Intrinsic disorder and conformational coexistence in auxin coreceptors.

AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) transcriptional repressor proteins and the TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB) proteins to which they bind act as auxin coreceptors. While the structure of TIR1 has been solved, structural characterization of the regions of the Aux/IAA protein responsible for auxin perception has been complicated by their predicted disorder. Here, we use NMR, CD and molecular dynamics simulation to investigate the N-terminal domains of the Aux/IAA protein IAA17/AXR3. We show that despite the conformational flexibility of the region, a critical W-P bond in the core of the Aux/IAA degron motif occurs at a strikingly high (1:1) ratio of cis to trans isomers, consistent with the requirement of the cis conformer for the formation of the fully-docked receptor complex. We show that the N-terminal half of AXR3 is a mixture of multiple transiently structured conformations with a propensity for two predominant and distinct conformational subpopulations within the overall ensemble. These two states were modeled together with the C-terminal PB1 domain to provide the first complete simulation of an Aux/IAA. Using MD to recreate the assembly of each complex in the presence of auxin, both structural arrangements were shown to engage with the TIR1 receptor, and contact maps from the simulations match closely observations of NMR signal-decreases. Together, our results and approach provide a platform for exploring the functional significance of variation in the Aux/IAA coreceptor family and for understanding the role of intrinsic disorder in auxin signal transduction and other signaling systems.

Chemical induction of hypocotyl rooting reveals extensive conservation of auxin signalling controlling lateral and adventitious root formation.

Upon exposure to light, etiolated Arabidopsis seedlings form adventitious roots (AR) along the hypocotyl. While processes underlying lateral root formation are studied intensively, comparatively little is known about the molecular processes involved in the initiation of hypocotyl AR. AR and LR formation were studied using a small molecule named Hypocotyl Specific Adventitious Root INducer (HYSPARIN) that strongly induces AR but not LR formation. HYSPARIN does not trigger rapid DR5-reporter activation, DII-Venus degradation or Ca2+ signalling. Transcriptome analysis, auxin signalling reporter lines and mutants show that HYSPARIN AR induction involves nuclear TIR1/AFB and plasma membrane TMK auxin signalling, as well as multiple downstream LR development genes (SHY2/IAA3, PUCHI, MAKR4 and GATA23). Comparison of the AR and LR induction transcriptome identified SAURs, AGC kinases and OFP transcription factors as specifically upregulated by HYSPARIN. Members of the SAUR19 subfamily, OFP4 and AGC2 suppress HYS-induced AR formation. While SAUR19 and OFP subfamily members also mildly modulate LR formation, AGC2 regulates only AR induction. Analysis of HYSPARIN-induced AR formation uncovers an evolutionary conservation of auxin signalling controlling LR and AR induction in Arabidopsis seedlings and identifies SAUR19, OFP4 and AGC2 kinase as novel regulators of AR formation.

Structural basis of carnitine monooxygenase CntA substrate specificity, inhibition, and intersubunit electron transfer.

Microbial metabolism of carnitine to trimethylamine (TMA) in the gut can accelerate atherosclerosis and heart disease, and these TMA-producing enzymes are therefore important drug targets. Here, we report the first structures of the carnitine oxygenase CntA, an enzyme of the Rieske oxygenase family. CntA exists in a head-to-tail α3 trimeric structure. The two functional domains (the Rieske and the catalytic mononuclear iron domains) are located >40 Å apart in the same monomer but adjacent in two neighboring monomers. Structural determination of CntA and subsequent electron paramagnetic resonance measurements uncover the molecular basis of the so-called bridging glutamate (E205) residue in intersubunit electron transfer. The structures of the substrate-bound CntA help to define the substrate pocket. Importantly, a tyrosine residue (Y203) is essential for ligand recognition through a π-cation interaction with the quaternary ammonium group. This interaction between an aromatic residue and quaternary amine substrates allows us to delineate a subgroup of Rieske oxygenases (group V) from the prototype ring-hydroxylating Rieske oxygenases involved in bioremediation of aromatic pollutants in the environment. Furthermore, we report the discovery of the first known CntA inhibitors and solve the structure of CntA in complex with the inhibitor, demonstrating the pivotal role of Y203 through a π-π stacking interaction with the inhibitor. Our study provides the structural and molecular basis for future discovery of drugs targeting this TMA-producing enzyme in human gut.

