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1.
PLoS Pathog ; 20(8): e1012401, 2024 Aug.
Article in English | MEDLINE | ID: mdl-39093898

ABSTRACT

Sphingolipids are ubiquitous in membranes of eukaryotes and are associated with important cellular functions. Although sphingolipids occur scarcely in bacteria, for some of them they are essential and, in other bacteria, they contribute to fitness and stability of the outer membrane, such as in the well-studied α-proteobacterium Caulobacter crescentus. We previously defined five structural genes for ceramide synthesis in C. crescentus, among them the gene for serine palmitoyltransferase, the enzyme that catalyzes the committed step of sphingolipid biosynthesis. Other mutants affected in genes of this same genomic region show cofitness with a mutant deficient in serine palmitoyltransferase. Here we show that at least two phosphosphingolipids are produced in C. crescentus and that at least another six gene products are needed for the decoration of ceramide upon phosphosphingolipid formation. All eleven genes participating in phosphosphingolipid formation are also required in C. crescentus for membrane stability and for displaying sensitivity towards the antibiotic polymyxin B. The genes for the formation of complex phosphosphingolipids are also required for C. crescentus virulence on Galleria mellonella insect larvae.


Subject(s)
Caulobacter crescentus , Sphingolipids , Caulobacter crescentus/metabolism , Caulobacter crescentus/genetics , Virulence , Animals , Sphingolipids/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Serine C-Palmitoyltransferase/metabolism , Serine C-Palmitoyltransferase/genetics , Moths/microbiology
2.
Plant J ; 118(4): 1136-1154, 2024 May.
Article in English | MEDLINE | ID: mdl-38341846

ABSTRACT

Rhizobial phosphatidylcholine (PC) is thought to be a critical phospholipid for the symbiotic relationship between rhizobia and legume host plants. A PC-deficient mutant of Sinorhizobium meliloti overproduces succinoglycan, is unable to swim, and lacks the ability to form nodules on alfalfa (Medicago sativa) host roots. Suppressor mutants had been obtained which did not overproduce succinoglycan and regained the ability to swim. Previously, we showed that point mutations leading to altered ExoS proteins can reverse the succinoglycan and swimming phenotypes of a PC-deficient mutant. Here, we report that other point mutations leading to altered ExoS, ChvI, FabA, or RpoH1 proteins also revert the succinoglycan and swimming phenotypes of PC-deficient mutants. Notably, the suppressor mutants also restore the ability to form nodule organs on alfalfa roots. However, nodules generated by these suppressor mutants express only low levels of an early nodulin, do not induce leghemoglobin transcript accumulation, thus remain white, and are unable to fix nitrogen. Among these suppressor mutants, we detected a reduced function mutant of the 3-hydoxydecanoyl-acyl carrier protein dehydratase FabA that produces reduced amounts of unsaturated and increased amounts of shorter chain fatty acids. This alteration of fatty acid composition probably affects lipid packing thereby partially compensating for the previous loss of PC and contributing to the restoration of membrane homeostasis.


Subject(s)
Fatty Acids , Medicago sativa , Phosphatidylcholines , Plant Root Nodulation , Sinorhizobium meliloti , Symbiosis , Sinorhizobium meliloti/physiology , Sinorhizobium meliloti/genetics , Medicago sativa/microbiology , Medicago sativa/genetics , Plant Root Nodulation/genetics , Fatty Acids/metabolism , Fatty Acids/biosynthesis , Phosphatidylcholines/metabolism , Phosphatidylcholines/biosynthesis , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Root Nodules, Plant/microbiology , Root Nodules, Plant/genetics , Root Nodules, Plant/metabolism , Mutation , Polysaccharides, Bacterial/metabolism , Polysaccharides, Bacterial/biosynthesis , Nitrogen Fixation
4.
Trends Microbiol ; 31(4): 323-325, 2023 04.
Article in English | MEDLINE | ID: mdl-36813608

ABSTRACT

In addition to glycerophospholipids, bacterial membranes often include amino acid-containing acyloxyacyl lipids. The functional implications of these aminolipids are largely unknown. However, a recent study by Stirrup et al. expands our understanding and shows that they are major determinants for membrane properties and the relative abundance of distinct membrane proteins in bacterial membranes.


