Eur. J. Biochem. 160,69-75 (1986) 0 FEBS 1986

A cell-free system from Rhizobium meliloti to study the specific expression of nodulation genes Ilona DUSHA ’, Joachim SCHRoDER*, Peter PUTNOKY ’, Zsofia BANFALVI and Adam KONDOROSI ’ ’ Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, Szeged

Biologisches Institut 11, Lehrstuhl firr Biochemie der Pflanzen, Universitat Freiburg

(Received January 31/June 3,1986) - EJB 86 0146

An in vitro transcription-translation system was developed using cell-free extracts from the symbiotic nitrogen- fixing bacterium Rhizobium meliloti strain 41. Conditions for preparation of the 30000 x g supernatant extract and for measurement of protein-synthesizing activity were determined and compared to the activity of an Eschericha coli cell-free system. Genes expressed in the free-living or in the symbiotic state were studied. The product of a recA-like gene (41-kDa protein) was synthesized both in R. meliloti and E. coli extracts, although less efficiently in the heterologous system. In agreement with earlier results obtained in E. coli minicells, three proteins (44, 28.5 and 23 kDa) were synthesized from a cloned 3.3 x 103-base DNA region carrying genes for nodulation (nod). However, differences in the transcription-translation of nod and host specificity (hsn) genes were observed when protein expression was compared in R. meliloti and E. coli cell-free extracts, and the possible explanations of these findings are discussed.

Rhizobium meliloti, a member of the family Rhizobiaceae, is able to establish a symbiotic relationship with leguminous plants. As a result of the interaction of plant and bacterial genes, root nodules are formed which are able to fix atmo- spheric nitrogen.

Bacterial genes required for nodule formation have been identified in R. meliloti. The nodulation genes, described so far, are located on a large plasmid and are closely linked to nif genes [I - 51. Nodulation genes are arranged into two clusters: early nodulation functions, which are conserved in many Rhizobium species, form one cluster (‘common’ nod genes) [4, 6, 71, while genes determining host specificity are located in another cluster [6]. Protein-coding regions of ‘common’ nod genes (nodABC) were mapped [8]. The DNA sequences of the nodABC genes of R. meliloti 41 [9] and of R. meliloti 201 1 [lo, 111 were published. Recently the presence of a nodD gene close to nodABC was reported [ l l ] (and GZittfert et al., unpublished results).

Studies on the expression and regulation of genes es- pecially of those participating in symbiotic processes are of great interest. Wellcharacterized in vitro and in vivo systems are available for the determination of gene products and for regulation studies such as the cell-free coupled transcription- translation systems of Escherichia coli [12, 131, and E. coli minicells and maxicells [14, 151. These systems were suc- cessfully used to study genes of various origins [8, 16, 171. However, in certain cases the use of these E. coli systems did

Correspondence to I. Dusha, Genetikai I n t k t , Szegedi Biol6giai Kczpont, Magyar Tudominyos Akademia, Postafibk 521, H-6701 Szeged, Hungary

Abbreviations. S-30,30000 x g supernatant; Tc, tetracycline; Ap, ampicillin; Cm, chloramphenicol; kb, lo3 bases; nod, nodulation; hsn, host specificity of nodulation.

Enzyme. Lysozyme (EC 3.2.1.17).

not provide satisfactory results, and the need for specific systems emerged. Difficulties concerning the expression of cloned Ti plasmid DNA fragments [I 81 and of DNA segments of Rhodopseudomonas sphaeroides [19] or of cloned chloro- plast DNA from Euglena gracilis [16] were reported. To ex- plain the lack or decreased level of protein synthesis from some heterologous DNA templates in the E. coli system, differences in sequence signals involved in transcription and translation processes were invoked.

Recently we published the observation that SmR of trans- poson Tn5 is expressed in vivo in R. meliloti although it is not detectable in E. coli [20]. This finding also supports the idea that differences either on the transcriptional or translational level or both between the two species may exist. To circumvent these difficulties our aim was to establish an in vitro protein- synthesizing system from R. meliloti, which would enable us to study specific expression of Rhizobium genes.