A Glycolipid Glycosyltransferase with Broad Substrate Specificity from the Marine Bacterium "Candidatus Pelagibacter sp." Strain HTCC7211.

In the marine environment, phosphorus availability significantly affects the lipid composition in many cosmopolitan marine heterotrophic bacteria, including members of the SAR11 clade and the Roseobacter clade. Under phosphorus stress conditions, nonphosphorus sugar-containing glycoglycerolipids are substitutes for phospholipids in these bacteria. Although these glycoglycerolipids play an important role as surrogates for phospholipids under phosphate deprivation, glycoglycerolipid synthases in marine microbes are poorly studied. In the present study, we biochemically characterized a glycolipid glycosyltransferase (GTcp) from the marine bacterium "Candidatus Pelagibacter sp." strain HTCC7211, a member of the SAR11 clade. Our results showed that GTcp is able to act as a multifunctional enzyme by synthesizing different glycoglycerolipids with UDP-glucose, UDP-galactose, or UDP-glucuronic acid as sugar donors and diacylglycerol (DAG) as the acceptor. Analyses of enzyme kinetic parameters demonstrated that Mg2+ notably changes the enzyme's affinity for UDP-glucose, which improves its catalytic efficiency. Homology modeling and mutational analyses revealed binding sites for the sugar donor and the diacylglycerol lipid acceptor, which provided insights into the retaining mechanism of GTcp with its GT-B fold. A phylogenetic analysis showed that GTcp and its homologs form a group in the GT4 glycosyltransferase family. These results not only provide new insights into the glycoglycerolipid synthesis mechanism in lipid remodeling but also describe an efficient enzymatic tool for the future synthesis of bioactive molecules. IMPORTANCE The bilayer formed by membrane lipids serves as the containment unit for living microbial cells. In the marine environment, it has been firmly established that phytoplankton and heterotrophic bacteria can replace phospholipids with nonphosphorus sugar-containing glycoglycerolipids in response to phosphorus limitation. However, little is known about how these glycoglycerolipids are synthesized. Here, we determined the biochemical characteristics of a glycolipid glycosyltransferase (GTcp) from the marine bacterium "Candidatus Pelagibacter sp." strain HTCC7211. GTcp and its homologs form a group in the GT4 glycosyltransferase family and can synthesize neutral glycolipids (monoglucosyl-1,2-diacyl-sn-glycerol [MGlc-DAG] and monogalactosyl [MGal]-DAG) and monoglucuronic acid diacylglycerol (MGlcA-DAG). We also uncovered the key residues for DAG binding through molecular docking, site-direct mutagenesis, and subsequent enzyme activity assays. Our data provide new insights into the glycoglycerolipid synthesis mechanism in lipid remodeling.

Seedling developmental defects upon blocking CINNAMATE-4-HYDROXYLASE are caused by perturbations in auxin transport.

The phenylpropanoid pathway serves a central role in plant metabolism, providing numerous compounds involved in diverse physiological processes. Most carbon entering the pathway is incorporated into lignin. Although several phenylpropanoid pathway mutants show seedling growth arrest, the role for lignin in seedling growth and development is unexplored. We use complementary pharmacological and genetic approaches to block CINNAMATE-4-HYDROXYLASE (C4H) functionality in Arabidopsis seedlings and a set of molecular and biochemical techniques to investigate the underlying phenotypes. Blocking C4H resulted in reduced lateral rooting and increased adventitious rooting apically in the hypocotyl. These phenotypes coincided with an inhibition in AUX transport. The upstream accumulation in cis-cinnamic acid was found to be likely to cause polar AUX transport inhibition. Conversely, a downstream depletion in lignin perturbed phloem-mediated AUX transport. Restoring lignin deposition effectively reestablished phloem transport and, accordingly, AUX homeostasis. Our results show that the accumulation of bioactive intermediates and depletion in lignin jointly cause the aberrant phenotypes upon blocking C4H, and demonstrate that proper deposition of lignin is essential for the establishment of AUX distribution in seedlings. Our data position the phenylpropanoid pathway and lignin in a new physiological framework, consolidating their importance in plant growth and development.

Light-Activated Electron Transfer and Catalytic Mechanism of Carnitine Oxidation by Rieske-Type Oxygenase from Human Microbiota.

