Novel pathway for phosphatidylcholine biosynthesis in bacteria associated with eukaryotes.

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and can be synthesised by either of two pathways, the CDP-choline pathway or the methylation pathway. Many prokaryotes lack PC, but it can be found in significant amounts in membranes of distantly related bacteria such as Rhizobacteria and Spirochetes. Enzymatic methylation of phosphatidylethanolamine via the methylation pathway was thought to be the only biosynthetic pathway to yield PC in bacteria. However, a novel choline-dependent pathway for PC biosynthesis has been discovered in Sinorhizobium meliloti. In this pathway, a novel enzymatic activity, PC synthase, condenses choline directly with CDP-diacylglyceride to form PC in one step. Surprisingly, genomes of some pathogens (Pseudomonas aeruginosa, Borrelia burgdorferi and Legionella pneumophila) contain genes similar to the sinorhizobial gene for phosphatidylcholine synthase. We, therefore, suggest that the new PC synthase pathway is present in a number of bacteria displaying symbiotic or pathogenic associations with eukaryotes and that the eukaryotic host functions as the provider of choline for this pathway.

Phosphatidylcholine levels in Bradyrhizobium japonicum membranes are critical for an efficient symbiosis with the soybean host plant.

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.

The nodulation protein NodG shows the enzymatic activity of an 3-oxoacyl-acyl carrier protein reductase.

The acyl carrier protein NodF is required for the synthesis of unusual polyunsaturated fatty acids that confer specificity to lipochitin oligosaccharide nodulation (Nod) factors of Rhizobium leguminosarum. In this study, homogeneous NodF protein was used as a ligand to identify proteins of R. leguminosarum that specifically interact with NodF and presumably are involved in the biosynthesis or transfer of the unusual fatty acids. The N-terminal amino acid sequence of a 29-kDa protein that interacts strongly with NodF revealed high similarity to NodG of Rhizobium sp. N33 and to NodG of Sinorhizobium meliloti We cloned and sequenced the gene coding for the NodG-like protein of R. leguminosarum and found it to be the product of the constitutively expressed gene fabG. FabG is the 3-oxoacyl-acyl carrier protein reductase that catalyzes the first reduction step in each cycle of fatty acid elongation. FabG of R. leguminosarum and NodG of Rhizobium sp. N33 were expressed in Escherichia coli. In both cases, the purified protein showed 3-oxoacyl-acyl carrier protein reductase activity in vitro. Therefore, NodG has the same biochemical function as FabG, and the high degree of similarity at the protein and DNA level suggest that nodG is a duplication of the housekeeping genefabG.

Inactivation of the gene for phospholipid N-methyltransferase in Sinorhizobium meliloti: phosphatidylcholine is required for normal growth.

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.

Disruption of a gene essential for sulfoquinovosyldiacylglycerol biosynthesis in Sinorhizobium meliloti has no detectable effect on root nodule symbiosis.

The sulfolipid sulfoquinovosyldiacylglycerol is commonly found in the thylakoid membranes of photosynthetic bacteria and plants. While there is a good correlation between the occurrence of sulfolipid and photosynthesis, a number of exceptions are known. Most recently, sulfoquinovosyldiacylglycerol was discovered in the non-photosynthetic, root nodule-forming bacterium Sinorhizobium meliloti. This discovery raised the questions of the phylogenetic origin of genes essential for the biosynthesis of this lipid in S. meliloti and of a function of sulfolipid in root nodule symbiosis. To begin to answer these questions, we isolated and inactivated the sqdB gene of S. meliloti. This gene and two other genes located directly 3' of sqdB are highly similar to the sqdB, sqdC, and sqdD genes known to be essential for sulfolipid biosynthesis in the photosynthetic, purple bacterium Rhodobacter sphaeroides. This observation confirms the close phylogenetic kinship between these two species. Furthermore, the reduced similarity of sqdB to the plant ortholog SQD1 of Arabidopsis thaliana does not support a previous sqd gene transfer from the plant as a consequence of close symbiosis. A sulfolipid-deficient mutant of S. meliloti disrupted in sqdB is capable of inducing functional nodules and does not show an obvious disadvantage under different laboratory culture conditions. Thus far, no specific function can be assigned to bacterial sulfolipid, in either nodule-associated or free-living cells. S. meliloti contains a rich set of polar membrane lipids some of which, including sulfolipid, may become critical only under growth conditions that still need to be discovered.

