Characterization of a Rhizobium meliloti fixation gene (fixF) located ...

JOURNAL OF BACTERIOLOGY, Oct. 1985, p. 245-254

Vol. 164, No. 1

0021-9193/85/100245-10$02.00/0 Copyright © 1985, American Society for Microbiology

Characterization of a Rhizobium meliloti Fixation Gene (fixF) Located near the Common Nodulation Region 0.

MARIO AGUILAR, DIETER KAPP, AND ALFRED PUHLER*

Lehrstuhl fur Genetik, Fakultat fur Biologie, Universitat Bielefeld, D4800 Bielefeld 1, Federal Republic of Germany Received 30 April 1985/Accepted 9 July 1985

Rhizobium meliloti 2011 DNA from pRmSL26, a plasmid which is known to carry genes involved in the early was used to construct Tn5 mutations by site-directed TnS mutagenesis. TnS mutations located within an 8.7 kilobase EcoRI fragment defined two adjacent loci encoding functions for nodulation (nod) and symbiotic N2 fixation (fix). We investigated the organization and regulation of thefix locus and the characteristics of alfalfa nodules induced by these Fix- mutants. By monitoring expression in Escherichia coli minicells, we determined that thefix locus encoded a 36-kilodalton polypeptide. The gene corresponding to this locus was designatedfixF. Morphological and ultrastructural studies of the ineffective nodules formed by R. melilotifixF mutants showed infected host cells similar to those of the wild type. The ineffective nodules were able to accumulate leghemoglobin, but at lower levels than those found in the wild-type nodules. Expression of the niJHDK operon was unaffected by TnS insertions in the fixF gene. Expression of the fixF gene was monitored in E. coli by using translational lacZ fusions. It was shown that transcription of thefixF gene in E. coli could be activated by Klebsiella pneumoniae nifA and the R. meliloti nifA-like regulatory gene products. Expression of thefixF gene was also studied in free-living and symbiotic R. meliloti cells. It was found that the fixF gene was transcribed in the symbiotic state. stages of nodulation,

The soil bacterium Rhizobium meliloti fixes N2 in symbiotic association with alfalfa (Medicago sativa). The symbiotic process begins on the root surface, proceeds with the bacterial invasion of root hairs, and culminates in the development of an effective nitrogen-fixing nodule. This complex process occurs by a multistage sequence of interdependent steps requiring the expression of specific genes in both symbionts. Understanding of the molecular basis of R. meliloti symbiotic genes has significantly progressed in the recent years. It has been shown that the symbiotic genes reside on large, indigenous plasmids (3, 37). Some of the genes that are involved in the early steps of the nodulation process and also the structural genes for the nitrogenase enzyme complex have been identified and cloned in Escherichia coli (24, 38). Recently, a regulatory fix gene resembling the Klebsiella pneumoniae nifA gene has been found in the main nif-fix cluster (45). In K. pneumoniae, a free-living, N2-fixing microorganism, positive regulation of nif genes by the nifA gene product has been demonstrated (11). In R. meliloti, a similar regulation seems to operate since both K. pneumoniae nifA (44) and the R. meliloti nifA-like regulatory gene products (45) activate expression from the R. meliloti nifH promoter. Similar results were obtained by Weber et al. (G. Weber, H. Reilander, and A. Piihler, submitted for publication), who designated the R. meliloti nifA-like gene fixD to avoid nonspecific terminology. Because of the complexity of events leading to the activation of symbiotic nitrogen fixation, little is known about the organization and regulation of symbiotic genes. In the work described here, we analyzed an R. meliloti DNA fragment originated from plasmid pRmSL26 (24) and found two symbiotic loci associated with the Nod- and Fixphenotypes. The Fix- R. meliloti mutants in this region generated by site-directed transposon TnS mutagenesis were analyzed, and we report here the morphological, biochemical, and genetic aspects of this analysis. In addition, we *

present evidence indicating that the fix locus contained a single gene, designated fixF. Finally, by constructing trans-

