Functional and topological analysis of phosphatidylcholine synthase from Sinorhizobium meliloti

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of eubacteria. It can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation pathway or the phosphatidylcholine synthase (Pcs) pathway. Pcs belongs to the CDP-alcohol phosphotransferase superfamily and synthesizes PC and CMP in one step from CDP-diacylglycerol and choline. In this study, we aligned sequences of characterized Pcs enzymes to identify conserved amino acid residues. Alanine scanning mutagenesis was performed on 55 of these conserved residues. The mutation of nine residues caused a drastic to complete loss (<20% of wild type activity) of Pcs activity. Six of these essential residues were subjected to further mutagenesis studies replacing them by amino acids with similar properties or size. A topological analysis of sinorhizobial Pcs showed the presence of eight transmembrane helices, with the C- and N-terminus located in the cytoplasm. The majority of the conserved residues is predicted to be either located within the cytoplasmic loops or on the cytoplasmic side of the membrane which can be expected for an enzyme using one membrane-associated and one soluble substrate.     

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.     

土壤假单胞菌593使用Pcs途径合成磷酯酰胆碱

【目的】原核生物有两条代谢途径N-甲基化途径(Pmt途径)和磷脂酰胆碱合酶途径(Pcs途径)合成磷脂酰胆碱(PC)。本文对土壤细菌Pseudomonas sp.593的磷脂酰胆碱合成进行了研究,试图弄清楚假单胞菌的磷脂酰胆碱的合成途径。【方法】通过氨基酸序列比较,获得已报道的磷脂酰胆碱合酶(Pcs)的氨基酸保守序列,并设计简并引物,从Pseudomonas sp.593总DNA中PCR扩增出磷脂酰胆碱合酶基因(pcs)的片段,然后用扩增的DNA片段作探针,对Pseudomonas sp.593基因组DNA亚克隆文库进行菌落原位杂交,获得pcs全基因序列;利用同源重组原理进行活体突变,获得Pseudomonas sp.593 pcs-突变体;采用薄层层析(TLC)法分析细菌总磷脂,检测PC含量以及pcs基因活性。【结果】TLC分析显示Pseudomonas sp.593细菌仅在添加外源胆碱的M9或LB培养基中生长时才合成PC;从Pseudomonas sp.593细菌中克隆出894 bp的DNA序列,编码的蛋白具有磷脂酰胆碱合酶活性;活体缺失pcs基因后,Pseudomonas sp.593 pcs-突变体在添加或不添加胆碱的条件下都不能合成PC。【结论】Pcs途径是土壤Pseudomonas sp.593乃至其它假单胞菌合成磷脂酰胆碱的唯一途径。     

Phosphatidylcholine synthesis in adult Dirofilaria immitis females

Crude homogenates of adult Dirofilaria immitis females were able to incorporate choline into phosphatidylcholine (PC) and also were able to methylate phosphatidyl (N,N-dimethyl)-ethanolamine, using S-adenosylmethionine as the methyl donor, to form PC. The finding of choline phosphotransferase (EC 2·7·8·2) and phosphatidyl (N,N-dimethyl) ethanolamine methyltransferase activity in the paniculate (mainly microsomal) fraction of the homogenates provided further evidence that adult D. immitis females can synthesize PC by way of choline and cytidine 5'-diphosphocholine (Kennedy pathway) and also by way of S-adenosylmethionine-mediated sequential methylation of phosphatidylethanolamine (Bremer-Greenberg pathway).     

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.     

The human LL-37 peptide exerts antimicrobial activity against Legionella micdadei interacting with membrane phospholipids

Legionella micdadei is responsible for community- or nosocomial-acquired pneumonia as well as the influenza-like illness Pontiac fever. The aim of this study was to investigate the ability of L. micdadei to utilize extracellular choline for phosphatidylcholine (PC) synthesis and its consequences for the phospholipid composition of its membrane system and the interaction with the human LL-37 peptide. Comparative analysis of the PC content using isotopic labeling revealed that in presence of exogenous choline 98% of the total PC was synthesized via the Pcs pathway while the remaining 2% were generated via the PE-methylation (PmtA) pathway. PC species were to a greater extent defined by the Pcs pathway in the outer membrane than in the inner membrane. While no major changes in the bacterial lipid content were observed using ³¹P NMR, indication for utilization of longer acyl chains and slight increase of PG in response to choline addition was observed by a top-down lipidomics screen. The LL-37 peptide inhibited L. micdadei growth in a dose-dependent manner. Bacteria cultured with exogenous choline were more sensitive to the LL-37 peptide when compared to the standard culture condition. Our biophysical investigations show that the peptide perturbs bacterial-derived phospholipid monolayers and this interaction is dependent on the molar portion of PC. This interaction is responsible for the observed changes in the anti-L. micdadei activity of the LL-37 peptide.     

