Free-living cells of the fast-growing cowpea Rhizobium NGR234 were able to grow on a variety of carbon substrates at growth rates varying from 2.5 h on glucose or fumarate to 15.6 h on phydroxybenzoate. Free-living cells constitutively oxidized glucose, glutamate and aspartate but were inducible for all the other systems investigated. Bacteroids from root nodules of snake bean, however, were only capable of oxidizing C,-dicarboxylic acids and failed to oxidize any other carbon sources. Free-living cells of NGR234 possess inducible fructose and succinate uptake systems. These substrates are accumulated by active processes since accumulation is in hi bi ted by azide, 2,4-dini trop hen01 and carbonyl cyanide m-chlorophenyl hydrazone. Bacteroids failed to take up fructose although they actively accumulated succinate, suggesting that the latter substrate is significant in the development of an effective symbiosis. I N T R O D U C T I O NAn understanding of the survival of rhizobia in soil and of the complex interactions between Rhizobium and legume required for the establishment of an effective symbiosis is likely only when the physiology of these organisms has been carefully studied. Although recent advances in the genetics and molecular biology of Rhizobium have been rapid, advances in understanding its physiology have been much slower.Previous work from this laboratory has examined various aspects of the carbon metabolism of Rhizobium leguminosarum (e.g. Glenn et al., Glenn & Dilworth, 1981 a ; Dilworth et al., 1983). In general, inducible oxidation systems for carbon compounds were found to be relatively uncommon for R. leguminosarum; most oxidation systems were produced at significant levels in the absence of the particular substrate. In a survey of the uptake and hydrolysis of disaccharides by rhizobia (Glenn & Dilworth, 1981 b), it was found that while R . leguminosarum synthesized significant levels of the uptake systems and hydrolytic enzymes for a range of disaccharides in the absence of the substrate, such systems in R. meliloti and cowpea Rhizobium NGR234 were inducible.Cowpea Rhizobium NGR234 is an unusual strain in that it is fast-growing and forms effective nodules on legumes typically nodulated by slow-growing bradyrhizobia. In addition, it has a single megaplasmid responsible for its symbiotic properties (Morrison et al., 1983). Because of these unusual properties, the general interest in the strain by workers in plasmid biology and some clear differences in physiology in comparison with R . leguminosarurn MNF3841 (Glenn & Dilworth, 1981a), the carbon metabolism of NGR234 has now been investigated in greater detail. METHODS Organism.Cowpea Rhizobium NGR234 was obtained from Dr M. J. Trinick, CSIRO, Perth, Western Australia. This strain is a typical fast-grower (Broughton & Dilworth, 1971) and nodulates both cowpea (Vigna unguiculata (L.) Walp. ssp. unguiculata) and snake bean ( V . unguiculata ssp. sesquipedalis (L.) Verdc.).

L-Arabinose is broken down by Rhizobium leguminosarum MNF300 via 2-oxoglutarate semialdehyde. Enzyme activities in cells grown on succinate, mannitol or arabinose indicated much greater modulation of arabinonate dehydratase, 2-keto-3-deoxyarabinonate dehydratase and 2-oxoglutarate semialdehyde dehydrogenase than of arabinose dehydrogenase or of arabinono-y-lactonase. In cowpea Rhizobium NGR234, all the enzymes of L-arabinose metabolism except L-arabinono-y-lactonase were inducible. Assays for such enzymes in snake bean bacteroids indicated that L-arabinose did not reach the bacteroids in large quantities. The Tn5-induced mutant MNF3045 of R . legurninosarum was unable to grow on L-arabinose and accumulated L-arabinono-y-lactone and L-arabinonate. Product accumulation and enzyme assays suggested that this mutant was defective in L-arabinonate dehydratase. It nodulated peas and the nodules fixed N2, indicating that the supply of L-arabinose is not essential for bacteroid function. Another Tn5-induced mutant of R . leguminosarum, MNF3041, lacked ribokinase and was unable to grow on D-ribose; this mutant was also able to nodulate peas and fix N,. I N T R O D U C T I O NRhizobia can efficiently utilize a variety of pentoses for growth. It is therefore possible that the supply of such compounds by host legumes may be important to nodule formation or function. One approach to the identification of compounds which are important to symbiotic N2 fixation in the legume nodule has been the study of rhizobial mutants blocked in particular steps in catabolism. A mutant of Rhizobium meliloti unable to grow on L-arabinose was identified as defective in the synthesis of 2-oxoglutarate dehydrogenase (Duncan & Fraenkel, 1979); it continued, however, to degrade L-arabinose at the same rate as the wild-type strain. A similar Tn5-induced mutant also metabolized L-arabinose without growth, and either nodulated later than the wild-type or formed no nodules at all (Duncan, 1981). Mutants of R . meliloti which were unable to grow on ribose or on xylose were normal in both nodulation and N2 fixation, and still possessed the capacity to degrade these pentoses (Duncan, 1981). While these data suggest that metabolic intermediates were accumulating, none was identified.The routes of pentose utilization in rhizobia are not well defined. In Brudyrhizobium juponicum, L-arabinose appears to be degraded to L-2-keto-3-deoxyarabinonate (KDA), which is then cleaved to pyruvate and glycolaldehyde (Fig. 1) via an aldolase reaction (Pedrosa & Zancan, 1974). While pyruvate and glycolaldehyde were identified as products of 2-keto-3-deoxyarabinonate, the alternative route, dehydration to 2-oxoglutarate semialdehyde, was not explored. It was subsequently reported (Duncan, 1979) that B. japonicum 61A76 and cowpea Bradyrhizobium 32H 1 not only possessed KDA aldolase but also 2-oxoglutarate semialdehyde dehydrogenase. The presence of this enzyme suggests that KDA dehydratase may also have Abbreviation: KDA, 2-keto-3-deoxyarabinonate. 0001-3402 0 1986 SGM

In sugar-grown cells of cowpea Rhizobium strain NGR234 activities for enzymes of the EntnerDoudoroff and pentose phosphate pathways were present while the virtual absence of phosphofructokinase and fructose-bisphosphate aldolase indicated that the Embden-Meyerhof-Parnas pathway was unlikely to be significant. Invertase, fructokinase, glucose-6-phosphate dehydrogenase and the Entner-Doudoroff enzymes were present at only low activities in succinate grown cells, but were induced in sugar-grown cells. Isolated snakebean bacteroids contained very low activities of these four enzymes. Although C,-dicarboxylic acids exerted some repressive effect on induction of these enzymes, there was substantial enzyme activity induced in cells grown on sucrose plus a C, dicarboxylic acid. The data suggest that the peribacteroid membrane may be relatively impermeable to sugars and so dictate the carbon source(s) available to the bacteroids.