Enhancing Escherichia coli abiotic stress resistance through ornithine lipid formation.

Escherichia coli is a common host for biotechnology and synthetic biology applications. During growth and fermentation, the microbes are often exposed to stress conditions, such as variations in pH or solvent concentrations. Bacterial membranes play a key role in response to abiotic stresses. Ornithine lipids (OLs) are a group of membrane lipids whose presence and synthesis have been related to stress resistance in bacteria. We wondered if this stress resistance could be transferred to bacteria not encoding the capacity to form OLs in their genome, such as E. coli. In this study, we engineered different E. coli strains to produce unmodified OLs and hydroxylated OLs by expressing the synthetic operon olsFC. Our results showed that OL formation improved pH resistance and increased biomass under phosphate limitation. Transcriptome analysis revealed that OL-forming strains differentially expressed stress- and membrane-related genes. OL-producing strains also showed better growth in the presence of the ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP), suggesting reduced proton leakiness in OL-producing strains. Furthermore, our engineered strains showed improved heterologous violacein production at phosphate limitation and also at low pH. Overall, this study demonstrates the potential of engineering the E. coli membrane composition for constructing robust hosts with an increased abiotic stress resistance for biotechnology and synthetic biology applications. KEY POINTS: • Ornithine lipid production in E. coli increases biomass yield under phosphate limitation. • Engineered strains show an enhanced production phenotype under low pH stress. • Transcriptome analysis and CCCP experiments revealed reduced proton leakage.

Crossing the lipid divide.

Archaeal membrane lipids are structurally different from bacterial and eukaryotic membrane lipids, but little is known about the enzymes involved in their synthesis. In a recent study, Exterkate et al. identified and characterized a cardiolipin synthase from the archaeon Methanospirillum hungatei. This enzyme can synthesize archaeal, bacterial, and mixed archaeal/bacterial cardiolipin species from a wide variety of substrates, some of which are not even naturally occurring. This discovery could revolutionize synthetic lipid biology, being used to construct a variety of lipids with nonnatural head groups and mixed archaeal/bacterial hydrophobic chains.

Identification of the Flavobacterium johnsoniae cysteate-fatty acyl transferase required for capnine synthesis and for efficient gliding motility.

Sulfonolipids (SLs) are bacterial lipids that are structurally related to sphingolipids. Synthesis of this group of lipids seems to be mainly restricted to Flavobacterium, Cytophaga and other members of the phylum Bacteroidetes. These lipids have a wide range of biological activities: they can induce multicellularity in choanoflagellates, act as von Willebrand factor receptor antagonists, inhibit DNA polymerase, or function as tumour suppressing agents. In Flavobacterium johnsoniae, their presence seems to be required for efficient gliding motility. Until now, no genes/enzymes involved in SL synthesis have been identified, which has been limiting for the study of some of the biological effects these lipids have. Here, we describe the identification of the cysteate-fatty acyl transferase Fjoh_2419 required for synthesis of the SL precursor capnine in F. johnsoniae. This enzyme belongs to the α-oxoamine synthase family similar to serine palmitoyl transferases, 2-amino-3-oxobutyrate coenzyme A ligase and 8-amino-7-oxononanoate synthases. Expression of the gene fjoh_2419 in Escherichia coli caused the formation of a capnine-derived molecule. Flavobacterium johnsoniae mutants deficient in fjoh_2419 lacked SLs and were more sensitive to many antibiotics. Mutant growth was not affected in liquid medium but the cells exhibited defects in gliding motility.

Bacillus toyonensis COPE52 Modifies Lipid and Fatty Acid Composition, Exhibits Antifungal Activity, and Stimulates Growth of Tomato Plants Under Saline Conditions.

