Ladderane Lipids Research Articles

Loss of nitrogen via anaerobic ammonium oxidation (anammox) in the California Current system during the late Quaternary

Abstract. The California Current system (CCS) hosts one of the largest oxygen minimum zones (OMZs) in the world: the eastern North Pacific (ENP) OMZ, which is dissociated into subtropical and tropical regions (i.e. the ESTNP and ETNP). In the modern ENP OMZ, bioavailable nitrogen (N) is lost via denitrification and anaerobic ammonium oxidation (anammox). Even so, paleo-reconstructions of N loss have focused solely on denitrification. Fluctuations in bulk sedimentary δ15N over glacial–interglacial cycles have been interpreted to reflect variations in denitrification rates in response to ETNP OMZ intensity changes. This δ15N signal is thought to be transported northwards to the ESTNP OMZ. Here, we present the first CCS sedimentary record of ladderane lipids, biomarkers for anammox, located within the ESTNP OMZ (32° N, 118° W). Over the last two glacial terminations (∼ 160 kyr cal BP), ladderane concentrations were analysed in combination with the index of ladderanes with five cyclobutane moieties (NL5), short-chain (SC) ladderane degradation products, and productivity proxies. This shows that (1) ladderanes were derived from anammox bacteria living within the ESTNP OMZ water column; (2) ladderanes were continuously present, with relatively high concentrations during both glacial and interglacial periods, showcasing that the ESTNP OMZ must have retained an anoxic core in which N loss occurred; and (3) anammox abundance appears to have been driven by both organic matter (OM) remineralization and advection changes, which regulated nutrient and oxygen levels. Our study shows that anammox was an important feature in the CCS, and it provides a more holistic picture of N-loss dynamics and the development of the ESTNP OMZ over glacial–interglacial cycles. Lastly, ladderanes and their SC products were also detected in 160–500 kyr cal BP sediments (15.7–37.5 m b.s.f., metres below sea floor; analysed at a low temporal resolution), highlighting their potential as anammox biomarkers in relatively deeper buried sediments for future studies.

Structures of an unusual 3-hydroxyacyl dehydratase (FabZ) from a ladderane-producing organism with an unexpected substrate preference

The genomes of anaerobic ammonium-oxidizing (anammox) bacteria contain a gene cluster comprising genes of unusual fatty acid biosynthesis enzymes that were suggested to be involved in the synthesis of the unique "ladderane" lipids produced by these organisms. This cluster encodes an acyl carrier protein (denoted as "amxACP") and a variant of FabZ, an ACP-3-hydroxyacyl dehydratase. In this study, we characterize this enzyme, which we call anammox-specific FabZ ("amxFabZ"), to investigate the unresolved biosynthetic pathway of ladderane lipids. We find that amxFabZ displays distinct sequence differences to "canonical" FabZ, such as a bulky, apolar residue on the inside of the substrate binding tunnel, where the canonical enzyme has a glycine. Additionally, substrate screens suggest that amxFabZ efficiently converts substrates with acyl chain lengths of up to eight carbons, whereas longer substrates are converted much more slowly under the conditions used. We also present crystal structures of amxFabZs, mutational studies and the structure of a complex between amxFabZ and amxACP, which show that the structures alone cannot explain the apparent differences from canonical FabZ. Moreover, we find that while amxFabZ does dehydrate substrates bound to amxACP, it does not convert substrates bound to canonical ACP of the same anammox organism. We discuss the possible functional relevance of these observations in the light of proposals for the mechanism for ladderane biosynthesis.

