From the
Unité de Génétique des Génomes Bactériens, Institut Pasteur, 75724 Paris Cedex 15, France,
¶ Laboratoire de Biochimie, Ecole Polytechnique, 91128 Palaiseau Cedex, France,
|| Atelier de Bio-informatique, Université Paris 6, 12 rue Cuvier, 75005 Paris, France,
** HKU-Pasteur Research Centre, Dexter HC Man Building, 8 Sassoon Road, Pokfulam, Hong Kong, China,
Laboratoire de Dynamique, Evolution et Expression de Génomes de Microorganismes, FRE 2326, Université Louis Pasteur/CNRS, 67083 Strasbourg Cedex, France
Received for publication, November 19, 2002
, and in revised form, February 24, 2003.
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ABSTRACT |
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INTRODUCTION |
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The increasing number of sequencing projects has recently revealed the existence of several H-NS-related proteins in Gram-negative bacteria with different life style such as the human pathogenic bacteria Yersinia pestis (8) and Pasteurella multocida (9) and the plant pathogenic bacteria Xylella fastidiosa (10) and Ralstonia solanacearum (11). Some of these proteins have been studied in detail, e.g. those from Vibrio cholerae (12) and Bordetella pertussis (13). All H-NS proteins share the same structural and functional organization in two modules (14, 15). In enterobacteria, the N-terminal domain of H-NS has been recently shown to contain three -helices (16), whereas the C-terminal three-dimensional structure of the protein resolved by NMR consists of a mix of
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structures (17). The H-NS protein forms oligomers via its N-terminal part and is able to bind curved and AT-rich DNA fragments via its C-terminal domain (18), both essential properties of H-NS and related proteins (19). Nevertheless, the role of most proteins of the H-NS family in bacterial physiology remains unknown (15). In contrast, in Escherichia coli and Salmonella typhimurium H-NS seem to be involved in bacterial nucleoid organization and in the regulation of various genes involved in adaptation to environmental challenges (20, 21). A variety of phenotypes has been associated with a mutation in hns, in particular an increase in pH resistance (21, 22) and a loss of motility (23, 24). Finally, in enterobacteria and related micro-organisms, H-NS proteins are cold shock proteins (12, 25), which could explain the susceptibility to low temperature of E. coli hns mutant (26).
To further investigate the structure-function-evolution relationship of H-NS-related proteins, we identified and characterized orthologous proteins in bacteria isolated from extreme environments, i.e. a psychrotrophic bacterium Acinetobacter spp. isolated from Lake Baikal in Siberia1 and a psychrophilic bacterium Psychrobacter spp. collected from Antarctica (27). Like the protein of E. coli, H-NS-like proteins of Psychrobacter and Acinetobacter were both able to complement H-NS-related phenotypes in E. coli at 30 °C. Surprisingly, the Psychrobacter H-NS protein was no longer able to reverse the effects of H-NS deficiency at 37 °C. In vivo and in vitro experiments demonstrated the crucial role of the N-terminal part on the thermal stability of this unusual H-NS protein and give new insight concerning the structural and functional organization of the proteins of this family.
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MATERIALS AND METHODS |
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Metabolism of -glucosides was tested on MacConkey agar indicator plates with 1% salicin as a carbon source. Tryptone swarm plates containing 1% Bacto-tryptone, 0.5% NaCl, and 0.3% Bacto-agar were used to test bacterial motility as previously described (23). When required, ampicillin or chloramphenicol was added at 100 µg/ml and 20 µg/ml, respectively. Ultracompetent XL1-Blue (Stratagene) cells were used to construct the genomic library of Acinetobacter 20 and Psychrobacter TAD1 strains. All experiments were performed in accordance with the European requirements for the contained use of genetically modified organisms of Group-I (agreement number 2735) and Group-II (agreement 2736 CAI).
