Ladderane lipid distribution in four genera of anammox bacteria

Intact ladderane phospholipids and core lipids were studied in four species of anaerobic ammonium oxidizing (anammox) bacteria, each representing one of the four known genera. Each species of anammox bacteria contained C(18) and C(20) ladderane fatty acids with either 3 or 5 linearly condensed cyclobutane rings and a ladderane monoether containing a C(20) alkyl moiety with 3 cyclobutane rings. The presence of ladderane lipids in all four anammox species is consistent with their putative physiological role to provide a dense membrane around the anammoxosome, the postulated site of anammox catabolism. In contrast to the core lipids, large variations were observed in the distribution of ladderane phospholipids, i.e. different combinations of hydrophobic tail (ladderane, straight chain and methyl branched fatty acid) types attached to the glycerol backbone sn-1 position, in combination with different types of polar headgroup (phosphocholine, phosphoethanolamine or phosphoglycerol) attached to the sn-3 position. Intact ladderane lipids made up a high percentage of the lipid content in the cells of "Candidatus Kuenenia stuttgartiensis", suggesting that ladderane lipids are also present in membranes other than the anammoxosome. Finally, all four investigated species contained a C(27) hopanoid ketone and bacteriohopanetetrol, which, indicates that hopanoids are anaerobically synthesised by anammox bacteria.     

16S rRNA gene and lipid biomarker evidence for anaerobic ammonium-oxidizing bacteria (anammox) in California and Nevada hot springs

Anammox, the oxidation of ammonium with nitrite to dinitrogen gas under anoxic conditions, is an important process in mesophilic environments such as wastewaters, oceans and freshwater systems, but little is known of this process at elevated temperatures. In this study, we investigated anammox in microbial mats and sediments obtained from several hot springs in California and Nevada, using geochemical and molecular microbiological methods. Anammox bacteria-specific ladderane core lipids with concentrations ranging between 0.3 and 52 ng g⁻¹ sediment were detected in five hot springs analyzed with temperatures up to 65 °C. In addition, 16S rRNA gene analysis showed the presence of genes phylogenetically related to the known anammox bacteria Candidatus Brocadia fulgida, Candidatus Brocadia anammoxidans and Candidatus Kuenenia stuttgartiensis (96.5–99.8% sequence identity) in three hot springs with temperatures up to 52 °C. Our data indicate that anammox bacteria may be able to thrive at thermophilic temperatures and thus may play a significant role in the nitrogen cycle of hot spring environments.     

Uptake and utilization of n-octacosane and n-nonacosane by Arthrobacter nicotianae KCC B35

Arthrobacter nicotianae KCC B35 isolated from blue-green mats densely covering oil sediments along the Arabian Gulf coast grew well on C₁₀ to C₄₀ n -alkanes as sole sources of carbon and energy. Growth on C₂₀ to C₄₀ alkanes was even better than on C₁₀ to C₁₈ alkanes. Biomass samples incubated for 6 h with n -octacosane (C₂₈) or n -nonacosane (C₂₉) accumulated these compounds as the predominant constituent alkanes of the cell hydrocarbon fractions. The even chain hexadecane C₁₆ and the odd chain pentadecane C₁₅ were the second dominant constituent alkanes in C₂₈ and C₂₉ incubated cells, respectively. n -Hexadecane-incubated cells accumulated in their lipids higher proportions of C₁₆-fatty acids than control cells not incubated with hydrocarbons. On the other hand, C₂₈ and C₂₉-incubated cells did not contain any fatty acids with the equivalent chain lengths, but the fatty acid patterns of the cell lipids suggest that there should have been mid-chain oxidation of these very long chain alkanes. This activity qualifies A. nicotianae KCC B35 to be used in cocktails for bioremediating environments polluted with heavy oil sediments.     

Biophysical properties of membrane lipids of anammox bacteria: II. Impact of temperature and bacteriohopanoids

Anammox bacteria possess unique membranes that are mainly comprised of phospholipids with extraordinary “ladderane” hydrocarbon chains containing 3 to 5 linearly concatenated cyclobutane moieties that have been postulated to form relatively impermeable membranes. In a previous study, we demonstrated that purified ladderane phospholipids form fluid-like mono- and bilayers that are tightly packed and relatively rigid. Here we studied the impact of temperature and the presence of bacteriohopanoids on the lipid density and acyl chain ordering in anammox membranes using Langmuir monolayer and fluorescence depolarization experiments on total lipid extracts. We showed that anammox membrane lipids of representatives of Candidatus “Kuenenia stuttgartiensis”, Candidatus “Brocadia fulgida” and Candidatus “Scalindua” were closely packed and formed membranes with a relatively high acyl chain ordering at the temperatures at which the cells were grown. Our findings suggest that bacteriohopanoids might play a role in maintaining the membrane fluidity in anammox cells.     