Anammox Bacterial S-Adenosyl-l-Methionine Dependent Methyltransferase Crystal Structure and Its Interaction with Acyl Carrier Proteins

Ladderane lipids (found in the membranes of anaerobic ammonium-oxidizing [anammox] bacteria) have unique ladder-like hydrophobic groups, and their highly strained exotic structure has attracted the attention of scientists. Although enzymes encoded in type II fatty acid biosynthesis (FASII) gene clusters in anammox bacteria, such as S-adenosyl-l-methionine (SAM)-dependent enzymes, have been proposed to construct a ladder-like structure using a substrate connected to acyl carrier protein from anammox bacteria (AmxACP), no experimental evidence to support this hypothesis was reported to date. Here, we report the crystal structure of a SAM-dependent methyltransferase from anammox bacteria (AmxMT1) that has a substrate and active site pocket between a class I SAM methyltransferase-like core domain and an additional α-helix inserted into the core domain. Structural comparisons with homologous SAM-dependent C-methyltransferases in polyketide synthase, AmxACP pull-down assays, AmxACP/AmxMT1 complex structure predictions by AlphaFold, and a substrate docking simulation suggested that a small compound connected to AmxACP could be inserted into the pocket of AmxMT1, and then the enzyme transfers a methyl group from SAM to the substrate to produce branched lipids. Although the enzymes responsible for constructing the ladder-like structure remain unknown, our study, for the first time, supports the hypothesis that biosynthetic intermediates connected to AmxACP are processed by SAM-dependent enzymes, which are not typically involved in the FASII system, to produce the ladder-like structure of ladderane lipids in anammox bacteria.

Amino acid (acyl carrier protein) ligase-associated biosynthetic gene clusters reveal unexplored biosynthetic potential.

This study aimed to evaluate the postulated cellular function of a novel family of amino acid (acyl carrier protein) ligases (AALs) in natural product biosynthesis. Here, we analyzed the manually curated, putative, aal-associated natural product biosynthetic gene clusters (NP BGCs) using two computational platforms for NP prediction, antiSMASH-BiG-SCAPE-CORASON and DeepBGC. The detected BGCs included a diversity of type I polyketide/nonribosomal peptide (PKS/NRPS) hybrid BGCs, exemplified by the guadinomine BGC, which suggested a dedicated function of AALs in the biosynthesis of rare (2S)-aminomalonyl-ACP extension units. Besides modular PKS/NRPSs and NRPSs, AAL-associated BGCs were predicted to assemble arylpolyenes, ladderane lipids, phosphonates, aminoglycosides, β-lactones, and thioamides of both nonribosomal and ribosomal origins. Additionally, we revealed a frequent association of AALs with putative, seldom observed transglutaminase-like and BtrH-like transferases of the cysteine protease superfamily, which may form larger families of ACP-dependent amide bond catalysts used in NP synthesis. Our results disclosed an exceptional chemical novelty and biosynthetic potential of the AAL-associated BGCs in NP biosynthesis. The presented in silico evidence supports the initial hypothesis and provides an important foundation for future experimental studies aimed at discovering novel pharmaceutically relevant active compounds.

Inorganic quantum dots - anammox consortia hybrid for stable nitrogen elimination under high-intensity solar-simulated irradiation

External stimulus such as light irradiation is able to deteriorate intracellular redox homeostasis and induce photooxidative damage to non-photogenic bacteria. Exploiting effective strategies to help bacteria resisting infaust stress is meaningful for achieving a stable operation of biological treatment system. In this work, selenium-doped carbon quantum dots (Se-CQDs) were blended into anaerobic ammonia oxidation (anammox) bacteria and an inorganic nanoparticle-microbe hybrid was successfully fabricated to evaluate its nitrogen removal performance under solar-simulated irradiation. It was found that the specific anammox activity decreased by 29.7 ± 5.2% and reactive oxygen species (ROS) content increased by 134.8 ± 4.1% under 50,000 lux light. Sludge activity could be completely recovered under the optimum dosage of 0.42 mL·(g volatile suspended solid) -1 Se-CQDs. Hydroxyl radical (·OH) and superoxide anion radical (·O2−) were identified as the leading ROS inducing lipid peroxidation and antioxidase function detriment. Also, the structure of ladderane lipids located on anammoxosome was destroyed by ROS and functional genes abundances declined accordingly. Although cell surface coated Se-CQDs could absorb ultraviolet light and partially mitigated the photoinhibition, the direct scavenging of ROS by intracellular Se-CQDs primarily contributed to the cellular redox homeostasis, antioxidase activity recovery and sludge activity improvement. The findings of this work provide in-depth understanding the metabolic response mechanism of anammox consortia to light irradiation and might be valuable for a more stable and sustainable nitrogen removal technology, i.e., algal-bacterial symbiotic system, development.