Oxidation of quaternary ammonium substrate, carnitine by non-heme iron containing Acinetobacter baumannii (Ab) oxygenase CntA/reductase CntB is implicated in the onset of human cardiovascular disease. Herein, we develop a blue-light (365 nm) activation of NADH coupled to electron paramagnetic resonance (EPR) measurements to study electron transfer from the excited state of NADH to the oxidized, Rieske-type, [2Fe-2S]2+ cluster in the AbCntA oxygenase domain with and without the substrate, carnitine. Further electron transfer from one-electron reduced, Rieske-type [2Fe-2S]1+ center in AbCntA-WT to the mono-nuclear, non-heme iron center through the bridging glutamate E205 and subsequent catalysis occurs only in the presence of carnitine. The electron transfer process in the AbCntA-E205A mutant is severely affected, which likely accounts for the significant loss of catalytic activity in the AbCntA-E205A mutant. The NADH photo-activation coupled with EPR is broadly applicable to trap reactive intermediates at low temperature and creates a new method to characterize elusive intermediates in multiple redox-centre containing proteins.

Beyond oil degradation: enzymatic potential of Alcanivorax to degrade natural and synthetic polyesters.

Pristine marine environments are highly oligotrophic ecosystems populated by well-established specialized microbial communities. Nevertheless, during oil spills, low-abundant hydrocarbonoclastic bacteria bloom and rapidly prevail over the marine microbiota. The genus Alcanivorax is one of the most abundant and well-studied organisms for oil degradation. While highly successful under polluted conditions due to its specialized oil-degrading metabolism, it is unknown how they persist in these environments during pristine conditions. Here, we show that part of the Alcanivorax genus, as well as oils, has an enormous potential for biodegrading aliphatic polyesters thanks to a unique and abundantly secreted alpha/beta hydrolase. The heterologous overexpression of this esterase proved a remarkable ability to hydrolyse both natural and synthetic polyesters. Our findings contribute to (i) better understand the ecology of Alcanivorax in its natural environment, where natural polyesters such as polyhydroxyalkanoates (PHA) are produced by a large fraction of the community and, hence, an accessible source of carbon and energy used by the organism in order to persist, (ii) highlight the potential of Alcanivorax to clear marine environments from polyester materials of anthropogenic origin as well as oils, and (iii) the discovery of a new versatile esterase with a high biotechnological potential.

Pinstatic Acid Promotes Auxin Transport by Inhibiting PIN Internalization.

Polar auxin transport plays a pivotal role in plant growth and development. PIN-FORMED (PIN) auxin efflux carriers regulate directional auxin movement by establishing local auxin maxima, minima, and gradients that drive multiple developmental processes and responses to environmental signals. Auxin has been proposed to modulate its own transport by regulating subcellular PIN trafficking via processes such as clathrin-mediated PIN endocytosis and constitutive recycling. Here, we further investigated the mechanisms by which auxin affects PIN trafficking by screening auxin analogs and identified pinstatic acid (PISA) as a positive modulator of polar auxin transport in Arabidopsis (Arabidopsis thaliana). PISA had an auxin-like effect on hypocotyl elongation and adventitious root formation via positive regulation of auxin transport. PISA did not activate SCFTIR1/AFB signaling and yet induced PIN accumulation at the cell surface by inhibiting PIN internalization from the plasma membrane. This work demonstrates PISA to be a promising chemical tool to dissect the regulatory mechanisms behind subcellular PIN trafficking and auxin transport.

Jasmonic Acid Inhibits Auxin-Induced Lateral Rooting Independently of the CORONATINE INSENSITIVE1 Receptor.

Plant root systems are indispensable for water uptake, nutrient acquisition, and anchoring plants in the soil. Previous studies using auxin inhibitors definitively established that auxin plays a central role regulating root growth and development. Most auxin inhibitors affect all auxin signaling at the same time, which obscures an understanding of individual events. Here, we report that jasmonic acid (JA) functions as a lateral root (LR)-preferential auxin inhibitor in Arabidopsis (Arabidopsis thaliana) in a manner that is independent of the JA receptor, CORONATINE INSENSITIVE1 (COI1). Treatment of wild-type Arabidopsis with either (-)-JA or (+)-JA reduced primary root length and LR number; the reduction of LR number was also observed in coi1 mutants. Treatment of seedlings with (-)-JA or (+)-JA suppressed auxin-inducible genes related to LR formation, diminished accumulation of the auxin reporter DR5::GUS, and inhibited auxin-dependent DII-VENUS degradation. A structural mimic of (-)-JA and (+)-coronafacic acid also inhibited LR formation and stabilized DII-VENUS protein. COI1-independent activity was retained in the double mutant of transport inhibitor response1 and auxin signaling f-box protein2 (tir1 afb2) but reduced in the afb5 single mutant. These results reveal JAs and (+)-coronafacic acid to be selective counter-auxins, a finding that could lead to new approaches for studying the mechanisms of LR formation.