MATERIALS AND METHODS

Preparation of R. meliloti cellqree extract

Strain AK631, a prototrophic, symbiotically effective de- rivative of R. meliloti 41 with compact colony morphology, was used for preparation of the 30000 x g supernatant (S-30) extract. Cells were grown with shaking at 170 rpm at 32°C in TY medium [21] to A600 = 0.7-1.0. Cultures were cooled rapidly in an ice/water bath and cells were collected by cen- trifugation at 6000 rpm for 20 min at 4°C. The bacterial pellet was washed twice with buffer A containing 20 mM Trislace- tate (pH 8.2), 10 mM magnesium acetate, 40 mM potassium acetate, 1 mM dithiothreitol. Bacteria were stored at - 20°C until used. For the preparation of S-30 extract 8 - 10 g frozen cells were thoroughly suspended in 9 - 11 ml ice-cold buffer

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A. Lysozyme, dissolved in 0.1 ml of the same buffer, was added to a final concentration of 0.05mg/ml and the suspension was kept on ice for 30 - 50 min. Cells were dis- rupted by two passages in a French pressure cell first at 69 MPa, then at 83 MPa. Immediately after disruption 150 pl 0.1 M dithiothreitol was added. The suspension was centrifuged twice at 15500 rpm for 30 rnin at 4°C in SM24 Sorvall rotor. After the second run 75% of the supernatant was removed carefully by pipette, and was preincubated in the following way: to 1 ml supernatant 90 p1 preincubation mixture was added containing 0.79 M Tris/acetdte (pH 8.4), 15 mM magnesium acetate, 4.4 mM ATP, 42 mM phospho- enolpyruvate, 5 mM dithiothreitol, 0.15 mM of each of 19 amino acids, 0.1 mM methionine and 0.05 mg/ml pyruvate kinase. The mixture was incubated for 2 h at 30°C in the dark. It was then dialysed for 4 h against four changes of 200 ml buffer A at 4°C in the dark. After this an aliquot was immediately tested for in vitro transcription-translation activi- ty, and the rest was distributed into 0.1-ml portions, rapidly frozen in liquid nitrogen and stored at - 70°C until used. The protein concentration of the extracts was about 7 - 10 mg/ml.

DNA-directed protein synthesis in R. meliloti cell-free extract

The standard reaction mixture of 25 pl contained 66 mM Tris/acetate, pH 8.4, 1.5 mM dithiothreitol, 55 mM potas- sium acetate, 0.55 mM CTP, GTP and UTP, 2.2 mM ATP, 21 mM trisodium phosphoenolpyruvate, 27 mM ammonium acetate, 14 mM magnesium acetate, 7.4 mM calcium chloride, 27 pg/ml folinic acid, 2% poly(ethyleneglyco1) 6000,0.15 mM each of 19 amino acids, 10- 15 pCi [35S]methionine and 1 pg template DNA. The reaction was started by the addition of 10 p1 cell-free extract. After 30 min or 60 min incubation at 30°C the reaction was stopped by adding 55 p1 dissociation buffer (0.08 M Tris/HCl, pH 6.8,2% sodium dodecyl sulfate, 10% glycerol and 0.004% bromophenolblue). Samples were heated for 5 min at 95"C, then either a 2 0 4 aliquot was removed to measure the incorporation of radioactive meth- ionine, or 60 pl was analyzed by polyacrylamide gel electro- phoresis.

DNA-directed protein synthesis in E. coli cell-free extract

The method of Zubdy et al. [12] was used with modifications [22, 231. Reaction mixtures were preincubated for 3 min at 37 "C before adding cell-free extract. After 30 min at 37 "C the reaction was terminated by the addition of 25 pl dissociation buffer.

