Expression and physiological relevance of Agrobacterium tumefaciens phosphatidylcholine biosynthesis genes

Phosphatidylcholine (PC), or lecithin, is the major phospholipid in eukaryotic membranes, whereas only 10% of all bacteria are predicted to synthesize PC. In Rhizobiaceae, including the phytopathogenic bacterium Agrobacterium tumefaciens, PC is essential for the establishment of a successful host-microbe interaction. A. tumefaciens produces PC via two alternative pathways, the methylation pathway and the Pcs pathway. The responsible genes, pmtA (coding for a phospholipid N-methyltransferase) and pcs (coding for a PC synthase), are located on the circular chromosome of A. tumefaciens C58. Recombinant expression of pmtA and pcs in Escherichia coli revealed that the individual proteins carry out the annotated enzyme functions. Both genes and a putative ABC transporter operon downstream of PC are constitutively expressed in A. tumefaciens. The amount of PC in A. tumefaciens membranes reaches around 23% of total membrane lipids. We show that PC is distributed in both the inner and outer membranes. Loss of PC results in reduced motility and increased biofilm formation, two processes known to be involved in virulence. Our work documents the critical importance of membrane lipid homeostasis for diverse cellular processes in A. tumefaciens.     

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.     

Phosphatidylcholine biosynthesis and function in bacteria

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of the domain Bacteria. Usually, PC can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation (Pmt) pathway or the phosphatidylcholine synthase (Pcs) pathway. The three subsequent enzymatic methylations of phosphatidylethanolamine are performed by a single phospholipid N-methyltransferase in some bacteria whereas other bacteria possess multiple phospholipid N-methyltransferases each one performing one or several distinct methylation steps. Phosphatidylcholine synthase condenses choline directly with CDP-diacylglycerol to form CMP and PC. Like in eukaryotes, bacterial PC also functions as a biosynthetic intermediate during the formation of other biomolecules such as choline, diacylglycerol, or diacylglycerol-based phosphorus-free membrane lipids. Bacterial PC may serve as a specific recognition molecule but it affects the physicochemical properties of bacterial membranes as well. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Membrane‐binding mechanism of a bacterial phospholipid N‐methyltransferase

The membrane lipid phosphatidylcholine (PC) is crucial for stress adaptation and virulence of the plant pathogen Agrobacterium tumefaciens. The phospholipid N‐methyltransferase PmtA catalyzes three successive methylations of phosphatidylethanolamine to yield PC. Here, we asked how PmtA is recruited to its site of action, the inner leaflet of the membrane. We found that the enzyme attaches to the membrane via electrostatic interactions with anionic lipids, which do not serve as substrate for PmtA. Increasing PC concentrations trigger membrane dissociation suggesting that membrane binding of PmtA is negatively regulated by its end product PC. Two predicted alpha‐helical regions (αA and αF) contribute to membrane binding of PmtA. The N‐terminal helix αA binds anionic lipids in vitro with higher affinity than the central helix αF. The latter undergoes a structural transition from disordered to α‐helical conformation in the presence of anionic lipids. The basic amino acids R8 and K12 and the hydrophobic amino acid F19 are critical for membrane binding by αA as well as for activity of full‐length PmtA. We conclude that a combination of electrostatic and hydrophobic forces is responsible for membrane association of the phospholipid‐modifying enzyme.     

Diverse pathways of phosphatidylcholine biosynthesis in algae as estimated by labeling studies and genomic sequence analysis

Phosphatidylcholine (PC) is an almost ubiquitous phospholipid in eukaryotic algae and plants but is not found in a few species, for example Chlamydomonas reinhardtii. We recently found that some species of the genus Chlamydomonas possess PC. In the universal pathway, PC is synthesized de novo by methylation of phosphatidylethanolamine (PE) or transfer of phosphocholine from cytidine diphosphate (CDP)‐choline to diacylglycerol. Phosphocholine, the direct precursor to CDP‐choline, is synthesized either by methylation of phosphoethanolamine or phosphorylation of choline. Here we analyzed the mechanism of PC biosynthesis in two species of Chlamydomonas (asymmetrica and sphaeroides) as well as in a red alga, Cyanidioschyzon merolae. Comparative genomic analysis of enzymes involved in PC biosynthesis indicated that C. merolae possesses only the PE methylation pathway. Radioactive tracer experiments using [³²P]phosphate showed delayed labeling of PC with respect to PE, which was consistent with the PE methylation pathway. In Chlamydomonas asymmetrica, labeling of PC was detected from the early time of incubation with [³²P]phosphate, suggesting the operation of phosphoethanolamine methylation pathway. Genomic analysis indeed detected the genes for the phosphoethanolamine methylation pathway. In contrast, the labeling of PC in C. sphaeroides was slow, suggesting that the PE methylation pathway was at work. These results as well as biochemical and computational results uncover an unexpected diversity of the mechanisms for PC biosynthesis in algae. Based on these results, we will discuss plausible mechanisms for the scattered distribution of the ability to biosynthesize PC in the genus Chlamydomonas.     

