Intracellular accumulation of glycine betaine has been shown to confer an enhanced level of osmotic stress tolerance in Rhizobium meliloti. In this study, we used a physiological approach to investigate the mechanism by which glycine betaine is accumulated in osmotically stressed R. meliloti. Results from growth experiments, 14C labeling of intermediates, and enzyme activity assays are presented. The results provide evidence for the pathway of biosynthesis and degradation of glycine betaine and the osmotic effects on this pathway. High osmolarity in the medium decreased the activities of the enzymes involved in the degradation of glycine betaine but not those of enzymes that lead to its biosynthesis from choline. Thus, the concentration of the osmoprotectant glycine betaine is increased in stressed cells. This report demonstrates the ability of the osmolarity of the growth medium to regulate the use of glycine betaine as a carbon and nitrogen source or as an osmoprotectant. The mechanisms of osmoregulation in R. meliloti and Escherichia coli are compared.

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Specificty in m-ay plant-pathogen interactions is determined by single genes In pathogen and host. The single locus for host-selective pathogenicity (TOX2) HC-toxin, the host-selective toxin produced by C. carbonum race 1 that is required for pathogenicity of this fungus on maize, is a cyclic tetrapeptide with the structure cyclo(DPro-L-Ala-D-Ala-L-Aeo), where Aeo is 2-amino-9,10-epoxy-8-oxodecanoic acid (4-6). We have identified and purified two enzymes involved in biosynthesis ofHC-toxin (7,8). One enzyme, HC-toxin synthetase 1 (HTS-1), has a molecular mass of =220 kDa, catalyzes ATP/PPi exchange in the presence of L-proline, and epimerizes L-proline to D-proline. The second enzyme, HTS-2, has an apparent molecular mass of 160 kDa, catalyzes L-alanine-dependent and D-alaninedependent ATP/PPj exchange, and epimerizes L-alanine to D-alanine. Both of these enzymes are detected only in race 1 (Tox+) isolates of C. carbonum and their activities segregate genetically with TOX2 (7). We have undertaken a molecular genetic analysis of HC-toxin biosynthesis with the goals of understanding the nature ofthe economically important TOX loci ofCochliobolus and the evolution ofnew races in this and related pathogens. MATERIALS AND METHODSNucleic Acid Manipulations. Isolation of fungal DNA and construction of the genomic DNA library in phage AEMBL3 were as described (9). Subcloning was done into pBluescript (Stratagene) or pUC18 (BRL). Probes were labeled with 32p by random priming and were present in hybridizations at 2 x 105 cpm/ml. Hybridizations were done overnight at 650C in 5x SSPE (lx SSPE = 150 mM NaCl/10 mM NaH2PO4/1 mM EDTA, pH 7.4)/7% SDS/0.5% nonfat dry milk/0.1 mg of denatured salmon sperm DNA per ml. Blots were washed in 2x SSPE/0.1% SDS; the final wash was at 650C for 1 hr.

As a first step towards the elucidation of the molecular mechanisms responsible for the utilization of choline and glycine betaine (betaine) either as carbon and nitrogen sources or as osmoprotectants in Sinorhizobium meliloti, we selected a T n 5 mutant, LTS23-1020, which failed to grow on choline but grew on betaine. The mutant was deficient in choline dehydrogenase (CDH) activity, failed to oxidize [methyl-14C]choline to [methyl-I4C]betaine, and did not use choline, but still used betaine, as an osmoprotectant. The T n 5 mutation in LTS23-1020 was complemented by plasmid pCH034, isolated from a genomic bank of 5. meliloti 102F34. Subcloning and DNA sequencing showed that pCH034 harbours two ORFs which showed 60% and 57% identity with the Escherichia coli betB gene encoding betaine-aldehyde dehydrogenase (BADH) and betA gene encoding CDH, respectively. In addition to the homology with E. coli genes, the deduced sequence of the sinorhizobial BADH protein displays consensus sequences also found in plant BADHs. The deduced sequence of the sinorhizobial CDH protein shares only 21 O/ O identical residues with choline oxidase from Atthrobacter globiformis. The structural organization of the betBA genes in S. meliloti differs from that described in E. coli: (i) the two ORFs are separated by a 210 bp sequence containing inverted repeats ressembling a putative rhoindependent transcription terminator, and (ii) no sequence homologous to betT (high-aff inity choline transport system) or bet/ (regulator) was found in the vicinity of the sinorhizobial betBA genes. Evidence is also presented that the S. meliloti betBA genes are not located on the megaplasmids.