On anammox activity at low temperature: Effect of ladderane composition and process conditions

The application of partial nitritation-anammox (PN/A) under mainstream conditions can enable substantial cost savings at wastewater treatment plants (WWTPs), but how process conditions and cell physiology affect anammox performance at psychrophilic temperatures below 15 °C remains poorly understood. We tested 14 anammox communities, including 8 from globally-installed PN/A processes, for (i) specific activity at 10–30 °C, (ii) composition of membrane lipids, and (iii) microbial community structure. We observed that membrane composition and cultivation temperature were closely related to the activity of anammox biomasses. The size of ladderane lipids and the content of bacteriohopanoids were key physiological components related to anammox performance at low temperatures. We also indicate that the adaptation of mesophilic cultures to psychrophilic regime necessitates months, but in some cases can take up to 5 years. Interestingly, biomass enriched in the marine genus “Candidatus Scalindua” displayed outstanding potential for nitrogen removal from cold streams. Collectively, our comprehensive study provides essential knowledge of cold adaptation mechanism, will enable more accurate modelling and suggests highly promising target anammox genera for inoculation and set-up of anammox reactors, in particular for mainstream WWTPs.

Effect of temperature on the compositions of ladderane lipids in globally surveyed anammox populations

The adaptation of bacteria involved in anaerobic ammonium oxidation (anammox) to low temperatures will enable more efficient removal of nitrogen from sewage across seasons. At lower temperatures, bacteria typically tune the synthesis of their membrane lipids to promote membrane fluidity. However, such adaptation of anammox bacteria lipids, including unique ladderane phospholipids and especially shorter ladderanes with absent phosphatidyl headgroup, is yet to be described in detail. We investigated the membrane lipids composition (UPLC–HRMS/MS) and dominant anammox populations (16S rRNA gene amplicon sequencing, Fluorescence in situ hybridization) in 14 anammox enrichments cultivated at 10–37 °C. “Candidatus Brocadia” appeared to be the dominant organism in all but two laboratory enrichments of “Ca. Scalindua” and “Ca. Kuenenia”. At lower temperatures, the membranes of all anammox populations were composed of shorter [5]-ladderane ester (reduced chain length demonstrated by decreased fraction of C20/(C18 + C20)). This confirmed the previous preliminary evidence on the prominent role of this ladderane fatty acid in low-temperature adaptation. “Ca. Scalindua” and “Ca. Kuenenia” had distinct profile of ladderane lipids compared to “Ca. Brocadia” biomasses with potential implications for adaptability to low temperatures. “Ca. Brocadia” membranes contained a much lower amount of C18 [5]-ladderane esters than reported in the literature for “Ca. Scalindua” at similar temperature and measured here, suggesting that this could be one of the reasons for the dominance of “Ca. Scalindua” in cold marine environments. Furthermore, we propose additional and yet unreported mechanisms for low-temperature adaptation of anammox bacteria, one of which involves ladderanes with absent phosphatidyl headgroup. In sum, we deepen the understanding of cold anammox physiology by providing for the first time a consistent comparison of anammox-based communities across multiple environments.

Bacteriohopanetetrol-<i>x</i>: constraining its application as a lipid biomarker for marine anammox using the water column oxygen gradient of the Benguela upwelling system