Measurement qf radioactivity incorporated into proteins

To 20 pl reaction mixture 50 p1 0.1 M NaOH containing 3 mg/ml unlabelled methionine was added. After 10 min at 56 "C proteins were precipitated with 10% trichloroacetic acid, the precipitate was washed with acetone, dissolved in 25 pl dissociation buffer and measured in 5 ml liquid scintilla- tion cocktail containing one part Triton X-100 and two parts toluene and fluors, supplied by Amersham.

Polyacrylamide gel electrophoresis

Radioactive samples were analyzed by electrophoresis on a 12% polyacrylamide gel containing 0.1 % dodecyl sulfate according to the method of Laemmli [24]. I4C-labelled pro-

teins (Amersham) were used as molecular mass markers. Gels were kept overnight in 50% trichloroacetic acid, washed, soaked with EnjHance (Amersham) for 1 h, washed with distilled water for 1 h, dried and exposed at - 70°C with Kodak X-Omat S film.

Preparation of plasmid DNA

For large-scale preparation the method of Birnboim and Doly [25] was used. Plasmids were purified by two density- gradient centrifugations in a cesium chloride/ethidium bro- mide gradient.

Molecular cloning

Restriction endonucleases, purchased from Bethesda Re- search Laboratories or Boehringer (Mannheim), were used to obtain DNA fragments, which were separated by agarose gel electrophoresis. Fragments were electroeluted as described by Maniatis et al. [26], then ligated into digested and alkaline- phosphatase-treated vectors. The following plasmids were used for cloning: pBR322 [27] and pBR325 [28]. Other re- combinant plasmids are described [8] or were constructed by Horvath et al. [29]. E. Cali strain HBlOl was transformed with recombinant plasmids [30]. The method of Ish-Horowicz and Burke [31] was used to purify DNA from transformants, and the orientation of the inserted fragment was determined by restriction endonuclease analysis.

Chemicals Chemicals for the reaction mixtures of in vitro trans-

cription-translation were obtained from Sigma (CTP, GTP, UTP, ATP, phosphoenolpyruvate, folinic acid and amino acids) or from Merck (Tris, magnesium acetate, potassium acetate, ammonium acetate, calcium chloride and dithiothrei- tol). [35S]Methionine was the product of Amersham, N,N'- methylenebisacrylamide and four-times-crystallized acryl- amide were from Bio-Rad.

Plasmids

cription-translation systems are listed in Table 1. Plasmids used as DNA templates in the cell-free trans-

RESULTS

Characterization of the coupled transcription-translation system of R. meliloti

The composition of the reaction mixture for in vitro pro- tein synthesis is described under Materials and Methods. Our reaction mixture contained basically the same components as the E. coli system [23] with minor modifications in concentra- tions. In addition, 2% poly(ethyleneglyco1) 6000 was used, as recently described for the E. coli system [34]. Various cofactors and tRNA, used in the E. coli system, were omitted from the Rhizobium protein-synthesizing mixture. Some of the components (nucleotide triphosphates and phosphoenol- pyruvate) were added to the R. meliloti reaction mixtures in higher concentrations, as compared to the A. tumejuciens system [18].

The activity of a cell-free extract depends on the MgZf concentration of the reaction mixture and the optimum con- centration may vary in different preparations [23]. Fig. 1 A

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Mg2+ conc incub time

Fig. 1. Dependence ofpmtein-synthesizing activity on Mgz+ ion concen- tration ( A ) and incubation time ( B ) . Reactions were measured in the presence of 1 pg pACYC184 DNA (0-0) or without added DNA (0-0) at 30°C in R. meliloti cell-free extract. 2 0 4 ali- quots of the reaction mixtures were used to determine radioactivity as described in Materials and Methods

pBR322 pBR325

pACYC184 pIN-II-A2

Fig. 2. Expression of antihiotic resistance markers coded by dfferent plasmids in R. meliloti extract. 1 pg p ~ ~ 3 2 2 (lane 21, p ~ ~ 3 2 5 (lane 31, pACYC184 (lane 4) and pIN-II-A2 (lane 6) DNA was added to the standard reaction mixture. Lanes 1 and 5 show control saniplcs without added DNA. Protein products were scparated on a 12% sodium dodecyl sulfate/polyacrylamide gel. CAT, chloramphenicol transacetylase