Subject(s)
Membrane Lipids , Membrane Proteins , Membrane Lipids/metabolism , Membrane Proteins/metabolism , Glycerophospholipids/metabolism , Cell Membrane/metabolism
5.
Front Microbiol ; 13: 961041, 2022.
Article in English | MEDLINE | ID: mdl-35992722

ABSTRACT

Serine palmitoyltransferase (SPT) catalyzes the first and committed step in sphingolipid biosynthesis condensating L-serine and acyl-CoA to form 3-oxo-sphinganine. Whenever the structural gene for SPT is present in genomes of Rhodobacteria (α-, ß-, and γ-Proteobacteria), it co-occurs with genes coding for a putative acyl carrier protein (ACP) and a putative acyl-CoA synthetase (ACS). In the α-proteobacterium Caulobacter crescentus, CC_1162 encodes an SPT, whereas CC_1163 and CC_1165 encode the putative ACP and ACS, respectively, and all three genes are known to be required for the formation of the sphingolipid intermediate 3-oxo-sphinganine. Here we show that the putative ACP possesses a 4'-phosphopantetheine prosthetic group, is selectively acylated by the putative ACS and therefore is a specialized ACP (AcpR) required for sphingolipid biosynthesis in Rhodobacteria. The putative ACS is unable to acylate coenzyme A or housekeeping ACPs, but acylates specifically AcpR. Therefore, it is a specialized acyl-ACP synthetase (AasR). SPTs from C. crescentus, Escherichia coli B, or Sphingomonas wittichii use preferentially acyl-AcpR as thioester substrate for 3-oxo-sphinganine synthesis. Whereas acyl-AcpR from C. crescentus is a good substrate for SPTs from distinct Rhodobacteria, acylation of a specific AcpR is achieved by the cognate AasR from the same bacterium. Rhodobacteria might use this more complex way of 3-oxo-sphinganine formation in order to direct free fatty acids toward sphingolipid biosynthesis.

6.
Front Plant Sci ; 12: 678976, 2021.
Article in English | MEDLINE | ID: mdl-34367203

ABSTRACT

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.

7.
Environ Microbiol ; 23(1): 143-159, 2021 01.
Article in English | MEDLINE | ID: mdl-33063925

ABSTRACT

Sphingolipids are essential and common membrane components in eukaryotic organisms, participating in many important cellular functions. Only a few bacteria are thought to harbour sphingolipids in their membranes, among them the well-studied α-proteobacterium Caulobacter crescentus, a model organism for asymmetric cell division and cellular differentiation. Here, we report that C. crescentus wild type produces several molecular species of dihydroceramides, which are not produced in a mutant lacking the structural gene for serine palmitoyltransferase (spt). Whereas growth of a spt-deficient mutant and wild type are indistinguishable during the exponential phase of growth, survival of the spt-deficient mutant is much reduced, in comparison with wild type, during stationary phase of growth, especially at elevated temperatures. The structural gene for spt is located within a genomic cluster, comprising another 16 genes and which, like spt, are important for fitness of C. crescentus. Mutants deficient in genes linked to spt by high cofitness were unable to produce dihydroceramide or to survive in stationary phase of growth at elevated temperatures. At least five structural genes are required for dihydroceramide biosynthesis in C. crescentus and sphingolipid biosynthesis is needed for survival of this bacterium and the integrity of its outer membrane.


Subject(s)
Bacterial Proteins/metabolism , Caulobacter crescentus/growth & development , Caulobacter crescentus/metabolism , Ceramides/biosynthesis , Bacterial Proteins/genetics , Caulobacter crescentus/genetics , Cell Membrane/genetics , Cell Membrane/metabolism , Mutation , Serine C-Palmitoyltransferase/genetics , Serine C-Palmitoyltransferase/metabolism , Sphingolipids/biosynthesis
8.
Syst Appl Microbiol ; 43(6): 126133, 2020 Nov.
Article in English | MEDLINE | ID: mdl-32998072

