J. Biosci., Vol. 20, Number 1, January 1995, 1728. Printed in India.

Pleiotropic effects of purine auxotrophy in Rhizobium meliloti on cell surface molecules

S SWAMYNATHAN* and AQBAL SINGH
Biotechnology Centre, Indian Agricultural Research Institute, New Delhi 110 012, India *Present Address: Centre for Cellular and Molecular Biology, Hyderabad 500 007, India MS received 30 April 1994; revised 24 October 1994 Abstract. Rhizobial purine auxotrophs have earlier been shown to be defective in symbiosis, though the exact reason for this failure is not clear. Using various dyes that specifically bind different cell surface molecules, we show that there are multiple changes in the cell surface molecules associated with different purine auxotrophs. Affected molecules in different purine auxotrophs that were tested include (i) acidic exopolysaccharides, (ii) cellulose fibrils, and (iii) beta (13) glucans. Our results show that the symbiotic deficiency of purine auxotrophs is likely to be a result of these associated changes on the cell surface. Keywords. Rhizobium meliloti; Medicago saliva; purine biosynthesis; exopolysaccharides; symbiosis.

1. Introduction Successful development of nodules by Rhizobium spp. on legume roots involves surface interactions between the two interacting species at many different stages of development (Long 1989; Brewin 1991). Rhizobial surface components play an important role in deciding the host compatibility and in bringing about the infection leading to nodulation and nitrogen fixation. These surface components include acidic exopolysaccharides, neutral beta glucans, lipopolysaccharides and to an extent, the elements responsible for motility of the rhizobial cells (Dudman 1984; Gray and Rolfe 1990; Reuber et al 1991). Acidic exopolysaccharides, identified by their ability to bind calcofluor, are needed for a successful infection (Leigh et al 1985; Finan et al 1985). Genes controlling their biosynthesis in R. meliloti are clustered on the megaplasmid pRmSu47b (Batut et al 1985; Finan 1986). Doherty et al (1988), found two genes which negatively regulate the synthesis of R. meliloti exopolysaccharides. Cellulose fibrils produced by rhizobia, identified by their capacity to bind congo red dye, are thought to be useful in entangling the bacteria on the surface of the plant root, thus leading to an increase in the efficiency of infection. A major type of curdlans, beta (13) glucans can be specifically stained with aniline blue. These molecules are normally not found on rhizobial surfaces but may be found in the related genus Agrobacterium (Nakanishi et al 1976). Another important class of molecules on the rhizobial surface is that of cyclic beta (12) glucans, needed for flagellum motility (Geremia et al 1987). Purine auxotrophs of Rhizobium spp. have earlier been reported to be defective

Corresponding author.

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in symbiosis (Noel et al 1987; Swamynathan 1990; Swamynathan and Singh 1992). We had also shown that these mutants are capable of forming infection threads and nodules that were not able to fix nitrogen. However, a specific reason for the failure of symbiotic nitrogen fixation by rhizobial purine auxotrophs is still not known. One of the several possible ways by which purine auxotrophy can manifest its effect on nitrogen fixation is by altering the surface characters involved in the interaction of Rhizobium with the host plant. In this paper, we demonstrate that there are multiple changes in the cell surface components associated with a set of six purine auxotrophs that we had earlier reported (Swamynathan 1990; Swamynathan and Singh 1992). Our results suggest that the failure of symbiosis by the purine auxotrophs is most likely due to these associated pleiotropic changes on the cell surface. 2. 2.1 Materials and methods Bacterial strains and plasmid constructs

Bacterial strains and the plasmid constructs used are listed in table 1. Rmd201, the wild type strain used in this study is a streptomycin resistant derivative of the
Table 1. Bacterial strains and plasmid constructs used.