Table 1. Plasmids used as DNA templales in cell-free transcription- translation systems

Plasmids Vector and characteristics Refer-

ApR, TcR

Clones carrying a rec gene of R. meliloti

pPP358 pPP395

Clones carrying common nod genes of R. meliloti

pJS120.1 pJSl20.2

pJS201.1 pJS201.2

Clones carrying hsn gencs of R. meliloti

pJS401 .I pJS401.2

pJS402.1 pJS402.2

pJS405.1 pJS405.2

pJS302.1 pJS302.2

pID500.1 pID500.2

pBR322, ApR, Tc', 5.3 kb BamHI fragment in two paper oricn tations

this

pACYCl84, TcR, CmS, 3.3 kb BglII-EcoR1 fragment in two orientations

Bglll-EcoR1 fragment in two orientations

[8]

pIN-II-A2, A$, 3.3 kb PI

pIN-II-A2, ApR, 4.2 kb ~ 9 1 BumHI fragment in two orientations

BamHI fragment in two orientations

SphI fragment in two orientations pACYC184, TcR, CmS, 3.5 kb SphI fragment in two orientations pBR325, ApR, TcR, CmS, 3.5 kb this SphI fragment in two Paper orientations

PIN-II-A~, ApR, 2 kb I291

pIN-II-A2, ApR, 3.5 kb 1291

[29]

obtained without added DNA, also increased threefold. A temperature of 30°C was, therefore, chosen for our measure- ments.

Fig. 1 B shows that the amount of proteins synthesized increases linearly for at least 60 min in the standard reaction mixture at 30°C.

Using pACYC184 plasmid as a template, it was found that increasing the DNA concentration beyond 1 pg/25 pI did not result in a proportional increase of the amount of labeled chloramphenicol transacetylase protein (data not shown).

Expression of antibiotic resistance genes in the in vitro transcription-translation system from R. meliloti

Plasmids carrying different antibiotic resistance markers were used to test the protein-synthesizing activity of the cell- free R. meliloti system. As shown in Fig. 2, the protein prod- ucts from the ApR gene are expressed using either pBR322 or pBR325 plasmids as a template. Expression of the ApK gene from pIN-II-A2 [32] is stronger, than from pBR325 or pBR322. No gene product corresponding to the TcR (tetracycline resistance) gene of pBR322, pBR325 or pACYC184 could be identified. Since chloramphenicol trans- acetylase is expressed well both from pACYC184 and pBR325, plasmid DNA coding for this resistance marker was used to optimize the Rhizobium cell-free extract.

Expression of R. meliloti genes in the R. meliloti cell-free extract

The first template of Rhizobium origin to be tested was a DNA fragment, carrying a gene expressed in the free-living

shows the protein-synthesizing activity of the extract mea- state of R. meliloti. A DNA fragment containing the recA sured at increasing Mg2+ concentrations. Other components gene of R. meliloti 41 was identified by interspecific of the standard reaction mixture were added as indicated in complementation of a recA- E. coli strain using the method Materials and Methods. of Better and Helinski [35]. Restriction analysis and DNA

Increasing the temperature of the reaction from 30°C to hybridization (unpublished data) showed homology between 37 "C resulted in a twofold increase of total incorporated the presumptive recA clone of R. meliloti 41 and the published radioactivity (not shown). However, the background activity, recA clone of R. meliloti 102F34 [35].