Fatty acid and lipid compositions of Conidiobolus

The fatty acid composition of the total lipids from two Conidiobolus species was studied by gas—liquid chromatography. The major fatty acids of C. lamprauges were palmitic acid (C_16:0), oleic acid (C_18:1), linolenic acid (C_18:3), and arachidonic acid (C_20:4). For C. eurymitus , myristic acid (C_14:0), C_16:0, and linoleic acid (C_18:2) were the most abundant acids. The fatty acid composition of C. eurymitus was quite different from that of the Conidiobolus species as mentioned in other reports. The lipid composition of the total lipids of C. lamprauges and C. eurymitus was also studied by thinchrography on quartz rods. Triglycerides and phospholipids were the major components in the two Conidiobolus species.     

Effect of non-target cells on the sensitivity of the PCR for Escherichia coli O157 : H7

The fatty acid composition from mycelia of Streptomyces hygroscopicus strains was studied. A significant proportion of C_{18 : 2} was found in cultures. High levels of C_{16 : 0}, iso-C_{16 : 0} and C_{18 : 1} were also detected in all S. hygroscopicus strains. The different representatives of S. hygroscopicus had almost the same proportion of unsaturated fatty acids. Certain shifts in the amount of iso, anteiso and straight-chain fatty acids in some cultures were revealed. This might be explained by the adaptation capability of strains belonging to one species to form a variety of available fatty acids determined by particular cell membrane composition favouring certain antibiotic biosynthesis.     

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.     

A hypersaline microbial mat from the Pacific Atoll Kiritimati: insights into composition and carbon fixation using biomarker analyses and a 13C-labeling approach

Modern microbial mats are widely recognized as useful analogs for the study of biogeochemical processes relevant to paleoenvironmental reconstruction in the Precambrian. We combined microscopic observations and investigations of biomarker composition to investigate community structure and function in the upper layers of a thick phototrophic microbial mat system from a hypersaline lake on Kiritimati (Christmas Island) in the Northern Line Islands, Republic of Kiribati. In particular, an exploratory incubation experiment with ¹³C-labeled bicarbonate was conducted to pinpoint biomarkers from organisms actively fixing carbon. A high relative abundance of the cyanobacterial taxa Aphanocapsa and Aphanothece was revealed by microscopic observation, and cyanobacterial fatty acids and hydrocarbons showed ¹³C-uptake in the labeling experiment. Microscopic observations also revealed purple sulfur bacteria (PSB) in the deeper layers. A cyclic C_19:0 fatty acid and farnesol were attributed to this group that was also actively fixing carbon. Background isotopic values indicate Calvin–Benson cycle-based autotrophy for cyc C_19:0 and farnesol-producing PSBs. Biomarkers from sulfate-reducing bacteria (SRB) in the top layer of the mat and their ¹³C-uptake patterns indicated a close coupling between SRBs and cyanobacteria. Archaeol, possibly from methanogens, was detected in all layers and was especially abundant near the surface where it contained substantial amounts of ¹³C-label. Intact glycosidic tetraether lipids detected in the deepest layer indicated other archaea. Large amounts of ornithine and betaine bearing intact polar lipids could be an indicator of a phosphate-limited ecosystem, where organisms that are able to substitute these for phospholipids may have a competitive advantage.     

Carbon metabolism of Listeria monocytogenes growing inside macrophages

The intracellular metabolism of Listeria monocytogenes was studied by ¹³C-isotopologue profiling using murine J774A.1 macrophages as host cells. Six hours after infection, bacteria were separated from the macrophages and hydrolyzed. Amino acids were converted into tert-butyl-dimethylsilyl derivatives and subjected to gas chromatography/mass spectrometry. When the macrophages were supplied with [U-¹³C₆]glucose prior to infection, but not during infection, label was detected only in Ala, Asp and Glu of the macrophage and bacterial protein with equal isotope distribution. When [U-¹³C₆]glucose was provided during the infection period, ¹³C label was found again in Ala, Asp and Glu from host and bacterial protein, but also in Ser, Gly, Thr and Val from the bacterial fraction. Mutants of L. monocytogenes defective in the uptake and catabolism of the C₃-metabolites, glycerol and/or dihydroxyacetone, showed reduced incorporation of [U-¹³C₆]glucose into bacterial amino acids under the same experimental settings. The ¹³C pattern suggests that (i) significant fractions (50–100%) of bacterial amino acids were provided by the host cell, (ii) a C₃-metabolite can serve as carbon source for L. monocytogenes under intracellular conditions and (iii) bacterial biosynthesis of Asp, Thr and Glu proceeds via oxaloacetate by carboxylation of pyruvate.     