Abstract. Interpreting lipid biomarkers in the sediment archive requires a good understanding of their application and limitations in modern systems. Recently it was discovered that marine bacteria performing anaerobic ammonium oxidation (anammox), belonging to the genus Ca. Scalindua, uniquely synthesize a stereoisomer of bacteriohopanetetrol (“BHT-x”). The ratio of BHT-x over total bacteriohopanetetrol (BHT, ubiquitously synthesized by diverse bacteria) has been suggested as a proxy for water column anoxia. As BHT has been found in sediments over 50 Myr old, BHT-x has the potential to complement and extend the sedimentary biomarker record of marine anammox, conventionally constructed using ladderane lipids. Yet, little is known about the distribution of BHT-x in relation to the distribution of ladderanes and to the genetic evidence of Ca. Scalindua in modern marine systems. Here, we investigate the distribution of BHT-x and the application of the BHT-x ratio in relation to distributions of ladderane intact polar lipids (IPLs), ladderane fatty acids (FAs) and Ca. Scalindua 16S rRNA genes in suspended particulate matter (SPM) from the water column of the Benguela upwelling system (BUS), sampled across a large oxygen gradient. In BUS SPM, high BHT-x abundances were restricted to the oxygen-deficient zone on the continental shelf (at [O2] < 45 µmol L−1, in all but one case). High BHT-x abundances co-occurred with high abundances of the Ca. Scalindua 16S rRNA gene (relative to the total number of bacterial 16S rRNA genes) and ladderane IPLs. At shelf stations with [O2] > 50 µmol L−1, the BHT-x ratio was < 0.04 (in all but one case). In apparent contradiction, ladderane FAs and low abundances of BHT and BHT-x (resulting in BHT-x ratios > 0.04) were also detected in oxygenated offshore waters ([O2] up to 180 µmol L−1), whereas ladderane IPLs were undetected. The index of ladderane lipids with five cyclobutane rings (NL5) correlates with in situ temperature. NL5-derived temperatures suggested that ladderane FAs in the offshore waters were not synthesized in situ but were transported down-slope from warmer shelf waters. Thus, in sedimentary archives of systems with known lateral organic matter transport, such as the BUS, relative BHT and BHT-x abundances should be carefully considered. In such systems, a higher BHT-x ratio may act as a safer threshold for deoxygenation and/or Ca. Scalindua presence: our results and previous studies indicate that a BHT-x ratio of ≥ 0.2 is a robust threshold for oxygen-depleted waters ([O2] < 50 µmol kg−1). In our data, ratios of ≥ 0.2 coincided with Ca. Scalindua 16S rRNA genes in all samples (n=62), except one. Lastly, when investigating in situ anammox, we highlight the importance of using ladderane IPLs over BHT-x and/or ladderane FAs; these latter compounds are more recalcitrant and may derive from transported fossil anammox bacteria remnants.

Adaptation of anammox bacteria to low temperature via gradual acclimation and cold shocks: Distinctions in protein expression, membrane composition and activities

Anammox bacteria enable efficient removal of nitrogen from sewage in processes involving partial nitritation and anammox (PN/A) or nitrification, partial denitrification, and anammox (N-PdN/A). In mild climates, anammox bacteria must be adapted to ≤15 °C, typically by gradual temperature decrease; however, this takes months or years. To reduce the time necessary for the adaptation, an unconventional method of ‘cold shocks’ is promising, involving hours-long exposure of anammox biomass to extremely low temperatures. We compared the efficacies of gradual temperature decrease and cold shocks to increase the metabolic activity of anammox (fed batch reactor, planktonic “Ca. Kuenenia”). We assessed the cold shock mechanism on the level of protein expression (quantitative shot-gun proteomics, LCHRMS/MS) and the structure of membrane lipids (UPLCHRMS/MS). The shocked culture was more active (0.66±0.06 vs 0.48±0.06 kg-N/kg-VSS/d) and maintained the relative content of N-respiration proteins at levels consistent levels with the initial state, whereas the content of these proteins decreased in gradually acclimated culture. Cold shocks also induced a more efficient expression of potential cold shock proteins (e.g. ppiD, UspA, pqqC), while putative cold shock proteins CspB and TypA were upregulated in both cultures. Ladderane lipids characteristic for anammox evolved to a similar end-point in both cultures; this confirms their role in anammox bacteria adaptation to cold and indicates a three-pronged adaptation mechanism (ladderane alkyl length, introduction of shorter non-ladderane alkyls, polar headgroup). Overall, we show the outstanding potential of cold shocks for low-temperature adaptation of anammox bacteria and provide yet unreported detailed mechanisms of anammox adaptation to low temperatures.

Purification of the key enzyme complexes of the anammox pathway from DEMON sludge.

