Legionella shows a diverse secondary metabolism dependent on a broad spectrum Sfp-type phosphopantetheinyl transferase.

Several members of the genus Legionella cause Legionnaires' disease, a potentially debilitating form of pneumonia. Studies frequently focus on the abundant number of virulence factors present in this genus. However, what is often overlooked is the role of secondary metabolites from Legionella. Following whole genome sequencing, we assembled and annotated the Legionella parisiensis DSM 19216 genome. Together with 14 other members of the Legionella, we performed comparative genomics and analysed the secondary metabolite potential of each strain. We found that Legionella contains a huge variety of biosynthetic gene clusters (BGCs) that are potentially making a significant number of novel natural products with undefined function. Surprisingly, only a single Sfp-like phosphopantetheinyl transferase is found in all Legionella strains analyzed that might be responsible for the activation of all carrier proteins in primary (fatty acid biosynthesis) and secondary metabolism (polyketide and non-ribosomal peptide synthesis). Using conserved active site motifs, we predict some novel compounds that are probably involved in cell-cell communication, differing to known communication systems. We identify several gene clusters, which may represent novel signaling mechanisms and demonstrate the natural product potential of Legionella.

Identification of a triacylglycerol lipase in the diatom Phaeodactylum tricornutum.

Diatoms accumulate triacylglycerols (TAGs) as storage lipids, but the knowledge about the molecular mechanisms of lipid metabolism is still sparse. Starting from a partial sequence for a putative TAG-lipase of the diatom Phaeodactylum tricornutum retrieved from the data bases, we have identified the full length coding sequence, tgl1. The gene encodes an 813 amino acid sequence that shows distinct motifs for so called "true" TAG-lipases [EC 3.1.1.3] that have been functionally characterized in model organisms like Arabidopsis thaliana and Saccharomyces cerevisiae. These lipases mediate the first initial step of TAG breakdown from storage lipids. To test whether Tgl1 can act as a TAG-lipase, a His-tagged version was overexpressed in Escherichia coli and the protein indeed showed esterase activity. To identify the TAG degrading function of Tgl1 in P. tricornutum, knock-down mutant strains were created using an antisense RNA approach. In the mutant cell lines the relative tgl1-mRNA-level was reduced up to 20% of that of the wild type, accompanied by a strong increase of TAG in the lipid extracts. In spite of the TAG accumulation, the polar lipid species pattern appeared to be unchanged, confirming the TAG-lipase function of Tgl1.

An iso-15 : 0 O-alkylglycerol moiety is the key structure of the E-signal in Myxococcus xanthus.

The E-signal is one of five intercellular signals (named A- to E-signal) guiding fruiting body development in Myxococcus xanthus, and it has been shown to be a combination of the branched-chain fatty acid (FA) iso-15 : 0 and the diacylmonoalkyl ether lipid TG1. Developmental mutants HB015 (Δbkd MXAN_4265::kan) and elbD (MXAN_1528::kan) are blocked at different stages of fruiting body and spore formation as they cannot form the required iso-FA or the actual ether lipid, respectively. In order to define the structural basis of the E-signal, different mono- and triglycerides containing ether or ester bonds were synthesized and used for complementation of these mutants. Here, the monoalkylglyceride dl-1-O-(13-methyltetradecyl)glycerol exhibited comparably high levels of complementation in both mutants, restoring fruiting body and spore formation, identifying iso-15 : 0 O-alkylglycerol, part of the natural lipid TG1, as the 'signalophore' of E-signalling.

Neutral and Phospholipids of the Myxococcus xanthus Lipodome during Fruiting Body Formation and Germination.

Myxobacteria are well-known for their complex life cycle, including the formation of spore-filled fruiting bodies. The model organism Myxococcus xanthus exhibits a highly complex composition of neutral and phospholipids, including triacylglycerols (TAGs), diacylglycerols (DAGs), phosphatidylethanolamines (PEs), phosphatidylglycerols (PGs), cardiolipins (CLs), and sphingolipids, including ceramides (Cers) and ceramide phosphoinositols (Cer-PIs). In addition, ether lipids have been shown to be involved in development and signaling. In this work, we describe the lipid profile of M. xanthus during its entire life cycle, including spore germination. PEs, representing one of the major components of the bacterial membrane, decreased by about 85% during development from vegetative rods to round myxospores, while TAGs first accumulated up to 2-fold before they declined 48 h after the induction of sporulation. Presumably, membrane lipids are incorporated into TAG-containing lipid bodies, serving as an intermediary energy source for myxospore formation. The ceramides Cer(d-19:0/iso-17:0) and Cer(d-19:0/16:0) accumulated 6-fold and 3-fold, respectively, after 24 h of development, identifying them to be novel putative biomarkers for M. xanthus sporulation. The most abundant ether lipid, 1-iso-15:0-alkyl-2,3-di-iso-15:0-acyl glycerol (TG1), exhibited a lipid profile different from that of all TAGs during sporulation, reinforcing its signaling character. The absence of all these lipid profile changes in mutants during development supports the importance of lipids in myxobacterial development. During germination of myxospores, only the de novo biosynthesis of new cell membrane fatty acids was observed. The unexpected accumulation of TAGs also during germination might indicate a function of TAGs as intermediary storage lipids during this part of the life cycle as well.

A multifunctional enzyme is involved in bacterial ether lipid biosynthesis.

Fatty acid-derived ether lipids are present not only in most vertebrates but also in some bacteria. Here we describe what is to our knowledge the first gene cluster involved in the biosynthesis of such lipids in myxobacteria that encodes the multifunctional enzyme ElbD, which shows similarity to polyketide synthases. Initial characterization of elbD mutants in Myxococcus xanthus and Stigmatella aurantiaca showed the importance of these ether lipids for fruiting body formation and sporulation.

