Modification of the protein expression patterninduced in the nitrogen-fixing actinomyceteFrankia sp. strain ACN14a-tsr by root exudates ofits symbiotic host Alnus glutinosa and cloning ofthe sodF gene

Y. Hammad, J. Maréchal, B. Cournoyer, P. Normand, and A.-M. Domenach

Abstract: Two-dimensional (2-D) polyacrylamide gel electrophoresis was used to detect proteins induced inFrankia sp.strain ACN14a-tsr by root exudates of its symbiotic host,Alnus glutinosa. The 5 most prominent proteins were purifiedfrom 2-D gels and characterized by N-terminal sequencing. All of these proteins had a high percentage of similaritywith known stress proteins. One protein match was the Fe superoxide dismutase (Fe-SOD), another was a tellurite re-sistance protein (Ter), the third was a bacterioferritin comigratory protein (Bcp); and two matches, differing only bytheir isoelectric point, were the same small heat shock protein (Hsp), a major immune reactive protein found in myco-bacteria. This suggests that the symbiotic microorganismFrankia, first responds with a normal stress response to toxicroot products of its symbiotic host plant. To confirm its identity, the gene corresponding to the Fe-SOD protein,sodFwas isolated from a genomic library by a PCR-approach and sequenced. It is the first stress response gene character-ized in Frankia.

Key words: Frankia, Alnus glutinosa, root-exudates, superoxide dismutase, tellurite resistance, bacterioferritincomigratory protein, heat shock protein.

Résumé: La technique d’électrophorèse sur gel de polyacrylamide en deux dimensions a été utilisée pour détecter lesprotéines induites dans la souche ACN14a-tsr du genreFrankia par les exsudats des racines de son hôte symbiotique,Alnus glutinosa. Les 5 plus importantes protéines ont été purifiées à partir de gels 2-D et caractérisées par séquençagede l’extrémité N-terminal. Toutes ces protéines montraient un fort pourcentage de similitude avec des protéines destress connues. Une de ces protéines correspondait à la superoxyde dismutase ferrique (Fe-SOD), une autre à la pro-téine de résistance au tellurite (Ter), la troisième à la protéine comigratoire de la bacterioferritine (Bcp); et les deuxautres, différant seulement par leur pI, correspondaient toutes deux à la petite protéine de choc thermique (Hsp), uneprotéine immunoréactive majeure retrouvée chez les mycobactéries. Ceci indique que le micro-organisme symbiotiqueFrankia possède une réponse initiale normale face au stress généré par les produits toxiques des racines de sa plante-hôte symbiotique. Afin de confirmer son identité, le gène correspondant à la protéine Fe-SOD,sodF, a été isolé à partirde la banque génomique par une approche PCR et séquencé. C’est le premier gène de réponse au stress caractériséchezFrankia.

Mots clés: Frankia, Alnus glutinosa, exsudats de racines, superoxyde dismutase, résistance au tellurite, protéinecomigratoire de la bactérioferritine, protéine de choc thermique.

[Traduit par la Rédaction] 547

Hammad et al.Introduction

Frankia is a Gram-positive eubacterial genus of slow-growing actinomycetes that form nitrogen-fixing root nodule

symbioses with plants belonging to 8 families of woodydicotyledonous plants.

Even though a similar root hair curling step is observedwith both Frankia andRhizobiumprior to nodule formation,the genes involved in the early stages of host recognition be-tweenFrankia and its plant host are unknown. There is pres-ently no solid evidence for the existence inFrankia ofnodulation genes homologous to the nod genes ofRhizobium(Reddy et al. 1995; Cérémonie et al. 1998). Moreover, nohomologs of the legume early nodulin genes described byScheres et al. (1990) have been isolated from actinorhiza,which suggests that different genetic programs are inducedduring the infection processes in both types of nitrogen-fixing root nodule symbioses.

Can. J. Microbiol.47: 541–547 (2001) © 2001 NRC Canada

541

DOI: 10.1139/cjm-47-6-541

Received August 1, 2000. Accepted December 5, 2000.Published on the NRC Research Press Web site on June 6,2001.

Y. Hammad, J. Maréchal, B. Cournoyer, P. Normand,1

and A.-M. Domenach.Ecologie Microbienne, UMR CNRS5557, Université LYON 1, 43 Boulevard du 11 Novembre1918, 69622 Villeurbanne, CEDEX, France.

1Corresponding author(e-mail: normand@biomserv.univ-lyon1.fr).

