The effect of methylation on some biological parameters in Salmonella enterica serovar Typhimurium

Pathologie Biologie 59 (2011) 192–198

Original article

The effect of methylation on some biological parameters in Salmonella enterica serovar Typhimurium Effets de la méthylation sur quelques paramètres biologiques de Salmonella enterica Typhimurium A. Aloui a,*, J. Tagourti a, A. El May a, D. Joseleau Petit b, A. Landoulsi a b

a Laboratory of Biochemistry, Faculty of science Bizerte, Tunisia Molécules de stress, institut Jacques Monod, université Paris-7, 2, place Jussieu, 75005 Paris, France

Received 27 November 2008; accepted 18 March 2009 Available online 23 May 2009

Abstract Cell growth is tightly coupled to DNA replication and its methylation [Proc Natl Acad Sci U S A 93 (1996) 12206–12211]. In a culture medium, growing of Salmonella Typhimurium (S. Typhimurium) mutant cells (dam ) decreased (2.5 fold) relative to thewild type strain (dam+). In this study, we show that the reason for this growth deficiency is due to the DNA methylation. The absence of a Dam methyltransferase protein results in poor growth efficiency and disturbs the synchrony of replication initiation in vivo, as evaluated by flow cytometry. On the other hand, we show that lack of methylation could increase the DNA response to thermal stress (decreasing the DNA melting temperature, Tm), and the reason for this effect is due to the methylation status and not to the number of guanine and cytosine bases (G + C) in the duplex DNA. Our results show that methylation is an epigenetic factor that may play a key role in the cell growth, the synchrony of DNA replication [C R Biologies 330 (2007) 576–580] and the stress protection. # 2009 Published by Elsevier Masson SAS. Keywords: Dam methylase; S. Typhimurium; Methylation; DNA replication; Bacterial growth; Melting temperature

Résumé La croissance cellulaire est étroitement liée à la réplication de l’ADN et à sa méthylation [Proc Natl Acad Sci U S A 93 (1996) 12206–12211]. Dans un milieu de culture, la croissance de salmonella Typhimurium (dam ) est inférieure (2,5 fois) par rapport au type sauvage (dam+). Dans cette étude, nous avons montré que la raison pour cette différence de croissance est due à la méthylation de l’ADN. L’absence de la protéine Dam méthyltransférase a comme conséquence une déficience de croissance et altère la synchronie de l’initiation de la réplication in vivo, comme cela a été évalué par cytométrie de flux. Par ailleurs, nous avons montré que l’absence de méthylation pourrait augmenter la réponse de l’ADN au stress thermique (en diminuant la température de fusion de l’ADN, Tm), et la raison pour cet effet est due à l’état de méthylation et non pas au nombre de bases de guanine et de cytosine (G + C) dans le duplex d’ADN. Nos résultats prouvent que la méthylation est un facteur épigénétique qui peut jouer un rôle principal dans la croissance cellulaire, la synchronie de la réplication d’ADN [C R Biologies 330 (2007) 576–580] et la protection contre les stress. # 2009 Publié par Elsevier Masson SAS. Mots clés : Dam méthylase ; S. Typhimurium ; Méthylation ; Réplication de l’ADN ; Croissance bactérienne ; Température de fusion

1. Introduction Much of our research is focused on Salmonella which causes diseases ranging from food and blood poisoning to

* Corresponding author. E-mail address: [email protected] (A. Aloui). 0369-8114/$ – see front matter # 2009 Published by Elsevier Masson SAS. doi:10.1016/j.patbio.2009.03.011

typhoid fever and heart disease [30]. S. Typhimurium accounts for approximately 17% of worldwide intestinal Salmonella infections reported yearly [1]. It’s an important pathogen responsible for many of all the reported cases of enterocolitis and the second most frequent cause of bacterial food-borne disease with an estimated 1.4 million cases per year in the US alone [34]. The rationale for examining Salmonella as a model system to study microbial pathogenesis is that it provides a