Plasmid pDIA572 was isolated from a Psychrobacter TAD1 genomic library (see below) and carries a DNA fragment of 1330 bp. The insert nucleotide sequence was determined on both strands. The DNA fragment of pDIA572 contains the hns gene of Psychrobacter TAD1 and its flanking regions (accession number AJ310993 [GenBank] ). Plasmid pDIA585 carries a 2673-bp DNA fragment containing the hns gene of Acinetobacter strain 20 and its flanking regions (accession number AJ458445 [GenBank] ). To overproduce the H-NS-His6 proteins of E. coli, Acinetobacter 20, and Psychrobacter TAD1 strains, their structural gene was amplified from genomic DNA using primers 5'-GGAGGTTCATATGAGCGAAGCACTTAAAAT-3' and 5'-CCGCTCGAGTTGCTTGATCAGGAAATCGT-3', primers 5'-GGAGGTTCATATGCCAGATATTAGTAATTTATCTG-3' and 5'-CCGCTCGAGGATGAGGAAGTCTTCCAGTTTCGCACC-3', and primers 5'-GGAGGTTCATATGACTAATAACACTACTAT-3' and 5'-CCGCTCGAGTACAGTAAAACTTTCTAGGT-3', respectively. These pairs of primers introduced a NdeI cloning site and a XhoI cloning site at 5'- and 3'-end, respectively. The PCR products were inserted into the NdeI and XhoI sites of the pET-22b vector (Novagen), giving rise to plasmids pDIA569 (which contains the hns gene of E. coli), pDIA588 (which contains the orthologous gene of Acinetobacter strain 20), and pDIA568 (which contains the orthologous gene of Psychrobacter TAD1).
The genes coding for the H-NS chimeric proteins of E. coli and Psychrobacter TAD1 were constructed as follows. The promoter region and the 5'-end of the hns gene of E. coli were amplified from plasmid pDIA547 (19) using primers 5'-GTTTTCCCAGTCACGAC-3' and 5'-AGATTTAACGGCAGCAAGGC-3' and its 3'-end using primers 5'-TAGAAGAGATTTTGAAGGCTGGCACCAAAGCTAAACGTGC-3' and 5'-AGCGGATAACAATTTCACACAGGA-3'; the 5'-end of the hns gene of Psychrobacter spp. was amplified from plasmid pDIA580 using primers 5'-ATGACTAATAACACTAC-3' and 5'-AGCCTTCAAAATCTCTTCTA-3' and its 3'-end using primers 5'-GCCTTGCTGCCGTTAAATCTGGTGAAAGCCTAGAGAAAAAACG-3' and 5'-CCCAAGCTTGGGTTATACAGTAAAACTTTCTAGG-3'. All constructs were inserted into the HindIII and EcoRI restriction sites of plasmid pDIA547 and gave rise to plasmids pDIA580, pDIA581, and pDIA582 (Table III).
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Construction of Genomic DNA LibrariesGenomic DNA was isolated from Acinetobacter 20 and Psychrobacter TAD1 bacterial strains. The Psychrobacter genomic library was constructed in plasmid pcDNA 2.1 (Invitrogen) as previously described (15), whereas the Acinetobacter genomic library was constructed in plasmid pDIA561 (21). About 60,000 clones were selected on LB plates supplemented with 100 µg/ml ampicillin or 20 µg/ml chloramphenicol and pooled. Large scale plasmid DNA isolation was carried out using the JETstar kit (GENOMED).
Protein PurificationThe recombinant H-NS-His6 proteins of E. coli and Psychrobacter TAD1 strains were purified from E. coli BL21 (DE3) (Stratagene) carrying pDIA17 and pDIA569 or pDIA568 using NiSO4 chelation columns (Qiagen), as previously described (19).
Gel Retardation ExperimentsGel retardation experiments were performed as previously described (23), with H-NS-purified protein of Psychrobacter TAD1 either at 4 or 37 °C. Restriction fragments derived from plasmid pDIA525 that contain flhDC or bla DNA fragments of E. coli were used as competitors.
Protein-Protein Cross-linkingCross-linking experiments were performed as previously described (12) with 25 µM H-NS of Psychrobacter TAD1 used in each reaction. After adding cross-linking reagents, i.e. 200 mM 1-ethyl-3(3-dimethylaminopropyl)carbodiimide and 50 mM N-hydroxysuccinimide, the reaction mixtures were incubated for 1 h either at 4 °Corat37 °C, loaded onto a SDS-14% Prosieve acrylamide gel, and silver-stained.