Lack of a genetic analysis system inFrankia (this bacteriahas not yet been transformed) has restricted progress in un-derstanding the nature of the signals exchanged betweenboth partners of the actinorhizal symbiosis, using standardgenetic complementation approaches.

Molecular approaches, whether based on the isolation ofmessenger RNA or from expressed proteins, may be helpfulin understanding the infection process. For example, a nod-ule-specific gene encoding a subtilisin-type protease ex-pressed in the early stages of nodule development was foundin cDNA libraries fromAlnus glutinosaroot nodules (Ribeiroet al. 1995) and its expression in nodules was followed by insitu hybridization. Similarly, using SDS–PAGE protein sepa-ration (sodium dodecyl sulfate–polyacrylamide gel electro-phoresis) followed by N-terminal microsequencing, it wasshown that a 48 kDa protein with no homolog in data bankswas induced in a Casuarina-infectiveFrankia strain, by plantflavonoids (El-Morsy 1995). It is well known that flavonoidssecreted by the legume host plant induce nod genes expres-sion inRhizobium(Schultze et al. 1994; Dénarié et al. 1996;Spaink 1996).

The resolving power of SDS–PAGE is not very importantbecause this technique separates proteins on the basis onlyof size. Two-dimensional (2-D) gel electrophoresis allowsthe simultaneous resolution of hundreds of proteins in a sin-gle procedure and can be used to investigate proteins ex-pressed byFrankia and actinorhizal plants during the earlysteps of their interactions. This method was used for taxo-nomic purposes onFrankia strains (Benson et al. 1984) butthis approach has not been applied to physiological studies.

Although 2-D electrophoresis has been used for nearly20 y (O’Farrell 1975), the relatively recent introduction ofimmobilized pH gradients in the first-dimension strips inpreformed gels (Görg et al. 1988) and easily programmablepower supplies, allow the technique to be reproducible.

In this paper, we sought to characterize proteins inducedin Frankia by plant root exudates. These proteins were de-tected by comparison of 2-D protein profiles of bacteriagrown in the presence and absence of plant exudates. Someof these were characterized and found to correspond to bac-terial stress response proteins. One protein was homologousto superoxide dismutase (SOD), a protein already describedin the actinorhizal symbiosis, and we set out to clone andcharacterize the corresponding gene.

Materials and methods

Bacterial strains, culture media, and growth conditionsThe thiostrepton-resistant derivative (Cournoyer and Normand

1994) of theAlnus-infectiveFrankia strain ACN14a (Normand andLalonde 1982) was grown in nitrogen-free BAP medium (Murry etal. 1984). When the ACN14a-tsr cultures were at the end of the ex-ponential phase, they were syringed 4× to homogeneity using a21G needle.

Protein quantification was done by a bicinchoninic acid (BCA)protein assay (Pierce, Rockford, Ill.) according to the manufac-turer’s protocol, after centrifugation of an aliquot of the culture,washing twice with distilled water, then resuspending in water andsonicating on ice (6 × 1 mn).

Four cultures were inoculated with an equivalent content of6 mg·L–1 of mycelial protein. They were grown at 28°C using 2 Lflasks containing 1 L of nitrogen-free BAP medium and stirred at

200 rpm with a 3 cm longcross-type magnetic bar (Schwencke1991). After 3 days of growth, 10% v/v of N-free Fähraeus me-dium (Vincent 1970), two with and two withoutA. glutinosarootexudates were added into theFrankia cultures.

Root exudates were obtained using surface sterilized seeds incu-bated in the dark at 4°C for 4 days to release dormancy. After4 days at 26°C, germinated seedlings were transferred to bioassayslides (40 plantlets in 120 mL of medium) as described byBhuvaneswari and Solheim (1985). The slide chambers were filledwith Fähraeus medium and incubated in the growth chambers for1 month. All handlings were carried out under sterile conditions.Fähraeus medium containing exudates were harvested and addedinto two culture flasks ofFrankia cells. After 4 days in the pres-ence of the exudates, the cultures were harvested when they hadreached the end of the exponential phase under our growth condi-tions. The proteins were then analyzed by 2-D electrophoresis.