12

Fig. 3. Expression of cloned R. meliloti recA gene in R. meliloti ( A ) and E. coli ( B ) extract. pPP358 (lanes 3,8) and pPP395 (lanes 4,9) contain the same 5.3-kb BamHI fragment in two orientations in pBR322. Lane 5 shows the expression from pPP358: :Tn5. Lanes 1 and 6 are controls without added DNA, lanes 2 and 7 show the fl-laetamase expressed from pBR322. The results of three separate experiments are summarized in the figure

8.5kb EcoRl fragment carrying ,,common' 6.0kb EcoRl fragment carrying nod genes of R-meliloti 41 hsn genes of R.mclilo1i 41 - -

I I

'pJS120; 1 I

-I PJS201 pJS405. pJS302 DID 500 Clones carrying .,common' A genes

pJS120.2 I

pACYC 184

Ipqp'ac Po pJS2012 I rl

PIN-FA2

1kb w

Clones carrying @ genes

* 4 pJS401.2 IP&?CP"

PIN-P- A2

IppPlac Po I pJS402.2

plN-U-AZ

Ip$lacPO t ---L. pJS405.2

PIN4 -A2

C,R t pJS302.2

~ A C V C itx

c mR plD500.2

p0R325

Fig.4. Diagrammatic representation of plasmids used as templates for in vitro transcription-translation. The proteins synthesized in R. meliloti extract are represented by arrows. E, EcoRI; Bg, BglII; B, BamHI; Sp, SphI; lpp, lipoprotein promoter; lacP", lac promoter-operator

pPP358 and pPP395 were obtained by cloning the recA- homologous DNA of R. meliloti 41 into pBR322 in two orientations. Both plasmids expressed a protein of 41 kDa in the R. meliloti cell-free extracts (Fig. 3 A). To prove that the 41-kDa protein corresponds to the recA gene product, derivatives of pPP358 carrying Tn5 insertions were isolated. One derivative was no longer capable of complementing the recA- phenotype of E. coli. When this plasmid (pPP358 : : Tn5) was used as a template in a R. meliloti cell- free extract, no protein band of 41 kDa was observed (Fig. 3 A). When the Tn5-mutated fragment was homoge- notized back into the wild-type genome of R. meliloti, the mutant derivative exhibited an increased sensitivity to methyl

methanesulfonate and to ultraviolet light as compared to the wild-type bacteria, indicating that the mutation abolished a function of the recA product (data not shown). Since Tn5 insertions do not show a strong polar effect in R. meliloti [29, 361, it is likely that the 41-kDa protein is the product of a recA-homologous gene.

Plasmids pPP358 and pPP395 were also examined in E. coli cell-free extract (Fig. 3 B) and both plasmids were found to direct the synthesis of a 41-kDa protein. Moreover, in both orientations (lanes 8 and 9) an additional protein band near to the 41-kDa protein appeared, the significance of which is not yet clear. When the expression of the Rhizobium recA gene was compared to that of the ApR marker of the vector plasmid,

73

Fig. 5. Proteins synthesizedfrom the 3.3-kb EcoRI-Bglllfragment of the common nod region in R. meliloti ( A ) and E. coli ( B ) extract. Lanes 1 , 4 and 8 are controls without added DNA. In lanes 2 (pJS120.2) and 3 (pJS120.1) the nod fragment was expressed from pACYC184 vector, in lanes 6 and 9 (pJS201.1) and lanes 7 and 10 (pJS201.2) from PIN-11-A2 vector. Lane 5 shows the polypeptides expressed from PIN-11-A2

it was observed that the vector protein was expressed best in E. coli extracts, whereas the Rhizobium gene was expressed best in the Rhizobium system.

Expression of 'common' nodulation genes in the R . meliloti cell-free extract

In E. coli minicells three proteins (23 kDa, 28.5 kDa and 44 kDa) are expressed from plasmids carrying nodABC genes [S]. Protein synthesis is observed only when the genes are placed under the control of strong E. coli promoters.

The same cloned fragments (see Table1 and Fig.4) were studied in R. meliloti and E. coli cell-free extracts. In plasmids pJS120.1 and pJS120.2 the 3.3-kb BglII-EcoRI subfragment was cloned into the EcoRI site of pACYC184, whereas pJS201.2 and pJS2Ol.l contained the same fragment in PIN- 11-A2. The PIN-11-A-type cloning vehicles are the derivatives of pBR322, carrying both the lipoprotein promoter and the lac promoter-operator region [32]. These vectors allow a high level of expression of the inserted DNA fragments.