Cloning and characterization of the gene for phosphatidylcholine synthase.

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and can be synthesized by either of two pathways, the CDP-choline pathway or the methylation pathway. In prokaryotes only the methylation pathway was thought to occur. Recently, however, we could demonstrate (de Rudder, K. E. E., Sohlenkamp, C., and Geiger, O. (1999) J. Biol. Chem. 274, 20011-20016) that a second pathway for phosphatidylcholine biosynthesis exists in Sinorhizobium (Rhizobium) meliloti involving a novel enzymatic activity, phosphatidylcholine synthase, that condenses choline and CDP-diacylglyceride in one step to form PC and CMP. Using a colony autoradiography method we have isolated mutants of S. meliloti deficient in phosphatidylcholine synthase and which are no longer able to incorporate radiolabeled choline into PC. Complementation of such mutants with a sinorhizobial cosmid gene bank, subcloning of the complementing fragment, and sequencing of the subclone led to the identification of a gene coding for a presumptive CDP-alcohol phosphatidyltransferase. Amplification of this gene and its expression in Escherichia coli demonstrates that it codes for phosphatidylcholine synthase. Genomes of some pathogens (Pseudomonas aeruginosa and Borrelia burgdorferi) contain genes similar to the sinorhizobial gene (pcs) for phosphatidylcholine synthase. Although pcs-deficient S. meliloti knock-out mutants show wild type-like growth and lipid composition, they are unable to perform rapid PC biosynthesis that normally is achieved via the phosphatidylcholine synthase pathway in S. meliloti wild type.

Olanzapine use in the elderly: a retrospective analysis.

OBJECTIVE: To evaluate the efficacy and safety of olanzapine in a hospitalized geriatric population that had previously failed to respond to, or tolerate, numerous trials with other antipsychotic medications. METHOD: A retrospective chart analysis was conducted on 58 elderly patients with psychotic symptoms who were given a trial on olanzapine. Data was collected regarding patients' psychiatric diagnoses, duration of illness, duration of hospitalization, prior response to psychotropic therapies, concomitant psychotropic agents, side effects, and clinically determined changes over time. RESULTS: Results indicated that 60.3% of this refractory group of patients improved on olanzapine. Side effects were reported for 38% of the patients, with delirium, extrapyramidal symptoms (EPS), and drowsiness or lethargy being the most common. CONCLUSIONS: The reported level of improvement in this group of refractory elderly psychotic patients indicates that olanzapine can make an important contribution to the mental health of elderly patients with similar characteristics.

Metaphors and the mental lexicon.

In two experiments we investigate the possibility that lexicalization can account for the distinction between literal and metaphorical language. In both experiments, sentence contexts were presented with the final word missing. When subjects signaled they understood the context, two possible final words were presented and subjects were required to decide which was more appropriate. Semantic decision times over five different types of stimuli investigating literal and metaphorical word usage were consistent with a modified class inclusion model in which both the lexicalized meaning of words and the context in which they are presented make essential contributions to understanding.

Plant-exuded choline is used for rhizobial membrane lipid biosynthesis by phosphatidylcholine synthase.

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.

The regulator gene phoB mediates phosphate stress-controlled synthesis of the membrane lipid diacylglyceryl-N,N,N-trimethylhomoserine in Rhizobium (Sinorhizobium) meliloti.

Bacteria react to phosphate starvation by activating genes involved in the transport and assimilation of phosphate as well as other phosphorous compounds. Some soil bacteria have evolved an additional mechanism for saving phosphorous. Under phosphate-limiting conditions, they replace their membrane phospholipids by lipids not containing phosphorus. Here, we show that the membrane lipid pattern of the free-living microsymbiotic bacterium Rhizobium (Sinorhizobium) meliloti is altered at low phosphate concentrations. When phosphate is growth limiting, an increase in sulpholipids, ornithine lipids and the de novo synthesis of diacylglyceryl trimethylhomoserine (DGTS) lipids is observed. Rhizobium meliloti phoCDET mutants, deficient in phosphate uptake, synthesize DGTS constitutively at low or high medium phosphate concentrations, suggesting that reduced transport of phosphorus sources to the cytoplasm causes induction of DGTS biosynthesis. Rhizobium meliloti phoU or phoB mutants are unable to form DGTS at low or high phosphate concentrations. However, the functional complementation of phoU or phoB mutants with the phoB gene demonstrates that, of the two genes, only intact phoB is required for the biosynthesis of the membrane lipid DGTS.