lational fusions between the fixF and E. coli lacZ genes, we have studied the expression of thefixF gene in E. coli and in free-living and bacteroid forms of R. meliloti. MATERIALS AND METHODS Bacteria and plasmids. Bacteria and plasmids used in this work are listed in Table 1. Media and growth conditions. E. coli strains were grown at 37°C in Luria broth or Penassay broth (Difco Laboratories) medium. R. meliloti was grown in TY medium (1% tryptone, 0.1% yeast extract, 2 mM CaCl2) at 30°C. The prototrophic phenotype of Tn5-induced R. meliloti mutants was determined in a medium containing, per liter, 10 g of mannitol, 2.05 g of K2HPO4, 1.45 g of KH2PO4, 0.15 g of NaCl, 0.7 g of NH4NO3, 0.5 g of MgSO4 * 7H20, 0.01 g of CaCl2, 6.7 mg of iron citrate, and 1 ml of a solution of vitamins (1 mg/ml each of biotin, thiamin, and calcium panthothenate) was added. Antibiotics were added at the following final concentrations ([Lg/ml): ampicillin, 200; chloramphenicol, 50; streptomycin, 400; and tetracycline, 5. To determine TnSencoded resistance, kanamycin (25 ,ug/ml) for E. coli and neomycin (100 ,ug/ml) for R. meliloti were added. DNA biochemistry. Plasmid DNA was isolated by the method of Ish-Horowicz and Burke (19) adapted to a largescale preparation. DNA was purified by dye buoyantgradient ultracentrifugation (4). To map the Tn5 insertions in multicopy plasmids, plasmid DNA was obtained by the rapid isolation method described by Birnboim (5). R. meliloti total DNA was prepared essentially as described by Meade et al. (27). Restriction endonucleases were used as suggested by the manufacturers. Calf alkaline phosphatase treatment, exonuclease Bal 31 digestions, filling-in reactions, and ligation of restricted plasmid DNA were performed as described by Maniatis et al. (26). Plasmid constructions. (i) Construction of plasmids for Tn5 mutagenesis (pMA1-1, pMA21, and pMA22). Plasmids

Corresponding author. 245

246

J. BACTERIOL.

AGUILAR ET AL. TABLE 1. Bacteria and plasmids Strain

E. coli hsdR pro 294 294: :TnS-Mob 294, TnS-Mob chromosomally integrated 294, recA RP4 derivative chroS17-1 mosomally integrated; Tpr C600 met::TnS S605 minicell-producing strain DS410

MC1000 R. meliloti 2011

Plasmids pACYC177 pACYC184 pIN-I-A pUC4K pSVB20 pSUP102 pSUP202

References

Relevant characteristics

F. Schoffl 40 41

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Kmr Apr Cmr Tcr Apr Apr, multiple cloning site Apr, multiple cloning site pACYC184-Mob pBR325-Mob pACYC177-C pACYC177-Cmr Kmr Aps lacZY Apr pMC1430 R. meliloti nifHD Tcr pRmR2 R. meliloti nifK Tcr pRmW53 R. meliloti nifD Tcr pRmW61 pRmW541-10 R. meliloti nifA-like regulatory gene, Cmr K. pneumoniae nifLA Cmr pWK130 K. pneumoniae nifA Cmr pWK131 Nodulation genes pRmSL26 Tcr Apr Cm, pMA1-1 Tcr Cmr Aps pMA21 Tcr Cms pMA22 Apr, pUC4K pMA159 Apr, pSVB20 pMA169 Tcr Cms pMA161 pMA161.2, .3 Cmr Ap5 Kms Apr, plN-I-A3 pMA164.1 Apr, pIN-I-A2 pMA165.2 Apr lacZ fusion pMA50, 51, 52, 53 Apr, derivative of pMA51 pMA51.4

9 9 29 49 This laboratory This laboratory 41 This laboratory 8 38 G. Weber G. Weber G. Weber 34 34 24 This work This work This work This work This work This work This work This work This work This work