Protein sample preparationFrankia cells were harvested from 7-day-old cultures. After a

first centrifugation step, they were washed 3× with distilled waterand resuspended in 8 mL of water. The suspension was sonicatedat 700 W (cumulative value) for 6 × 1 mn. Thesonically treatedmaterial was centrifuged at 1000 ×g for 20 min of to remove celldebris, then the protein concentration was measured by BCA pro-tein assay. For each sample, one aliquot of 100µg was precipitatedwith 5 volumes of acetone, then mixed with 50µL of samplebuffer: 25 mL of 9 M urea, 130µL of triton ×100 (Sigma, St.Louis, Mo.), 250 mg of DTT, 0.5 mL of pharmalyte 4-7(Pharmacia, Uppsala, Sweden) and approximately 5 mg of bromo-phenol blue, for loading on the first-dimensional IPG strips.

2-D polyacrylamide gel electrophoresisA Pharmacia Biotech Multiphor II Electrophoresis Unit

equipped with a multidrive gradient power supply was used to per-form the electrofocusing. Precast Immobiline Dry Strips, pH 4-7linear, 110 mm (Pharmacia), were rehydrated with rehydration so-lution (25 mL of 8 M urea, 130µL of triton ×100, 50 mg of DTT,130µL of Pharmalite 4-7, 0.5% v/v Nonidet P40, and a few grainsof orange G) overnight in a reswelling cassette (Pharmacia). TheMulti Temp III (Pharmacia) cooling bath was loaded at the anodic(acidic) end using sample cups, then allowed to focus under a lowviscosity oil with a programmed voltage gradient (300 V during6 h and 3500 V during 10 h 30 min). After focusing in the first di-mension, IEF gels were each equilibrated twice for 17 min in 2 ×10 mL of equilibration buffer (6 M urea, 30% v/v glycerol, 3% w/vSDS, 0.05 M Tris–HCl buffer pH 6.8). Ten mg·mL–1 DTT wereadded for the first equilibration step and 45 mg·mL–1 of iodo-acetamide and a few grains of bromophenol blue for the 2nd step.The second dimension is a vertical 12% w/v SDS–PAGE as de-scribed by Laëmmli (1970). The cooling bath was set at 15°C andthe gels run at 150 V for 5 h. A silver staining method (Pharmacia)was used because of its high sensitivity.

MicrosequencingFive milligrams of protein were dissolved into the rehydration

solution and IPG strips were rehydrated in the Immobiline DryStrip Reswelling Tray (Pharmacia). From 2-D gels, proteins weretransferred to PVDF membranes (Schleicher & Schuell, Dusseldorf,Germany) as described by Matsudaira (1987), stained by Coomassieblue, and sequenced in a 492 Protein Sequencer Procise AppliedBiosystems (Perkin Elmer, Norwalk, Conn.). The proteins werecompared to the NCBI and SWISS-prot data banks using the NCBIserver <http://www.ncbi.nlm.nih.gov/blast> and the BLAST-Pfunction (Altschul et al. 1997).

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542 Can. J. Microbiol. Vol. 47, 2001

Cloning of the sodF geneA set of primers targeting thesodF gene was made using a set

of aligned sodF homologous protein sequences from variousActinobacteria (AF141866, Q59519, X81385, S52361, P53644,S52366, JC4351, P53651, P13367, I39745, X91650, P80293,JC4396, I40858). The resulting primers were CCACGTACACGC-TnCCnGA targeting the conserved ATYTLPD motif found bymicrosequencing at the N-terminal end and AGGTAGAAnGCr-TGyTCCCA which is complementary to the conserved motifWEHAFYL. The resulting amplicon was sequenced and used todesign a 2nd generation of primers specific toFrankia that did notcross-react withEsherichia coli. The Frankia strain ACN14a-tsrgene library (Cournoyer and Lavire 1999) built in the low-copyvector pRK404 (Ditta et al. 1985) was screened with these primersand a clone designated pFAQ350 was isolated and partially se-quenced aroundsodF. Sequence analysis (ORF, start/stop, codonusage) was made using the MACVECTOR software, v. 4 (Kodak,Rochester, N.Y.).

Results

Two 2-D polyacrylamide electrophoresis gels of total pro-tein extracts ofFrankia strain ACN14a-tsr growing in N-freemedium are shown in Fig. 1. The majority of visible proteinsmigrated to an acidic position in the isoelectric focusing di-mension. One hundred to 200 spots per gel were revealed.The protein patterns produced by 2-D-PAGE were reproduc-ible when cultures were grown under identical conditionsand did not change dramatically in the absence or in thepresence ofA. glutinosa root exudates. However, severalprotein spots showed intensity differences. Spots that exhib-ited a clear increase between the 2 conditions or were absentwithout exudates and present in the presence of exudates,were selected and named Fre1 to 4′ (Frankia Response toExudates). Under these conditions, 5 major spots indicatedby an arrow in Fig. 1 were chosen and about 30 amino acidsat their N-terminal ends were sequenced (Table 1). Supple-mentary proteins were visualized in analytical silver stainedgels but disappeared in preparative gels colored with Co-omassie brilliant blue and therefore could not be analyzedfurther.