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well-characterized genetic system to understand its infection mechanism [37]. Methylation is carried out by the dam gene product [31]. dam mutants have been isolated that contain no detectable DNA methylase activity and yet are viable [20]. However, since the first dam mutants of S. Typhimurium were isolated [46], the biological functions of these methylated sites have been widely investigated by numerous workers, and several important roles have been deduced [44–45]. The first solitary DNA adenine methyltransferase that was found to play a key role in biological functions was the Dam enzyme of Escherichia coli (E. coli) [30]. Recently, Dam methylase and its homologs were characterized among bacteria in the gamma and alpha subdivision of Proteobacteria, such as S. Typhimurium [17], Yersinia pseudotuberculosis and Vibrio cholera [24], and E. coli [40]. The Salmonella gene encoding the dam protein has homologs in almost every prokaryotic organism and cell that has been examined to date, ranging from E. coli to many other species. This ubiquity and evolutionary conservation indicate that it may play a fundamental physiological role. The DNA methylation by this dam protein at GATC sites has been shown to have enormous impact on nucleoid stability, replication, the cell cycle, mismatch repair, and gene expression [23]. Many enterobacterial species have a central metabolite: S-adenosylmethionine (SAM) which is synthesized from methionine and ATP by the enzyme SAM synthetase [9], it is the major methyl donor in metabolism. S. Typhimurium strain has a DNA adenine methyltransferase (the Dam enzyme) which catalyzes the adenine methylation at N6 in the sequence GATC in the duplex DNA [41,16,19], a reaction in which SAM is the methyl group donor [22] and also an allosteric effector [4]. The aim of this work was to examine if salmonella dam could grow equally to salmonella dam+ on a nutrient broth. It was also of interest to see whether the type and the composition of medium (minimal or complete) enhanced the survival and growth of these strain. For the Tm, we decided to examine the relationship between the melting temperature of intracellular DNA and its methylation and to see if the difference observed between dam+ and dam– is due to the number of (G + C) or to the absence of methylation. Our future plans are to understand the molecular basis of how Dam controls the bacterial growth and the synchronous initiation of chromosome replication. For the second parameter (Tm) it will be of interest to see if there is a clear correlation between Tm, number of (G + C) and methylation. 2. Material and methods 2.1. Bacterial strains One isogenic strain of Salmonella enterica serovar Typhimurium was used in this study and both strains and their relevant genotype are listed in Table 1. SL1344 reference strains and SV1610 (contains dam disrupted gene) were kindly provided by Dr. Francisco Ramos-Morales (Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Spain).

193

Table 1 Strains used in this study. Bacterium

Strain

Relevant genotype or alternate designation

Reference or sourcea

S. Typhimurium

SL1344

Wild type

S. Typhimurium

SV1610

SL1344 dam-228::MudJ Km r

Portillo et al. [13] Portillo et al. [13]

a

Omitted for strains described in this study.

2.2. Bacterial culture and growth conditions Cultures were prepared by inoculating liquid media with a single colony from nutrient agar. Bacterial growth were done aerobically in a shaker water bath at 37 8C in Erlenmeyer flasks (250 ml) containing 50 ml volumes of a fresh sterile liquid medium and aerated by shaking during the experimental procedure. SL1344 and SV1610 were grown exponentially in the following media, which yield different growth rates: NB (nutrient broth) and M9 (M9ZB) media were prepared as described by Miller (1972) [37]. The sterile complete medium is prepared with NB (Pronadisa, Hispanlab) 8 g l–1 and the sterile minimal medium M9ZB is formed by 200 ml of a saline solution M9 (5  concentrated) supplemented with 20 ml of a glucose solution 20% as carbon source, with 2 ml of MgSO4 1 M, and 0,1 ml of CaCl2 1 M. Media were autoclaved at 120 8C for 30 min. The speed of growth of a bacterial culture can be appreciated by measuring its absorbance (optical density [OD]) at 600 nm using a spectrophotometer. In exponentially growing cultures, the doubling time was calculated for each experiment: samples are taken every 60 min and OD was measured. Spectrophotometric measurements are consigned on a graph expressing the OD600 according to time. 2.3. Coulter counter analysis Coulter counter analysis was done according to Vinella et al., 1992 [51], with some modifications by which we use the new following experimental conditions:  usual sample dilution: 0.2 ml sample (bacterial culture) in 9.8 ml diluents (NB liquid medium);  typical settings:  lower threshold: 10,  upper threshold: 99.9 (out),  manometer: 500 ml,  current: 100. Cells/ml in original suspension = number of counts  2 (for 500 ml)  50 (200 ml in 10 ml) 2.4. Flow cytometry analysis An overnight culture of bacteria in M9 supplemented with glucose (0.2%), Casamino acids (0.5%), tryptophan (40 mg ml 1), and vitamin B1 (1 mg ml 1) was diluted 500fold into the same fresh medium and cultivated at 37 8C until