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strain AK631, which effectively nodulates Medicago sativa (Khanuja and Kumar 1989). Apart from streptomycin resistance, the strains Rmd201 and AK631 are identical with respect to their nodulation ability and symbiotic efficiency. 2.2 Media and culture conditions

Complete medium (TY) and minimal medium (RMM) for Rhizobium have been described earlier (Khanuja and Kumar 1988). Amino imidazole carboxamide riboside (AICAR), obtained from Sigma, was used at a final concentration of 01 mM. Inosine monophosphate (IMP), thiamine pyrophosphate (TPP), adenine or guanine was used at a final concentration of 01 mM, with RMM or the plant growth substrate, as and when required. Rhizobium and . coli cultures were grown at 30C and 37C, respectively. Tetracycline, streptomycin and ampicillin were used at a final concentration of 10, 30 and 200 g/ml respectively. Spontaneous prototrophic revertants were obtained by spreading a high density of auxotrophs on RMM plates. Reversion frequency was enhanced by spotting 25 g of NTG on a filter disc at the centre of the plate already spread with mutant cells. The revertants of purine auxotrophs used in this study also regain their symbiotic efficiency (Swamynathan and Singh 1992). 2.3 Transfer of plasmids Plasmids pD2, pD34, pD56 and pEX312 were transferred to rhizobial purine auxotrophs from . coli HB 101 by conjugation, done on TY plates. Exconjugants were identified by replica plating the mating mix after 36 h of growth at 30C, on to TY plates containing tetracycline and streptomycin. 2.4 Nodulation and acerylene reduction ability assays Tests for determining the symbiotic efficiency of the mutants were done as reported earlier (Khanuja and Kumar 1989). The acetylene reduction ability (ARA) reported here is an average of six replications. 2.5 Tests for production of exopolysaccharides Bacterial strains to be tested were streaked on YEMA plates having aniline blue or congo red or calcofluor and incubated at 30C for three days. Calcofluor staining was observed as fluorescence under long wavelength UV rays while other stains were observed in visible light. 2.6 Test for lipopolysaccharide production The strains to be tested were spotted on TY plates having sodium deoxycholate (DOC, 1 mg/ml) and incubated at 30C for two days. The strains that grew on these plates were considered to be capable of producing lipopolysaccharides. 2.7 Test for motility

The bacterial strains to be tested were spoiled on swarm plates (TY plates with

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03% agar) and incubated at 3Co for two days. The motility of the bacterial strains were determined by the spread of the colonies in the swarm plates. 3. Results 3.1 Acidic exopolysaccharides

An examination under UV light of strains grown on YEMA plates containing calcofluor showed that Rmd l l02 produced less acidic exopolysaccharides than the wild type Rmd201 (figure 1). The other five purine auxtotrophs studied were found to be unaffected in the production of acidic exopolysaccharides. Revertants of Rmd 1102, selected by their ability to grow on RMM in the absence of exogenous supply of purines, were found to be producing an equal amount of acidic exopolysaccharides as the wild type, Rmd201 (Figure 1). This indicates that the Exo phenotype of Rmdl 102 is a pleiotropic effect of the point mutation responsible for purine auxotrophy. We then tested if this deficiency of Rmdl 102 in acidic exopolysaccharide production can be complemented by one of the several genes that are known to

Figure 1. Rind 1102 produces less calcoflour binding exopolysaccharides than Rmd20l as evidenced by a lower fluorescence under UV when grown on a YEMA plate containing calcoflour. Note that the Exo phenotype of Rmd 1102 is nor complemented by any of the known exo genes. P. Rmd201 Q, Rmdl 102; R, Revertant of Rmd 1102; S. Rmd 1l02: pD2; T. Rmd l102: pD34; U. Rmd l102. pD36: V, Rmdll02: pEx312.

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be involved in rhizobial exopolysaccharide production. Our results show that the exo genes present on pD2, pD34, pD56 and pEX312 do not complement the Exophenotype of Rmd ll02 (figure 1). In addition, these plasmids failed to complement the symbiotic defect of Rmd 1102 (table 2).
Table 2. Symbiotic ability of Rmd ll02 carrying various exo clones as compared to the wild type.

