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.

The Phospholipid N-Methyltransferase and Phosphatidylcholine Synthase Pathways and the ChoXWV Choline Uptake System Involved in Phosphatidylcholine Synthesis Are Widely Conserved in Most, but Not All Brucella Species.

The brucellae are facultative intracellular bacteria with a cell envelope rich in phosphatidylcholine (PC). PC is abundant in eukaryotes but rare in prokaryotes, and it has been proposed that Brucella uses PC to mimic eukaryotic-like features and avoid innate immune responses in the host. Two PC synthesis pathways are known in prokaryotes: the PmtA-catalyzed trimethylation of phosphatidylethanolamine and the direct linkage of choline to CDP-diacylglycerol catalyzed by the PC synthase Pcs. Previous studies have reported that B. abortus and B. melitensis possess non-functional PmtAs and that PC is synthesized exclusively via Pcs in these strains. A putative choline transporter ChoXWV has also been linked to PC synthesis in B. abortus. Here, we report that Pcs and Pmt pathways are active in B. suis biovar 2 and that a bioinformatics analysis of Brucella genomes suggests that PmtA is only inactivated in B. abortus and B. melitensis strains. We also show that ChoXWV is active in B. suis biovar 2 and conserved in all brucellae except B. canis and B. inopinata. Unexpectedly, the experimentally verified ChoXWV dysfunction in B. canis did not abrogate PC synthesis in a PmtA-deficient mutant, which suggests the presence of an unknown mechanism for obtaining choline for the Pcs pathway in Brucella. We also found that ChoXWV dysfunction did not cause attenuation in B. suis biovar 2. The results of these studies are discussed with respect to the proposed role of PC in Brucella virulence and how differential use of the Pmt and Pcs pathways may influence the interactions of these bacteria with their mammalian hosts.

PsrA positively regulates the unsaturated fatty acid synthesis operon fabAB in Azotobacter vinelandii.

In Pseudomonas spp. PsrA, a transcriptional activator of the rpoS gene, regulates fatty acid catabolism by repressing the fadBA5 ß-oxidation operon. In Azotobacter vinelandii, a soil bacterium closely related to Pseudomonas species, PsrA is also an activator of rpoS expression, although its participation in the regulation of lipid metabolism has not been analyzed. In this work we found that inactivation of psrA had no effect on the expression of ß-oxidation genes in this bacterium, but instead decreased expression of the unsaturated fatty acid biosynthetic operon fabAB (3-hydroxydecanoyl-ACP dehydratase/isomerase and 3-ketoacyl-ACP synthase I). This inactivation also reduced the unsaturated fatty acid content, as revealed by the thin-layer chromatographic analysis, and confirmed by gas chromatography; notably, there was also a lower content of cyclopropane fatty acids, which are synthesized from unsaturated fatty acids. The absence of PsrA has no effect on the growth rate, but showed loss of cell viability during long-term growth, in accordance with the role of these unsaturated and cyclopropane fatty acids in the protection of membranes. Finally, an electrophoretic mobility shift assay revealed specific binding of PsrA to the fabA promoter region, where a putative binding site for this regulator was located. Taken together, our data show that PsrA plays an important role in the regulation of unsaturated fatty acids metabolism in A. vinelandii by positively regulating fabAB.

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.

Structure and diversity of native bacterial communities in soils contaminated with polychlorinated biphenyls.

Persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs) are a group of high-risk synthetic substances for human and environmental health. Currently, the study of sites contaminated by the spillage of equipment PCBs containing have been considered targeted areas for the study of bacterial communities with potential for PCBs degradation. There in isolation of bacterial strains is vital for use in biodegradable processes, such as bacterial bioaugmentation, which accelerates the development of phenomena such as natural attenuation of contaminated sites. The objective of this study was to assess biodiversity of bacteria contained in anthropogenic contaminated soils (HS and HP) with PCBs compared to a control sample without contaminant and the modified forest (F) and agricultural (A) soil in the laboratory with 100 mg L-1 PCB. For the analysis of 16S rRNA genes amplified from DNA extracted from the soils evaluated, the latest generation of Illumina Miseq and Sanger sequencing for the cultivable strains were detected. The bacteria identified as the most abundant bacterial phyla for HS and HP soil was Proteobacteria (56.7%) and Firmicutes (22.9%), which decreased in F and A soils. The most abundant bacterial genera were Burkholderia, Bacillus, Acinetobacter, Comamonas and Cupriavidus. Several species identified in this study, such as Bacillus cereus, Burkholderia cepacia, Comamonas testosteroni and Acinetobacter pittii have been reported as PCBs degraders. Finally, by means of a principal component analysis (PCA), a correlation between the physical and chemical characteristics of the soils in relation to the relative abundances of the bacteria identified was obtained. The C/N ratio was directly related to the control soil (without contaminant), while SOM maintained a relationship with F and A soils and the bacterial abundances were directly related to Hs and Hp soils due to the presence of aroclor 1260. Bacteria with the ability to tolerate high concentrations of this pollutant are considered for future use in biostimulation and bioaugmentation processes in contaminated soils.

