small bacteriocin was isolated from the culture broth of the gram-negative bacterium Rhizobium leguminosarum, which forms symbiotic nitrogen-fixing root nodules on a number of leguminous plants. The structure of the molecule was elucidated by spectroscopic methods and identified as N-(3R-hydroxy-7-cis-tetradecanoyl)-L-homoserine lactone. The absolute configuration of both asymmetric carbon atoms in the molecule was determined by the use of the chiral solvating agents S-(؉)-and R-(؊)-2,2,2-trifluoro-1-(9-anthryl)-ethanol. small bacteriocin is structurally related to the quorum sensing co-transcription factors for genes from other bacteria such as Vibrio fischeri, Pseudomonas aeruginosa, Erwinia carotovora, and Agrobacterium tumefaciens which are involved in animal-microbe or plant-microbe interactions. The mechanism of regulation of such interactions by this kind of co-transcription factors is still unknown in R. leguminosarum.small bacteriocin (small) is produced by strains of all three biovars of Rhizobium leguminosarum and inhibits the growth of R. leguminosarum bv. viciae 248 and several other strains of this species which, like strain 248, contain a self-transmissible plasmid (12,17). Two genes located close to the transfer (tra) genes (17) on the Sym plasmid pRL1JI of strain 248 are responsible for the fact that this strain does not produce small (rps [repression production small] gene) and that it is sensitive to small (sbs [small bacteriocin sensitivity] gene) (12,17). When strain 248 is cured of its Sym plasmid, pRL1JI (as, for example, in strain RBL1390), it is insensitive to small and also produces small (12, 17). To our knowledge, small is produced only by R. leguminosarum strains, and in nonproducing strains of this species, a gene for small production like that in strain 248 is present (12,17). Therefore, the presence of small is considered a characteristic of this species. Since another typical property of this species is symbiotic root nodule formation on certain leguminous plants, it is possible that both properties are related. However, a strain with a Tn5 insertion in the small gene(s) could induce formation of normal root nodules, which shows that the small gene is not required for root nodule formation (16). However, this does not exclude an ecological link between small production and the interaction of Rhizobium spp. with plants, since other genes may complement the lost function or the function is not essential. In order to detect its biological significance, small was extracted from the bacterial culture medium with chloroform (16) and identified chemically. The small molecule appeared to contain an N-acyl homoserine lactone structure, as known from quorum sensing signal molecules, which function as co-transcription factors in bacteria that often interact with higher organisms (reviewed by Fuqua et al. [10]). The implications of this finding are discussed. MATERIALS AND METHODSBacterial strains and growth conditions. The bacterial strains used in this study were the small-sensitive strain...

Phosphatidylcholine (PC), the major membrane phospholipid in eukaryotes, is found in only some bacteria including members of the family Rhizobiaceae. For this reason, it has long been speculated that rhizobial PC might be required for a successful interaction of rhizobia with their legume host plants in order to allow the formation of nitrogen-fixing root nodules. A major pathway for PC formation in prokaryotes involves a threefold methylation of the precursor phosphatidylethanolamine (PE). Here, we report on the isolation of a Bradyrhizobium japonicum gene (pmtA) encoding the phospholipid N-methyltransferase PmtA. Upon expression of the bradyrhizobial pmtA gene in Escherichia coli, predominantly monomethylphosphatidylethanolamine was formed from PE. PmtA-deficient B. japonicum mutants still produced low levels of PC by a second methylation pathway. The amount of PC formed in such mutants (6% of total phospholipids) was greatly decreased compared with the wild type (52% of total phospholipids). Root nodules of soybean plants infected with B. japonicum pmtA mutants showed a nitrogen fixation activity of only 18% of the wild-type level. The interior colour of the nodules was beige instead of red, suggesting decreased amounts of leghaemoglobin. Moreover, ultrastructure analysis of these nodules demonstrated a greatly reduced number of bacteroids within infected plant cells. These data suggest that the biosynthesis of wild-type amounts of PC are required to allow for an efficient symbiotic interaction of B. japonicum with its soybean host plant.