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0.2 OD (600 nm). After addition of rifampicin (150 mg ml 1) and cephalexin (12 mg ml 1), followed by cultivation for 120 min, bacteria were collected by centrifugation and analysed according to a standard procedure (Skarstad et al., 1986) [47] with flow cytometer. 2.5. DNA manipulation The total DNA of the reporter strains was extracted and purified by the Bost Rod (l989) procedure [6], before the heating operation. Genomic DNA was purified with a purity greater than 90% as judged by R value = 1.86 (R = A260 nm/A280 nm and 1.8 < R < 2).The DNA concentration was determined by ultraviolet spectrophotometry or by comparison with known standards after gel electrophoresis. Samples were heated in a Polystat24 thermostat water bath and temperature values increased linearly at 10 8C from 25 to 110 8C using an empty pan as reference. This heating rate was chosen arbitrarily, after preliminary tests showed that it yielded well-separated and clearly defined thermogram graph. The optic density was measured in UV spectrophotometer at 260 nm. The measurements are consigned on a graph expressing the absorbance at 260 nm according to the temperature. 3. Results and discussion 3.1. Growth defects of the dam deficient strain 3.1.1. Growth deficiency In order to check the effect of methylation on the growth of our bacterial strains, we carried out an analysis of the biomass in two different liquid culture media: a complete medium (nutrient broth) and a minimal medium (M9ZB). The ability of S. Typhimurium dam strain to grow in different media was examined over 1 day and the results are represented on Figs. 1 and 2 (the average results of three experiments are presented on these figures). The follow-up of the evolution of the biomass by measuring the OD600 and the establishment of the curve (OD = f (t)) showed a significant altered growth (Student test P < 0.01) of the mutant strain compared to the wild type. This alteration starts at the beginning of the exponential phase and continues in stationary

Fig. 1. Growth curves for the wild-type strain of S. Typhimurium (filled triangles) and its derivative dam mutant (filled circles). Cultures were grown in NB complete medium as described in Methods. Experiments were done three times, and the mean value  standard error of the mean (SEM) was calculated.

Fig. 2. Growth curves for the wild-type strain of S. Typhimurium (filled triangles) and its derivative dam mutant (filled circles). Cultures were grown in M9ZB minimal medium as described in Methods. Experiments were done three times, and the mean value  standard error of the mean (SEM) was calculated.

phase, in the complete or in the minimal medium. However, other studies have been performed by Badie et al., 2007 [55] with dam mutant and Damop derivatives of different isolates of S. Typhimurium which mention that no significant growth rates differences are found with the wild-type strain. Thus, the growth difference is specific for our strains. Also, the establishment of a competitive index (CI) confirms the growth advantage (data not shown). Moreover, the knowledge of the microbial speed growth during the cell cycle (exponential phase) would be especially useful and essential in elucidating this difference and the influence of the methylation on the growth of our bacterial strains. For that, we determined the generation times of wild-type and mutant strains in the two culture media using the following mathematical formula: g = 1/k; k = n/t = (log Nt – log N0)/0.301  t (N0: initial number cells; Nt: population at t time; n: number of generation at t time; t: time for biomass doubling [43]) and we obtained the values marked in Table 2. The analysis of these results shows that the doubling time of the wild strain was lower compared to that of the dam deficient mutant that makes the double time of parental strain, in both nutrient broth medium and M9ZB minimal medium at 37 8C (data not shown). However, the dam mutant, gave smaller colonies on NB plates (at 37 8C), reflecting a slight growth disadvantage compared to the control strain (data not shown). The unique explanation for these differences must be the lack of methylation and not the medium composition. Because SL1344 and SV1610 are isogenic except for their dam loci, the difference in their growth rates must be solely due to the dam gene which is expressed under growth rate control [44,41]. 3.1.2. Coulter counter analysis The electronic Coulter counting technique, for both strains and at the same OD600, offers the following results marked in Table 3. Coulter counter analysis of growing cultures of wildtype and dam mutants showed that dam cell volume is higher than dam+ (30%). In addition, dam cells are heterogeneous and present varied cell volume distributions from one clone to another (heterogeneity estimation: dam+: 0.87 and dam : 1.05). These differences could be due to induction of SOS-associated division inhibitor SfiA, since enterobacterial dam mutants are partially induced for the SOS response (such as E. coli [42]).