Ornithine Lipids in Burkholderia spp. Pathogenicity.

The genus Burkholderia sensu lato is composed of a diverse and metabolically versatile group of bacterial species. One characteristic thought to be unique for the genus Burkholderia is the presence of two forms each (with and without 2-hydroxylation) of the membrane lipids phosphatidylethanolamine (PE) and ornithine lipids (OLs). Here, we show that only Burkholderia sensu stricto strains constitutively form OLs, whereas all other analyzed strains belonging to the Burkholderia sensu lato group constitutively form the two forms of PE, but no OLs. We selected two model bacteria to study the function of OL in Burkholderia sensu lato: (1) Burkholderia cenocepacia wild-type which constitutively forms OLs and its mutant deficient in the formation of OLs and (2) Robbsia andropogonis (formerly Burkholderia andropogonis) which does not form OL constitutively, and a derived strain constitutively forming OLs. Both were characterized under free-living conditions and during pathogenic interactions with their respective hosts. The absence of OLs in B. cenocepacia slightly affected bacterial growth under specific abiotic stress conditions such as high temperature and low pH. B. cenocepacia lacking OLs caused lower mortality in Galleria mellonella larvae while R. andropogonis constitutively forming OLs triggers an increased formation of reactive oxygen species immediately after infection of maize leaves, suggesting that OLs can have an important role during the activation of the innate immune response of eukaryotes.

Headgroup hydroxylation by OlsE occurs at the C4 position of ornithine lipid and is widespread in proteobacteria and bacteroidetes.

Amino acid-containing acyloxyacyl lipids are composed of a 3-hydroxy fatty acid amide-bound to the α-amino group of an amino acid. A second fatty acid is ester-linked to the 3-hydroxy group of the first fatty acid. Most commonly, ornithine is the headgroup of these lipids, but glycine, serineglycine, glutamine and lysine have also been described in bacteria. Ornithine lipids (OL) can be synthesized by about 50% of the sequenced bacterial species, and several covalent modifications of its basic structure have been described. The OL hydroxylase OlsE is widespread in Rhizobium and Agrobacterium species and is responsible for introducing a hydroxyl group at a hence unknown position within the ornithine headgroup causing the formation of the OL named S2. Using NMR on purified OL S2, we show that the OlsE-mediated hydroxylation takes place at the C-4 position of the ornithine headgroup. Furthermore, we identify a hydroxylase in the genome of Pseudopedobacter saltans, distantly related to OlsE from α-proteobacteria, able to hydroxylate the headgroup of both ornithine lipids and lysine lipids. A homology search with the amino acid sequence of this hydroxylase allows us to predict that OL headgroup hydroxylation is not restricted to a few α-proteobacteria, but is apparently also common in many genera belonging to the Cytophaga-Flavobacterium-Bacteroidetes (CFB) group of bacteria.

Identification of lptA, lpxE, and lpxO, Three Genes Involved in the Remodeling of Brucella Cell Envelope.

The brucellae are facultative intracellular bacteria that cause a worldwide extended zoonosis. One of the pathogenicity mechanisms of these bacteria is their ability to avoid rapid recognition by innate immunity because of a reduction of the pathogen-associated molecular pattern (PAMP) of the lipopolysaccharide (LPS), free-lipids, and other envelope molecules. We investigated the Brucella homologs of lptA, lpxE, and lpxO, three genes that in some pathogens encode enzymes that mask the LPS PAMP by upsetting the core-lipid A charge/hydrophobic balance. Brucella lptA, which encodes a putative ethanolamine transferase, carries a frame-shift in B. abortus but not in other Brucella spp. and phylogenetic neighbors like the opportunistic pathogen Ochrobactrum anthropi. Consistent with the genomic evidence, a B. melitensis lptA mutant lacked lipid A-linked ethanolamine and displayed increased sensitivity to polymyxin B (a surrogate of innate immunity bactericidal peptides), while B. abortus carrying B. melitensis lptA displayed increased resistance. Brucella lpxE encodes a putative phosphatase acting on lipid A or on a free-lipid that is highly conserved in all brucellae and O. anthropi. Although we found no evidence of lipid A dephosphorylation, a B. abortus lpxE mutant showed increased polymyxin B sensitivity, suggesting the existence of a hitherto unidentified free-lipid involved in bactericidal peptide resistance. Gene lpxO putatively encoding an acyl hydroxylase carries a frame-shift in all brucellae except B. microti and is intact in O. anthropi. Free-lipid analysis revealed that lpxO corresponded to olsC, the gene coding for the ornithine lipid (OL) acyl hydroxylase active in O. anthropi and B. microti, while B. abortus carrying the olsC of O. anthropi and B. microti synthesized hydroxylated OLs. Interestingly, mutants in lptA, lpxE, or olsC were not attenuated in dendritic cells or mice. This lack of an obvious effect on virulence together with the presence of the intact homolog genes in O. anthropi and B. microti but not in other brucellae suggests that LptA, LpxE, or OL ß-hydroxylase do not significantly alter the PAMP properties of Brucella LPS and free-lipids and are therefore not positively selected during the adaptation to intracellular life.