Rapid determination of the amino acid configuration of xenotetrapeptide.

An E. coli strain with deletions in five transaminases (ΔaspC ΔilvE ΔtyrB ΔavtA ΔybfQ) was constructed to be unable to degrade several amino acids. This strain was used as an expression host for the analysis of the amino acid configuration of nonribosomally synthesized peptides, including the novel peptide "xenotetrapeptide" from Xenorhabdus nematophila, by using a combination of labeling experiments and mass spectrometry. Additionally, the number of D-amino acids in the produced peptide was assigned following simple cultivation of the expression strain in D2 O.

Two lipid signals guide fruiting body development of Myxococcus xanthus.

UNLABELLED: Myxococcus xanthus produces several extracellular signals that guide fruiting body morphogenesis and spore differentiation. Mutants defective in producing a signal may be rescued by codevelopment with wild-type cells or cell fractions containing the signal. In this paper, we identify two molecules that rescue development of the E signal-deficient mutant LS1191 at physiological concentrations, iso15:0 branched-chain fatty acid (FA) and 1-iso15:0-alkyl-2,3-di-iso15:0-acyl glycerol (TG1), a development-specific monoalkyl-diacylglycerol. The physiological concentrations of the bioactive lipids were determined by mass spectrometry from developing wild-type cells using chemically synthesized standards. Synthetic TG1 restored fruiting body morphogenesis and sporulation and activated the expression of the developmentally regulated gene with locus tag MXAN_2146 at physiological concentrations, unlike its nearly identical tri-iso15:0 triacylglycerol (TAG) counterpart, which has an ester linkage instead of an ether linkage. iso15:0 FA restored development at physiological concentrations, unlike palmitic acid, a straight-chain fatty acid. The addition of either lipid stimulates cell shortening, with an 87% decline in membrane surface area, concomitantly with the production of lipid bodies at each cell pole and in the center of the cell. We suggest that cells produce triacylglycerol from membrane phospholipids. Bioactive lipids may be released by programmed cell death (PCD), which claims up to 80% of developing cells, since cells undergoing PCD produce lipid bodies before lysing. IMPORTANCE: Like mammalian adipose tissue, many of the M. xanthus lipid body lipids are triacylglycerols (TAGs), containing ester-linked fatty acids. In both systems, ester-linked fatty acids are retrieved from TAGs with lipases and consumed by the fatty acid degradation cycle. Both mammals and M. xanthus also produce lipids containing ether-linked fatty alcohols with alkyl or vinyl linkages, such as plasmalogens. Alkyl and vinyl linkages are not hydrolyzed by lipases, and no clear role has emerged for lipids bearing them. For example, plasmalogen deficiency in mice has detrimental consequences to spermatocyte development, myelination, axonal survival, eye development, and long-term survival, though the precise reasons remain elusive. Lipids containing alkyl- and vinyl-linked fatty alcohols are development-specific products in M. xanthus. Here, we show that one of them rescues the development of E signal-producing mutants at physiological concentrations.

Biosynthesis of the natural fluorophore legioliulin from legionella.

Let it shine: The biosynthesis of the UV fluorophore legioliulin (1) from Legionella spp. was elucidated and the phenylalanine ammonium lyase LglD responsible for the formation of the starter unit cinnamic acid was biochemically characterized. Additionally, two novel derivatives differing in the starter unit have been identified by mutasynthesis experiments.

The endogenous tryptophan metabolite and NAD+ precursor quinolinic acid confers resistance of gliomas to oxidative stress.

Quinolinic acid is a product of tryptophan degradation and may serve as a precursor for NAD(+), an important enzymatic cofactor for enzymes such as the DNA repair protein PARP. Pathologic accumulation of quinolinic acid has been found in neurodegenerative disorders including Alzheimer and Huntington disease, where it is thought to be toxic for neurons by activating the N-methyl-D-aspartate (NMDA) receptor and inducing excitotoxicity. Although many tumors including gliomas constitutively catabolize tryptophan, it is unclear whether quinolinic acid is produced in gliomas and whether it is involved in tumor progression. Here, we show that quinolinic acid accumulated in human gliomas and was associated with a malignant phenotype. Quinolinic acid was produced by microglial cells, as expression of the quinolinic acid-producing enzyme 3-hydroxyanthranilate oxygenase (3-HAO) was confined to microglia in glioma tissue. Human malignant glioma cells, but not nonneoplastic astrocytes, expressed quinolinic acid phosphoribosyltransferase (QPRT) to use quinolinic acid for NAD(+) synthesis and prevent apoptosis when de novo NAD(+) synthesis was blocked. Oxidative stress, temozolomide, and irradiation induced QPRT in glioma cells. QPRT expression increased with malignancy. In recurrent glioblastomas after radiochemotherapy, QPRT expression was associated with a poor prognosis in two independent datasets. Our data indicate that neoplastic transformation in astrocytes is associated with a QPRT-mediated switch in NAD(+) metabolism by exploiting microglia-derived quinolinic acid as an alternative source of replenishing intracellular NAD(+) pools. The elevated levels of QPRT expression increase resistance to oxidative stress induced by radiochemotherapy, conferring a poorer prognosis. These findings have implications for therapeutic approaches inducing intracellular NAD(+) depletion, such as alkylating agents or direct NAD(+) synthesis inhibitors, and identify QPRT as a potential therapeutic target in malignant gliomas.