Phosphatidylcholine is required for the efficient formation of photosynthetic membrane and B800-850 light-harvesting complex in Rhodobacter sphaeroides

No phosphatidylcholine (PC) was detected in the membrane of Rhodobacter sphaeroides pmtA mutant (PmtA1) lacking phosphatidylethanolamine (PE) N-methyltransferase, whereas PE in the mutant was increased up to the mole % comparable to the combined level of PE and PC of wild type. Neither the fatty acid composition nor the fluidity of membrane was altered by pmtA mutation. Consistently, aerobic and photoheterotrophic growth of PmtA1 were not different from wild type. However, PmtA1 showed an extended lag phase (15 h) after the growth transition from aerobic to photoheterotrophic conditions, indicating the PC requirement for the efficient formation of intracytoplasmic membrane (ICM). Interestingly, the B800-850 complex of PmtA1 was decreased more than twofold in comparison with wild type, whereas the level of the B875 complex comprising the fixed photosynthetic unit was not changed. Since puc expression is not affected by pmtA mutation, PC appears to be required for the proper formation of the B800-850 complex in the ICM of R. sphaeroides.     

S-adenosylmethionine-binding properties of a bacterial phospholipid N-methyltransferase

The presence of the membrane lipid phosphatidylcholine (PC) in the bacterial membrane is critically important for many host-microbe interactions. The phospholipid N-methyltransferase PmtA from the plant pathogen Agrobacterium tumefaciens catalyzes the formation of PC by a three-step methylation of phosphatidylethanolamine via monomethylphosphatidylethanolamine and dimethylphosphatidylethanolamine. The methyl group is provided by S-adenosylmethionine (SAM), which is converted to S-adenosylhomocysteine (SAH) during transmethylation. Despite the biological importance of bacterial phospholipid N-methyltransferases, little is known about amino acids critical for binding to SAM or phospholipids and catalysis. Alanine substitutions in the predicted SAM-binding residues E58, G60, G62, and E84 in A. tumefaciens PmtA dramatically reduced SAM-binding and enzyme activity. Homology modeling of PmtA satisfactorily explained the mutational results. The enzyme is predicted to exhibit a consensus topology of the SAM-binding fold consistent with cofactor interaction as seen with most structurally characterized SAM-methyltransferases. Nuclear magnetic resonance (NMR) titration experiments and (14)C-SAM-binding studies revealed binding constants for SAM and SAH in the low micromolar range. Our study provides first insights into structural features and SAM binding of a bacterial phospholipid N-methyltransferase.     

Virulence of Agrobacterium tumefaciens requires phosphatidylcholine in the bacterial membrane

Phosphatidylcholine (PC, lecithin) has long been considered a solely eukaryotic membrane lipid. Only a minority of all bacteria is able to synthesize PC. The plant‐transforming bacterium Agrobacterium tumefaciens encodes two potential PC forming enzymes, a phospholipid N‐methyltransferase (PmtA) and a PC synthase (Pcs). We show that PC biosynthesis and tumour formation on Kalanchoë plants was impaired in the double mutant. The virulence defect was due to a complete lack of the type IV secretion machinery in the Agrobacterium PC mutant. Our results strongly suggest that PC in bacterial membranes is an important determinant for the establishment of host–microbe interactions.     