Salinity is one of the most important factors that limit the productivity of agricultural soils. Certain plant growth-promoting bacteria (PGPB) have the ability to stimulate the growth of crop plants even under salt stress. In the present study, we analysed the potential of PGPB Bacillus toyonensis COPE52 to improve the growth of tomato plants and its capacity to modify its membrane lipid and fatty acid composition under salt stress. Thus, strain COPE52 increased the relative amount of branched chain fatty acids (15:0i and 16:1∆9) and accumulation of an unknown membrane lipid, while phosphatidylethanolamine (PE) levels decreased during growth with 100 and 200 mM NaCl. Importantly, direct and indirect plant growth-promoting (PGP) mechanisms of B. toyonensis COPE52, such as indole-3-acetic acid (IAA), protease activity, biofilm formation, and antifungal activity against Botrytis cinerea, remained unchanged in the presence of NaCl in vitro, compared to controls without salt. In a greenhouse experiment, tomato plants (Lycopersicon esculentum 'Saladette') showed increased shoot and root length, higher dry biomass, and chlorophyll content when inoculated with B. toyonensis COPE52 at 0 and 100 mM NaCl. In summary, these results indicate that Bacillus toyonensis COPE52 can modify cell membrane lipid components as a potential protecting mechanism to maintain PGP traits under saline-soil conditions.

1,2-Diacylglycerol choline phosphotransferase catalyzes the final step in the unique Treponema denticola phosphatidylcholine biosynthesis pathway.

Treponema denticola synthesizes phosphatidylcholine through a licCA-dependent CDP-choline pathway identified only in the genus Treponema. However, the mechanism of conversion of CDP-choline to phosphatidylcholine remained unclear. We report here characterization of TDE0021 (herein designated cpt) encoding a 1,2-diacylglycerol choline phosphotransferase homologous to choline phosphotransferases that catalyze the final step of the highly conserved Kennedy pathway for phosphatidylcholine synthesis in eukaryotes. T. denticola Cpt catalyzed in vitro phosphatidylcholine formation from CDP-choline and diacylglycerol, and full activity required divalent manganese. Allelic replacement mutagenesis of cpt in T. denticola resulted in abrogation of phosphatidylcholine synthesis. T. denticola Cpt complemented a Saccharomyces cerevisiae CPT1 mutant, and expression of the entire T. denticola LicCA-Cpt pathway in E. coli resulted in phosphatidylcholine biosynthesis. Our findings show that T. denticola possesses a unique phosphatidylcholine synthesis pathway combining conserved prokaryotic choline kinase and CTP:phosphocholine cytidylyltransferase activities with a 1,2-diacylglycerol choline phosphotransferase that is common in eukaryotes. Other than in a subset of mammalian host-associated Treponema that includes T. pallidum, this pathway is found in neither bacteria nor Archaea. Molecular dating analysis of the Cpt gene family suggests that a horizontal gene transfer event introduced this gene into an ancestral Treponema well after its divergence from other spirochetes.

OlsG (Sinac_1600) Is an Ornithine Lipid N-Methyltransferase from the Planctomycete Singulisphaera acidiphila.

Ornithine lipids (OLs) are phosphorus-free membrane lipids widespread in bacteria but absent from archaea and eukaryotes. In addition to the unmodified OLs, a variety of OL derivatives hydroxylated in different structural positions has been reported. Recently, methylated derivatives of OLs were described in several planctomycetes isolated from a peat bog in Northern Russia, although the gene/enzyme responsible for the N-methylation of OL remained obscure. Here we identify and characterize the OL N-methyltransferase OlsG (Sinac_1600) from the planctomycete Singulisphaera acidiphila. When OlsG is co-expressed with the OL synthase OlsF in Escherichia coli, methylated OL derivatives are formed. An in vitro characterization shows that OlsG is responsible for the 3-fold methylation of the terminal δ-nitrogen of OL. Methylation is dependent on the presence of the detergent Triton X-100 and the methyldonor S-adenosylmethionine.

A bifunctional glycosyltransferase from Agrobacterium tumefaciens synthesizes monoglucosyl and glucuronosyl diacylglycerol under phosphate deprivation.

Glycolipids are mainly found in phototrophic organisms (like plants and cyanobacteria), in Gram-positive bacteria, and a few other bacterial phyla. Besides the function as bulk membrane lipids, they often play a role under phosphate deprivation as surrogates for phospholipids. The Gram-negative Agrobacterium tumefaciens accumulates four different glycolipids under phosphate deficiency, including digalactosyl diacylglycerol and glucosylgalactosyl diacylglycerol synthesized by a processive glycosyltransferase. The other two glycolipids have now been identified by mass spectrometry and nuclear magnetic resonance spectroscopy as monoglucosyl diacylglycerol and glucuronosyl diacylglycerol. These two lipids are synthesized by a single promiscuous glycosyltransferase encoded by the ORF atu2297, with UDP-glucose or UDP-glucuronic acid as sugar donors. The transfer of sugars differing in their chemistry is a novel feature not observed before for lipid glycosyltransferases. Furthermore, this enzyme is the first glucuronosyl diacylglycerol synthase isolated. Deletion mutants of Agrobacterium lacking monoglucosyl diacylglycerol and glucuronosyl diacylglycerol or all glycolipids are not impaired in growth or virulence during infection of tobacco leaf discs. Our data suggest that the four glycolipids and the nonphospholipid diacylglyceryl trimethylhomoserine can mutually replace each other during phosphate deprivation. This redundancy of different nonphospholipids may represent an adaptation mechanism to enhance the competitiveness in nature.