A BLAST-P search with the five peptides listed in Table 1yielded matches in all cases. The Fre1 amino acid sequencehas 76% similarity with the Fe superoxide dismutase (Fe-SOD) of Mycobacterium smegmatis. The Fre2 amino acidsequence had 92% similarity with the tellurite resistance(Ter) protein of Streptomyces coelicolor. The Fre3 aminoacid sequence had 67% similarity with a bacterioferritincomigratory protein (Bcp) ofE. coli. The last two up-regulated proteins (Fre4 and Fre4′) with the same molecularweight, but different isoelectric points (pI, difference of 0.5pH units approximately), matched with the same heat shockprotein: the first 22 amino acid residues of Fre4 and Fre4′spots showed 81% similarity with the 18 kDa Hsp ofMyco-bacterium leprae.

Five pools of theFrankia strain ACN14a-tsr genomic li-brary (500 clones per pool of 96 clones each) when screenedwith the sodF specific primers gave two PCR positive sig-nals, one of which was isolated and characterized furtherand found to correspond to a single clone with an insert ofabout 10 kb. Sequencing was started with thesodF primers

and continued until the whole of the coding sequence andsurrounding regions was obtained.

The sequence of thesodFgene, deposited to EMBL underaccession number AJ277540, is given in Fig. 2. It is 621 nu-cleotides long and starts with a GTG. An alternative GTGstart site situated 50 nt upstream was discarded given thelack of a suitable ribosome binding site and the fact thatmost published homologous sequences start at about thesame residue. The closest neighbor isS. coelicolor(AL138662.1)sodF at 83% similarity in the DNA sequenceand 63% in the polypeptide sequence. The codon usageshows a strong bias toward G- and C-ending codons (194/206G- or C-ending for 94.2%, against a 66% G+C overall) withthe notable exception of the counter-selected GGG glycinecodon. Two differences were found between the peptide(Fig. 1) and the DNA sequence, both at the end of the pep-tide where the background noise was beginning to rise. Theamino acid sequence was found not to be processed, startingimmediately after the first methionine residue. The aminoacid sequence was aligned with homologoussodFandsodAsequences to determine if the conserved residues were pres-ent, for instance the D-x-W-E-H-(STA)-(FY) motif given byPROSITE <www.expasy.ch/cgi-bin/> as the main metalligand region. The last of these positions was not conservedbut replaced by a tryptophan. The four metal ligand sites (3histidine and 1 aspartic acid) are conserved. No obvious pro-moter or transcription stop was detected.

Discussion

Two-dimensional protein electrophoresis has been shownto permit a global view of proteins induced in response toenvironmental factors and thus of the physiological eventstaking place. It appears particularly appropriate as an ap-proach for a microorganism refractory to classical genetictechniques. However,Frankia has a slow growth rate and areduced metabolism in nutrient poor medium (Schwencke1991). Moreover, the high lipid content of some bacteria,which is the case forFrankia (Berry et al. 1993), was shownto reduce protein solubility and electrofocusing gel quality(Rabilloud 1996). Appropriate sample preparation must bedetermined empirically, for instance, the choice of detergentsin sample buffer and equilibration conditions. Choice of op-timal isoelectric focusing conditions is also crucial and theconditions described here were found to yield reproduciblepatterns. This approach allowed us to identify 5 proteins in-duced by root exudates that were named Fre1 to Fre4′ andidentified as well characterized stress proteins.

SOD is considered as one of the key enzymes in the oxi-dative defense system of aerobic organisms, destroying oxy-gen radicals such as the superoxide ion (O2

•–) that areproduced within the cells from the normal step-wise respira-tory electron transfer to O2 and also through the enzymexanthine oxidase as part of the host response to pathogens(Umezawa et al. 1997). Superoxide is toxic to biologicalsystems in general (Kim et al. 1998), and particularly to4Fe-4S proteins (Touati 1997). Enzymes in the latter cate-gory comprise general metabolic enzymes such as fer-redoxins and cytochromes, but also enzymes specificallyexpressed in the actinorhizal symbiosis such as nitrogenaseand hydrogenase. SOD is thus important forFrankia metab-

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Hammad et al. 543

olism in general and crucial under N2-fixing conditions,which may explain why its level inFrankia is among thehighest reported in prokaryotes (Steele and Stowers 1986).The highest SOD activity was observed whenFrankia wascultivated in N-free medium (Puppo et al. 1989) and wasconcentrated in the diazo-vesicles suggesting an importantrole for SOD in the protection of the oxygen-labile nitrogen-fixing machinery. The increased SOD level in N-free me-dium in the presence ofAlnusroot exudates suggests that anoxidative stress is induced by exuded plant compounds. Thisis supported by the fact thatPseudomonas putida sodmu-tants, especially the Fe-SOD mutants, are impaired forgrowth in the rhizosphere of bean (Kim et al. 2000).