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pMA1-1 and pMA21 were constructed by cloning the 8.7kilobase (kb) EcoRI fragment and the 5.5-kb PstI fragment of pRmSL26 into the EcoRI and the PstI cloning sites of the pSUP202 vector. Plasmid pMA22 was constructed in the following way. The 5.5-kb PstI fragment was cloned into the polylinker sequence of pUC4K for the addition of flanking BamHI and EcoRI sites. The original 5.5-kb PstI fragment was then excised as an EcoRI fragment and cloned into the EcoRI site of the pSUP102 vector. (ii) Construction of plasmids for expression in E. coli minicells (pMA161, pMA162, pMA161.2, pMA161.3, pMA164.1, and pMA165.2). To study the expression of the fix locus in E. coli minicells, the 2.6-kb PstI-BamHI fragment of pRmSL26 was excised from pMA22 as a BamHI fragment, inserted into pUC4K by replacing the existing BamHI fragment to obtain flanking EcoRI sites, and cloned (as an EcoRI fragment) into the unique EcoRI site present in the cat (chloramphenicol acetyltransferase) gene of pACYC184. For convenience, we designated the BamHI site approximately 2.6-kb away from the PstI site as site 1 and the BamHI site immediately adjacent to the PstI site as site 2. The new plasmids, containing thefix locus downstream from

the cat gene, were designated pMA161 (correct orientation with respect to the cat promoter) and pMA162 (opposite orientation). Plasmids pMA161.2 and pMA161.3 were derived from plasmid pMA161 as follows. The EcoRI fragment containing the 2.6-kb PstI-BamHI fragment was cut out from pMA161 and cloned into the multiple cloning site of the pSVB20 vector to yield pMA169. In the vector part of pMA169, there were two restriction sites located immediately adjacent to the PstI and BamHI sites. A BstEII site flanked BamHI site 1, whereas BamHI site 2 flanked the PstI end. The presence of these recognition sites was useful for subsequent steps. Plasmid pMA169 was linearized with BstEII and digested with Bal 31 to delete the region adjacent to R. meliloti BamHI site 1, and the remaining R. meliloti fragment was excised by cutting on the other side with BamHI in site 2. These deleted fragments, each with a blunt end on one side formed by Bal 31 and a cohesive end on the other side formed by BamHI, were then ligated into the SmaI-BamHI sites of the gene coding for the Kmr phenotype of the pACYC177-C vector. Plasmids pMA164.1 and pMA165.2 were constructed by cloning the R. meliloti 2.2-kb HindIlI fragment in both orientations with respect to the lipoprotein (lpp) gene promoter of the pIN-I-Al, pIN-I-A2, and pIN-I-A3 vectors. The six hybrid plasmids were assayed in minicells, and only those of particular interest are shown (see Fig. 2A). (iii) Construction of lacZ fusion plasmids (pMA50, pMA51, pMA52, and pMA53). Plasmids pMA161 and pMC1403 were used as sources of DNA. Plasmids pMA50, pMA52, and pMA53 were constructed by cloning, respectively, the small BglII-EcoRI, SmaI-EcoRI, and EcoRI fragments of pMA161 into the cloning sites of lacZ plasmid pMC1403. To construct pMA51, pMA161 and pMC1403 were first completely digested with KpnI and BamHI, respectively. The linearized plasmids were cut with EcoRI, mixed, and ligated with T4 DNA ligase. The ligation mixture was used to transform E. coli MC1000. Although BamHI and KpnI enzymes produce incompatible cohesive ends, several E. coli transformants were obtained. One of these transformants was X-Gal (5bromo-4-chloro-galactoside) positive, resulting in a darkblue colony. A detailed restriction analysis of the plasmid isolated from the blue colony and designated pMA51 indicated the presence of all restriction sites originally mapped in pMC1403 and in the cloned R. meliloti DNA, except for the KpnI and BamHI restriction sites. The ligation of incompatible cohesive ends which resulted in fusion of R. meliloti DNA to the lacZ gene rendered the junction resistant to BamHI and KpnI enzymes. Fragment-specific TnS mutagenesis of R. meliloti DNA. Transposon TnS mutagenesis of R. meliloti DNA inserts in plasmids pMAl-1, pMA21, and pMA22 was carried out in E. coli S605 by the procedure for multicopy plasmids described by Weber and Puhler (50). It was found that the majority of analyzed pMA21::Tn5 plasmids carried the transposon located in vector pSUP202, which is a derivative of pBR325. This was not the case for pMA22, in which vector pSUP102 was a derivative of pACYC184. Bacterial matings between wild-type R. meliloti 2011 and mobilizing E. coli S17-1 containing the Tn5-mutated plasmids were performed as described by Simon et al. (41). Replacement of the R. meliloti wild-type fragments with the mutated fragments was performed in two steps. First, R. meliloti transconjugants were selected on TY medium supplemented with streptomycin, neomycin, and tetracycline (selection for integration of the whole plasmid by a single crossover). In a second step,

