Phosphatidylcholine-deficient suppressor mutant of Sinorhizobium meliloti, altered in fatty acid synthesis, partially recovers nodulation ability in symbiosis with alfalfa (Medicago sativa).

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.

Membrane lipid composition defines membrane protein spectrum.

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.

Specialized acyl carrier protein used by serine palmitoyltransferase to synthesize sphingolipids in Rhodobacteria.

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.

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.

Rhizobial Volatiles: Potential New Players in the Complex Interkingdom Signaling With Legumes.

Bacteria release a wide range of volatile compounds that play important roles in intermicrobial and interkingdom communication. Volatile metabolites emitted by rhizobacteria can promote plant growth and increase plant resistance to both biotic and abiotic stresses. Rhizobia establish beneficial nitrogen-fixing symbiosis with legume plants in a process starting with a chemical dialog in the rhizosphere involving various diffusible compounds. Despite being one of the most studied plant-interacting microorganisms, very little is known about volatile compounds produced by rhizobia and their biological/ecological role. Evidence indicates that plants can perceive and respond to volatiles emitted by rhizobia. In this perspective, we present recent data that open the possibility that rhizobial volatile compounds have a role in symbiotic interactions with legumes and discuss future directions that could shed light onto this area of investigation.

Five structural genes required for ceramide synthesis in Caulobacter and for bacterial survival.

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.

Paraburkholderia lycopersici sp. nov., a nitrogen-fixing species isolated from rhizoplane of Lycopersicon esculentum Mill. var. Saladette in Mexico.

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).

Role of Sinorhizobium meliloti and Escherichia coli Long-Chain Acyl-CoA Synthetase FadD in Long-Term Survival.

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.

Expression, purification, and characterization of a metagenomic thioesterase from activated sludge involved in the degradation of acylCoA-derivatives.

Metagenomic libraries are a novel and powerful approach to seek for pathways involved in xenobiotic degradation, since this technique abolishes the need for cultivating microorganisms that otherwise would be overlooked if they cannot grow on standard laboratory media and conditions. In this paper, we describe the expression, purification and characterization of a novel metagenomic thioesterase which was described to be involved in phenylacetic acid degradation (A. Sánchez-Reyes, R. Batista-García, G. Valdés-García E. Ortiz, L. Perezgasga, A. Zárate-Romero, N. Pastor, J. L. Folch-Mallol, A Family 13 thioesterase isolated from an activated sludge metagenome: insights into aromatic compounds metabolism, Proteins 85 (2017) 1222-1237). According to similarity and phylogenetic analyses, the enzyme seems to belong to an Actinobacterium. Nevertheless, after a process of denaturation and refolding, the protein expressed in E. coli was obtained in an active form. New data concerning the substrate preferences for this enzyme are presented which suggest that this thioesterase could be involved in breaking the ester bond in the CoA-linear acyl derivatives of the phenylacetic acetic pathway.

2-Tridecanone impacts surface-associated bacterial behaviours and hinders plant-bacteria interactions.

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.

Bacterial lipid diversity.

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.

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.

Defining Substrate Specificities for Lipase and Phospholipase Candidates.

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. Alternatively, other (phospho)lipases may be physically associated with the producing organism causing a turnover of intrinsic lipids and frequently giving rise to a remodeling of the cellular membranes. Although potential (phospho)lipases can be predicted with a number of algorithms when the gene/protein sequence is available, experimental proof of the enzyme activities, substrate specificities, and potential physiological functions has frequently not been obtained. This manuscript describes the optimization of assay conditions for prospective (phospho)lipases with unknown substrate specificities and how to employ these optimized conditions in the search for the natural substrate of a respective (phospho)lipase. Using artificial chromogenic substrates, such as p-nitrophenyl derivatives, may help to detect a minor enzymatic activity for a predicted (phospho)lipase under standard conditions. Having encountered such a minor enzymatic activity, the distinct parameters of an enzyme assay can be varied in order to obtain a more efficient hydrolysis of the artificial substrate. After having determined the conditions under which an enzyme works well, a variety of potential natural substrates should be assayed for their degradation, a process that can be followed employing distinct chromatographic methods. The definition of substrate specificities for new enzymes, often provides hypotheses for a potential physiological role of these enzymes, which then can be tested experimentally. Following these guidelines, we were able to identify a phospholipase C (SMc00171) that degrades phosphatidylcholine to phosphocholine and diacylglycerol, in a crucial step for the remodeling of membranes in the bacterium Sinorhizobium meliloti upon phosphorus-limiting conditions of growth. For two predicted patatin-like phospholipases (SMc00930 and SMc01003) of the same organism, we could redefine their substrate specificities and clarify that SMc01003 is a diacylglycerol lipase.

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.

Cupriavidus alkaliphilus sp. nov., a new species associated with agricultural plants that grow in alkaline soils.

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)).

FadD is required for utilization of endogenous fatty acids released from membrane lipids.

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.

The dioxygenase-encoding olsD gene from Burkholderia cenocepacia causes the hydroxylation of the amide-linked fatty acyl moiety of ornithine-containing membrane lipids.

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.

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.