Genetic analysis of the cytoplasmic domain of the PfRh2b merozoite invasion protein of Plasmodium falciparum

Apicomplexan parasites employ multiple adhesive ligands for recognition and entry into host cells. The Duffy binding-like (DBL) and the reticulocyte binding protein-like (RBL) families are central to the invasion of erythrocytes by the malaria parasite. These type-1 transmembrane proteins are composed of large ectodomains and small conserved cytoplasmic tail domains. The cytoplasmic tail domain of the micronemal DBL protein EBA-175 is required for a functional ligand-receptor interaction, but not for correct trafficking and localisation. Here we focus on the cytoplasmic tail domain of the rhoptry-localised Plasmodium falciparum RBL PfRh2b. We have identified a conserved sequence of six amino acids, enriched in acidic residues, in the cytoplasmic tail domains of RBL proteins from Plasmodium spp. Genetic analyses reveal that the entire cytoplasmic tail and the conserved motif within the cytoplasmic tail are indispensable for invasion P. falciparum. Site-directed mutagenesis of the conserved moiety reveals that changes in the order of the amino acids of the conserved moiety, but not the charge of the sequence, can be tolerated. Shuffling of the motif has no effect on either invasion phenotype or PfRh2b expression and trafficking. Although the PfRh2b gene can be readily disrupted, our results suggest that modification of the PfRh2b cytoplasmic tail results in strong dominant negative activity, highlighting important differences between the PfRh2b and EBA-175 invasion ligands.     

Nucleotide-Specific Recognition of Iron-Responsive Elements by Iron Regulatory Protein 1

IRP1 [iron regulatory protein (IRP) 1] is a bifunctional protein with mutually exclusive end-states. In one mode of operation, IRP1 binds iron-responsive element (IRE) stem–loops in messenger RNAs encoding proteins of iron metabolism to control their rate of translation. In its other mode, IRP1 serves as cytoplasmic aconitase to correlate iron availability with the energy and oxidative stress status of the cell. IRP1/IRE binding occurs through two separate interfaces, which together contribute about two-dozen hydrogen bonds. Five amino acids make base-specific contacts and are expected to contribute significantly to binding affinity and specificity of this protein:RNA interaction. In this mutagenesis study, each of the five base-specific amino acids was changed to alter binding at each site. Analysis of IRE binding affinity and translational repression activity of the resulting IRP1 mutants showed that four of the five contact points contribute uniquely to the overall binding affinity of the IRP1:IRE interaction, while one site was found to be unimportant. The stronger-than-expected effect on binding affinity of mutations at Lys379 and Ser681, residues that make contact with the conserved nucleotides G16 and C8, respectively, identified them as particularly critical for providing specificity and stability to IRP1:IRE complex formation. We also show that even though the base-specific RNA-binding residues are not part of the aconitase active site, their substitutions can affect the aconitase activity of holo-IRP1, positively or negatively.     

Topology and active site of PlsY: the bacterial acylphosphate:glycerol-3-phosphate acyltransferase

The most widely distributed biosynthetic pathway to initiate phosphatidic acid formation in bacterial membrane phospholipid biosynthesis involves the conversion of acyl-acyl carrier protein to acylphosphate by PlsX and the transfer of the acyl group from acylphosphate to glycerol 3-phosphate by an integral membrane protein, PlsY. The membrane topology of Streptococcus pneumoniae PlsY was determined using the substituted cysteine accessibility method. PlsY has five membrane-spanning segments with the amino terminus and two short loops located on the external face of the membrane. Each of the three larger cytoplasmic domains contains a highly conserved sequence motif. Site-directed mutagenesis revealed that each conserved domain was critical for PlsY catalysis. Motif 1 had an essential serine and arginine residue. Motif 2 had the characteristics of a phosphate-binding loop. Mutations of the conserved glycines in motif 2 to alanines resulted in a Km defect for glycerol 3-phosphate binding leading to the conclusion that this motif corresponded to the glycerol 3-phosphate binding site. Motif 3 contained a conserved histidine and asparagine that were important for activity and a glutamate that was critical to the structural integrity of PlsY. PlsY was noncompetitively inhibited by palmitoyl-CoA. These data define the membrane architecture and the critical active site residues in the PlsY family of bacterial acyltransferases.     