Methodological considerations for the identification of choline and carnitine-degrading bacteria in the gut.

The bacterial formation of trimethylamine (TMA) has been linked to cardiovascular disease. This review focuses on the methods employed to investigate the identity of the bacteria responsible for the formation of TMA from dietary choline and carnitine in the human gut. Recent studies have revealed the metabolic pathways responsible for bacterial TMA production, primarily the anaerobic glycyl radical-containing, choline-TMA lyase, CutC and the aerobic carnitine monooxygenase, CntA. Identification of these enzymes has enabled bioinformatics approaches to screen both human-associated bacterial isolate genomes and whole gut metagenomes to determine which bacteria are responsible for TMA formation in the human gut. We centre on several key methodological aspects for identifying the TMA-producing bacteria and report how these pathways can be identified in human gut microbiota through bioinformatics analysis of available bacterial genomes and gut metagenomes.

Manganese Is Essential for PlcP Metallophosphoesterase Activity Involved in Lipid Remodeling in Abundant Marine Heterotrophic Bacteria.

In vast areas of the ocean, microbes must adapt to the availability of scarce nutrients, and a key strategy for reducing the cellular phosphorus (P) quota is to remodel membranes by replacing phospholipids with non-P surrogate lipids. A metallophosphoesterase, PlcP, is essential for lipid remodeling in cosmopolitan marine bacteria of the Roseobacter (e.g., Phaeobacter sp. strain MED193) and SAR11 (e.g., Pelagibacter sp. strain HTCC7211) clades, and transcription of plcP is known to be induced by P limitation. In order to better understand PlcP-mediated lipid remodeling, we sought to characterize PlcP for its metal ion requirement and to determine its selectivity for native bacterial phospholipids. Here, we report the occurrence of a highly conserved binuclear ion center in PlcPs from MED193 and HTCC7211 and show that manganese is the preferred metal for metallophosphoesterase activity. PlcP displayed high activity towards the major bacterial phospholipids, e.g., phosphatidylglycerol but also phosphatidic acid, a key intermediate in phospholipid biosynthesis. In contrast, phosphatidylserine and phosphatidylinositol, both of which are rare lipids in bacteria, are not preferred substrates. These data suggest that PlcP undertakes a generic lipid remodeling role during the cellular response of marine bacteria to P deficiency and that manganese availability may play a key role in regulating the lipid remodeling process.IMPORTANCE Membrane lipids form the structural basis of all cells. In the marine environment, it is well established that phosphorus availability significantly affects lipid composition in cosmopolitan marine bacteria, whereby non-phosphorus-containing lipids are used to replace phospholipids in response to phosphorus stress. Central to this lipid remodeling pathway is a newly identified phospholipase C-type metallophosphoesterase (PlcP). However, little is known about how PlcP activity is regulated. Here, we determined the role of metal ions in regulating PlcP activity and compared PlcP substrate specificities in PlcP enzymes from two model marine bacteria from the marine Roseobacter clade and the SAR11 clade. Our data provide new insights into the regulation of lipid remodeling in these marine bacteria.

Auxin molecular field maps define AUX1 selectivity: many auxin herbicides are not substrates.