Selective expression of light-harvesting complexes alters phospholipid composition in the intracytoplasmic membrane and core complex of purple phototrophic bacteria

Phospholipid–protein interactions play important roles in regulating the function and morphology of photosynthetic membranes in purple phototrophic bacteria. Here, we characterize the phospholipid composition of intracytoplasmic membrane (ICM) from Rhodobacter (Rba.) sphaeroides that has been genetically altered to selectively express light-harvesting (LH) complexes. In the mutant strain (DP2) that lacks a peripheral light-harvesting (LH2) complex, the phospholipid composition was significantly different from that of the wild-type strain; strain DP2 showed a marked decrease in phosphatidylglycerol (PG) and large increases in cardiolipin (CL) and phosphatidylcholine (PC) indicating preferential interactions between the complexes and specific phospholipids. Substitution of the core light-harvesting (LH1) complex of Rba. sphaeroides strain DP2 with that from the purple sulfur bacterium Thermochromatium tepidum further altered the phospholipid composition, with substantial increases in PG and PE and decreases in CL and PC, indicating that the phospholipids incorporated into the ICM depend on the nature of the LH1 complex expressed. Purified LH1–reaction center core complexes (LH1–RC) from the selectively expressing strains also contained different phospholipid compositions than did core complexes from their corresponding wild-type strains, suggesting different patterns of phospholipid association between the selectively expressed LH1–RC complexes and those purified from native strains. Effects of carotenoids on the phospholipid composition were also investigated using carotenoid-suppressed cells and carotenoid-deficient species. The findings are discussed in relation to ICM morphology and specific LH complex–phospholipid interactions.     

Phospholipid turnover and acyl chain remodeling in the yeast ER

The turnover of phospholipids plays an essential role in membrane lipid homeostasis by impacting both lipid head group and acyl chain composition. This review focusses on the degradation and acyl chain remodeling of the major phospholipid classes present in the ER membrane of the reference eukaryote Saccharomyces cerevisiae, i.e. phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Phospholipid turnover reactions are introduced, and the occurrence and important functions of phospholipid remodeling in higher eukaryotes are briefly summarized. After presenting an inventory of established mechanisms of phospholipid acyl chain exchange, current knowledge of phospholipid degradation and remodeling by phospholipases and acyltransferases localized to the yeast ER is summarized. PC is subject to the PC deacylation-reacylation remodeling pathway (PC-DRP) involving a phospholipase B, the recently identified glycerophosphocholine acyltransferase Gpc1p, and the broad specificity acyltransferase Ale1p. PI is post-synthetically enriched in C18:0 acyl chains by remodeling reactions involving Cst26p. PE may undergo turnover by the phospholipid: diacylglycerol acyltransferase Lro1p as first step in acyl chain remodeling. Clues as to the functions of phospholipid acyl chain remodeling are discussed.     

Proteomics reveals differential expression of proteins related to a variety of metabolic pathways by genistein-induced Bradyrhizobium japonicum strains

The rhizobia-legume symbiosis requires a coordinated molecular interaction between the symbionts, initiated by seed and root exudation of several compounds, mainly flavonoids, that trigger the expression of nodulation genes in the bacteria. Since the role of flavonoids seems to be broader than the induction of nodulation genes, we aimed at characterizing genistein-induced proteins of Bradyrhizobium japonicum CPAC 15 (= SEMIA 5079), used in commercial soybean inoculants in Brazil, and of two genetically related strains grown in vitro. Whole-cell proteins were extracted both from induced (1 μM genistein) and from non-induced cultures of the three strains, and separated by two-dimensional electrophoresis. Spot profiles were compared between the two conditions and selected spots were excised and identified by mass spectrometry. Forty-seven proteins were significantly induced by genistein, including several hypothetical proteins, the cytoplasmic flagellar component FliG, periplasmic ABC transporters, a protein related to biosynthesis of exopolysaccharides (ExoN), and proteins involved in redox-state maintenance. Noteworthy was the induction of the PhyR-σ^EcfG regulon, recently demonstrated to be involved in the symbiotic efficiency of, and general stress response in B. japonicum. Our results confirm that the role of flavonoids, such as genistein, can go far beyond the expression of nodulation-related proteins in B. japonicum.     

Phosphatidylethanolamine synthesis is required for optimal virulence of Brucella abortus

The Brucella cell envelope contains the zwitterionic phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Synthesis of PC occurs exclusively via the PC synthase pathway, implying that the pathogen depends on the choline synthesized by the host cell to form PC. Notably, PC is necessary to sustain a chronic infection process, which suggests that the membrane lipid content is relevant for Brucella virulence. In this study we investigated the first step of PE biosynthesis in B. abortus, which is catalyzed by phosphatidylserine synthase (PssA). Disruption of pssA abrogated the synthesis of PE without affecting the growth in rich complex medium. In minimal medium, however, the mutant required choline supplementation for growth, suggesting that at least PE or PC is necessary for Brucella viability. The absence of PE altered cell surface properties, but most importantly, it impaired several virulence traits of B. abortus, such as intracellular survival in both macrophages and HeLa cells, the maturation of the replicative Brucella-containing vacuole, and mouse colonization. These results suggest that membrane phospholipid composition is critical for the interaction of B. abortus with the host cell.     