Anaerobic Ammonium Oxidation ("anammox") is a bacterial process in which nitrite and ammonium are converted into nitrogen gas and water, yielding energy for the cell. Anammox is an important branch of the global biological nitrogen cycle, being responsible for up to 50% of the yearly nitrogen removal from the oceans. Strikingly, the anammox process uniquely relies on the extremely reactive and toxic compound hydrazine as a free intermediate. Given its global importance and biochemical novelty, there is considerable interest in the enzymes at the heart of the anammox pathway. Unfortunately, obtaining these enzymes in sufficiently large amounts for biochemical and structural studies is problematic, given the slow growth of pure cultures of anammox bacteria when high cell densities are required. However, the anammox process is being applied in wastewater treatment to remove nitrogenous waste in processes like DEamMONification (DEMON). In plants using such processes, which rely on a combination of aerobic ammonia-oxidizers and anammox organisms, kilogram amounts of anammox bacteria-containing sludge are readily available. Here, we report a protein isolation protocol starting from anammox cells present in DEMON sludge from a wastewater treatment plan that readily yields pure preparations of key anammox proteins in the tens of milligrams, including hydrazine synthase HZS and hydrazine dehydrogenase (HDH), as well as hydroxylamine oxidoreductase (HAO). HDH and HAO were active and of sufficient quality for biochemical studies and for HAO, the crystal structure could be determined. The method presented here provides a viable way to obtain materials for the study of proteins not only from the central anammox metabolism but also for the study of other exciting aspects of anammox bacteria, such as for example, their unusual ladderane lipids.

Ladderane records over the last century in the East China sea: Proxies for anammox and eutrophication changes

Anaerobic ammonium oxidation (anammox), an important process for converting fixed nitrogen to N2, plays an important role in the present-day marine nitrogen cycle. However, little is known about anammox activities in the past, especially in regions that were strongly affected by human activities, evidenced by eutrophication and hypoxia, which promote anammox bacteria growth. In this study, ladderanes have been measured in a sediment core and suspended particulate matter (SPM) in the East China Sea (ECS), to reconstruct the anammox record and to evaluate its responses to eutrophication and hypoxia. The detection and distribution of different ladderane lipids in SPM provide additional evidence that ladderanes were mostly produced in the water column and could reflect anammox activities. Summed ladderane content from the core varied between 11 and 300 ng/g dry weight (dw) sediment, with C20-[5]-ladderane fatty acid methyl esters (FAME) as the predominant compound (5–150 ng/g dw), followed by C20-[3]-ladderane FAME (1–110 ng/g dw), C18-[3]-ladderane FAME (1–32 ng/g dw) and C18 -[5]-ladderane FAME (3–11 ng/g dw). The detection of ladderanes over the last century indicate the existence of anammox in the past. The rapidly increasing trend of ladderanes since the 1960s correlates with an increase in phytoplankton biomarkers (Σ(B + D + A), brassicasterol (B), dinosterol (D) and C37 alkenones (A)), indicating that eutrophication exacerbated anammox growth. The co-variation between our ladderane record and published records of low-oxygen tolerant foraminiferal microfossils and hypoxia events over the past 60 years suggested that sediment ladderanes are a useful indicator for past changes of oxygen depletion or hypoxia in the ECS.

Description of “Candidatus Jettenia ecosi” sp. nov., a New Species of Anammox Bacteria