Pseudomonas aeruginosa synthesizes phosphatidylcholine by use of the phosphatidylcholine synthase pathway

Phosphatidylcholine (PC) is a ubiquitous membrane lipid in eukaryotes but has been found in only a limited number of prokaryotes. Both eukaryotes and prokaryotes synthesize PC by methylating phosphatidylethanolamine (PE) by use of a phospholipid methyltransferase (Pmt). Eukaryotes can synthesize PC by the activation of choline to form choline phosphate and then CDP-choline. The CDP-choline then condenses with diacylglycerol (DAG) to form PC. In contrast, prokaryotes condense choline directly with CDP-DAG by use of the enzyme PC synthase (Pcs). PmtA was the first enzyme identified in prokaryotes that catalyzes the synthesis of PC, and Pcs in Sinorhizobium meliloti was characterized. The completed release of the Pseudomonas aeruginosa PAO1 genomic sequence contains on open reading frame predicted to encode a protein that is highly homologous (35% identity, 54% similarity) to PmtA from Rhodobacter sphaeroides. Moreover, the P. aeruginosa PAO1 genome encodes a protein with significant homology (39% amino acid identity) to Pcs of S. meliloti. Both the pcs and pmtA homologues were cloned from PAO1, and homologous sequences were found in almost all of the P. aeruginosa strains examined. Although the pathway for synthesizing PC by use of Pcs is functional in P. aeruginosa, it does not appear that this organism uses the PmtA pathway for PC synthesis. We demonstrate that the PC synthesized by P. aeruginosa PAO1 localized to both the inner and outer membranes, where it is readily accessible to its periplasmic, PC-specific phospholipase D.     

CLC Anion Channel Regulatory Phosphorylation and Conserved Signal Transduction Domains

The signaling mechanisms that regulate CLC anion channels are poorly understood. Caenorhabditis elegans CLH-3b is a member of the CLC-1/2/Ka/Kb channel subfamily. CLH-3b is activated by meiotic cell-cycle progression and cell swelling. Inhibition is brought about by GCK-3 kinase-mediated phosphorylation of S742 and S747 located on a ∼176 amino acid disordered domain linking CBS1 and CBS2. Much of the inter-CBS linker is dispensable for channel regulation. However, deletion of a 14 amino acid activation domain encompassing S742 and S747 inhibits channel activity to the same extent as GCK-3. The crystal structure of CmCLC demonstrated that CBS2 interfaces extensively with an intracellular loop connecting membrane helices H and I, the C-terminus of helix D, and a short linker connecting helix R to CBS1. Point mutagenesis of this interface identified two highly conserved aromatic amino acid residues located in the H-I loop and the first α-helix (α1) of CBS2. Mutation of either residue to alanine rendered CLH-3b insensitive to GCK-3 inhibition. We suggest that the dephosphorylated activation domain normally interacts with CBS1 and/or CBS2, and that conformational information associated with this interaction is transduced through a conserved signal transduction module comprising the H-I loop and CBS2 α1.     

Regulation of lipid droplet size and phospholipid composition by stearoyl-CoA desaturase

Fatty acid desaturation regulates membrane function and fat storage in animals. To determine the contribution of stearoyl-CoA desaturase (SCD) activity on fat storage and development in the nematode Caenorhabditis elegans, we analyzed the lipid composition and lipid droplet size in the fat-6;fat-7 desaturase mutants independently and in combination with mutants disrupted in conserved lipid metabolic pathways. C. elegans with impaired SCD activity displayed both reduced fat stores and decreased lipid droplet size. Mutants in the daf-2 (insulin-like growth factor receptor), rsks-1 (homolog of p70S6kinase, an effector of the target of rapamycin signaling pathway), and daf-7 (transforming growth factor β) displayed high fat stores, the opposite of the low fat observed in the fat-6;fat-7 desaturase mutants. The metabolic mutants in combination with fat-6;fat-7 displayed low fat stores, with the exception of the daf-2;fat-6;fat-7 triple mutants, which had increased de novo fatty acid synthesis and wild-type levels of fat stores. Notably, SCD activity is required for the formation of large-sized lipid droplets in all mutant backgrounds, as well as for normal ratios of phosphatidylcholine (PC) to phosphatidylethanolamine (PE). These studies reveal previously uncharacterized roles for SCD in the regulation of lipid droplet size and membrane phospholipid composition.     