Phosphatidylcholine is a major lipid of eukaryotic membranes, but found in only few prokaryotes. Enzymatic methylation of phosphatidylethanolamine by phospholipid N-methyltransferase was thought to be the only biosynthetic pathway to yield phosphatidylcholine in bacteria. However, mutants of the microsymbiotic soil bacterium Sinorhizobium (Rhizobium) meliloti, defective in phospholipid N-methyltransferase, form phosphatidylcholine in wild type amounts when choline is provided in the growth medium. Here we describe a second bacterial pathway for phosphatidylcholine biosynthesis involving the novel enzymatic activity, phosphatidylcholine synthase, that forms phosphatidylcholine directly from choline and CDP-diacylglycerol in cell-free extracts of S. meliloti. We further demonstrate that roots of host plants of S. meliloti exude choline and that the amounts of exuded choline are sufficient to allow for maximal phosphatidylcholine biosynthesis in S. meliloti via the novel pathway.

In phosphatidylcholine (PC)‐containing prokaryotes, only the methylation pathway of PC biosynthesis was thought to occur. However, a second choline‐dependent pathway for PC formation, the PC synthase (Pcs) pathway, exists in Sinorhizobium (Rhizobium) meliloti in which choline is condensed with CDP‐diacylglyceride. Here, we characterize the methylation pathway of PC biosynthesis in S. meliloti. A mutant deficient in phospholipid N‐methyltransferase (Pmt) was complemented with a S. meliloti gene bank and the complementing DNA was sequenced. A gene coding for a S‐adenosylmethionine‐dependent N‐methyltransferase was identified as the sinorhizobial Pmt, which showed little similarity to the corresponding enzyme from Rhodobacter sphaeroides. Upon expression of the sinorhizobial Pmt, besides phosphatidylcholine, the methylated intermediates of the methylation pathway, monomethylphosphatidylethanolamine and dimethylphosphatidylethanolamine, are also formed. When Pmt‐deficient mutants of S. meliloti are grown on minimal medium, they cannot form PC, and they grow significantly more slowly than the wild type. Growth of the Pmt‐deficient mutant in the presence of choline allows for PC formation via the Pcs pathway and restores wild‐type‐like growth. Double knock‐out mutants, deficient in Pmt and in Pcs, are unable to form PC and show reduced growth even in the presence of choline. These results suggest that PC is required for normal growth of S. meliloti.

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes. In addition to this structural function, PC is thought to play a major role in lipid turnover and signalling in eukaryotic systems. In prokaryotes, only some groups of bacteria, among them the members of the family Rhizobiaceae, contain PC. To understand the role of PC in bacteria, we have studied Rhizobium meliloti 1021, which is able to form nitrogen-fixing nodules on its legume host plants and therefore has a very complex phenotype. R. meliloti was mutagenized with N-methyl-N-nitro-N-nitrosoguanidine, and potential mutants defective in phospholipid N-methyltransferase were screened by using a colony autoradiography procedure. Filters carrying lysed replicas of mutagenized colonies were incubated with S-adenosyl-L-[methyl-14 C]methionine. Enzymatic transfer of methyl groups to phosphatidylethanolamine (PE) leads to the formation of PC and therefore to the incorporation of radiolabel into lipid material. Screening of 24,000 colonies for reduced incorporation of radiolabel into lipids led to the identification of seven mutants which have a much-reduced specific activity of phospholipid N-methyltransferase. In vivo labelling of mutant lipids with [ 14 C]acetate showed that the methylated PC biosynthesis intermediates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine are no longer detectable. This loss is combined with a corresponding increase in the potential methyl acceptor PE. These results indicate that PC biosynthesis via the methylation pathway is indeed blocked in the mutants isolated. However, mass spectrometric analysis of the lipids shows that PC was still present when the mutants had been grown on complex medium and that it was present in the mutants in wild-type amounts. In vivo labelling with [methyl-14 C]methionine shows that in phospholipid N-methyltransferasedeficient mutants, the choline moiety of PC is not formed by methylation. These findings suggest the existence of a second pathway for PC biosynthesis in Rhizobium.