ABSTRACT

A survey of our in-house bacterial collection identified a group of six strains isolated from the tomato rhizoplane that possessed 16S rRNA gene sequences with 98.2% sequence similarity to Paraburkholderia pallida, suggesting that these strains represented a novel species. Multilocus sequence analysis using gltB, lepA and recA gene sequences showed the clustering of the strains and the BOX-PCR patterns were similar among these strains. The average nucleotide identity and the DNA-DNA virtual hybridization of strain TNe-862T was <89% and <34%, respectively, to the genomes of any sequenced Paraburkholderia species. The genome of strain TNe-862T possessed all the genes necessary for nitrogen fixation and biosynthesis of indoleacetic acid and antimicrobials terpenes, phosphonates and bacteriocins. It also contained genes for metal resistance, xenobiotic degradation, and hydrolytic enzymes such as a putative chitinase and isoamylase. Even though the strain contained potential genes for degradation of cellulose and starch, the bacterium was unable to utilize these substrates in culture medium. The genome encoded flagella and pili as well as multiple chemotaxis systems. In addition, genes encoding for the type I, II, IV, V and VI secretion systems were also present. The strains grow up to 42°C and 5% NaCl. The optimum growth pH was 8. The major cellular fatty acids were C16:0 and C18:1 ω7c. Based on this polyphasic analysis, these strains represent a novel species in the genus Paraburkholderia, for which the name Paraburkholderia lycopersici sp. nov. is proposed. The type strain is TNe-862T (=LMG 26415T=CIP 110323T).


Subject(s)
Burkholderiaceae/classification , Nitrogen Fixation , Phylogeny , Soil Microbiology , Solanum lycopersicum/microbiology , Bacterial Typing Techniques , Base Composition , Burkholderiaceae/isolation & purification , DNA, Bacterial/genetics , Fatty Acids/chemistry , Genes, Bacterial , Mexico , Multilocus Sequence Typing , Nucleic Acid Hybridization , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA
9.
Microorganisms ; 8(4)2020 Mar 26.
Article in English | MEDLINE | ID: mdl-32225039

ABSTRACT

FadD is an acyl-coenzyme A (CoA) synthetase specific for long-chain fatty acids (LCFA). Strains mutated in fadD cannot produce acyl-CoA and thus cannot grow on exogenous LCFA as the sole carbon source. Mutants in the fadD (smc02162) of Sinorhizobium meliloti are unable to grow on oleate as the sole carbon source and present an increased surface motility and accumulation of free fatty acids at the entry of the stationary phase of growth. In this study, we found that constitutive expression of the closest FadD homologues of S. meliloti, encoded by sma0150 and smb20650, could not revert any of the mutant phenotypes. In contrast, the expression of Escherichia coli fadD could restore the same functions as S. meliloti fadD. Previously, we demonstrated that FadD is required for the degradation of endogenous fatty acids released from membrane lipids. Here, we show that absence of a functional fadD provokes a significant loss of viability in cultures of E. coli and of S. meliloti in the stationary phase, demonstrating a crucial role of fatty acid degradation in survival capacity.

10.
Environ Microbiol ; 20(6): 2049-2065, 2018 06.
Article in English | MEDLINE | ID: mdl-29488306

ABSTRACT

Surface motility and biofilm formation are behaviours which enable bacteria to infect their hosts and are controlled by different chemical signals. In the plant symbiotic alpha-proteobacterium Sinorhizobium meliloti, the lack of long-chain fatty acyl-coenzyme A synthetase activity (FadD) leads to increased surface motility, defects in biofilm development and impaired root colonization. In this study, analyses of lipid extracts and volatiles revealed that a fadD mutant accumulates 2-tridecanone (2-TDC), a methylketone (MK) known as a natural insecticide. Application of pure 2-TDC to the wild-type strain phenocopies the free-living and symbiotic behaviours of the fadD mutant. Structural features of the MK determine its ability to promote S. meliloti surface translocation, which is mainly mediated by a flagella-independent motility. Transcriptomic analyses showed that 2-TDC induces differential expression of iron uptake, redox and stress-related genes. Interestingly, this MK also influences surface motility and impairs biofilm formation in plant and animal pathogenic bacteria. Moreover, 2-TDC not only hampers alfalfa nodulation but also the development of tomato bacterial speck disease. This work assigns a new role to 2-TDC as an infochemical that affects important bacterial traits and hampers plant-bacteria interactions by interfering with microbial colonization of plant tissues.


Subject(s)
Bacterial Proteins/metabolism , Ketones/metabolism , Ketones/pharmacology , Medicago sativa/microbiology , Sinorhizobium meliloti/drug effects , Sinorhizobium meliloti/metabolism , Bacterial Proteins/genetics , Biofilms/drug effects , Gene Expression Profiling , Gene Expression Regulation, Bacterial , Mutation , Phenotype , Sinorhizobium meliloti/genetics , Symbiosis
11.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1862(11): 1287-1299, 2017 Nov.
Article in English | MEDLINE | ID: mdl-27760387