Characterization of a novel acyl carrier protein, RkpF, encoded by an operon involved in capsular polysaccharide biosynthesis in Sinorhizobium meliloti.

Rhizobial capsular polysaccharides (RKPs) play an important role in the development of a nitrogen-fixing symbiosis with the plant host and in Sinorhizobium meliloti AK631 functional rkpABCDEF genes are required for the production of RKPs. After cloning the rkpF gene, we overexpressed and purified the derived protein product (RkpF) in Escherichia coli. Like acyl carrier protein (ACP), the RkpF protein can be labeled in vivo with radioactive beta-alanine added to the growth medium. If homogeneous RkpF protein is incubated with radiolabeled coenzyme A in the presence of purified holo-ACP synthase from E. coli, an in vitro transfer of 4'-phosphopantetheine to the RkpF protein can be observed. The conversion from apo-RkpF protein to holo-RkpF protein seems to go along with a major conformational change of the protein structure, because the holo-RkpF protein runs significantly faster on native polyacrylamide gel electrophoresis than the apo-RkpF protein. Electrospray mass spectrometric analysis reveals a mass of 9,585 for the apo-RkpF protein and a mass of 9,927 for the holo-RkpF protein. Our data show that RkpF is a novel ACP.

The presence of Canadian hospitals on the World Wide Web: an empirical analysis.

As the World Wide Web expands, hospitals are considering how they may benefit from establishing Web sites of their own. We examined the Web sites of 20 Canadian hospitals to identify and compare their features. We developed two instruments for this assessment: a quantitative Features Checklist containing 67 items and a qualitative Categorical Rating Scale using 15 dimensions. Two of us (O.G. and D.A.M.) assessed each site. At most sites the most fully implemented feature was the provision of basic contact information, although development was inconsistent. Few sites took full advantage of multimedia capabilities. There was a strong correlation (r = 0.82, P < 0.001) between the number of features observed at a site and its score on the Categorical Rating Scale.

NodFE-dependent fatty acids that lack an alpha-beta unsaturation are subject to differential transfer, leading to novel phospholipids.

In Rhizobium leguminosarum, the nodABC and nodFEL operons are involved in the production of lipo-chitin oligosaccharide signals that mediate host specificity. A nodFE-determined, highly unsaturated C18:4 fatty acid (trans-2, trans-4, trans-6, cis-11-octadecatetraenoic acid) is essential for the ability of the signals to induce nodule meristems and pre-infection thread structures on the host plant Vicia sativa. Of the nod genes, induction of only nodFE is sufficient to modify fatty acid biosynthesis to yield trans-2, trans-4, trans-6, cis-11-octadecatetraenoic acid, with an absorbance maximum of 303 nm. This unusual C18:4 fatty acid is not only found in the lipo-chitin oligosaccharides but is also associated with the phospholipids (O. Geiger, J. E. Thomas-Oates, J. Glushka, H. P. Spaink, and B. J. J. Lugtenberg, 1994, J. Biol. Chem. 269:11090-11097). Here we report that the phospholipids can contain other nodFE-derived fatty acids, a C18:3 trans-4, trans-6, cis-11-octadecatrienoic acid that has a characteristic absorption maximum at 225 nm, and a C18:2 octadecadienoic acid. Neither this C18:3 nor this C18:2 fatty acid has to date been observed attached to lipo-chitin oligosaccharides, suggesting that an as yet unknown acyl transferase (presumably NodA), responsible for the transfer of the fatty acyl chain to the glycan backbone of the lipo-chitin oligosaccharides, does not transfer all fatty acids synthesized by the action of NodFE to the lipo-chitin oligosaccharides. Rather, it must have a preference for alpha-beta unsaturated fatty acids during transfer.

Rhizobium meliloti mutants deficient in phospholipid N-methyltransferase still contain phosphatidylcholine.

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-14C]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 [14C]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-14C]methionine shows that in phospholipid N-methyltransferase-deficient 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.

NMR investigations of the structural properties of the nodulation protein, NodF, from Rhizobium leguminosarum and its homology with Escherichia coli acyl carrier protein.