Discovery of a bifunctional acyltransferase responsible for ornithine lipid synthesis in Serratia proteamaculans.

Ornithine lipids (OLs) are phosphorus-free membrane lipids that can be formed by many bacteria but that are absent from archaea and eukaryotes. A function for OLs in stress conditions and in host-bacteria interactions has been shown in some bacteria. Some bacterial species have been described that can form OLs, but lack the known genes (olsBA) involved in its biosynthesis, which implied the existence of a second pathway. Here we describe the bifunctional protein OlsF from Serratia proteamaculans involved in OL formation. Expression of OlsF and its homologue from Flavobacterium johnsoniae in Escherichia coli causes OL formation. Deletion of OlsF in S. proteamaculans caused the absence of OL formation. Homologues of OlsF are widely distributed among γ-, δ- and ε-Proteobacteria and in the Cytophaga-Flavobacterium-Bacteroidetes group of bacteria, including several well-studied pathogens for which the presence of OLs has not been suspected, such as for example Vibrio cholerae and Klebsiella pneumonia. Using genomic data, we predict that about 50% of bacterial species can form OLs.

Fatty acid-releasing activities in Sinorhizobium meliloti include unusual diacylglycerol lipase.

Phospholipids are well known for their membrane-forming properties and thereby delimit any cell from the exterior world. In addition, membrane phospholipids can act as precursors for signals and other biomolecules during their turnover. Little is known about phospholipid signalling, turnover and remodelling in bacteria. Recently, we showed that a FadD-deficient mutant of Sinorhizobium meliloti, unable to convert free fatty acids to their coenzyme A derivatives, accumulates free fatty acids during the stationary phase of growth. Enzymatic activities responsible for the generation of these free fatty acids were unknown in rhizobia. Searching the genome of S. meliloti, we identified a potential lysophospholipase (SMc04041) and two predicted patatin-like phospholipases A (SMc00930, SMc01003). Although SMc00930 as well as SMc01003 contribute to the release of free fatty acids in S. meliloti, neither one can use phospholipids as substrates. Here we show that SMc01003 converts diacylglycerol to monoacylglycerol and a fatty acid, and that monoacylglycerol can be further degraded by SMc01003 to another fatty acid and glycerol. A SMc01003-deficient mutant of S. meliloti transiently accumulates diacylglycerol, suggesting that SMc01003 also acts as diacylglycerol lipase (DglA) in its native background. Expression of the DglA lipase in Escherichia coli causes lysis of cells in stationary phase of growth.

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.

The calcium-stimulated lipid A 3-O deacylase from Rhizobium etli is not essential for plant nodulation.

The lipid A component of lipopolysaccharide from the nitrogen-fixing plant endosymbiont, Rhizobium etli, is structurally very different from that found in most enteric bacteria. The lipid A from free-living R. etli is structurally heterogeneous and exists as a mixture of species which are either pentaacylated or tetraacylated. In contrast, the lipid A from R. etli bacteroids is reported to consist exclusively of tetraacylated lipid A species. The tetraacylated lipid A species in both cases lack a beta-hydroxymyristoyl chain at the 3-position of lipid A. Here, we show that the lipid A modification enzyme responsible for 3-O deacylation in R. etli is a homolog of the PagL protein originally described in Salmonella enterica sv. typhimurium. In contrast to the PagL proteins described from other species, R. etli PagL displays a calcium dependency. To determine the importance of the lipid A modification catalyzed by PagL, we isolated and characterized a R. etli mutant deficient in the pagL gene. Mass spectrometric analysis confirmed that the mutant strain was exclusively tetraacylated and radiochemical analysis revealed that 3-O deacylase activity was absent in membranes prepared from the mutant. The R. etli mutant was not impaired in its ability to form nitrogen-fixing nodules on Phaseolus vulgaris but it displayed slower nodulation kinetics relative to the wild-type strain. The lipid A modification catalyzed by R. etli PagL, therefore, is not required for nodulation but may play other roles such as protecting bacterial endosymbionts from plant immune responses during infection.