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Table 2 Generation time of bacterial strains. Medium culture Strains Generation time

NB

NB +

dam 0,4 h

1

(24 min)/gen

dam 1,0 h

M9ZB

M9ZB

+

1

(60min)/gen

dam 0,5 h

1

(30 min)/gen

dam 1,3 h

1

(78 min)/gen

h: hour; min: minute; gen: generation. The generation times of wild-type and mutants cells growing exponentially at 37 8C in NB and M9ZB media were 24/30 min (dam+) and 60/78 min (dam ), respectively.

Table 3 Bacterial cells count. Bacterial strains +

dam dam

OD600

Cells/ml

0,8 0,8

9,4  10 8 8,9  10 8

Cells/ml in original suspension = number of counts  2 (for 500 ml)  50 (200 ml in 10 ml).

The cell volume distribution of a double mutant (dam-13 sfiA100::TnS) [10] was also heterogeneous and varied from one clone to another, suggesting that SfiA does not completely account for the partial cell division inhibition in the enterobacterial dam mutant [42]. This type of SOS-independent division inhibition has been previously observed under conditions in which initiation or elongation of DNA synthesis or chromosome decatenation was defective [21,38]. Genetic test by introduction of a sulA mutation (or a lexA [Ind- ] mutation) reduces this heterogeneity (data not shown). 3.1.3. Mutation of dam gene disrupts replication synchrony In wild-type strains, DNA initiation is well regulated, occurring simultaneously at all origins within each cell, exactly once per cell cycle in steady-state growth [27]. When rounds of replication are allowed to run to completion, the number of chromosomes per cell is 2n (n = 0, 1, 2, 3, etc). When initiations are asynchronous, as in dnaA (Ts) initiation mutants at the permissive temperature and in the E. coli dam mutant [8,48], the presence of a different number of chromosome equivalents (three, five, six, etc.) was detected by flow cytometry. The presence of cells containing a number of chromosomes different from 2n suggests that the dam mutant has a defect in the synchrony of replication initiation. Both strains growing exponentially in glucose–CAA medium were treated with rifampicin and cephalexin, which block initiation of replication and cell division respectively. Wild-type cells initiated replication synchronously (number of chromosomes per cell is 2n). The appearance of cells with chromosome numbers other than 2n indicates a moderate asynchrony of initiation. So, flow cytometer analysis of our dam mutants has shown that replication initiation is asynchronous and can occur throughout the cell cycle, not only at the normal cell age for initiation. The most likely reason for this asynchrony phenotype is that secondary initiations occurred at newly replicated origins in dam mutants, due to lack of methylation and inadequate sequestration [3]. We showed that initiation synchrony was dependent on intact GATC methylation sites. This loss of synchrony affected culture growth rates and cell size

distributions only slightly. The above results are in marked accord to those obtained by Vinella et al., 1992 [52] with E. coli strains and suggest that dam mutants have a slight defect in synchronizing replication initiation. All these results suggest that DNA methylation plays a role in preventing the occurrence of multiple initiations at a single origin in the same replication cycle. However, using flow cytometry, we found that the asynchrony of initiation, which is one of the phenotypes of the dam mutation, was returned to almost normal in the dam null mutant (SV1610) harboring pTP166 (it’s a ColE1 derivative harboring the wild-type dam gene under the control of a tac promoter [31]) producing the dam protein, but not with the vector plasmid (data not shown). 3.1.4. Conclusion Like many enterobacterial species such as E. coli, strains deficient in Dam methylase are not well grouted in minimal and complete medium. Because of its key role in DNA replication [2], we can say that there is a link between DNA methylation, replication and cells growth. Boye et al. [7] showed that, in wild-type strain, the cell mass at initiation of replication is the same for all cells in a given culture, reflecting a tight coupling between mass increase and replication. On the other hand, the finding that Dam methylation modulates the growth in minimal and complete medium in E. coli led us to examine whether it could play the same role in S. Typhimurium growth. Our study implies a connection between Dam methylation, DNA replication and cell growth. Our data clearly indicate that the dam mutant is also affected when grown in a minimal or a complete medium. Because SL1344 and SV1610 are isogenic except for their dam loci, the difference in their growth rate must be solely due to the dam gene and not to the medium composition. Therefore, further study and characterization of molecular basis of how Dam controls growth is required to ascertain which gene products are essential for optimum growth. A report by Heitoff et al. [17] proposes that a large number of Dam-regulated genes exist in S. Typhimurium, thus supporting our view that the effects of DNA adenine methylase mutation on Salmonella growth are defective but not pleiotropic. 3.2. The dam mutant DNA is sensitive to thermal stress The correlation between the melting temperature of intracellular DNA and its guanine + cytosine (G + C) content was examined for many species of bacteria. The guanine + cytosine content of bacterial DNA varies between about 25 and 75 mol%. The value is constant for a given organism and is