Heteronuclear NMR methods have been used to elucidate the secondary structure and the general tertiary fold of the protein NodF from Rhizobium leguminosarum. A similarity to acyl carrier proteins of the fatty acid synthase system had been suggested by the presence of a phosphopantetheine prosthetic group and a short stretch of sequence homology near the prosthetic group attachment site. NMR results suggest that the structural homology extends well beyond this region. Both proteins have three well-formed helices which fold in a parallel-antiparallel fashion and a prosthetic group attachment site near the beginning of the second helix.

Serine residue 45 of nodulation protein NodF from Rhizobium leguminosarum bv. viciae is essential for its biological function.

A system for testing the role of the Rhizobium nodF gene in the production of host-specific lipochitin oligosaccharides and in nodulation was developed. We show that a mutant nodF gene, in which the codon for serine residue 45 was changed to that for threonine, still expresses NodF, which, however, is no longer functional.

Phospholipids of Rhizobium contain nodE-determined highly unsaturated fatty acid moieties.

In Rhizobium leguminosarum the nodABC and nod-FEL operons are involved in the production of lipooligosaccharide signals, which mediate host specificity. A nodE-determined highly unsaturated C18:4 fatty acid (trans-2,trans-4,trans-6,cis-11-octadecatetraenoic acid) is essential for the ability of the signals to induce nodule primordia (Spaink, H. P., Sheeley, D. M., van Brussel, A. A. N., Glushka, J., York, W.S., Tak, T., Geiger, O., Kennedy, E. P., Reinhold, V. N., and Lugtenberg, B. J. J. (1991) Nature 354, 125-130) and preinfection thread structures (van Brussel, A. A. N., Bakhuizen, R., van Spronsen, P. C., Spaink, H. P., Tak, T., Lugtenberg, B. J. J., and Kijne, J. W. (1992) Science 257, 70-72) on the host plant Vicia sativa. We presently focus on the question of how these lipo-oligosaccharide signals are synthesized in Rhizobium. Here we show that after the induction of the nodFE genes, even in the absence of the nodABC genes, the trans-2,trans-4,trans-6,cis-11-octadecatetraenoic acid, which has a characteristic absorbance maximum of 303 nm, is synthesized; this shows that the biosynthesis of the unusual fatty acid is not dependent on the synthesis of the lipooligosaccharides. This finding also suggests that the biosynthesis of the unusual fatty acid is completed before it is linked to the sugar backbone of the lipooligosaccharide. In an attempt to identify the lipid fraction with which the unusual C18:4 fatty acid is associated, we found that it is linked to the sn-2 position of the phospholipids. Even when lipooligosaccharide signals are produced in a wild type Rhizobium cell, a fraction of the unusual fatty acid is still bound to all major phospholipids. These findings offer interesting possibilities. 1) The phospholipids might be biosynthetic intermediates for the synthesis of lipooligosaccharide signals, and 2) phospholipids, containing nodFE-derived fatty acids, might have a signal function of their own.

Membrane-derived oligosaccharides affect porin osmoregulation only in media of low ionic strength.

Gram-negative bacteria grown under conditions of low osmolarity accumulate significant amounts of periplasmic glucans, membrane-derived oligosaccharides (MDO) in Escherichia coli and cyclic glucans in members of the family Rhizobiaceae. It was reported previously (W. Fiedlder and H. Rotering, J. Biol. Chem. 263:14684-14689, 1988) that mdoA mutants unable to synthesize MDO show a number of altered phenotypes, among them a decreased expression of OmpF and an increased expression of OmpC, when grown in a Bacto Peptone medium of low osmolarity and low ionic strength. Although we confirm the findings of Fiedler and Rotering, we find that the regulation of OmpF and OmpC expression in mdoA mutants is normal in cells grown on other low-osmolarity media, eliminating the possibility that MDO itself might control porin expression. Our data suggest that a certain minimal ionic strength in the periplasm is needed for normal porin regulation. In media containing very low levels of salt, this may be contributed by anionic MDO.

A novel highly unsaturated fatty acid moiety of lipo-oligosaccharide signals determines host specificity of Rhizobium.

In Rhizobium leguminosarum biovar viciae, the nodABC and nodFEL operons are involved in the production of lipo-oligosaccharide signals which mediate host specificity. The structure of these metabolites and those produced in nod mutants links the nodE and nodL genes to specific chemical features of the signal molecules. A nodE-determined, highly unsaturated fatty acid and a nodL-determined O-acetyl substituent are essential for the ability of the signals to induce nodule meristems on the host plant Vicia sativa.