Fig. 5A shows that the expression from the pACYC184 clones was very weak (lanes 2, 3) and the three nod proteins are synthesized only from an E. coli promoter (lane 2). In contrast, using pJS201.1 and pJS201.2, protein synthesis is observed in both orientations (lanes 6,7).

Fig. 5 B shows that the expression of the nod proteins from pJS201.1 and pJS201.2 in E. coli cell-free extracts differs mark- edly from the results obtained in the R. meliloti extract. In the E. coli system proteins are synthesized only in orientation .2.

Fig.6. Proteins coded by the 4.2-kb and the 2-kb BamHI fragment of the hsn region expressed in R. meliloti cell-free extract. Lane 1, control without DNA; lane 2 (pJS401.1) and lane 3 (pJS401.2) show the protein products of the 4.2-kb fragment; lane 4 (pJS402.1) and lane 5 (pJS402.2) show the polypeptides synthesized from the 2-kb fragment

Protein synthesis from a DNA region coding for host specificity (hsn) functions

In R. meliloti 41 a 6.8-kb EcoRI fragment, determining host-range specificity, was identified. The hsn fragment was physically mapped and the exact position of the protein- coding regions-was determined usingdifferent methods, which will be published elsewhere [29]. fragments in the Rhizobium protein-sinthesizing system, we

Three subclones of the 6.8-kb EcoRI fragment were stud- ied in the R. meliloti and E. coli cell-free extracts. The cloned fragments are shown in Fig. 4. Plasmids pJS401.1 and pJS401.2 carry the 4.2-kb BamHI fragment of the hsn region in PIN-11-A2 vector (Table 1). A protein of 43 kDa is expressed in both orientations (Fig.6). The other BamHI fragment of the hsn region was cloned into PIN-II-A2 yielding plasmids pJS402.1 and pJS402.2 (Table 1). Using these plasmids as templates, a protein of 28 kDa was expressed by both clones in the R. meliloti extract.

To confirm the data obtained bv the cloned BamHI

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Fig. I. Proteins encoded by the Sphl fragment of the hsn region expressed in R. meliloti extract ( A ) and in E. coli extract (B) . Templates in the reaction mixtures were the following clones in pACYC184: in lanes 2 and 6, pJS302.1; in lanes 3 and 5, pJS302.2. Lanes 1 and 4 are controls without DNA

compared the expression of the middle SphI fragment of the hsn region in three different vector plasmids in both orientations. As shown in Fig.7A, 43-kDa and 28-kDa polypeptides are synthesized at a very low level in both orientations from pACYC184 clones pJS302.1 and pJS302.2 (lanes 2 and 3). The same results were obtained by using PIN-11-A2 clones (pJS405.1 and pJS405.2) or pBR325 clones (pID500.1, pID500.2) (data not shown).

The same six plasmids were studied in E. coli cell-free extract. The results obtained with the two pACYC184 clones are represented in Fig. 7 B (other data not shown). The 43-kDa protein is expressed only in orientation .1, that is, when an E. coli promoter is located in front of the gene. The 28-kDa protein is present only in orientation .2, suggesting an opposite direction of expression from the two coding regions.

DISCUSSION

A DNA-directed coupled transcription-translation system has been developed from the cell-free extract of R. meliloti 41. Our earlier attempts, based on the published preparation techniques [12, 181, failed to produce extracts with satisfying activity. By varying the conditions for the preparation of the cell-free extract (growth, pretreatment and disruption of bacteria, preincubation etc.) a protein-synthesizing system of reproducible activity was obtained.

The composition of the Rhizobium protein-synthesizing reaction mixtures and the concentration of some of the components were slightly different when compared to those of E. toli and A. tumefaciens systems. A lower incubation temperature (30 "C) for the transcription-translation was necessary in the Rhizobium system.

