline]
Derbise, A., Lesic, B., Dacheux, D., Ghigo, J. M. & Carniel, E. (2003). A rapid and simple method for inactivating chromosomal genes in Yersinia. FEMS Immunol Med Microbiol 38, 113–116.[CrossRef][Medline]
Falker, S., Schmidt, M. A. & Heusipp, G. (2006). Altered Ca2+ regulation of Yop secretion in Yersinia enterocolitica after DNA adenine methyltransferase overproduction is mediated by Clp-dependent degradation of LcrG. J Bacteriol 188, 7072–7081.
Handa, N. & Kobayashi, I. (1999). Post-segregational killing by restriction modification gene complexes: observations of individual cell deaths. Biochimie 81, 931–938.[Medline]
Handa, N., Ichige, A., Kusano, K. & Kobayashi, I. (2000). Cellular responses to post-segregational killing by restriction-modification genes. J Bacteriol 182, 2218–2229.
Handa, N., Nakayama, Y., Sadykov, M. & Kobayashi, I. (2001). Experimental genome evolution: large-scale genome rearrangements associated with resistance to replacement of a chromosomal restriction-modification gene complex. Mol Microbiol 40, 932–940.[CrossRef][Medline]
Heithoff, D. M., Sinsheimer, R. L., Low, D. A. & Mahan, M. J. (1999). An essential role for DNA adenine methylation in bacterial virulence. Science 284, 967–970.
Heithoff, D. M., Enioutina, E. Y., Daynes, R. A., Sinsheimer, R. L., Low, D. A. & Mahan, M. J. (2001). Salmonella DNA adenine methylase mutants confer cross-protective immunity. Infect Immun 69, 6725–6730.
Heitman, J. (1993). On the origins, structures and functions of restriction-modification enzymes. Genet Eng (N Y) 15, 57–108.[Medline]
Heusipp, G., Falker, S. & Alexander Schmidt, M. (2007). DNA adenine methylation and bacterial pathogenesis. Int J Med Microbiol 297, 1–7.[CrossRef][Medline]
Hinchliffe, S. J., Isherwood, K. E., Stabler, R. A., Prentice, M. B., Rakin, A., Nichols, R. A., Oyston, P. C., Hinds, J., Titball, R. W. & Wren, B. W. (2003). Application of DNA microarrays to study the evolutionary genomics of Yersinia pestis and Yersinia pseudotuberculosis. Genome Res 13, 2018–2029.
Ibanez, M., Alvarez, I., Rodriguez-Pena, J. M. & Rotger, R. (1997). A ColE1-type plasmid from Salmonella enteritidis encodes a DNA cytosine methyltransferase. Gene 196, 145–158.[CrossRef][Medline]
Jeltsch, A. (2003). Maintenance of species identity and controlling speciation of bacteria: a new function for restriction/modification systems?. Gene 317, 13–16.[CrossRef][Medline]
Jeltsch, A. & Pingoud, A. (1996). Horizontal gene transfer contributes to the wide distribution and evolution of type II restriction-modification systems. J Mol Evol 42, 91–96.[Medline]
Jeltsch, A., Kroger, M. & Pingoud, A. (1995). Evidence for an evolutionary relationship among type-II restriction endonucleases. Gene 160, 7–16.[CrossRef][Medline]
Julio, S. M., Heithoff, D. M., Provenzano, D., Klose, K. E., Sinsheimer, R. L., Low, D. A. & Mahan, M. J. (2001). DNA adenine methylase is essential for viability and plays a role in the pathogenesis of Yersinia pseudotuberculosis and Vibrio cholerae. Infect Immun 69, 7610–7615.
Julio, S. M., Heithoff, D. M., Sinsheimer, R. L., Low, D. A. & Mahan, M. J. (2002). DNA adenine methylase overproduction in Yersinia pseudotuberculosis alters YopE expression and secretion and host immune responses to infection. Infect Immun 70, 1006–1009.
Kessler, C. & Manta, V. (1990). Specificity of restriction endonucleases and DNA modification methyltransferases: a review (Edition 3). Gene 92, 1–248.[CrossRef][Medline]
Kobayashi, I. (2001). Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution. Nucleic Acids Res 29, 3742–3756.