R. MELILOTI fixF GENE

VOL. 164, 1985

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FIG. 1. Physical and genetic map of a symbiotic region of R. meliloti 2011. The location of the analyzed region ( ) of the insert of plasmid pRmSL26 (Liii) with respect to the nifHDK operon is indicated. Plasmid pMA1-1 carries an 8.7-kb EcoRI insert, whereas plasmids pMA21 and pMA22 carry 5.5-kb PstI fragments (see text for details). Vertical lines indicate the positions of TnS insertions. dn, Delayed nodulation. Restriction endonucleases are abbreviated as follows: B, BamHI; Bg, BglII; K, KpnI; R, EcoRI; P, PstI.

Smr, Nmr, and Tcs clones were isolated (second crossover and loss of the vector molecule); to increase the ratio of Tcs clones, an enrichment procedure with ampicillin, based on that described by Simon et al. (41), was included. The Tn5 location in the genomes of these R. meliloti mutants was confirmed by Southern hybridization with a radioactive Tn5 probe. Expression in E. coli minicells. Methods for the isolation of minicells and for 35S labeling of polypeptides synthesized by minicells have been described by Weber and Puhler (50). Heme determination. Heme concentrations in alfalfa nodule cytosol were determined by the hemochrome method described by Bisseling et al. (6). Preparation of 32P-labeled total RNA from R. meliloti. Isolation of total RNA from free-living cells and bacteroids of alfalfa nodules was by the method of Krol et al. (23). Radioactive labeling of RNA with polynucleotide kinase and [_y-32P]ATP was by the method of Goldbach et al. (15). DNA-RNA hybridization. Digested plasmid DNA was separated in an agarose gel and transferred to nitrocellulose filters by the method described by Southern (42). Filters were prehybridized at 42°C for 3 h in the following mixture: 5x SSC (lx SSC is 15 mM sodium citrate and 150 mM NaCl), 0.1% sodium dodecyl sulfate (SDS), 50 mM Na3PO4 (pH 7.5), 200 Fg of yeast RNA per ml, and 50% formamide. Then the radioactively labeled RNA was added and incubated for an additional 18 h. The filters were washed four times with 2x SSC at 42°C and finally dried and autoradiographed. Antigenic detection of nitrogenase. R. meliloti nitrogenase components were detected by the Western blot procedure as described by Burnette (7). Antiserum against component 1 (CI) and component 2 (CII) of R. leguminosarum nitrogenase were kindly supplied by the Department of Molecular Biology, Wageningen, The Netherlands. Determination of ,I-galactosidase activity. 1-Galactosidase activity was measured by using the O-nitrophenyl-pgalactoside cleavage assay in SDS-chloroform-permea-