Fluorinated Aromatic Amino Acids Distinguish Cation-π Interactions from Membrane Insertion

Cation-π interactions, where protein aromatic residues supply π systems while a positive-charged portion of phospholipid head groups are the cations, have been suggested as important binding modes for peripheral membrane proteins. However, aromatic amino acids can also insert into membranes and hydrophobically interact with lipid tails. Heretofore there has been no facile way to differentiate these two types of interactions. We show that specific incorporation of fluorinated amino acids into proteins can experimentally distinguish cation-π interactions from membrane insertion of the aromatic side chains. Fluorinated aromatic amino acids destabilize the cation-π interactions by altering electrostatics of the aromatic ring, whereas their increased hydrophobicity enhances membrane insertion. Incorporation of pentafluorophenylalanine or difluorotyrosine into a Staphylococcus aureus phosphatidylinositol-specific phospholipase C variant engineered to contain a specific PC-binding site demonstrates the effectiveness of this methodology. Applying this methodology to the plethora of tyrosine residues in Bacillus thuringiensis phosphatidylinositol-specific phospholipase C definitively identifies those involved in cation-π interactions with phosphatidylcholine. This powerful method can easily be used to determine the roles of aromatic residues in other peripheral membrane proteins and in integral membrane proteins.     

Site-directed mutagenesis reveals roles for conserved amino acid residues in the hexameric DNA helicase DnaB from Bacillus stearothermophilus

Site-directed mutagenesis studies on conserved amino acid residues within motifs H1, H1a, H2 and H3 of the hexameric replicative helicase DnaB from Bacillus stearothermophilus revealed specific functions associated with these residues. In particular, residues that coordinate a bound Mg²⁺ in the active site (T217 and D320) are important for the function of the enzyme but are not required for the formation of stable hexamers. A conserved glutamic acid (E241) in motif H1a is likely to be involved in the activation of a water molecule for in line attack on the γ-phosphate of the bound nucleotide during catalysis. A conserved glutamine (Q362) in motif H3 acts as a γ-phosphate sensor and mediates the conformational coupling of nucleotide- and DNA-binding sites. The nature of the residue at this position is also important for the primase-mediated activation of DnaB, suggesting that primase uses the same conformational coupling pathway to induce its stimulatory effect on the activity of DnaB. Together, these mutations reveal a conservation of many aspects of biochemical activity in the active sites of monomeric and hexameric helicases.     

A pair of membrane-embedded acidic residues in the NuoK subunit of Escherichia coli NDH-1, a counterpart of the ND4L subunit of the mitochondrial complex I, are required for high ubiquinone reductase activity

The ND4L subunit of mitochondrial NADH:ubiquinone oxidoreductase (complex I) is an integral membrane protein that contains two highly conserved glutamates within putative trans-membrane helices. We employed complex I from Escherichia coli (NDH-1) to study the role of these residues by site-directed mutagenesis. The conserved glutamates of the NuoK subunit, E36 and E72, were replaced by either Asp or Gln residues, and the effects of the mutations on cell growth and catalysis of electron transfer from deamino-NADH to ubiquinone analogues were examined. Additional mutants that carried acidic residues at selected positions within this domain were also prepared and analyzed. The results indicated that two closely located membrane-embedded acidic residues in NuoK are essential for high rates of ubiquinone reduction, a prerequisite for the growth of cytochrome bo-deficient E. coli cells on malate as the main carbon source. The two acidic residues do not have to be on adjacent helices, and mutual location on the same helix, either helix 2 or 3, at an interval of three amino acids (about one turn of the putative helix), resulted in high activity and good growth phenotypes. Nevertheless, shifting only one of them, either E36 or E72, toward the periplasmic side of the membrane by about one turn of the helix severely hampered activity and growth, whereas moving both acidic residues together to that deeper membrane position stimulated the ubiquinone reductase activity of the enzyme but not cell growth on malate, suggesting impaired energy conservation in this mutant.     