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A. Aloui et al. / Pathologie Biologie 59 (2011) 192–198 Table 4 DNA melting temperature. DNA samples +

dam dam dam (remethylated)

Tm (8C) 96 82 96

Tm values were determined directly from thermograms. The average results of two or more experiments are presented above. Fig. 3. Melting DNA for the wild-type strain of S. Typhimurium (filled squares) and its derivative dam mutant (filled diamonds). The average results of two or more experiments were calculated.

therefore considered an essential item in the description of any species. The relative thermostability of a DNA duplex and its melting temperature increase with the increasing content of the mole fraction of (G + C) base pairs in this DNA [15]. The number can be estimated by Bolton and Mc Carty formula: Tm (8C) = 69,2 + 0,41 (%CG) [5] and the base composition can be determined directly by hydrolysis of a DNA sample followed by chromatographic separation of the bases, or indirectly from buoyant density or melting point data [11,25,29]. Although these methods are widely used they require pure DNA as the starting point and are therefore time-consuming to do. Technical problems sometimes arise because of the difficulty of lysing certain types of bacteria to extract DNA, or because of contamination of DNA samples with polysaccharides, RNA or other substances. The thermal denaturation of double-stranded DNA is accompanied by the disruption of Watson–Crick base pairs, unstacking of the bases, increasing viscosity and absorbance (hyperchromicity) and disordering of the B-form backbone. These types of structural change are highly correlated throughout the investigated temperature range of 20 to 93 8C. [11]. The temperature values measured during the experimental procedure led us to draw the following sigmoid denaturation thermograms presented on Fig. 3 (the average results of two or more experiments are presented on this figure) and relative Tm were determined directly from Fig. 3 graph and values are presented in Table 4. To our knowledge, this is the first study to elucidate the effect of methylation on S. Typhimurium genomic DNA melting temperature and the analysis of our data shows a significant difference (Student test P < 0,01) and it does not support the view that the Tm difference observed between dam+ and dam is due to the (G + C) basis number. The Tm difference between dam+ and dam DNA was magnified to a level of about 14.6%. According to this result, we suggest that the decrease of the Tm of dam DNA can be attributed directly to the absence of dam methylation, as after methylation of dam DNA in vitro using partially purified dam methylase (according to the method of Urieli-Shoval et al., 1983 [51]), no difference was observed between the Tm of dam+ DNA and remethylated dam DNA (data not shown). Because SL1344 and SV1610 are isogenic except for their dam loci we suggest that the methyl group increases the DNA melting, so it protects DNA from the thermal stress. We conclude that methylation is an additional