ABSTRACT

The glycerophospholipids phosphatidylethanolamine, phosphatidylglycerol (PG), and cardiolipin (CL) are major structural components of bacterial membranes. In some bacteria, phosphatidylcholine or phosphatidylinositol and its derivatives form part of the membrane. PG or CL can be modified with the amino acid residues lysine, alanine, or arginine. Diacylglycerol is the lipid anchor from which syntheses of phosphorus-free glycerolipids, such as glycolipids, sulfolipids, or homoserine-derived lipids initiate. Many membrane lipids are subject to turnover and some of them are recycled. Other lipids associated with the membrane include isoprenoids and their derivatives such as hopanoids. Ornithine-containing lipids are widespread in Bacteria but absent in Archaea and Eukarya. Some lipids are probably associated exclusively with the outer membrane of many bacteria, i.e. lipopolysaccharides, sphingolipids, or sulfonolipids. For certain specialized membrane functions, specific lipid structures might be required. Upon cyst formation in Azotobacter vinelandii, phenolic lipids are accumulated in the membrane. Anammox bacteria contain ladderane lipids in the membrane surrounding the anammoxosome organelle, presumably to impede the passage of highly toxic compounds generated during the anammox reaction. Considering that present knowledge on bacterial lipids was obtained from only a few bacterial species, we are probably only starting to unravel the full scale of lipid diversity in bacteria. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.


Subject(s)
Bacteria/metabolism , Diglycerides/biosynthesis , Glycerophospholipids/biosynthesis , Lipogenesis , Membrane Lipids/biosynthesis , Diglycerides/chemistry , Diglycerides/classification , Glycerophospholipids/chemistry , Glycerophospholipids/classification , Membrane Lipids/chemistry , Membrane Lipids/classification , Molecular Structure , Structure-Activity Relationship
12.
Mol Microbiol ; 103(5): 896-912, 2017 03.
Article in English | MEDLINE | ID: mdl-28009086

ABSTRACT

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.


Subject(s)
Biosynthetic Pathways , Diacylglycerol Cholinephosphotransferase/metabolism , Phosphatidylcholines/biosynthesis , Treponema denticola/metabolism , Alleles , Biosynthetic Pathways/genetics , Biosynthetic Pathways/physiology , Catalysis , Kinetics , Manganese/metabolism , Mutagenesis , Sequence Alignment , Treponema denticola/genetics
13.
Environ Microbiol ; 17(9): 3391-406, 2015 Sep.
Article in English | MEDLINE | ID: mdl-25711932

ABSTRACT

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.


Subject(s)
Diglycerides/metabolism , Fatty Acids/metabolism , Glycerol/metabolism , Lipoprotein Lipase/metabolism , Sinorhizobium meliloti/metabolism , Amino Acid Sequence , Escherichia coli/genetics , Escherichia coli/metabolism , Lipoprotein Lipase/genetics , Molecular Sequence Data , Phospholipids/metabolism , Sinorhizobium meliloti/enzymology , Sinorhizobium meliloti/genetics
14.
Biochim Biophys Acta ; 1831(3): 503-13, 2013 Mar.
Article in English | MEDLINE | ID: mdl-22922101

ABSTRACT

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.


Subject(s)
Agrobacterium tumefaciens/metabolism , Bacterial Proteins/metabolism , Phosphatidylcholines/biosynthesis , Sinorhizobium meliloti/metabolism , Animals , Choline/metabolism , Cytidine Diphosphate Diglycerides/metabolism , Cytidine Monophosphate/metabolism , Humans , Isoenzymes/metabolism , Methylation , Phosphatidyl-N-Methylethanolamine N-Methyltransferase/metabolism , Phosphatidylethanolamines/metabolism , Species Specificity , Transferases (Other Substituted Phosphate Groups)/metabolism
15.
Syst Appl Microbiol ; 35(5): 310-4, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22738764

ABSTRACT

A group of 20 bacterial strains was isolated from the rhizosphere of different agricultural plants growing in alkaline soils in the northeast of Mexico. The phylogenetic analysis of the 16S rRNA gene sequence from four strains showed that this novel group belonged to the Cupriavidus genus, with C. taiwanensis (∼98.9%) and C. necator (∼98.8%) as the closest species. However, DNA-DNA reassociation values were less than 20%. The novel group did not fix nitrogen and lacked nifH and nodA genes, unlike C. taiwanensis. Whole-cell protein patterns were highly similar among the 20 strains but different from the closest Cupriavidus species. BOX-PCR patterns were distinct among the 20 strains but also differed from other Cupriavidus type species. The major cellular fatty acids from strains ASC-732(T) and SLV-2362 were C(16:0), C(18:1) ω7c/12t/9t and C(16:1) ω7c and/or C(15:0) iso 2OH. The major polar lipids consisted of phosphatidylglycerol, cardiolipin, phosphatidylethanolamine, 2-hydroxylated-phosphatidylethanolamine and an unknown aminolipid. The DNA G+C content of strain ASC-732(T) was 66.8mol%. All 20 strains grew in the presence of 5-10mgmL(-1) arsenic, 1mgmL(-1) zinc, and 0.1mgmL(-1) copper. Consequently, the group of strains was considered to represent a novel species for which the name Cupriavidus alkaliphilus sp. nov. is proposed. The type strain is ASC-732(T) (=LMG 26294(T)=CIP 110330(T)).