3.2

Cellulose fibrils

The wild type strain Rmd201 produced red colonies when grown on a medium containing congo red. Rmd ll04, Rmd1105 and Rmd ll06 failed to form red colonies in the presence of congo red (figure 2a). However, their revertants were found to have regained the capacity to form red colonies on congo red plates, indicating again, the pleiotropic nature of purine auxotrophs (figure 2b). Colonies of acid producing strains and the medium surrounding them will turn deep purple if the strains are stored at 4C on congo red plates (Kneen and LaRue 1983). We found that Rmd ll04, Rmd 1105 and Rmd 1106 colonies and the medium surrounding them turned deep purple on storage for about a month at 4C (figure 3a). There was no such change in the parent strain and the revertants upon storage for the same period of time (figure 3b). 3.3 Beta glucans

Aniline blue is known to specifically bind the curdlan type of polysaccharides, of which beta (13) glucan is a major component (Nakanishi et al 1976). The parent strain Rmd201 does not produce beta (13) curdlans as indicated by its failure to be stained by aniline blue. It was, however, seen that all the purine auxotrophs, other than Rmd ll03, could be stained with aniline blue, indicating their new capability to produce the beta (13) curdlans (figure 4a). The revertants lost this staining ability, confirming that the production of beta (13) curdlans by the purine auxotrophs is a result of the pleiotropic effect of the mutation in pur genes (figure 4b). Absence of beta (12) glucans has been linked to a defective flagellum and the resulting absence of chemotactic response which in turn leads to formation of ineffective nodules (Geremia et al 1989). Results of our experiments with swarm plates show that all the six mutants are motile like the wild type, implying that the beta (12) glucan production is unhindered by the purine auxotrophy (figure 5).

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Figure 2 .

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It is also evident that calcofluor binding acidic exopolysaccharides, cellulose fibrils and beta (13) glucans have no critical role to play in the motility of a rhizobial cell. 3.4 Lipopolysaccharides Failure to produce lipopolysaccharides (LPS) results in an inability to form effective nodules (Noel et al 1989). Further, LPS confer on rhizobia, resistance to sodium deoxycholate (DOC). All our purine auxotrophs, like the wild type, were found to be equally tolerant to DOC in the medium. This indicates that there is no defect in LPS production associated with these purine auxotrophs in Rhizobium. 4. Discussion Rhizobial cell surface is a complex conglomerate of various polysaccharides that are known to play a major role in the development of symbiotic nodules. Since rhizobial purine auxotrophs have been found to be symbiotically defective, we wanted to test if there are any changes on the rhizobial cell surface associated with purine auxotrophy. We used various dyes that interact specifically with these molecules, for four purposes. Our results provide the first evidence for a pleiotropic change on the rhizobial cell surface associated with purine auxotrophy. The results presented here show that purine auxotrophy in Rhizobium influences the production of acidic exopolysaccharides, cellulose fibrils and beta (13) glucans but not that of lipopolysaccharides or beta (12) glucans. Since the known exo genes failed to complement the Exo phenotype of these auxotrophs, the defect in acidic exopolysaccharide synthesis is not due to a mutation in these structural genes encoding the enzymes regulating the exopolysaccharide biosynthesis pathway. On the other hand, a defect in the regulatory circuit of these exo genes can not be ruled out al this stage. Moreover, we have tested only the exo genes present on the megaplasmid pRmSu47B, for the ability to complement the Exo phenotype of Rmdll02. Thus, the locus affected in Rmdll02 may fall outside the exo cluster present on pRmSu47B; it could be elsewhere on the plasmid itself or on the chromosome and it may define a new regulatory element required for exopolysaccharide biosynthesis in R. meliloti. The fact that purine auxotrophy has its effects on such a wide range of unrelated phenotype is surprising. However, multifunctionality of purine biosynthetic enzymes is well known. There are precedence in bacteria, fission yeasts and plants, which point at the involvement of enzymes of de novo purine biosynthesis pathway in other, seemingly unrelated pathways (Moffatt and Somerville 1988; Speiser et al 1992). The pleitropic nature of our mutants is clear since the pur+ revertants of
Figure 2. Reaction of (a) purine auxotroph and (b) their revertants. with the dye congo red. Note that Rmdl 104. Rmdl 105 and Rmdl 106 produce white colonies while their revertants produce red colonies like wild type on YEMA plates containing congo red. 1, Rmd20l; 2, Rmd l101; 3, Rmd l102; 4, Rmdl103; 5, Rmdl104; 6, Rmdl 105. 7, Rmdl106. a, Rmd20l: b, Revertant of Rmd ll01. c, Revertant of Rmdll02; d, Revertant of Rmdll04: e, Revertant of Rmd l105; f, Revertant of Rmd l106; g, Rmd1102, for comparison.