Kong, H., Lin, L. F., Porter, N., Stickel, S., Byrd, D., Posfai, J. & Roberts, R. J. (2000). Functional analysis of putative restriction-modification system genes in the Helicobacter pylori J99 genome. Nucleic Acids Res 28, 3216–3223.
Kusano, K., Naito, T., Handa, N. & Kobayashi, I. (1995). Restriction-modification systems as genomic parasites in competition for specific sequences. Proc Natl Acad Sci U S A 92, 11095–11099.
Messer, W., Seufert, W., Schaefer, C., Gielow, A., Hartmann, H. & Wende, M. (1988). Functions of the DnaA protein of Escherichia coli in replication and transcription. Biochim Biophys Acta 951, 351–358.[Medline]
Murray, N. E. (2002). 2001 Fred Griffith review lecture. Immigration control of DNA in bacteria: self versus non-self. Microbiology 148, 3–20.
Naito, T., Kusano, K. & Kobayashi, I. (1995). Selfish behavior of restriction-modification systems. Science 267, 897–899.
Nobusato, A., Uchiyama, I. & Kobayashi, I. (2000a). Diversity of restriction-modification gene homologues in Helicobacter pylori. Gene 259, 89–98.[CrossRef][Medline]
Nobusato, A., Uchiyama, I., Ohashi, S. & Kobayashi, I. (2000b). Insertion with long target duplication: a mechanism for gene mobility suggested from comparison of two related bacterial genomes. Gene 259, 99–108.[CrossRef][Medline]
Noyer-Weidner, M. & Trautner, T. A. (1993). Methylation of DNA in prokaryotes. EXS 64, 39–108.[Medline]
Oshima, T., Wada, C., Kawagoe, Y., Ara, T., Maeda, M., Masuda, Y., Hiraga, S. & Mori, H. (2002). Genome-wide analysis of deoxyadenosine methyltransferase-mediated control of gene expression in Escherichia coli. Mol Microbiol 45, 673–695.[CrossRef][Medline]
Ostendorf, T., Cherepanov, P., de Vries, J. & Wackernagel, W. (1999). Characterization of a dam mutant of Serratia marcescens and nucleotide sequence of the dam region. J Bacteriol 181, 3880–3885.
Parkhill, J., Wren, B. W., Thomson, N. R., Titball, R. W., Holden, M. T., Prentice, M. B., Sebaihia, M., James, K. D., Churcher, C. & other authors (2001). Genome sequence of Yersinia pestis, the causative agent of plague. Nature 413, 523–527.[CrossRef][Medline]
Pouillot, F., Derbise, A., Kukkonen, M., Foulon, J., Korhonen, T. K. & Carniel, E. (2005). Evaluation of O-antigen inactivation on Pla activity and virulence of Yersinia pseudotuberculosis harbouring the pPla plasmid. Microbiology 151, 3759–3768.
Pucciarelli, M. G., Prieto, A. I., Casadesus, J. & Garcia-del Portillo, F. (2002). Envelope instability in DNA adenine methylase mutants of Salmonella enterica. Microbiology 148, 1171–1182.
Reed, L. J. & Muench, H. (1935). A simple method of estimating fifty per cent endpoints. Am J Hyg 27, 493–497.
Roberts, R. J. & Macelis, D. (1998). REBASE – restriction enzymes and methylases. Nucleic Acids Res 26, 338–350.
Robinson, V. L., Oyston, P. C. & Titball, R. W. (2005). A dam mutant of Yersinia pestis is attenuated and induces protection against plague. FEMS Microbiol Lett 252, 251–256.[CrossRef][Medline]
Sistla, S. & Rao, D. N. (2004). S-Adenosyl-L-methionine-dependent restriction enzymes. Crit Rev Biochem Mol Biol 39, 1–19.[CrossRef][Medline]
Taylor, V. L., Titball, R. W. & Oyston, P. C. (2005). Oral immunization with a dam mutant of Yersinia pseudotuberculosis protects against plague. Microbiology 151, 1919–1926.
Tock, M. R. & Dryden, D. T. (2005). The biology of restriction and anti-restriction. Curr Opin Microbiol 8, 466–472.[CrossRef][Medline]
Wion, D. & Casadesus, J. (2006). N6-methyl-adenine: an epigenetic signal for DNA-protein interactions. Nat Rev Microbiol 4, 183–192.[CrossRef][Medline]
Received 3 January 2007;
revised 28 February 2007;
accepted 2 March 2007.
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