Divergent signature motifs of nucleotide binding domains of ABC multidrug transporter, CaCdr1p of pathogenic Candida albicans, are functionally asymmetric and noninterchangeable

Nucleotide binding domains (NBDs) of the multidrug transporter of Candida albicans, CaCdr1p, possess unique divergent amino acids in their conserved motifs. For example, NBD1 (N-terminal-NBD) possesses conserved signature motifs, while the same motif is divergent in NBD2 (C-terminal-NBD). In this study, we have evaluated the contribution of these conserved and divergent signature motifs of CaCdr1p in ATP catalysis and drug transport. By employing site-directed mutagenesis, we made three categories of mutant variants. These included mutants where all the signature motif residues were replaced with either alanines or mutants with exchanged equipositional residues to mimic the conservancy and degeneracy in opposite domain. In addition, a set of mutants where signature motifs were swapped to have variants with either both the conserved or degenerated entire signature motif. We observed that conserved and equipositional residues of NBD1 and NBD2 and swapped signature motif mutants showed high susceptibility to all the tested drugs with simultaneous abrogation in ATPase and R6G efflux activities. However, some of the mutants displayed a selective increase in susceptibility to the drugs. Notably, none of the mutant variants and WT-CaCdr1p showed any difference in drug and nucleotide binding. Our mutational analyses show not only that certain conserved residues of NBD1 signature sequence (S304, G306, and E307) are important in ATP hydrolysis and R6G efflux but also that a few divergent residues (N1002 and E1004) of NBD2 signature motif have evolved to be functionally relevant and are not interchangeable. Taken together, our data suggest that the signature motifs of CaCdr1p, whether it is divergent or conserved, are nonexchangeable and are functionally critical for ATP hydrolysis.     

Transmembrane Segment II of NhaA Na+/H+ Antiporter Lines the Cation Passage, and Asp65 Is Critical for pH Activation of the Antiporter

The crystal structure of Escherichia coli NhaA determined at pH 4 has provided insights into the mechanism of activity of a pH-regulated Na⁺/H⁺ antiporter. However, because NhaA is activated at physiological pH (pH 5.5–8.5), many questions related to the active state of NhaA have remained elusive. Our experimental results at physiological pH and computational analyses reveal that amino acid residues in transmembrane segment II contribute to the cation pathway of NhaA and its pH regulation: 1) transmembrane segment II is a highly conserved helix and the conserved amino acid residues are located on one side of the helix facing either the cytoplasmic or periplasmic funnels of NhaA structure. 2) Cys replacements of the conserved residues and measuring their antiporter activity in everted membrane vesicles showed that D65C, L67C, E78C, and E82C increased the apparent K_m to Na⁺ and Li⁺ and changed the pH response of the antiporter. 3) Introduced Cys replacements, L60C, N64C, F71C, F72C, and E78C, were significantly alkylated by [¹⁴C]N-ethylmaleimide implying the presence of water-filled cavities in NhaA. 4) Several Cys replacements were modified by MTSES and/or MTSET, membrane impermeant, negatively and positively charged reagents, respectively, that could reach Cys replacements from the periplasm only via water-filled funnel(s). Remarkably, the reactivity of D65C to MTSES increased with increasing pH and chemical modification by MTSES but not by MTSET, decreased the apparent K_m of the antiporter at pH 7.5 (10-fold) but not at pH 8.5, implying the importance of Asp⁶⁵ negative charge for pH activation of the antiporter.     

Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. This study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.     

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.     

The Platform Protein Is Essential for Type IV Pilus Biogenesis

A systematic genetic analysis was performed to identify the inner membrane proteins essential for type IV pilus (T4P) expression in Pseudomonas aeruginosa. By inactivating the retraction aspect of pilus function, genes essential for T4P assembly were discriminated. In contrast to previous studies in the T4P system of Neisseria spp., we found that components of the inner membrane subcomplex consisting of PilMNOP were not essential for surface pilus expression, whereas the highly conserved inner membrane protein PilC was essential. Here, we present data that PilC may coordinate the activity of cytoplasmic polymerization (PilB) and depolymerization (PilT) ATPases via their interactions with its two cytoplasmic domains. Using in vitro co-affinity purification, we show that PilB interacts with the N-terminal cytoplasmic domain of PilC. We hypothesized that PilT similarly interacts with the PilC C-terminal cytoplasmic domain. Overexpression of that domain in the wild-type protein reduced twitching motility by ∼50% compared with the vector control. Site-directed mutagenesis of conserved T4P-specific residues in the PilC C-terminal domain yielded mutant proteins that supported wild-type pilus assembly but had a reduced capacity to support twitching motility, suggesting impairment of putative PilC-PilT interactions. Taken together, our results show that PilC is an essential inner membrane component of the T4P system, controlling both pilus assembly and disassembly.