bilized cells (28). E. coli and R. meliloti were grown with continuous shaking at 30°C in minimal medium (28) and TY medium, respectively. Cells from logarithmic-phase cultures were collected by centrifugation, suspended in Z-buffer (28), and assayed for ,-galactosidase activity. Bacteroids were isolated from alfalfa nodules as follows. Nodules were picked 4 weeks after inoculation, surface sterilized. with 80% ethanol, thoroughly rinsed with sterile distilled water, and crushed in a 0.5 M mannitol-30 mM Tris hydrochloride (pH 7.5) solution by using a mortar and pestle. The homogenate was transferred into an Eppendorf tube, and after 15 min at 0°C, the plant debris was further sedimented by pulse centrifugation in a bench top microfuge. Independent satmples were withdrawn from the supernatant containing bacteroids to assay 3-galactosidase activity, to obtain viable cell counts by plating on TY and TY-plus-neomycin medium, and to quantify total proteins by the method of Lowry et al. (25). Microscopy. Nodules were harvested 3 weeks after inoculation. For each strain, at least 12 nodules collected from different plants were investigated by light microscopy, and at least 6 nodules from different plants were investigated by electron microscopy. Whole and longitudinally cut nodules were fixed under reduced pressure in a 4% glutaraldehyde-0.05 M phosphate solution (pH 7.2) for 5 h. Then the nodules were washed in 0.05 M phosphate buffer (pH 7.2) and postfixed for 2 h in 2% OS04 which was prepared in the same phosphate buffer. Samples were rinsed with phosphate buffer and dehydrated in a graded ethanol series at 0°C. Finally, the samples were embedded in Spurr resin (43). For light microscopy, 0.5- to 1-,um sections were cut and stained with 0.5% toluidine blue in 0.5% borate buffer (pH 9). Light micrographs were taken with a research grade Zeiss microscope. For electron microscopy, ultrathin sections were collected on 300-mesh copper grids, stained with 2% uranyl acetate and Reynolds lead citrate (35), and examined in a Hitachi HS500 electron microscope operated at 75 kV. Plant inoculation tests. Alfalfa (M. sativa) seedlings were

248

AGUILAR ET AL.

J. BACTERIOL.

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FIG. 2. Mapping of coding regions located on the R. meliloti fragment containing the fix locus. (A) Analysis in E. coli minicells. Hybrid plasmids carrying thefix locus of R. meliloti were constructed as described in the text and were analyzed in E. coli minicells. Symbols: R. meliloti DNA inserts; , vector plasmids (only partially shown); +-, promoters of chloramphenicol (Cm) and kanamycin (Km) resistance genes or of the lipoprotein gene (lpp); FAd, deletions in plasmids pMA161.2 and pMA161.3; uzzi, gene products encoded by R. meliloti DNA; ruzzza, parts of the antibiotic resistance gene product. (B) Construction of the coding region map. Coding regions and direction of translation are indicated with arrows. The restriction map and the correlated genetic map, as described in the legend to Fig. 1, are also shown. .i, fixF coding region specifying a 36K polypeptide. For the coding regions specifying a 23K and a 34K polypeptide, the starting points have not been determined (jagged lines). Molecular sizes of R. meliloti gene products are indicated in kilodaltons (K). Restriction endonuclease sites are abbreviated as follows: B, BamHI; Bg, BglII; Hi HindIII; K, KpnI; P, PstI; R, EcoRI; S, SmaI.

aseptically grown on nitrogen-free agar by the method described by Rolfe et al. (36). Plants were harvested 4 weeks after inoculation, and nitrogenase activity in the nodulated plants was measured by the acetylene reduction assay. In those cases where nodulation did not occur after the initial 4 weeks, plants were inspected at intervals during an additional 4-week period to confirm this phenotype. RESULTS Analysis of a DNA fragment of R. meliloti 2011 coding for symbiotic functions. We have previously reported that sorne random TnS-induced Nod- mutants were positively complemented by subfragments of pRmSL26 cloned into replicative vectors (1). A detailed analysis of the complementing region present in pRmSL26 was performed by TnS site-directed mutagenesis. The 8.7-kb EcoRI and 5.5-kb PstI fragments from pRmSL26 were used to construct 15 different R. meliloti strains, designated MAl to MA15, carrying TnS insertions in a DNA region of approximately 6 kb (see above for details). These R. meliloti: :TnS mutant strains were inoculated onto sterile alfalfa seedlings and analyzed for their symbiotic properties. The results are summarized in Fig. 1. Two symbiotic regions containing genes for nodulation and nitrogen fixation were defined. TnS insertions 12, 13, 14, and 15 resulting in Nod- phenotypes were distributed within a region of approximately 1.5 kb. R. meliloti MA3, MA4, MA5, and MA6 formed nodules (Nod'), but failed to reduce acetylene (Fix-). TnS insertion 11 delayed nodulation by 1 week. The remaining TnS insertions resulted in Nod' Fix' phenotypes.