A new species of anammox bacteria, “Candidatus Jettenia ecosi,” was identified in the microbial community of a lab-scale vertical anaerobic upflow bioreactor fed with mineral medium and the biomass immobilized on a brush-shaped carrier. The reactor was inoculated with activated sludge from a denitrifying bioreactor of a municipal wastewater treatment station in the valley of the Mzymta River (Krasnodar krai, Russia). At constant increase of concentrations of the substrates for the anammox process ( $${\text{NH}}_{4}^{ + }$$ and $${\text{NO}}_{2}^{ - }$$ ) in the course of five years, a microbial community containing a new species of anammox bacteria “Candidatus Jettenia ecosi” developed in the bioreactor. Stable activity in a wide range of substrate concentrations (0.02 to 5.6 g N/L), рН (7.2 to 8.8), and under microaerophilic conditions (3% oxygen in the gas phase) were the remarkable features of the new species. Optimal growth temperature was 30°C. Doubling time of physiologically active anammox bacteria was 13 days. Cells of the new bacteria (~1 µm in diameter) exhibited the typical anammox morphology and ultrastructure. The cells had a tendency for attached growth and formation of biofilms. Hopanoids and ladderane lipids, which are among the key markers of this microorganisms, were found in the membranes of the new anammox bacteria. According to the results of the 16S rRNA gene sequencing, the new bacteria belong to the candidate genus “Candidatus Jettenia,” phylum Planctomycetes with the proposed name “Candidatus Jettenia ecosi” sp. nov.

Ladderane phospholipids form a densely packed membrane with normal hydrazine and anomalously low proton/hydroxide permeability

Ladderane lipids are unique to anaerobic ammonium-oxidizing (anammox) bacteria and are enriched in the membrane of the anammoxosome, an organelle thought to compartmentalize the anammox process, which involves the toxic intermediate hydrazine (N2H4). Due to the slow growth rate of anammox bacteria and difficulty of isolating pure ladderane lipids, experimental evidence of the biological function of ladderanes is lacking. We have synthesized two natural and one unnatural ladderane phosphatidylcholine lipids and compared their thermotropic properties in self-assembled bilayers to distinguish between [3]- and [5]-ladderane function. We developed a hydrazine transmembrane diffusion assay using a water-soluble derivative of a hydrazine sensor and determined that ladderane membranes are as permeable to hydrazine as straight-chain lipid bilayers. However, pH equilibration across ladderane membranes occurs 5-10 times more slowly than across straight-chain lipid membranes. Langmuir monolayer analysis and the rates of fluorescence recovery after photobleaching suggest that dense ladderane packing may preclude formation of proton/hydroxide-conducting water wires. These data support the hypothesis that ladderanes prevent the breakdown of the proton motive force rather than blocking hydrazine transmembrane diffusion in anammox bacteria.

Ladderane Phospholipids Form Dense Membranes with Low Proton Permeability

Anaerobic ammonium oxidizing (anammox) bacteria form a critical part of the nitrogen cycle by converting ammonium and nitrite to nitrogen gas. Anammox bacteria carry out their metabolism in the anammoxosome, a membrane-bound organelle that is comprised of the unique ladderane lipids. Ladderane lipids, named for the ladder-like structure of fused cyclobutane rings in their fatty acid tails, have not been found anywhere else in nature, suggesting that they play a critical role in the anammox metabolism. It has been hypothesized that ladderane lipids prevent the diffusion of hydrazine (an anammox intermediate), protons, or other species across the anammoxosome membrane. As anammox bacteria have not been grown in axenic culture and grow extremely slowly in enrichment cultures, researchers have not been able to isolate sufficient quantities of pure ladderane lipids to determine the biophysical properties of ladderane membranes. Without knowledge of the physical properties of ladderane lipids or genetic tools for studying the lipids in vivo, their biological function remains unknown. We have developed efficient synthetic routes to naturally occurring ladderane phospholipids and unnatural analogs. We show that ladderane lipids have physical properties that are distinct from conventional straight-chain lipids. Ladderane lipids form dense bilayers with slow lateral diffusion and dense monolayers with low compressibility. By varying the identities of the fatty acid tails, we establish structure-function relationships for the different ladderane structures. These physical properties result in membranes with much slower rates of transbilayer diffusion of protons, which are pumped across the anammoxosome membrane and used to produce ATP. These results suggest that ladderane lipids in the anammoxosome may prevent the dissipation of the proton gradient during the slow anammox metabolism. This role for ladderane lipids in the anammoxosome may partially explain why anammox bacteria evolved such unique lipids.