Effect of phophatidylcholine and phosphatidylethanolamine enrichment on the structure and function of yeast membrane

The phospholipid composition of yeast plasma membrane was manipulated by two different methods: (i) by using two auxotrophic strains KA101 (cho1) and MC13 (Cho⁺) which required phospholipid bases for growth and (ii) by supplementing Saccharomyces cerevisiae (3059) cells with high concentration of choline or ethanolamine. It was possible to enrich the plasma membrane with phosphatidylcholine (PC) or phosphatidylethanolamine (PE) by both methods. The uptake of amino acids, e.g., glycine, glutamic acid, leucine, lysine methionine, phenylalanine, proline and serine, was significantly reduced in PC- or PE-enriched cells. However, the extent of reduction in transport was variable among different strains. A fluorescent probe, 1-anilino-8-naphthalene sulfonate (ANS), was used to monitor the structural changes induced by altered phospholipid composition. It was observed that the relative fluorescence intensity of bound ANS was decreased as a consequence of PC or PE enrichment. The decrease in fluorescence was probably associated with reduced number of available binding sites (n) and increased apparent dissociation constant (K_d). Furthermore, our results also suggest that a critical level of PE or PC is required for proper functioning of yeast membrane.     

Symbiotic Expression of Cosmid-Borne Bradyrhizobium japonicum Hydrogenase Genes

The expression of cosmid-borne Bradyrhizobium japonicum hydrogenase genes in alfalfa, clover, and soybean nodules harboring Rhizobium transconjugants was studied. Cosmid pHU52 conferred hydrogen uptake (Hup) activity in both free-living bacteria and in nodules on the different plant hosts, although in nodules the instability of the cosmid resulted in low levels of Hup activity. In contrast, cosmid pHU1, which does not confer Hup activity on free-living bacteria, gave a Hup⁺ phenotype in nodules on alfalfa and soybean. Nodules formed by B. japonicum USDA 123Spc(pHU1) recycled about 90% of nitrogenase-mediated hydrogen evolution. Both subunits of hydrogenase (30- and 60-kilodalton polypeptides) were detected in enzyme-linked immunosorbent assays of bacteroid preparations from nodules harboring B. japonicum strains with pHU1 or pHU52. Neither pHU53 nor pLAFR1 conferred detectable Hup activity in either nodules or free-living bacteria. Based on the physical maps of pHU1 and pHU52, it is suggested that a 5.5-kilobase EcoRI fragment unique to pHU52 contains a gene or part of a gene required for Hup activity in free-living bacteria but not in nodules. This conclusion is supported by the observation that two Tn5 insertions in the chromosome of B. japonicum USDA 122DES obtained by marker exchange with Tn5-mutagenized pHU1 abolished Hup activity in free-living bacteria but not in nodules.     

Improving Soybean (Glycine max L.) N2 Fixation under Stress

Soybean is a major leguminous plant that has the ability to establish a symbiotic association with the N-fixing bacteria, Bradyrhizobium japonicum. Soils are usually subjected to stress including salinity, drought, acidity, and suboptimal root zone temperature, adversely affecting the symbiotic process between soybean and the bacteria. One of the important processes affecting the performance of soybean under stress is the inhibited exchange of symbiosis-related signaling molecules, specifically genistein, between the host legume and B. japonicum during the initiation of symbiosis. Interestingly, inoculation of B. japonicum with the signal molecule genistein can partially or completely alleviate the stress. Understanding the techniques and the precise molecular pathways, which may be influenced by the signaling molecules during the stress, can be useful to determine parameters that enhance the plant’s ability to cope with stress. For example, the use of proteomic techniques to identify proteins expressed under stress can help characterize those proteins and their involvement in stress. Biotechnological-genetic techniques, either breeding or transformation, are also among the effective methods of improving soybean’s ability to fix N₂ under stress. This can be achieved by identifying the genes, which may be expressed under stress in tolerant bacterial and plant species, and inserting them into the non-tolerant species. This article highlights some important advances in soybean N₂ fixation under different stress conditions, and reviews some of the techniques used to improve the ability of plants and bacteria to efficiently fix N₂ under stress.