A DNA-directed cell-free protein-synthesizing system was described for E. coli [12]. In later experiments evidence accumulated pointing to transcription-translational speci- ficity in different bacteria [37]. When expression of genes from other species was studied in E. coli, it was found that foreign

genes were in some cases either not expressed at all, or only very poorly. Genes from gram-positive bacteria [38] as well as genes from gram-negative bacteria [18, 391 failed to express efficiently in vivo in E. coli. Differences in specificity of pro- moter recognition or of translational starts were suggested to account for these observations [19, 40,411.

The cell-free system described in this paper allows the study of specific gene expression in R. meliloti. The data obtained in the homologous Rhizobium system were compared to the protein synthesis in the E. coli extract. In the case of nodABC proteins they were also compared to earlier in vivo results obtained in E. coli minicells [8].

The antibiotic determinants ApR and CmR, coded by plasmids pBR325, pBR322, PIN-11-A2 and pACYC184, are expressed well in the cell-free R. meliloti extract (Fig. 2). The molecular masses of the ApR gene product (32 kDa) and chloramphenicol transacetylase (24 kDa) correspond to the published values [42 - 441. In E. coli minicells the tetracycline determinant codes for polypeptides of 34 kDa, 26 kDa and 14 kDa [45]. In the in vitro protein-synthesizing system o f A. tumefaciens a faint protein band of 34 kDa was observed [18]. In the R. meliloti extract, however, no protein corresponding to the Tc gene products can be detected (Fig.2). Probably this is due to the very low level of protein synthesis and not to the lack of expression. (Rhizobium cells are able to express Tc resistance in vivo). The level of bla gene expression is also lower in R. meliloti extracts, as can be seen from the relative intensities of ApR gene product and recA protein bands, when compared in E. coli and R. meliloti extracts (Fig. 3).

Comparison of the expression of cloned R. meliloti nod and hsn genes in E. coli and R. meliloti S-30 extracts gives evidence for differences in transcription-translation capability of the two systems. DNA-directed protein synthesis occurs in the E. coli extract only if a promoter region is provided by the vector plasmid upstream of the cloned structural genes (Figs 5 and 7B). This is in agreement with earlier in vivo results obtained for nod proteins in E. coli minicells [8].

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Nodulation genes are expressed in vivo only at a very low level in free-living cells of Rhizobium species as was published recently [46 - 481. Translational lacZ fusions to different nod gene promoters demonstrated the role of a factor present in plant root exudates and of the nodR gene product in the induction of nod gene expression. However, when D N A fragments carrying nodABC genes or two hsn genes were used as templates in the in vitro cell-free system of R. meliloti, an orientation-independent protein synthesis was observed (Figs 5, 6, 7A). This can be explained either by the presence of another promoter-like sequence on the vector plasmids, or might be due to the expression from promoters of the cloned regions. Since nodABC genes are expressed only from the PIN-I1 clones but not from the pACYC184 clones in both orientations, the protein synthesis observed is probably directed by promoters present on the vector. The SphI frag- ment of the hsn region was tested in three different vectors (Fig. 7A, and other data not shown). The very-low-level, vector-independent expression of hsn genes in both orientations may suggest the transcription from their own promoter. However, it is worth emphasizing that the in vitro conditions for the protein synthesis in cell-free extracts may result in the less eficient control of gene expression, and may account for the low-level synthesis of host-specificity proteins.

The cell-free system described in this paper allows specific studying of gene expression in R. meliloti and can be useful in furthering our understanding of bacterial symbiotic functions.

The authors wish to express their gratitude to Prof. R. Ehring for her generous gift of B. coli extract and for her help and advice in the preparation of Rhizobium extracts. We whish to thank Prof. J. Schell the opportunity that part of this work was done in his department in the Max-Planck Institut (Koln). We thank M. John and J. Schmidt for plasmid preparations and B. Dusha for making the photos.

This work was supported by a joint grant of Deutsche Forschungsgemeinschajt and the Hungarian Academy of Sciences (436UNG-113/25/0).

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