Circular Dichroism (CD) SpectroscopyCD spectra were obtained with a Jobin-Yvon CD6 dichrograph equipped with a thermostatted cell holder. The CD spectra were recorded between 190 and 260 nm from 4 to 70 °C (after 10 min of temperature equilibration before recording the data). The results are the mean values of two successive spectra. CD spectra of purified proteins were determined in pure water with a protein concentration of 14 µM.
In Silico Sequence AnalysisThe ProtParam software on the Ex-PASy web site (www.expasy.org/tools/protparam.html) was used to determine the amino acid composition of proteins. The CLUSTALw method (32) was used for sequence alignments. Secondary structure prediction was performed using the PREDATOR method (33), available on the web site pbil.univ-lyon1.fr. The fold recognition method FROST (Fold Recognition Oriented Search Tool), available on the web site www-mig.jouy.inra.fr/mig/index.html, was used for fold assignments to H-NS protein sequences (34).
Nucleotide Sequence Accession NumbersThe nucleotide sequences of 16 S rRNA and hns genes from Psychrobacter TAD1 bacterial strain have been assigned EMBL nucleotide sequence data base accession numbers AJ310992 [GenBank] and AJ310993 [GenBank] , respectively. The 1379 nucleotide sequence of 16 S rRNA gene of Acinetobacter strain 20 was in accordance with the partial sequence in databases under accession number AJ222834 [GenBank] . The Acinetobacter spp. hns gene and its product have been assigned EMBL accession number AJ458445 [GenBank] .
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RESULTS |
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The second strain was collected from frozen continental water in Terre Adélie in Antarctica by C. Gerday, as previously mentioned (27). The morphological and biochemical characteristics of this strain, e.g. coccoid occurring in pairs, oxidase- and catalase-positive, nonmotile bacterium, and growth at low temperatures from 0 to 25 °C with an optimum growth temperature close to 15 °C, indicate that it could be classified either in Moraxella or Psychrobacter groups in agreement with recent data (36). Sequence analysis of the 16 S rDNA (accession number AJ310992 [GenBank] ) together with other characteristics of TAD1 bacterial strain such as a tolerance to 8% NaCl and a susceptibility to bile salt allowed us to refine the classification of this bacterium within the Psychrobacter genus and indicated that this strain could be related to Psychrobacter phenotypic group 2 (phenon 2), which is represented by Psychrobacter uratovorans (37).
Isolation and Characterization of Cold-adapted H-NS-like ProteinsTo isolate a putative hns-like gene from both bacterial strains, we took advantage of the serine susceptibility of hns mutants in E. coli (38). A genomic library was constructed for both strains (see "Materials and Methods"), and each of them was introduced into the hns E. coli strain BE1410. The selection was performed on minimal medium supplemented with serine, as previously described (12, 15). Several clones were screened at 20 °C for 3 additional phenotypes, i.e. swarming on semi-solid medium, -glucoside metabolism on MacConkey agar plate, and mucoidy on rich medium.
Analysis of the nucleotide sequence of different plasmid DNA inserts revealed the presence of a coding sequence of 321 bp, coding for a 107-amino acid protein with a predicted molecular mass of about 12 kDa and a pI of about 8 in both organisms. The analysis of flanking regions suggests that both genes are not part of a polycistronic operon. As compared with the E. coli H-NS, the modification in the amino acid composition, e.g. the proline content, of the Psychrobacter spp. orthologous protein (Table I) seemed to be similar to that commonly observed from mesophilic to psychrophilic proteins (39). Multiple alignment with various H-NS-related proteins revealed that both cold-adapted proteins share 40% amino acid identity in common; they also showed more than 30% identity with the H-NS-related proteins of Rhodobacter species, i.e. HvrA and SPB, and less than 20% with the E. coli H-NS amino acid sequence (Fig. 1). Despite this, the N-terminal part of these two new proteins was predicted to adopt an -helical structure, like H-NS in E. coli (data not shown). Moreover, the Acinetobacter H-NS protein displayed the H-NS consensus motif, i.e. YX6(G/S)-(E/D)X(0/2)TW(T/S)G(Q/R)G(R/K)XPX(4/5)AX(3/4)G (0/2, 4/5, and 3/4 indicate 0 or 2, 4 or 5, and 3 or 4 residue(s), respectively) (15), whereas the (G/S) residue was replaced by an asparagine in the Psychrobacter protein (Fig. 1). Finally, using FROST, both C-terminal domains were clearly predicted to share a similar three-dimensional structure with the E. coli H-NS protein, with an error rate lower than 1% as indicated by the normalized distances obtained, i.e. 7.5 and 8.1, respectively, for the Acinetobacter and Psychrobacter proteins (34).