Structural identification of ladderane and other membrane lipids of planctomycetes capable of anaerobic ammonium oxidation (anammox)

The membrane lipid composition of planctomycetes capable of the anaerobic oxidation of ammonium (anammox), i.e. Candidatus'Brocadia anammoxidans' and Candidatus'Kuenenia stuttgartiensis', was shown to be composed mainly of so-called ladderane lipids. These lipids are comprised of three to five linearly concatenated cyclobutane moieties with cis ring junctions, which occurred as fatty acids, fatty alcohols, alkyl glycerol monoethers, dialkyl glycerol diethers and mixed glycerol ether/esters. The highly strained ladderane moieties were thermally unstable, which resulted in breakdown during their analysis with GC. This was shown by isolation of a thermal product of these ladderanes and subsequent analysis with two-dimensional NMR techniques. Comprehensive MS and relative retention time data for all the encountered ladderane membrane lipids is reported, allowing the identification of ladderanes in other bacterial cultures and in the environment. The occurrence of ladderane lipids seems to be limited to the specific phylogenetic clade within the Planctomycetales able to perform anammox. This was consistent with their proposed biochemical function, namely as predominant membrane lipids of the so-called anammoxosome, the specific organelle where anammox catabolism takes place in the cell.     

Cellular fatty acid composition in moderately halophilic Gram-negative rods

The fatty acid compositions for 40 strains of moderately halophilic Gram-negative rods were determined by gas-liquid chromatography. The strains studied were included in the genera: Vibrio, Deleya, Alteromonas, Chromobacterium, Flavobac-terium and Pseudomonas. Although there were quantitative differences all strains showed more or less similar spectra of fatty acids ranging from C₁₂ to C₂₀ chain. The major fatty acid species were C_16:0, C_16:1 and C_18:1. Most striking was the predominance of the C_18:1 component, the major fatty acid in extracts of 29 of the 40 strains.     

Temperature-dependent changes in phospholipid and fatty acid composition and membrane lipid fluidity of Yersinia enterocolitica

Temperature-dependent compositional changes of phospholipids and their fatty acids were analysed in Yersinia enterocolitica grown at 5°, 25° and 37°C. The relative amounts of the four phospholipids, phosphatidylethanolamine (75–78%), phosphatidylglycerol (10–11%), cardiolipin (<7%) and lysophosphatidylethanolamine (<5%), were essentially the same at all growth temperatures. The degree of fatty acid unsaturation of the four phospholipids increased with decrease in growth temperature, mainly due to an increase of C_16:1 and C_18:1 and a corresponding decrease of C_16;0, C_18:0 and cyclo C_17:0. An electron spin resonance spectroscopic study of the membrane lipids showed that membrane lipid fluidity was enhanced by decreasing the growth temperatures. The changes in fatty acid composition of phospholipids in response to varied temperatures were consistent with the temperature-dependent changes in the membrane lipid fluidity of Y. enterocolitica , and were similar to those reported for other bacteria.     

Evidence for the synthesis of the multi-positional isomers of monounsaturated fatty acid in Methylococcus capsusatus by the anaerobic pathway

Abstract The biosynthesis of the positional isomers of the monounsaturated fatty acids of Methylococcus capsulatus (Bath) has been investigated by studying the incorporation of [2-¹⁴C]malonyl CoA into long-chain fatty acids in vitro. The major unsaturated products were Δ ⁹ 16:1 and Δ ¹¹ 18:1; however, Δ ⁸, Δ ¹⁰ and Δ ¹¹ 16:1, as well as, Δ ¹⁰, Δ ¹² and Δ ¹³ 18:1 were also synthesized. The exclusion of O₂ from the reaction vessel did not affect the synthesis of unsaturated fatty acids or the double bonds positions. Cerulenin inhibited the synthesis of unsaturated fatty acid more than saturated fatty acid. The use of both [1-¹⁴C] octanoate and [1-¹⁴C] decanoate as substrate resulted in the synthesis of long-chain fatty acids, however, unsaturates were only synthesized from octanoate. These results imply that the unique positional isomers of M. capsulatus are not synthesized by an aerobic mechanism.     