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.

The GacS/A-RsmA Signal Transduction Pathway Controls the Synthesis of Alkylresorcinol Lipids that Replace Membrane Phospholipids during Encystment of Azotobacter vinelandii SW136.

Azotobacter vinelandii is a soil bacterium that undergoes a differentiation process that forms cysts resistant to desiccation. During encystment, a family of alkylresorcinols lipids (ARs) are synthesized and become part of the membrane and are also components of the outer layer covering the cyst, where they play a structural role. The synthesis of ARs in A. vinelandii has been shown to occur by the activity of enzymes encoded in the arsABCD operon. The expression of this operon is activated by ArpR, a LysR-type transcriptional regulator whose transcription occurs during encystment and is dependent on the alternative sigma factor RpoS. In this study, we show that the two component response regulator GacA, the small RNA RsmZ1 and the translational repressor protein RsmA, implicated in the control of the synthesis of other cysts components (i.e., alginate and poly-ß-hydroxybutyrate), are also controlling alkylresorcinol synthesis. This control affects the expression of arsABCD and is exerted through the regulation of arpR expression. We show that RsmA negatively regulates arpR expression by binding its mRNA, repressing its translation. GacA in turn, positively regulates arpR expression through the activation of transcription of RsmZ1, that binds RsmA, counteracting its repressor activity. This regulatory cascade is independent of RpoS. We also show evidence suggesting that GacA exerts an additional regulation on arsABCD expression through an ArpR independent route.

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.

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.

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 mutants deficient in phosphatidylserine decarboxylase accumulate phosphatidylserine and are strongly affected during symbiosis with alfalfa.

Sinorhizobium meliloti contains phosphatidylglycerol, cardiolipin, phosphatidylcholine, and phosphatidylethanolamine (PE) as major membrane lipids. PE is formed in two steps. In the first step, phosphatidylserine synthase (Pss) condenses serine with CDP-diglyceride to form phosphatidylserine (PS), and in the second step, PS is decarboxylated by phosphatidylserine decarboxylase (Psd) to form PE. In this study we identified the sinorhizobial psd gene coding for Psd. A sinorhizobial mutant deficient in psd is unable to form PE but accumulates the anionic phospholipid PS. Properties of PE-deficient mutants lacking either Pss or Psd were compared with those of the S. meliloti wild type. Whereas both PE-deficient mutants grew in a wild-type-like manner on many complex media, they were unable to grow on minimal medium containing high phosphate concentrations. Surprisingly, the psd-deficient mutant could grow on minimal medium containing low concentrations of inorganic phosphate, while the pss-deficient mutant could not. Addition of choline to the minimal medium rescued growth of the pss-deficient mutant, CS111, to some extent but inhibited growth of the psd-deficient mutant, MAV01. When the two distinct PE-deficient mutants were analyzed for their ability to form a nitrogen-fixing root nodule symbiosis with their alfalfa host plant, they behaved strikingly differently. The Pss-deficient mutant, CS111, initiated nodule formation at about the same time point as the wild type but did form about 30% fewer nodules than the wild type. In contrast, the PS-accumulating mutant, MAV01, initiated nodule formation much later than the wild type and formed 90% fewer nodules than the wild type. The few nodules formed by MAV01 seemed to be almost devoid of bacteria and were unable to fix nitrogen. Leaves of alfalfa plants inoculated with the mutant MAV01 were yellowish, indicating that the plants were starved for nitrogen. Therefore, changes in lipid composition, including the accumulation of bacterial PS, prevent the establishment of a nitrogen-fixing root nodule symbiosis.