Developmental responses to auxin are regulated by facilitated uptake and efflux, but detailed molecular understanding of the carrier proteins is incomplete. We have used pharmacological tools to explore the chemical space that defines substrate preferences for the auxin uptake carrier AUX1. Total and partial loss-of-function aux1 mutants were assessed against wild-type for dose-dependent resistance to a range of auxins and analogues. We then developed an auxin accumulation assay with associated mathematical modelling to enumerate accurate IC50 values for a small library of auxin analogues. The structure activity relationship data were analysed using molecular field analyses to create a pharmacophoric atlas of AUX1 substrates. The uptake carrier exhibits a very high level of selectivity towards small substrates including the natural indole-3-acetic acid, and the synthetic auxin 2,4-dichlorophenoxyacetic acid. No AUX1 activity was observed for herbicides based on benzoic acid (dicamba), pyridinyloxyacetic acid (triclopyr) or the 6-arylpicolinates (halauxifen), and very low affinity was found for picolinic acid-based auxins (picloram) and quinolinecarboxylic acids (quinclorac). The atlas demonstrates why some widely used auxin herbicides are not, or are very poor substrates. We list molecular descriptors for AUX1 substrates and discuss our findings in terms of herbicide resistance management.

cis-Cinnamic Acid Is a Novel, Natural Auxin Efflux Inhibitor That Promotes Lateral Root Formation.

Auxin steers numerous physiological processes in plants, making the tight control of its endogenous levels and spatiotemporal distribution a necessity. This regulation is achieved by different mechanisms, including auxin biosynthesis, metabolic conversions, degradation, and transport. Here, we introduce cis-cinnamic acid (c-CA) as a novel and unique addition to a small group of endogenous molecules affecting in planta auxin concentrations. c-CA is the photo-isomerization product of the phenylpropanoid pathway intermediate trans-CA (t-CA). When grown on c-CA-containing medium, an evolutionary diverse set of plant species were shown to exhibit phenotypes characteristic for high auxin levels, including inhibition of primary root growth, induction of root hairs, and promotion of adventitious and lateral rooting. By molecular docking and receptor binding assays, we showed that c-CA itself is neither an auxin nor an anti-auxin, and auxin profiling data revealed that c-CA does not significantly interfere with auxin biosynthesis. Single cell-based auxin accumulation assays showed that c-CA, and not t-CA, is a potent inhibitor of auxin efflux. Auxin signaling reporters detected changes in spatiotemporal distribution of the auxin response along the root of c-CA-treated plants, and long-distance auxin transport assays showed no inhibition of rootward auxin transport. Overall, these results suggest that the phenotypes of c-CA-treated plants are the consequence of a local change in auxin accumulation, induced by the inhibition of auxin efflux. This work reveals a novel mechanism how plants may regulate auxin levels and adds a novel, naturally occurring molecule to the chemical toolbox for the studies of auxin homeostasis.

A cheminformatics review of auxins as herbicides.

Herbicides are an important asset in ensuring food security, especially when faced with an ever-increasing demand on food production to feed the global population. The current selection of herbicides is increasingly encountering resistance in agricultural weeds they once targeted effectively. It is imperative that new compounds or more effective modes of action are discovered in order to overcome this resistance. This cheminformatics review looks at current herbicides and evaluates their physiochemical properties on a class-by-class basis. We focus in particular on the synthetic auxin herbicides, Herbicide Resistance Action Committee class O, analyzing these against herbicides more generally and for class-specific features such as mobility in plant vasculature. We summarise the physiochemical properties of all 24 compounds used commercially as auxins and relate these results to ongoing approaches to novel auxin discovery. We introduce an interactive, open source cheminformatics tool known as DataWarrior for herbicide discovery, complete with records for over 300 herbicidal compounds. We hope this tool helps researchers as part of a rational approach to not only auxin discovery but agrochemical discovery in general.

The Allelochemical MDCA Inhibits Lignification and Affects Auxin Homeostasis.

The phenylpropanoid 3,4-(methylenedioxy)cinnamic acid (MDCA) is a plant-derived compound first extracted from roots of Asparagus officinalis and further characterized as an allelochemical. Later on, MDCA was identified as an efficient inhibitor of 4-COUMARATE-CoA LIGASE (4CL), a key enzyme of the general phenylpropanoid pathway. By blocking 4CL, MDCA affects the biosynthesis of many important metabolites, which might explain its phytotoxicity. To decipher the molecular basis of the allelochemical activity of MDCA, we evaluated the effect of this compound on Arabidopsis thaliana seedlings. Metabolic profiling revealed that MDCA is converted in planta into piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H), the enzyme directly upstream of 4CL. The inhibition of C4H was also reflected in the phenolic profile of MDCA-treated plants. Treatment of in vitro grown plants resulted in an inhibition of primary root growth and a proliferation of lateral and adventitious roots. These observed growth defects were not the consequence of lignin perturbation, but rather the result of disturbing auxin homeostasis. Based on DII-VENUS quantification and direct measurement of cellular auxin transport, we concluded that MDCA disturbs auxin gradients by interfering with auxin efflux. In addition, mass spectrometry was used to show that MDCA triggers auxin biosynthesis, conjugation, and catabolism. A similar shift in auxin homeostasis was found in the c4h mutant ref3-2, indicating that MDCA triggers a cross talk between the phenylpropanoid and auxin biosynthetic pathways independent from the observed auxin efflux inhibition. Altogether, our data provide, to our knowledge, a novel molecular explanation for the phytotoxic properties of MDCA.