The genetic determinant of the SOD protein was charac-terized to ascertain its identity: thesod gene in Frankiastrain ACN14a-tsr was found closest to the iron–protein (ECnumber = 1.15.1.1) gene inStreptomycesspp. namedsodFbecause it contains iron. Based on inhibition experiments,the protein present in anotherAlnus-infective Frankia strainwas deduced to contain iron (Alskog and Huss-Danell1997). This supports our opinion that theFrankia geneshould thus be namedsodF and notsodAas are called thedeterminants for the manganese-containing proteins in someof the phylogenetically relatedMycobacteriumspp., whichalso yield high scores with BLAST. Such enzymes are de-scribed as Mn-containing seemingly on the basis of similar-ity to sodA genes from other bacteria (Menendez et al.1995), which is a problem with gene annotation based onBLAST scores alone.

The Fe-SODs present inMycobacteriumspp. are de-scribed as extracellular (Kang et al. 1998) while nothing isknown about the location of Fe-SOD inFrankia. Neverthe-less, given that superoxide radicals cannot cross membranesunder physiological conditions (Touati 1997), the localiza-

tion of the protein in nodules will indicate whether its func-tion is to protect the superoxide-labile nitrogenase within thevesicles or to fight against host defenses extracellularly as isthe case for the pathogenicMycobacterium tuberculosis(Harth and Horwitz 1999).

As described by Orsaria et al. (1998) inS. coelicolor,SOD and Ter proteins are expressed together. These authorsproposed a joint regulation for bothsodand Ter genes (ter)similar to thesoxRSsystem that was shown inE. coli (Hi-dalgo et al. 1997) to control the expression of the oxidativestress regulon and the response to toxic heavy metals. Thepresent work opens the possibility of following the expres-sion pattern of this complex regulon, known to be triggeredby different and unrelated stimuli such as the presence ofheavy metals (Yoo et al. 1999), sporulation (Henriques et al.1998), and entry into stationary phase (Gort et al. 1999).

Until recently, Bcp was described simply based on the factthat it comigrated with bacterioferritin (Andrews et al.1991). A recent study (Jeong et al. 2000) showed thatE. colibcp null mutants are hypersensitive toward oxidating agentssuch as H2O2, t-butyl hydroperoxide and linoleic acid hydro-peroxide. Bcp-reduced hydroperoxides use thioredoxin as anelectron donor and thus Bcp was considered a new memberof the alkyl hydroperoxidase (AhpC) family. The co-induction with SOD of a peroxidase is all the more interest-ing given that the product of the SOD enzyme is hydrogenperoxide, which will spontaneously react in the Fenton reac-tion with ferrous iron (Fe2+) to form hydroxyl radical ionsthat are even more toxic for cell constituents than superoxideradicals (Touati 1997). This is presumably the reason whySOD is co-induced with catalase in nitrogen-fixingAlnusincana nodules (Alskog and Huss-Danell 1997).

The last two induced proteins with different pIs (differ-ence of 0.5 pH units approximately) but the same molecular

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544 Can. J. Microbiol. Vol. 47, 2001

Fig. 1. 2-D-electrophoresis gels ofFrankia sp. strain ACN14a-tsr grown in the absence (A) and presence (B) of plant exudates; ineach gel, the pH gradient extended from 4 to 7. The positions of marker proteins are indicated and correspond to kiloDaltons in de-scending order. Gels were stained with silver and the arrows point to proteins induced inFrankia by Alnus root exudates, named Fre1to Fre4′ and characterized by micro-sequencing.

weight were similar to the same small heat shock protein(sHsp). The fact that two spots had different pI but similarmass may be related to a post-translational modificationsuch as phosphorylation. The sHsp is one of the major im-mune reactive proteins inMycobacteria(Booth et al. 1993).In Streptomycesspp., sHsps appear to be involved in physio-

logical and morphological differentiation (Puglia et al. 1995)and their role in protein overproduction and secretion wasshown inE. coli (Wild et al. 1992). Few functions of sHsp’sare known, however, the role of Hsp18 in thermotolerancehas been reported inStreptomyces albus(Servant andMazodier 1995). It has been hypothesized that this proteinmay act as a molecular chaperone in protein folding and un-folding events (Jakob and Buchner 1994). The Hsp18 is nothomologous to the 18 kDa protein generated from Hsp58 en-coded by agroEL1-like gene;groEL is probably involved inthe folding or assembly of transcriptionally active NodD inRhizobium(Ogawa and Long 1995).