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Figure 3. For caption, see page 28.

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Figure 4. For caption, see page 28.

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Figure 5. Motility of R. melilott is not influenced by purine auxotrophy or the associated changes on the rhizobial surface. First two rows consist of purine auxotrophs as compared to the wild type (W). Next two rows consist of the revertants of purine auxotrophs as compared to the wild type (W).

these auxotrophs are similar to the wild type strain with respect to all the characters that we have studied, for which the purine auxotrophs are defective. Moreover, no partial revertants were isolated for any of these characters, in spite of multiple rounds of screening. A functional redundancy in rhizobial exopolysaccharides has been hypothesised earlier, according to which rhizobia have a capacity to produce more than one kind of exopolysaccharide with the same function, but under different conditions (Glazebrook and Walker 1989). Our data indicate the possibility of existence of such a mechanism, between beta (13) glucans and cellulose fibrils also. It is possible that there is a reciprocal regulation between these two molecules, which needs to be investigated. The purine auxotrophs we have used in this study are defective at different stages of the de novo purine biosynthetic pathway. These mutants also differ with respect to the aberrations at the surface, yet all of these show a symbiotic deficiency. These results suggest that purine auxotrophy manifests its effect on symbiosis by influencing the production of various surface components. We are now trying to purify these surface components from the wild type strain and study the effect of their incorporation in the plant growth substrate on symbiotic efficiency of the respective purine auxotrophs.

Cell surface defects in rhizobial purine auxotrophs Acknowledgements

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We are grateful to Prof. Sushil Kumar for his valuable suggestions during the work and in the course of manuscript preparation. This work was funded by Indo-US SSP grants. Financial assistance in the form of IARI junior research fellowship to SKS during the course of this work is gratefully acknowledged. References
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Swamynathan S 1990 Isolation and characterization of mutants of the purine biosynthetic pathway in Rhizobium meliloti, M.Sc. thesis, Indian Agricultural Research Institute, New Delhi Swamynathan S and Singh A 1992 Rhizobium melioti purine auxotrophs are nod+ but defective in nitrogen fixation; J. Genet. 75 1121

Figure 3. Effect of long term storage al 4oC on rhizobial strains grown on YEMA plates containing congo red. (a) The purine auxotrophs and (b), their revertants. Note that the colonies and the medium surrounding them has turned deep purple around Rmdl104, Rmdl105 and Rmd1106, while there is no such change in other mutants or the revertants. 1, Rmd201;, 2, Rmd1101; 3, Rmd1102; 4, Rmdll03; 5, Rmd l104; 6, Rmdll05; 7, Rmd l106. a, Rmd20l; b, Revertant of Rmdl101; c, Revertant of Rmd l102; d, Revertant of Rmd1104; e, Revertant of Rmdl105; f, Revertant of Rmdll06; g, Rmd l102, for comparison. Figure 4. Reaction of (a) purine auxotrophs and (b) their revertants with aniline blue. Note that Rmd201 forms white colonies while all the mutants other than Rmdll03 form blue colonies when grown on YEMA plates containing aniline blue. All the revertants form white colonies like the wild type. Rind201;, 2, Rmdll01; 3, Rmdll02; 4, Rmdl 103: 5, Rmd1104; 6. Rmdll05; 7, Rmd1106. a, Rmd20l; b, Revertant of Rmdl 101; c, Revertant of Rmdll02; d, Revertant of Rmdll04; e. Revertant of Rmdl 105; f, Revertant of Rmdl 106; g, Rmdl 102, for comparison.