Characterisation of an unusual cysteine pair in the Rieske carnitine monooxygenase CntA catalytic site.

Rieske monooxygenases undertake complex catalysis integral to marine, terrestrial and human gut-ecosystems. Group-I to -IV Rieske monooxygenases accept aromatic substrates and have well-characterised catalytic mechanisms. Nascent to our understanding are Group-V members catalysing the oxidation/breakdown of quaternary ammonium substrates. Phylogenetic analysis of Group V highlights a cysteine residue-pair adjacent to the mononuclear Fe active site with no established role. Following our elucidation of the carnitine monooxygenase CntA structure, we probed the function of the cysteine pair Cys206/Cys209. Utilising biochemical and biophysical techniques, we found the cysteine residues do not play a structural role nor influence the electron transfer pathway, but rather are used in a nonstoichiometric role to ensure the catalytic iron centre remains in an Fe(II) state.

Aminolipids elicit functional trade-offs between competitiveness and bacteriophage attachment in Ruegeria pomeroyi.

Lipids play a crucial role in maintaining cell integrity and homeostasis with the surrounding environment. Cosmopolitan marine roseobacter clade (MRC) and SAR11 clade bacteria are unique in that, in addition to glycerophospholipids, they also produce an array of amino acid-containing lipids that are conjugated with beta-hydroxy fatty acids through an amide bond. Two of these aminolipids, the ornithine aminolipid (OL) and the glutamine aminolipid (QL), are synthesized using the O-acetyltransferase OlsA. Here, we demonstrate that OL and QL are present in both the inner and outer membranes of the Gram-negative MRC bacterium Ruegeria pomeroyi DSS-3. In an olsA mutant, loss of these aminolipids is compensated by a concurrent increase in glycerophospholipids. The inability to produce aminolipids caused significant changes in the membrane proteome, with the membrane being less permeable and key nutrient transporters being downregulated while proteins involved in the membrane stress response were upregulated. Indeed, the import of 14C-labelled choline and dimethylsulfoniopropionate, as a proxy for the transport of key marine nutrients across membranes, was significantly impaired in the olsA mutant. Moreover, the olsA mutant was significantly less competitive than the wild type (WT) being unable to compete with the WT strain in co-culture. However, the olsA mutant unable to synthesize these aminolipids is less susceptible to phage attachment. Together, these data reveal a critical role for aminolipids in the ecophysiology of this important clade of marine bacteria and a trade-off between growth and avoidance of bacteriophage attachment.

Bacterial catabolism of membrane phospholipids links marine biogeochemical cycles.

In marine systems, the availability of inorganic phosphate can limit primary production leading to bacterial and phytoplankton utilization of the plethora of organic forms available. Among these are phospholipids that form the lipid bilayer of all cells as well as released extracellular vesicles. However, information on phospholipid degradation is almost nonexistent despite their relevance for biogeochemical cycling. Here, we identify complete catabolic pathways for the degradation of the common phospholipid headgroups phosphocholine (PC) and phosphorylethanolamine (PE) in marine bacteria. Using Phaeobacter sp. MED193 as a model, we provide genetic and biochemical evidence that extracellular hydrolysis of phospholipids liberates the nitrogen-containing substrates ethanolamine and choline. Transporters for ethanolamine (EtoX) and choline (BetT) are ubiquitous and highly expressed in the global ocean throughout the water column, highlighting the importance of phospholipid and especially PE catabolism in situ. Thus, catabolic activation of the ethanolamine and choline degradation pathways, subsequent to phospholipid metabolism, specifically links, and hence unites, the phosphorus, nitrogen, and carbon cycles.

A novel class of sulfur-containing aminolipids widespread in marine roseobacters.