The 48 kDa protein induced inFrankia by root exudatesof the host plantCasuarina equisetifoliawas not found inthe present study. Several spots of approximately 45–50 kDawere present and may obscure the expression of a homolo-gous protein but given that the band characterized by El-Morsy Selim (1995) was important, it is likely that the re-sponses of the two symbiotic systems inCasuarina andAlnus are different.

The five proteins induced by root exudates and character-ized in our study are known to be involved in response tostress. This may reflect an adaptation to root products as anormal first step of the symbiosis process. Prior to infection,bacteria have to be able to resist defense products of the hostplant such as plant phenolics. Among others, benzoic acidcauses an acid shock inE. coli (Lin et al. 1996) and resultsin the induction of two dozen stress proteins (Lambert et al.1997). Furthermore, benzoic, salicylic, acetic and acetyl-salicylic acids are also chemotactic repellents forE. coli(Rosner 1985). Flavonoid phytoalexins are part of the plantdefenses during hypersensitive reaction (HR) (Rosner 1985)and have wide activity spectra againstPlasmodium(Seehausand Tenhaken 1998) and viruses (Li et al. 1995). Suchphenolics have been shown to be present inA. glutinosa(Perradin 1982). Symbiotic microbes should therefore beable to respond to the normal challenge imposed by theplant on its rhizospheric microflora.

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Hammad et al. 545

Frankia protein Matching protein with highest BLAST score

Amino acid sequenceMW(kDa)a pIb Protein description

MW(kDa)c pId Identities Positives

Fre1:ATYTLPDLKYDYGALEPSIIGQIVELLHH(K)K

21.5 4.7 Fe-SOD, (AF061031),(Mycobacteriumsmegmatis)

22.8 5.31 23/30 (76%) 23/30 (76%)

Fre2:GVSLSKGGNVSLSKEAPGLTNILVGLGXDV

22.5 4.1 Ter, (CAB62770.1),(Streptomycescoelicolor)

20.4 4.77 26/30 (86%) 28/30 (92%)

Fre3:TVEVGSVAPDFSLKDQNNEVITLSXFRG(K)(K)(N)(N)(V)

18 4.8 Bcp, (AAC46233.1),(Escherichia coli)

17.6 4.86 12/28 (42%) 19/28 (67%)

Fre4:XLVRTDPFRELDRL(S)QQVL(G)(T)L

17.2 5.7 sHsp 18 5.9

Fre4′:MLV(R)TDPFRELDRLSQQVLGTLXGVARPSG

17.2 6.1 sHsp, (P12809), 18 kDaantigenic protein(Mycobacterium leprae)

18 5.9 20/29 (68%) 24/29 (81%)

a,bMolecular weight and isoelectric point estimated from gels, respectively.c,dMolecular weight and isoelectric point of closest homologue in databases, respectively.

Table 1. Protein sequences characterized from preparative 2-D gels and their comparison with databases<http://www.ncbi.nlm.nih.gov/blast>.

Fig. 2. Sequence of theFrankia strain ACN14a-tsrsodF gene.The double-underlined sequences at coord. 4–24 correspond tothe forward primer, those at coord. 484–504 to the reverseprimer, and the underlined sequence at coord. 4–93 correspondto the protein microsequence (Fig. 1). The 4 shaded residues (3histidines and 1 aspartic acid) correspond to the conserved metalligands. Selected restriction sites (underlined) are indicated abovethe sequence.

These five polypeptides are the most abundant, but theyare by no means the only response ofFrankia to the chemi-cal environment of the host plant.Frankia with a genome of7000 – 10 000 kb (An et al. 1985) is capable of expressingthousands of peptides, many of which are expected to be in-volved in the subsequent steps of symbiotic development.

Acknowledgement

We thank M.M. Boutillon (Institut de biologie et chimiedes proteines, CNRS, Lyon) for protein sequencing.

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