Characterization of IntA, a bidirectional site-specific recombinase required for conjugative transfer of the symbiotic plasmid of Rhizobium etli CFN42.

Site-specific recombination occurs at short specific sequences, mediated by the cognate recombinases. IntA is a recombinase from Rhizobium etli CFN42 and belongs to the tyrosine recombinase family. It allows cointegration of plasmid p42a and the symbiotic plasmid via site-specific recombination between attachment regions (attA and attD) located in each replicon. Cointegration is needed for conjugative transfer of the symbiotic plasmid. To characterize this system, two plasmids harboring the corresponding attachment sites and intA were constructed. Introduction of these plasmids into R. etli revealed IntA-dependent recombination events occurring at high frequency. Interestingly, IntA promotes not only integration, but also excision events, albeit at a lower frequency. Thus, R. etli IntA appears to be a bidirectional recombinase. IntA was purified and used to set up electrophoretic mobility shift assays with linear fragments containing attA and attD. IntA-dependent retarded complexes were observed only with fragments containing either attA or attD. Specific retarded complexes, as well as normal in vivo recombination abilities, were seen even in derivatives harboring only a minimal attachment region (comprising the 5-bp central region flanked by 9- to 11-bp inverted repeats). DNase I-footprinting assays with IntA revealed specific protection of these zones. Mutations that disrupt the integrity of the 9- to 11-bp inverted repeats abolish both specific binding and recombination ability, while mutations in the 5-bp central region severely reduce both binding and recombination. These results show that IntA is a bidirectional recombinase that binds to att regions without requiring neighboring sequences as enhancers of recombination.

The calcium-stimulated lipid A 3-O deacylase from Rhizobium etli is not essential for plant nodulation.

The lipid A component of lipopolysaccharide from the nitrogen-fixing plant endosymbiont, Rhizobium etli, is structurally very different from that found in most enteric bacteria. The lipid A from free-living R. etli is structurally heterogeneous and exists as a mixture of species which are either pentaacylated or tetraacylated. In contrast, the lipid A from R. etli bacteroids is reported to consist exclusively of tetraacylated lipid A species. The tetraacylated lipid A species in both cases lack a ß-hydroxymyristoyl chain at the 3-position of lipid A. Here, we show that the lipid A modification enzyme responsible for 3-O deacylation in R. etli is a homolog of the PagL protein originally described in Salmonella enterica sv. typhimurium. In contrast to the PagL proteins described from other species, R. etli PagL displays a calcium dependency. To determine the importance of the lipid A modification catalyzed by PagL, we isolated and characterized a R. etli mutant deficient in the pagL gene. Mass spectrometric analysis confirmed that the mutant strain was exclusively tetraacylated and radiochemical analysis revealed that 3-O deacylase activity was absent in membranes prepared from the mutant. The R. etli mutant was not impaired in its ability to form nitrogen-fixing nodules on Phaseolus vulgaris but it displayed slower nodulation kinetics relative to the wild-type strain. The lipid A modification catalyzed by R. etli PagL, therefore, is not required for nodulation but may play other roles such as protecting bacterial endosymbionts from plant immune responses during infection.

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.

Agrobacteria lacking ornithine lipids induce more rapid tumour formation.

Ornithine lipids (OLs) are phosphorus-free membrane lipids that are widespread among Gram-negative bacteria. Their basic structure consists of a 3-hydroxy fatty acyl group attached in amide linkage to the α-amino group of ornithine and a second fatty acyl group ester-linked to the 3-hydroxy position of the first fatty acid. It has been shown that OLs can be hydroxylated within the amide-linked fatty acyl moiety, the secondary fatty acyl moiety or within the ornithine moiety. These modifications have been related to increased stress tolerance and symbiotic proficiency in different organisms such as Rhizobium tropici or Burkholderia cenocepacia. Analysing the membrane lipid composition of the plant pathogen Agrobacterium tumefaciens we noticed that it forms two different OLs. In the present work we studied if OLs play a role in stress tolerance and pathogenicity in A. tumefaciens. Mutants deficient in the OLs biosynthesis genes olsB or olsE were constructed and characterized. They either completely lack OLs (ΔolsB) or only form the unmodified OL (ΔolsE). Here we present a characterization of both OL mutants under stress conditions and in a plant transformation assay using potato tuber discs. Surprisingly, the lack of agrobacterial OLs promotes earlier tumour formation on the plant host.