Marine roseobacter group bacteria are numerically abundant and ecologically important players in ocean ecosystems. These bacteria are capable of modifying their membrane lipid composition in response to environmental change. Remarkably, a variety of lipids are produced in these bacteria, including phosphorus-containing glycerophospholipids and several amino acid-containing aminolipids such as ornithine lipids and glutamine lipids. Here, we present the identification and characterization of a novel sulfur-containing aminolipid (SAL) in roseobacters. Using high resolution accurate mass spectrometry, a SAL was found in the lipid extract of Ruegeria pomeroyi DSS-3 and Phaeobacter inhibens DSM 17395. Using comparative genomics, transposon mutagenesis and targeted gene knockout, we identified a gene encoding a putative lyso-lipid acyltransferase, designated salA, which is essential for the biosynthesis of this SAL. Multiple sequence analysis and structural modeling suggest that SalA is a novel member of the lysophosphatidic acid acyltransferase (LPAAT) family, the prototype of which is the PlsC acyltransferase responsible for the biosynthesis of the phospholipid phosphatidic acid. SAL appears to play a key role in biofilm formation in roseobacters. salA is widely distributed in Tara Oceans metagenomes and actively expressed in Tara Oceans metatranscriptomes. Our results raise the importance of sulfur-containing membrane aminolipids in marine bacteria.

A novel ATP dependent dimethylsulfoniopropionate lyase in bacteria that releases dimethyl sulfide and acryloyl-CoA.

Dimethylsulfoniopropionate (DMSP) is an abundant and ubiquitous organosulfur molecule in marine environments with important roles in global sulfur and nutrient cycling. Diverse DMSP lyases in some algae, bacteria, and fungi cleave DMSP to yield gaseous dimethyl sulfide (DMS), an infochemical with important roles in atmospheric chemistry. Here, we identified a novel ATP-dependent DMSP lyase, DddX. DddX belongs to the acyl-CoA synthetase superfamily and is distinct from the eight other known DMSP lyases. DddX catalyses the conversion of DMSP to DMS via a two-step reaction: the ligation of DMSP with CoA to form the intermediate DMSP-CoA, which is then cleaved to DMS and acryloyl-CoA. The novel catalytic mechanism was elucidated by structural and biochemical analyses. DddX is found in several Alphaproteobacteria, Gammaproteobacteria, and Firmicutes, suggesting that this new DMSP lyase may play an overlooked role in DMSP/DMS cycles.

The global sulfur cycle is a collection of geological and biological processes that circulate sulfur-containing compounds through the oceans, rocks and atmosphere. Sulfur itself is essential for life and important for plant growth, hence its widespread use in fertilizers. Marine organisms such as bacteria, algae and phytoplankton produce one particular sulfur compound, called dimethylsulfoniopropionate, or DMSP, in massive amounts. DMSP made in the oceans gets readily converted into a gas called dimethyl sulfide (DMS), which is the largest natural source of sulfur entering the atmosphere. In the air, DMS is converted to sulfate and other by-products that can act as cloud condensation nuclei, which, as the name suggests, are involved in cloud formation. In this way, DMS can influence weather and climate, so it is often referred to as 'climate-active' gas. At least eight enzymes are known to cleave DMSP into DMS gas with a few by-products. These enzymes are found in algae, bacteria and fungi, and are referred to as lyases, for the way they breakdown their target compounds (DMSP, in this case). Recently, researchers have identified some bacteria that produce DMS from DMSP without using known DMSP lyases. This suggests there are other, unidentified enzymes that act on DMSP in nature, and likely contribute to global sulfur cycling. Li, Wang et al. set out to uncover new enzymes responsible for converting the DMSP that marine bacteria produce into gaseous DMS. One new enzyme called DddX was identified and found to belong to a superfamily of enzymes quite separate to other known DMSP lyases. Li, Wang et al. also showed how DddX drives the conversion of DMSP to DMS in a two-step reaction, and that the enzyme is found across several classes of bacteria. Further experiments to characterise the protein structure of DddX also revealed the molecular mechanism for its catalytic action. This study offers important insights into how marine bacteria generate the climatically important gas DMS from DMSP, leading to a better understanding of the global sulfur cycle. It gives microbial ecologists a more comprehensive perspective of these environmental processes, and provides biochemists with data on a family of enzymes not previously known to act on sulfur-containing compounds.