factor to base composition that affects the temperature of DNA melting within the bacterial cell. Our observation that unmethylated DNA has a lower Tm than methylated DNA is contrary to previously published data [56]. The reasons for these differences are unclear. Worcel and Burgi, 1972; Flink and Pettijohn, 1975 and Sjasted, 1982 [54,14,49] showed that variation in DNA melting might arise from stabilizing interactions with other molecules within the cell, e.g. polyamines, RNA or protein. However, in our experiments, DNA melting temperatures were determined to two isogenic strains (except for their dam loci) suggesting that interactions with other molecules were not an important influence. Alternatively, the heating could affect the thermostability of intracellular DNA for other reasons. The occurrence of mismatched regions of DNA, for example, might lower the melting point of DNA complex since the dam mutants are enable to repair DNA mismatch formed in the absence of Dam methylation. Mismatching could arise as a result of the absence of Dam methylase or possibly from the formation of DNA– RNA heteroduplex regions during cooling denaturated samples. An additional and probably more important source of variation is intracellular solute concentration which is known to vary quite widely between bacterial species. For example, the osmolarity of the cytoplasm of E. coli or S. Typhimurium is two to three times lower than that of Staphylococcus aureus or Streptococcus faecalis [36]. A lower value of 0.3 Os kg 1 for S. Typhimurium was obtained by vapour pressure equilibration and other methods [50]. In our case we used the same species: Salmonella enterica serovar Typhimurium, so osmolarity would not have been a source of Tm variability. Variations in growth conditions or method of sample preparation could influence DNA melting either by affecting internal salt concentration or for other, unknown reasons. Cells grown on complex media had higher Tm values than those grown on minimal media (data not shown). In our experiments cells were grown in the same conditions (NB complete media) and samples were prepared identically, so there is no ways of overcoming possible variability from these sources. This paper provides further evidence consistent with the view that the DNA methylation is a postreplicative change that is required for DNA protection from stressing conditions (osmolarity, acidity, temperature. . .). 4. General conclusion In the bacterial cell, DNA adenine methylation (Dam) modulates a variety of processes such as DNA replication, transcription of certain genes and bacterial growth [2–30,39].

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Deletion of the dam gene produces a variety of phenotypes in several bacteria, ranging from cell death [24] to attenuation of virulence [53], indicating multiple functions for GATC methylation in modulating gene expression, DNA mismatch repair, initiation of chromosome replication, and nucleoid stabilization. Given these multiple roles, it is not surprising that dam mutations are highly pleiotropic. However, the lack of Dam methylation does not impair viability. [19–37,46]. S. Typhimurium dam strain described here lacks N6-adenine methylation of GATC sequences and is more sensitive to this mutation than dam+ which shows the inverse. In addition, no difference between the dam mutant of S. Typhimurium and those of some enterobacterial species such as E. coli was observed with cell growth, replication synchrony or Tm. In conclusion, the role of Dam in the prokaryotic cellular processes such as the DNA replication, cell growth and stress protection is clear. So it may rely on its capacity as a global regulator of the gene expression (such as hilA, prgH and sipA) [13] during bacterial life, in vitro, in a similar manner as it does in vivo. Of special interest for future studies are: firstly, the growing list of genes governed by DNA adenine methylation in bacterial pathogens ; secondly, the finding of novel genes regulated by Dam methylation using high throughput analysis, and, thirdly, the evidence that DNA adenine methylation may regulate the expression of many unidentified genes involved in stress protection. Finally, our knowledge on the effects of DNA methylation in S. Typhimurium has considerably improved in the last decade. This fundamental research has several implications that will prove to be useful for the development of novel therapeutic approaches. But to date, therapeutic applications are still in their early experimental phases, but several recent studies provide promising results for future clinical developments. Acknowledgements The authors are extremely grateful to: Dr. Francisco RamosMorales (Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Spain) for the generous donation of the Salmonella Typhimurium wild and dam . This work was supported by the Tunisian Ministry of Higher Education, Scientific Research and Technology. References [1] Adkins JN, Mottaz HM, Norbeck AD, Gustin JK, Rue J, Clauss TR, et al. Analysis of the Salmonella Typhimurium proteome through environmental response towards infectious conditions. Mol Cell Proteomics 2006;5:1450–61. [2] Aloui A, Chatty A, El May A, Landoulsi A. The effect of methylation on DNA replication in Salmonella enterica serovar Typhimurium. C R Biologies (2007) 2007;330:576–80. [3] Bach T, Skarstad K. Re-replication from non-sequesterable origins generates three-nucleoid cells which divide asymmetrically. Molecular Microbiology 2004;51:1589–600. [4] Bergerat A, Guschlbauer W. The double role of methyl donor and allosteric effector of S-adenosy-methionine for Dam methylase of E. coli. Nucleic Acids Res 1990;18:4369–75. [5] Bolton ET, Mc Carty BJ. A general method for the isolation of RNA complementery to DNA. Proc Nat Acad Sc U S A 1962;48:1390.

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