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Effect of a Moderate Temperature Increase on in Vivo Biological Activity and in Vitro Biochemical Properties of the Psychrobacter H-NS ProteinBecause of the low growth temperature optimum of both Acinetobacter spp. and Psychrobacter spp. strains, the ability of their H-NS-like proteins to complement phenotypes of E. coli hns mutant was evaluated at various temperatures. Remarkably, the overexpression of Acinetobacter protein fully restored, like the E. coli H-NS protein, a wild-type phenotype with regard to -glucoside utilization and mucoidy at all temperatures tested, whereas the Psychrobacter H-NS protein was no longer able to reverse the effects of H-NS deficiency in an E. coli mutant strain above 30 °C (Table II). This loss of in vivo complementation observed above 30 °C did not result from the proteolysis of the protein. Indeed, the over-expressed H-NS protein of Psychrobacter was visualized on a polyacrylamide gel after extraction from E. coli hns cells grown at 37 °C (data not shown). This observation prompted us to further examine the biochemical properties of purified Psychrobacter H-NS protein at various temperatures.
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One of the most important functions of H-NS-like proteins is their ability to bind curved DNA (18). Therefore, the binding properties of a recombinant His-6 Psychrobacter H-NS protein was analyzed at 4 and 37 °C in gel retardation experiments. As compared with the 400-bp DNA fragment used as a control, a full retardation in the electrophoretic mobility of both flhDC and bla curved DNA fragments was observed at 4 °C at a protein concentration of 0.25 µM (Fig. 2). No such retardation was detected at 37 °C at the same concentration (Fig. 2). Nevertheless, an increase in the concentration of the protein up to 1 µM resulted in a specific retardation of both curved DNA fragments at 37 °C (Fig. 2).
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The H-NS activity also relies on its propensity to form oligomers (40). Therefore, the ability of the Psychrobacter H-NS protein to oligomerize in vitro was analyzed by cross-linking experiments at 4 and 37 °C (Fig. 3). After a 60-min incubation, the protein was able to form oligomers at 4 °C in the presence of linking reagents (Fig. 3). In contrast, only a residual amount of protein oligomerized as dimers at 37 °C. In addition, no higher order form was observed as compared with those visualized at 4 °C (Fig. 3).
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The Low Thermal Stability of the Psychrobacter H-NS Protein Depends on the N-terminal DomainThe loss of Psychrobacter H-NS protein activity in vivo could result from an altered conformation during a moderate temperature increase. To evaluate the role of the different domains of the Psychrobacter protein in its thermosensitivity, we constructed different chimeric proteins with either the N- or the C-terminal part of E. coli H-NS fused to the C- or N-terminal part of the Psychrobacter protein (see "Materials and Methods"). To rule out any effect of temperature above 30 °C on Psychrobacter hns gene expression, all constructs were placed under the same transcriptional control of the E. coli hns promoter, giving rise to three plasmids (Table III) pDIA580 (producing the Psychrobacter wild-type H-NS protein), pDIA581 (producing a chimeric protein containing the N-terminal part of the E. coli H-NS and the C-terminal domain of the Psychrobacter protein), and pDIA582 (producing a chimeric protein containing the N-terminal part of the Psychrobacter protein and the C-terminal domain of the E. coli H-NS).