Sinorhizobium meliloti phospholipase C required for lipid remodeling during phosphorus limitation.

Rhizobia are Gram-negative soil bacteria able to establish nitrogen-fixing root nodules with their respective legume host plants. Besides phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine, rhizobial membranes contain phosphatidylcholine (PC) as a major membrane lipid. Under phosphate-limiting conditions of growth, some bacteria replace their membrane phospholipids with lipids lacking phosphorus. In Sinorhizobium meliloti, these phosphorus-free lipids are sulfoquinovosyl diacylglycerol, ornithine-containing lipid, and diacylglyceryl trimethylhomoserine (DGTS). Pulse-chase experiments suggest that the zwitterionic phospholipids phosphatidylethanolamine and PC act as biosynthetic precursors of DGTS under phosphorus-limiting conditions. A S. meliloti mutant, deficient in the predicted phosphatase SMc00171 was unable to degrade PC or to form DGTS in a similar way as the wild type. Cell-free extracts of Escherichia coli, in which SMc00171 had been expressed, convert PC to phosphocholine and diacylglycerol, showing that SMc00171 functions as a phospholipase C. Diacylglycerol , in turn, is the lipid anchor from which biosynthesis is initiated during the formation of the phosphorus-free membrane lipid DGTS. Inorganic phosphate can be liberated from phosphocholine. These data suggest that, in S. meliloti under phosphate-limiting conditions, membrane phospholipids provide a pool for metabolizable inorganic phosphate, which can be used for the synthesis of other essential phosphorus-containing biomolecules. This is an example of an intracellular phospholipase C in a bacterial system; however, the ability to degrade endogenous preexisting membrane phospholipids as a source of phosphorus may be a general property of Gram-negative soil bacteria.

Cardiolipin synthesis in Pseudomonas fluorescens UM270 plays a relevant role in stimulating plant growth under salt stress.

Membrane cardiolipin (CL) phospholipids play a fundamental role in the adaptation of bacteria to various environmental conditions, including saline stress. Here, we constructed deletion mutants of two CL synthetase genes, clsA (UM270 ∆clsA) and clsB (UM270 ∆clsB), in the rhizobacterium Pseudomonas fluorescens UM270, and evaluated their role in plant growth promotion under salt stress. UM270 ∆clsA and UM270 ∆clsB mutants showed a significant reduction in CL synthesis compared to the P. fluorescens UM270 wild-type (UM270 wt) strain (58% ∆clsA and 53% ∆clsB), and their growth rate was not affected, except when grown at 100 and 200 mM NaCl. Additionally, the root colonization capacity of both mutant strains was impaired compared with that of the wild type. Concomitant with the deletion of clsA and clsB genes, some physiological changes were observed in the UM270 ∆clsA and UM270 ∆clsB mutants, such as a reduction in indole acetic acid and biofilm production. By contrast, an increase in siderophore biosynthesis was observed. Further, inoculation of the UM270 wt strain in tomato plants (Solanum lycopersicum) grown under salt stress conditions (100 and 200 mM NaCl) resulted in an increase in root and shoot length, chlorophyll content, and dry weight. On the contrary, when each of the mutants were inoculated in tomato plants, a reduction in root length was observed when grown at 200 mM NaCl, but the shoot length, chlorophyll content, and total plant dry weight parameters were significantly reduced under normal or saline conditions (100 and 200 mM NaCl), compared to UM270 wt-inoculated plants. In conclusion, these results suggest that CL synthesis in P. fluorescens UM270 plays an important role in the promotion of tomato plant growth under normal conditions, but to a greater extent, under salt-stress conditions.

Hydroxylated ornithine lipids increase stress tolerance in Rhizobium tropici CIAT899.