Subject(s)
Cupriavidus/classification , Cupriavidus/isolation & purification , Rhizosphere , Agriculture , Base Composition , Cupriavidus/genetics , Hydrogen-Ion Concentration , Mexico , Phylogeny , Soil/chemistry , Soil Microbiology
16.
J Bacteriol ; 193(22): 6295-304, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21926226

ABSTRACT

FadD is an acyl coenzyme A (CoA) synthetase responsible for the activation of exogenous long-chain fatty acids (LCFA) into acyl-CoAs. Mutation of fadD in the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti promotes swarming motility and leads to defects in nodulation of alfalfa plants. In this study, we found that S. meliloti fadD mutants accumulated a mixture of free fatty acids during the stationary phase of growth. The composition of the free fatty acid pool and the results obtained after specific labeling of esterified fatty acids with a Δ5-desaturase (Δ5-Des) were in agreement with membrane phospholipids being the origin of the released fatty acids. Escherichia coli fadD mutants also accumulated free fatty acids released from membrane lipids in the stationary phase. This phenomenon did not occur in a mutant of E. coli with a deficient FadL fatty acid transporter, suggesting that the accumulation of fatty acids in fadD mutants occurs inside the cell. Our results indicate that, besides the activation of exogenous LCFA, in bacteria FadD plays a major role in the activation of endogenous fatty acids released from membrane lipids. Furthermore, expression analysis performed with S. meliloti revealed that a functional FadD is required for the upregulation of genes involved in fatty acid degradation and suggested that in the wild-type strain, the fatty acids released from membrane lipids are degraded by ß-oxidation in the stationary phase of growth.


Subject(s)
Bacterial Proteins/metabolism , Coenzyme A Ligases/metabolism , Fatty Acids/metabolism , Membrane Lipids/metabolism , Sinorhizobium meliloti/enzymology , Bacterial Proteins/genetics , Biological Transport , Coenzyme A Ligases/genetics , Mutation , Sinorhizobium meliloti/genetics , Sinorhizobium meliloti/metabolism
17.
Biochemistry ; 50(29): 6396-408, 2011 Jul 26.
Article in English | MEDLINE | ID: mdl-21707055

ABSTRACT

Burkholderia cenocepacia is an important opportunistic pathogen, and one of the most striking features of the Burkholderia genus is the collection of polar lipids present in its membrane, including phosphatidylethanolamine (PE) and ornithine-containing lipids (OLs), as well as the 2-hydroxylated derivatives of PE and OLs (2-OH-PE and 2-OH-OLs, respectively), which differ from the standard versions by virtue of the presence of a hydroxyl group at C2 (2-OH) of an esterified fatty acyl residue. Similarly, a lipid A-esterified myristoyl group from Salmonella typhimurium can have a 2-hydroxy modification that is due to the LpxO enzyme. We thus postulated that 2-hydroxylation of 2-OH-OLs might be catalyzed by a novel dioxygenase homologue of LpxO. In B. cenocepacia, we have now identified two open reading frames (BCAM1214 and BCAM2401) homologous to LpxO from S. typhimurium. The introduction of bcam2401 (designated olsD) into Sinorhizobium meliloti leads to the formation of one new lipid and in B. cenocepacia of two new lipids. Surprisingly, the lipid modifications on OLs due to OlsD occur on the amide-linked fatty acyl chain. This is the first report of a hydroxyl modification of OLs on the amide-linked fatty acyl moiety. Formation of hydroxylated OLs occurs only when the biosynthesis pathway for nonmodified standard OLs is intact. The hydroxyl modification of OLs on the amide-linked fatty acyl moiety occurs only under acid stress conditions. An assay has been developed for the OlsD dioxygenase, and an initial characterization of the enzyme is presented.