These plasmids were used in in vivo complementation experiments at two different temperatures, i.e. 25 and 37 °C. The synthesis of a chimeric protein containing the N-terminal part of the Psychrobacter H-NS protein (i.e. from plasmid pDIA582) in the hns E. coli mutant strain restored the wild-type phenotypes at 25 °C but not at 37 °C, as observed with the wild-type Psychrobacter protein from plasmid pDIA580 (Tables II and III). In contrast, the synthesis of a chimeric protein containing the N-terminal part of the E. coli H-NS protein fused to the Psychrobacter C-terminal domain (i.e. from plasmid pDIA581) fully reversed the effects of H-NS deficiency in an E. coli hns mutant also at 37 °C (Table III).
Biophysical Properties of the Psychrobacter H-NS, a Thermosensitive ProteinThe influence of an increase in temperature on the conformation of the Psychrobacter H-NS protein was evaluated by CD spectroscopy (41). Below 25 °C, the spectra of the protein revealed the presence of 1 positive maximum band at 190 nm and of 2 negative maxima bands at 208 and 222 nm, indicating the existence of a large proportion of -helix structures (Fig. 4A). As a control, CD analyses of the E. coli H-NS protein also revealed the existence of a large portion of
-helix structures (data not shown), which mainly reflect the secondary structure of its N-terminal part, in agreement with recent results (40). In contrast, unlike both the E. coli (data not shown) and the Acinetobacter (Fig. 4B) H-NS proteins, a moderate increase in temperature led to a complete change in CD profile of the Psychrobacter H-NS protein. The presence of an isodichroic point at 202 nm indicated a transition from the
-helix structures toward random coils (Fig. 4A). In addition, the CD profile did not revert to the native spectrum after a temperature down-shift from 50 to 20 °C, and after cooling the CD profile remained similar to that observed above 30 °C (Fig. 4A), providing evidence that, unlike E. coli H-NS (40, 42) and Acinetobacter H-NS (Fig. 4B), the perturbation observed was irreversible. Finally, from the sigmoidal shape of the denaturation curve resulting from the CD spectra, we could determine a melting temperature (Tm) of 21 °C, which further supports the low thermal stability of the Psychrobacter H-NS protein (Fig. 5).
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DISCUSSION |
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The catalytic activity of psychrophilic enzymes is known to decrease dramatically as the temperature increases (4). For instance, the activity of a -amylase enzyme secreted by the Antarctic bacterial strain Alteromonas haloplanctis decreases above 25 °C because of a low thermal stability, whereas a homologous protein of pig pancreas displays an optimum activity at 60 °C (43). This low thermal stability in psychrophilic enzymes, which is often considered as a consequence of higher flexibility and catalytic activity, results from a decrease in electrostatic and hydrophobic interactions (5, 44). Usually various modifications are observed when psychrophilic proteins are compared with their mesophilic counterparts, such as a decrease of charged residues, a substitution of hydrophobic residues, e.g. Ala instead of Val, or a decrease in proline content (39). In this respect, as compared with the E. coli H-NS protein, the aspartate and valine content was decreased in the Psychrobacter H-NS protein sequence (Table I). Recent results show that the substitution of two alanines by two threonines and one alanine by one valine could create a psychrophilic-like subtilisin from a mesophilic enzyme (45). Interestingly, the threonine content increases in the Psychrobacter H-NS protein as compared with the orthologous protein in E. coli. In addition, the asparagine content, which is considered as a thermolabile residue (46), is increased in the Psychrobacter protein. No such differences were observed in the sequence of the Acinetobacter H-NS protein (Table I). Finally, the Psychrobacter H-NS protein has the lowest proline content, i.e. one proline residue (Fig. 1), as compared with all H-NS-related proteins identified so far. This proline residue, which is located in the C-terminal domain, corresponds to the Pro-115 residue in E. coli. In this organism, this proline has been suggested to play a crucial role in oligomer formation despite its location in the DNA binding domain (47). Although this proline residue could also be involved in the oligomerization of other H-NS proteins, its presence is not sufficient to confer a thermal stability to the H-NS protein of Psychrobacter. In this respect, we were not able to create a mesophilic-like protein from H-NS of Psychrobacter spp. by random mutagenesis experiments,2 suggesting that more than one amino acid modification is involved in its structural adaptation to low temperature.