Pulling on polyladderenes

Using mechanical force, rather than light or heat, to transform molecules from one form to another has gained traction in the past decade, creating the field of polymer mechanochemistry. Stanford University chemists have made a polymer that transforms from a nonconducting form to a semiconducting one in response to mechanical force. One day, such a material could find use in mimicking the sense of touch or hearing, in which a mechanical force is converted into an electrical signal. Yan Xia’s group spent two years trying to design the polymer without success. One day Xia was chatting with his Stanford colleague Noah Z. Burns, when Burns showed him the ladderane lipids his group had been synthesizing. The two realized these ladder-like fused cyclobutanes would be a great motif for the polymer Xia envisioned. When subjected to mechanical force, they imagined that the σ bonds in the cyclobutanes would unzip to form

Specificity of Intact Ladderane Lipids in Anaerobic Ammonium Oxidizing Bacteria from Four Reactors

Intact ladderane lipids are a special kind of phospholipids in the anammoxosome membrane of AnAOB.Phospholipid organic mixture was extracted respectively from four sludge samples in anaerobic EGSB reactor,aerobic SBR reactor,autotrophic nitrogen removal SBBR reactor and anaerobic ammonium oxidation EGSB reactor.Phospholipid extracts of sludge were detected by the HPLC-ESI-MS/MS method to determine the types of intact ladderane lipids,and the specificity of intact ladderane lipids was also analyzed,at the same time,the bacterial community structure of autotrophic nitrogen removal SBBR reactor and anaerobic ammonium oxidation EGSB reactor was determined by high-throughput sequencing technology.Analytical results of intact ladderane lipids showed that intact ladderane lipids were not detected from anaerobic EGSB reactor and aerobic SBR reactor,and were detected from autotrophic nitrogen removal SBBR reactor and anaerobic ammonium oxidation EGSB reactor.Five kinds of intact ladderane lipids were detected in the phospholipid extract of sludge from autotrophic nitrogen removal SBBR reactor,and seven kinds of intact ladderane lipids were detected in the phospholipid extract of sludge from anaerobic ammonium oxidation EGSB reactor.The results of high-throughput sequencing confirmed that the autotrophic nitrogen SBBR reactor contained AnAOB of the genera Candidatus Brocadia and Candidatus Jettenia,and the anaerobic ammonium oxidation EGSB reactor contained AnAOB of the genera Candidatus Kuenenia and Candidatus Jettenia.These results illustrated that intact ladderane lipids were the peculiar phospholipids in AnAOB.

Chemical Synthesis and Self-Assembly of a Ladderane Phospholipid.

Ladderane lipids produced by anammox bacteria constitute some of the most structurally fascinating yet poorly studied molecules among biological membrane lipids. Slow growth of the producing organism and the inherent difficulty of purifying complex lipid mixtures have prohibited isolation of useful amounts of natural ladderane lipids. We have devised a highly selective total synthesis of ladderane lipid tails and a full phosphatidylcholine to enable biophysical studies on chemically homogeneous samples of these molecules. Additionally, we report the first proof of absolute configuration of a natural ladderane.

Abundance and Diversity of Denitrifying and Anammox Bacteria in Seasonally Hypoxic and Sulfidic Sediments of the Saline Lake Grevelingen.

Denitrifying and anammox bacteria are involved in the nitrogen cycling in marine sediments but the environmental factors that regulate the relative importance of these processes are not well constrained. Here, we evaluated the abundance, diversity, and potential activity of denitrifying, anammox, and sulfide-dependent denitrifying bacteria in the sediments of the seasonally hypoxic saline Lake Grevelingen, known to harbor an active microbial community involved in sulfur oxidation pathways. Depth distributions of 16S rRNA gene, nirS gene of denitrifying and anammox bacteria, aprA gene of sulfur-oxidizing and sulfate-reducing bacteria, and ladderane lipids of anammox bacteria were studied in sediments impacted by seasonally hypoxic bottom waters. Samples were collected down to 5 cm depth (1 cm resolution) at three different locations before (March) and during summer hypoxia (August). The abundance of denitrifying bacteria did not vary despite of differences in oxygen and sulfide availability in the sediments, whereas anammox bacteria were more abundant in the summer hypoxia but in those sediments with lower sulfide concentrations. The potential activity of denitrifying and anammox bacteria as well as of sulfur-oxidizing, including sulfide-dependent denitrifiers and sulfate-reducing bacteria, was potentially inhibited by the competition for nitrate and nitrite with cable and/or Beggiatoa-like bacteria in March and by the accumulation of sulfide in the summer hypoxia. The simultaneous presence and activity of organoheterotrophic denitrifying bacteria, sulfide-dependent denitrifiers, and anammox bacteria suggests a tight network of bacteria coupling carbon-, nitrogen-, and sulfur cycling in Lake Grevelingen sediments.