Fix- mutants MA3, MA4, MA5, and MA6 were prototrophic, indicating that the phenotype was not due to an indirect effect of auxotrophy. Mapping of the R. meliotifixF coding region. To determine the coding properties of the fix region, we used the E. coli minicell system. Since there was no significant expression of cloned R. meliloti genes from their own promoters, it was necessary to construct hybrid plasmids in which R. meliloti genes were located downstream from a strong E. coli plasmid promoter. In particular, a translational fusion product was expected when the R. meliloti coding sequence was fused in phase to the coding region of a plasmid gene. A collection of hybrid plasmids was constructed as described above and analyzed in minicells. The 2.6-kb PstI-BamHI fragment containing the fix loci (Fig. 1) was cloned in both orientations with respect to the promoter of the cat gene of vector pACYC184. In addition to the vector-encoded polypeptides, two polypeptides with molecular weights of 29,000 (29K polypeptide) and 40,000 (40K polypeptide) were weakly expressed in one orientation (pMA161 [Fig. 2A]). No insert-specific product was detected in the reverse orientation (pMA162 [data not shown]). To map the location of the coding regions of the 29K and 40K polypeptides, we constructed plasmids pMA161.2 and pMA161.3, which carry deletions in the 2.6-kb PstI-BamHI fragment. In minicells containing pMA161.2, the 29K polypeptide was still weakly expressed, whereas a stronger expression occurred with plasmid pMA161.3, carrying a larger deletion (Fig. 3, lanes a and b). In the latter case, a 32K polypeptide resulted from a translational gene fusion

R. MELILOTI fixF GENE

VOL. 164, 1985

between 303 base pairs (bp) of the gene coding for the Kmr phenotype of the vector plasmid (33) and approximately 570 bp of the R. meliloti DNA insert (Fig. 2A). Thus, it was concluded that the 29K polypeptide was encoded by the left side of the PstI-BamHI fragment, where Tn5 insertions 3, 4, 5, and 6 cause a Fix- phenotype. Further analysis of the 29K polypeptide coding region was performed on a 2.2-kb HindIll fragment. This fragment overlapped the 2.6-kb PstI-BamHI fragment and extended to the left of the PstI site (Fig. 2A). The 2.2-kb HindIII fragment was cloned in both orientations in three different pIN-I-A vectors (29). These vectors transcribe insert DNA from the Ipp gene promoter. The vectors differ in the reading frame at the cloning site and are therefore helpful for the construction of gene fusions. In our analysis, minicells containing hybrid plasmid pMA164.1 (Fig. 2A [promoter at the right side of the cloned fragment, vector pIN-I-A3]) synthesized a 23K polypeptide (Fig. 3, lane d), whereas minicells containing pMA165.2 (Fig. 2A [promoter at the left side of the cloned fragment, vector pIN-I-A21) synthesized 15K and 34K polypeptides (Fig. 3, lane f). From the strong expression of 23K and 34K polypeptides, we assume that they represented translational fusions with the Ipp gene product. Since only five codons remained between the start codon of the Ipp gene and the HindIll cloning site, these fusion polypeptides were mostly encoded by R. meliloti DNA. In the case of the 23K polypeptide, the calculated coding region extended 200 bp to the left of the PstI site (Fig. 2A). From this experiment we conclude that there existed a coding region specifying a 36K polypeptide within the HindIII-BamHI fragment, which spanned the fix loci (Fig. 2A). In addition, by cloning the 2.6-kb PstI-BamHI fragment into the series of pIN-I-A vectors, we detected a 23K polypeptide which was expressed from the BamHI site to the left; this indicated the presence of an open reading frame upstream from the 40K polypeptide (data not shown). The results of this study are summarized in Fig. 2B. The gene-protein map of the HindIII-BamHI fragment is presented. Comparing this map with the genetic map of the TnS insertions, it seems evident that only the 36K coding region was involved in the Fix- phenotype. This coding region was designated fixF. Morphological characterization of alfalfa nodules induced by an R. melilotifixF mutant. R. meliloti Fix- mutants MA3, MA4, MA5, and MA6 induced nodule formation that was similar to that induced by the wild-type strains both in time of appearance of nodules and in distribution of nodules on alfalfa roots. ]However, no acetylene reduction was detected when plants were tested 3, 4, and 5 weeks after inoculation. Furthermore, after 4 weeks plants showed clear symptoms of nitrogen starvation. To establish the degree of development reached by this ineffective association, nodules induced byfixF mutant MA6 were examined by light and electron microscopy. Typical structures of effective and ineffective nodules are shown in Fig. 4. The structure of effective alfalfa nodules induced by R. meliloti 2011, which is shown in Fig. 4A, is consistent with previous reports (17, 20, 21, 32, 48). It was found that the ineffective nodules induced by mutant MA6 were usually smaller than those induced by the wild type, although they showed similarities in structure and development (Fig. 4). Indeed, no major differences were observed in the meristematic, the thread invasion, and the early and late symbiotic zones. Mutant rhizobia were released from the infection thread and subsequently were surrounded by the