The Acinetobacter H-NS protein fully restored to the wild type H-NS-dependent phenotypes at all temperatures tested although the Psychrobacter H-NS protein was no longer able to reverse the effects of H-NS deficiency in an E. coli mutant strain above 30 °C (Table II). Moreover, the Psychrobacter H-NS protein could no longer bind curved DNA fragments at 37 °C excepted at a concentration of 1 µM (Fig. 2). In contrast, no oligomer form could be visualized at the same temperature even with a protein concentration of 25 µM (Fig. 3). This suggests that, like the H-NS protein of E. coli (48), the Psychrobacter protein could bind curved DNA as a monomer although with a lower efficiency. More importantly, these observations suggest that the effect of an increase in temperature results in an alteration of the conformation of the N-terminal domain. The construction of chimeric proteins between the H-NS protein of E. coli and the orthologous protein of Psychrobacter supports this hypothesis. Indeed, the protein containing the N-terminal part of the Psychrobacter H-NS combined with the C-terminal part of the E. coli H-NS restored the wild-type phenotypes at 25 °C but not at 37 °C (Table III). In contrast, the protein containing the N-terminal part of E. coli H-NS fused to the C-terminal part of the Psychrobacter protein reversed the effects of H-NS deficiency in an hns E. coli mutant strain at 37 °C (Table III). These results clearly demonstrated that the organization of N-terminal domain of the Psychrobacter protein may account for the low thermal stability of this H-NS-like protein.
In S. typhimurium, the N-terminal domain of H-NS has been recently shown to contain three -helical regions (16). The authors proposed a model of global topological fold for the N-terminal domain; the third and longest
-helix forms the core of a coiled-coil configuration, whereas the two others stabilize the structure (16). Secondary structure prediction methods clearly suggest that the Psychrobacter H-NS protein characterized in the present study contain less than three
-helical regions (data not shown). It is, therefore, tempting to speculate that the amino acid modifications and the different
-helix organization observed in its N-terminal domain as compared with that of the orthologous E. coli protein reflect a structural adaptation to low temperature of the Psychrobacter H-NS protein. These features might, therefore, offer weaker intermolecular interactions as compared with other H-NS proteins such as those of Acinetobacter and E. coli (or S. typhimurium). CD analyses performed with the Psychrobacter H-NS protein in comparison with the E. coli and Acinetobacter H-NS proteins support this hypothesis (Fig. 4). Indeed, unlike the E. coli protein, which has been isolated more than 30 years ago as a heat-stable factor (49), and the Acinetobacter H-NS protein (Fig. 4B), the
-helical structure of the Psychrobacter protein was irreversibly denatured upon a moderate temperature increase (Fig. 4A). In addition, although the Tm of the E. coli H-NS protein was estimated around 60 °C (40, 42) and that of Acinetobacter H-NS around 40 °C (data not shown), we could clearly determine the Tm of the Psychrobacter H-NS protein, i.e. 21 °C (Fig. 5). This Tm value is significantly lower than that of both E. coli and Acinetobacter H-NS proteins and may explain the temperature effect observed in vitro and in vivo with the Psychrobacter H-NS protein. A moderate temperature increase could, therefore, easily disassociate oligomers with a subsequent and/or concomitant irreversible denaturation of the protein, as observed by CD analysis. We can hypothesize from these results that the organization of
-helix structures in the N-terminal domain is crucial for the structure and the function of H-NS-related proteins. Hence, the identification and characterization of proteins from micro-organisms phylogenetically distant and/or with very different live style, including extremophiles, may lead to a better understanding of the structure, function and evolution relationship of this family of enigmatic proteins.
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FOOTNOTES |
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Supported by Ministère de l'Enseignement Supérieur et de la Recherche grants.
To whom correspondence should be addressed: Laboratoire de Dynamique, Evolution et Expression de Génomes de Microorganismes, Université Louis Pasteur, 28 rue Goethe, 67000 Strasbourg, France. Tel.: 33-3-19-24-20-08; Fax: 33-3-19-24-20-28; E-mail: bertin{at}gem.u-strasbg.fr.
1 L. Denissova, unpublished data.
2 C. Tendeng, E. Krin, O. A. Soutourina, A. Marin, A. Danchin, and P. N. Bertin, unpublished results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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