Seasonal changes of organic matter origins and anammox activity in the Changjiang Estuary deduced from multi-biomarkers in suspended particulates

Abstract Human activity-induced eutrophication and harmful algal blooms are main causes of the expansion of the hypoxic zone in the Changjiang Estuary. Among the many changes in biogeochemical processes, anaerobic ammonium oxidation (anammox) is proposed to play an important role in the nitrogen cycle in hypoxic areas. Ladderane lipids have been used as biomarkers to indicate anammox activity in ecosystems, but the origins of anammox bacteria and ladderanes in suspended particulates are still unclear. In this study, we report the results of a suite of biomarker analyses of suspended particulates across a salinity gradient of the Changjiang Estuary in both the spring and summer to evaluate the origins of the ladderanes and their potential as proxies for anammox activity and hypoxia. The spatio-temporal variations in terrestrial biomarkers ( n -alkanes and n -alkanols), marine biomarkers (brassicasterol and dinosterol), and the Terrestrial and Marine Biomarker Ratio and Branched and Isoprenoid Tetraether indices reveal that marine organic matter was dominant in the particulates in both the spring (55%) and summer (86%) seasons. Correlations with both marine and terrestrial biomarkers suggest that ladderanes were mainly produced in the water column, and therefore that ladderane concentrations in suspended particulates in the Changjiang Estuary mainly reflect anammox activity in the water column, although changes in anammox bacterial assemblages may also have played a role in ladderane concentrations. Overall, ladderane results suggest that anammox activity was widespread in the Changjiang Estuary; but higher ladderane concentrations in the summer (especially in the upwelling zone) were correlated with lower dissolved oxygen concentrations, which suggest that they are useful proxies for hypoxia.

Investigation of Proposed Ladderane Biosynthetic Genes from Anammox Bacteria by Heterologous Expression in E. coli.

Ladderanes are hydrocarbon chains with three or five linearly concatenated cyclobutane rings that are uniquely produced as membrane lipid components by anammox (anaerobic ammonia-oxidizing) bacteria. By virtue of their angle and torsional strain, ladderanes are unusually energetic compounds, and if produced biochemically by engineered microbes, could serve as renewable, high-energy-density jet fuel components. The biochemistry and genetics underlying the ladderane biosynthetic pathway are unknown, however, previous studies have identified a pool of 34 candidate genes from the anammox bacterium, Kuenenia stuttgartiensis, some or all of which may be involved with ladderane fatty acid biosynthesis. The goal of the present study was to establish a systematic means of testing the candidate genes from K. stuttgartiensis for involvement in ladderane biosynthesis through heterologous expression in E. coli under anaerobic conditions. This study describes an efficient means of assembly of synthesized, codon-optimized candidate ladderane biosynthesis genes in synthetic operons that allows for changes to regulatory element sequences, as well as modular assembly of multiple operons for simultaneous heterologous expression in E. coli (or potentially other microbial hosts). We also describe in vivo functional tests of putative anammox homologs of the phytoene desaturase CrtI, which plays an important role in the hypothesized ladderane pathway, and a method for soluble purification of one of these enzymes. This study is, to our knowledge, the first experimental effort focusing on the role of specific anammox genes in the production of ladderanes, and lays the foundation for future efforts toward determination of the ladderane biosynthetic pathway. Our substantial, but far from comprehensive, efforts at elucidating the ladderane biosynthetic pathway were not successful. We invite the scientific community to take advantage of the considerable synthetic biology resources and experimental results developed in this study to elucidate the biosynthetic pathway that produces unique and intriguing ladderane lipids.