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peribacteroid membrane (Fig. 4B). This membrane remained intact during the early and late symbiotic stages of development. However, in contrast to the wild-type nodules, the ineffective nodules exhibited earlier senescence. The senescent zone was markedly increased (Fig. 4A). In addition, transition between the late symbiotic and senescent zones occurred abruptly. Another relevsnt feature of the ineffective nodules was the increased number and size of starch granules (Fig. 4C). High accumulation of starch granules was observed in the infected and interstitial cells within the region located between the early and late symbiotic zones. Cells of the inner nodule cortex and of the central cylinder were also found to contain starch granules. Molecular analysis of alfalfa nodules induced by an R. meliloti fixF mutant. Leghemoglobin is one of the most abundant proteins in effective nodules. Functionally, it facilitates the diffusion of oxygen to bacteroids and maintains a low oxygen pressure. Since biochemical studies suggest a bacterial origin for the heme group of leghemoglobin (2, 10), we explored the presence of leghemoglobin in ineffective nodules induced by strain MA6. SDS-polyacrylamide gel analysis of the soluble protein fraction contained the leghemoglobin polypeptides (data not shown). A significant amount of heme was also detected in the cytosol by the hemochrome assay. The heme content was found to be reduced to 40% (22 ,umol of heme per g of fresh nodule) compared with the heme content of wild-type nodules. To determine whether mutations in the fixF gene affected

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AGUILAR ET AL.

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FIG. 4. Light and electron microscopy of thin sections of nodules induced by the wild-type R. meliloti 2011 and the Fix- mutant R. meliloti MA6. (A) Light micrograph montage of median longitudinal sections of nodules induced by the wild type (left) and by the Fix- mutant (right). Zones illustrated are the meristem (M), early symbiotic zone (ES), late symbiotic zone (LS), and senescent zone (S). Bar, 200 Jim (x50). (B) Release (r) of rhizobia cells of the MA6 mutant from the infection thread (it) via an unwalled infection droplet (id). Bacteria enclosed by peribacteroid membrane are indicated by arrows. Bar, 1 pLm (x7,000). (C) Starch accumulation in nodules induced by the Fix- mutant R. meliloti MA6. Large amyloplasts in cells of the inner nodule cortex (INC) and infected cells (ic) are indicated by arrows. The endodermis is denoted by asterisks. Bar, 20 p.m (x500). (D) Detailed view of a plant cell infected by R. meliloti MA6. The infected cell from the late symbiotic zone shows mature bacteroids surrounded by peribacteroid membranes (arrows). Rough endoplasmatic reticulum (rer), mitochondria (m), and starch granules (sg) can also be seen. Bar, 1 p.m (x 14,000).

R. MELILOTI

VOL. 164, 1985

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