Ornithine lipids (OLs) are widespread among Gram-negative bacteria. Their basic structure consists of a 3-hydroxy fatty acyl group attached in amide linkage to the α-amino group of ornithine and a second fatty acyl group ester-linked to the 3-hydroxy position of the first fatty acid. OLs can be hydroxylated within the secondary fatty acyl moiety and this modification has been related to increased stress tolerance. Rhizobium tropici, a nodule-forming α-proteobacterium known for its stress tolerance, forms four different OLs. Studies of the function of these OLs have been hampered due to lack of knowledge about their biosynthesis. Here we describe that OL biosynthesis increases under acid stress and that OLs are enriched in the outer membrane. Using a functional expression screen, the OL hydroxylase OlsE was identified, which in combination with the OL hydroxylase OlsC is responsible for the synthesis of modified OLs in R. tropici. Unlike described OL hydroxylations, the OlsE-catalysed hydroxylation occurs within the ornithine moiety. Mutants deficient in OlsE or OlsC and double mutants deficient in OlsC/OlsE were characterized. R. tropici mutants deficient in OlsC-mediated OL hydroxylation are more susceptible to acid and temperature stress. All three mutants lacking OL hydroxylases are affected during symbiosis.

Altered lipid A structures and polymyxin hypersensitivity of Rhizobium etli mutants lacking the LpxE and LpxF phosphatases.

The lipid A of Rhizobium etli, a nitrogen-fixing plant endosymbiont, displays significant structural differences when compared to that of Escherichia coli. An especially striking feature of R. etli lipid A is that it lacks both the 1- and 4'-phosphate groups. The 4'-phosphate moiety of the distal glucosamine unit is replaced with a galacturonic acid residue. The dephosphorylated proximal unit is present as a mixture of the glucosamine hemiacetal and an oxidized 2-aminogluconate derivative. Distinct lipid A phosphatases directed to the 1 or the 4'-positions have been identified previously in extracts of R. etli and Rhizobium leguminosarum. The corresponding structural genes, lpxE and lpxF, respectively, have also been identified. Here, we describe the isolation and characterization of R. etli deletion mutants in each of these phosphatase genes and the construction of a double phosphatase mutant. Mass spectrometry confirmed that the mutant strains completely lacked the wild-type lipid A species and accumulated the expected phosphate-containing derivatives. Moreover, radiochemical analysis revealed that phosphatase activity was absent in membranes prepared from the mutants. Our results indicate that LpxE and LpxF are solely responsible for selectively dephosphorylating the lipid A molecules of R. etli. All the mutant strains showed an increased sensitivity to polymyxin relative to the wild-type. However, despite the presence of altered lipid A species containing one or both phosphate groups, all the phosphatase mutants formed nitrogen-fixing nodules on Phaseolus vulgaris. Therefore, the dephosphorylation of lipid A molecules in R. etli is not required for nodulation but may instead play a role in protecting the bacteria from cationic antimicrobial peptides or other immune responses of plants.

ExoS/ChvI Two-Component Signal-Transduction System Activated in the Absence of Bacterial Phosphatidylcholine.

Sinorhizobium meliloti contains the negatively charged phosphatidylglycerol and cardiolipin as well as the zwitterionic phosphatidylethanolamine (PE) and phosphatidylcholine (PC) as major membrane phospholipids. In previous studies we had isolated S. meliloti mutants that lack PE or PC. Although mutants deficient in PE are able to form nitrogen-fixing nodules on alfalfa host plants, mutants lacking PC cannot sustain development of any nodules on host roots. Transcript profiles of mutants unable to form PE or PC are distinct; they differ from each other and they are different from the wild type profile. For example, a PC-deficient mutant of S. meliloti shows an increase of transcripts that encode enzymes required for succinoglycan biosynthesis and a decrease of transcripts required for flagellum formation. Indeed, a PC-deficient mutant is unable to swim and overproduces succinoglycan. Some suppressor mutants, that regain swimming and form normal levels of succinoglycan, are altered in the ExoS sensor. Our findings suggest that the lack of PC in the sinorhizobial membrane activates the ExoS/ChvI two-component regulatory system. ExoS/ChvI constitute a molecular switch in S. meliloti for changing from a free-living to a symbiotic life style. The periplasmic repressor protein ExoR controls ExoS/ChvI function and it is thought that proteolytic ExoR degradation would relieve repression of ExoS/ChvI thereby switching on this system. However, as ExoR levels are similar in wild type, PC-deficient mutant and suppressor mutants, we propose that lack of PC in the bacterial membrane provokes directly a conformational change of the ExoS sensor and thereby activation of the ExoS/ChvI two-component system.