Subject(s)
Burkholderia cenocepacia/enzymology , Burkholderia cenocepacia/genetics , Dioxygenases/genetics , Fatty Acids/metabolism , Genes, Bacterial/genetics , Membrane Lipids/metabolism , Ornithine/analogs & derivatives , Acids/pharmacology , Amides/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Burkholderia cenocepacia/drug effects , Burkholderia cenocepacia/growth & development , Cardiolipins/metabolism , Cell Membrane/drug effects , Cell Membrane/enzymology , Dioxygenases/metabolism , Esterification/drug effects , Fatty Acids/chemistry , Hydroxylation/drug effects , Lipids/chemistry , Mass Spectrometry , Membrane Lipids/chemistry , Mutation/genetics , Ornithine/chemistry , Ornithine/metabolism , Phosphatidylethanolamines/metabolism , Phosphatidylglycerols/metabolism , Salmonella typhimurium/drug effects , Salmonella typhimurium/enzymology , Sequence Homology, Amino Acid , Sinorhizobium meliloti/drug effects , Sinorhizobium meliloti/metabolism
18.
Mol Microbiol ; 79(6): 1496-514, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21205018

ABSTRACT

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.


Subject(s)
Ornithine/analogs & derivatives , Rhizobium tropici/physiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Hydroxylation , Lipids/chemistry , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Molecular Structure , Mutation , Ornithine/chemistry , Ornithine/metabolism , Rhizobium tropici/chemistry , Rhizobium tropici/enzymology , Rhizobium tropici/genetics , Stress, Physiological
19.
Proc Natl Acad Sci U S A ; 107(1): 302-7, 2010 Jan 05.
Article in English | MEDLINE | ID: mdl-20018679

ABSTRACT

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.


Subject(s)
Bacterial Proteins/metabolism , Membrane Lipids/metabolism , Phosphorus/metabolism , Sinorhizobium meliloti/enzymology , Type C Phospholipases/metabolism , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Membrane Lipids/chemistry , Molecular Structure , Mutation , Phosphatidylcholines/metabolism , Phosphatidylethanolamines/metabolism , Sinorhizobium meliloti/cytology , Sinorhizobium meliloti/genetics , Triglycerides/chemistry , Triglycerides/metabolism , Type C Phospholipases/genetics
20.
FEMS Microbiol Lett ; 303(2): 123-31, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20030724

ABSTRACT

Phosphatidylcholine, the major phospholipid in eukaryotes, is found in rhizobia and in many other bacteria interacting with eukaryotic hosts. Phosphatidylcholine has been shown to be required for a successful interaction of Bradyrhizobium japonicum USDA 110 with soybean roots. Our aim was to study the role of bacterial phosphatidylcholine in the Bradyrhizobium-peanut (Arachis hypogaea) symbiosis. Phospholipid N-methyltransferase (Pmt) and minor phosphatidylcholine synthase (Pcs) activities were detected in crude extracts of the peanut-nodulating strain Bradyrhizobium sp. SEMIA 6144. Our results suggest that phosphatidylcholine formation in Bradyrhizobium sp. SEMIA 6144 is mainly due to the phospholipid methylation pathway. Southern blot analysis using pmt- and pcs-probes of B. japonicum USDA 110 revealed a pcs and multiple pmt homologues in Bradyrhizobium sp. SEMIA 6144. A pmtA knockout mutant was constructed in Bradyrhizobium sp. SEMIA 6144 that showed a 50% decrease in the phosphatidylcholine content in comparison with the wild-type strain. The mutant was severely affected in motility and cell size, but formed wild-type-like nodules on its host plant. However, in coinoculation experiments, the pmtA-deficient mutant was less competitive than the wild type, suggesting that wild-type levels of phosphatidylcholine are required for full competitivity of Bradyrhizobium in symbiosis with peanut plants.


Subject(s)
Arachis/microbiology , Bradyrhizobium/cytology , Bradyrhizobium/physiology , Phosphatidylcholines/analysis , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Blotting, Southern , Bradyrhizobium/chemistry , Bradyrhizobium/isolation & purification , DNA, Bacterial/genetics , Gene Dosage , Gene Knockout Techniques , Locomotion , Methyltransferases/genetics , Methyltransferases/metabolism , Molecular Sequence Data , Sequence Analysis, DNA , Symbiosis , Transferases (Other Substituted Phosphate Groups)/genetics , Transferases (Other Substituted Phosphate Groups)/metabolism , Virulence
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