Effect of short-chain fatty acids on the ethylene pathway in embryonic axes of Cicer arietinum during germination

The effect of short-chain saturated fatty acids (C₅–C₁₀) on the biosynthesis of ethylene in embryonic axes of chick-pea ( Cicer arietinum L.) seeds was investigated. The emergence of radicle and fresh weight of embryonic axes diminished with increasing number of carbons. The inhibition of germination caused by lower concentrations (1 m M ) of fatty acids (C₅–C₁₀) was partially reversed by exogenous 1-aminocyclopropane-1-carboxylic acid (ACC), whereas exogenous ethylene was able to overcome the inhibitory effect provoked by all concentrations (1–5 m M ) of applied fatty acids (C₅–C₁₀). Ethylene production rates, and enzyme activities of ACC synthase and ACC oxidase decreased concomitantly with the molecular mass and increasing concentration of fatty acids. The inhibitory effect of these acids on ethylene production seems to result not only from a decreased ACC synthesis, but also from an enhancement of 1-malonylamino)cyclopropane-1-carboxylic acid (MACC) synthesis.     

LIPID CLASS DISTRIBUTION OF HIGHLY UNSATURATED LONG CHAIN FATTY ACIDS IN MARINE DINOFLAGELLATES

The very long chain highly unsaturated C₂₈ fatty acids, octacosaheptaenoic [28:7(n-6)] and octacosaoctaenoic acid [28:8(n-3)], were found to be associated with phospholipids, obtained by fractionation of total lipid extracts into distinct lipid classes, in 4 and 6, respectively, of 16 examined dinoflagellates. An interfraction comparison of fatty acids associated with phospholipids and glycolipids has also shown that the phospholipid fractions contained the majority (over 75% in 12 of 16 strains) of docosahexaenoic acid [22:6(n-3)] and traces of tetracosanoic acid (24:0). By contrast, the highly unsaturated C₁₈ fatty acids octadecatetraenoic [18:4(n-3)] and octadecapentaenoic acid [18:5(n-3)] were primarily recovered from a chloroplast-associated glycolipid fraction comprised of monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol. In 12 of 16 strains, an interfraction comparison showed that over 90% of 18:5(n-3) was found to be associated with glycolipids. These findings indicate that the C₂₈ fatty acids are located and probably synthesized in the cytoplasm or in an organelle other than the chloroplast, possibly with 22:6(n-3) and 24:0 as precursors, whereas the C₁₈ fatty acids 18:4(n-3) and 18:5(n-3) are glycolipid constituents apparently synthesized within the chloroplast. The function(s) of these C₂₈ fatty acids as components of phospholipids in cellular membranes is currently unknown.     

Arabidopsis CER8 encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis

Plant cuticle is an extracellular lipid-based matrix of cutin and waxes, which covers aerial organs and protects them from many forms of environmental stress. We report here the characterization of CER8 / LACS1 , one of nine Arabidopsis long-chain acyl-CoA synthetases thought to activate acyl chains. Mutations in LACS1 reduced the amount of wax in all chemical classes on the stem and leaf, except in the very long-chain fatty acid (VLCFA) class wherein acids longer than 24 carbons (C₂₄) were elevated more than 155%. The C₁₆ cutin monomers on lacs1 were reduced by 37% and 22%, whereas the C₁₈ monomers were increased by 28% and 20% on stem and leaf, respectively. Amounts of wax and cutin on a lacs1-1 lacs2-3 double mutant were much lower than on either parent, and lacs1-1 lacs2-3 had much higher cuticular permeability than either parent. These additive effects indicate that LACS1 and LACS2 have overlapping functions in both wax and cutin synthesis. We demonstrated that LACS1 has synthetase activity for VLCFAs C₂₀–C₃₀, with highest activity for C₃₀ acids. LACS1 thus appears to function as a very long-chain acyl-CoA synthetase in wax metabolism. Since C₁₆ but not C₁₈ cutin monomers are reduced in lacs1 , and C₁₆ acids are the next most preferred acid (behind C₃₀) by LACS1 in our assays, LACS1 also appears to be important for the incorporation of C₁₆ monomers into cutin polyester. As such, LACS1 defines a functionally novel acyl-CoA synthetase that preferentially modifies both VLCFAs for wax synthesis and long-chain (C₁₆) fatty acids for cutin synthesis.     

Induction of acetoacetate decarboxylase in Clostridium acetobutylicum

Abstract Factors that may initiate the biosynthesis of acetoacetate decarboxylase were investigated in resting cells of Clostridium acetobutylicum . Linear acids from C₁ to C₄ were inducers, whereas branched acids and linear acids from C₅ to C₇ were not inducers of acetoacetate decarboxylase biosynthesis. Induction of acetoacetate decarboxylase was maximal at pH 4.8 in the presence of acid concentrations comparable with those found during fermentation. In growth conditions repression of acetoacetate decarboxylase biosynthesis was found. This fact explains that acetone production by Clostridium acetobutylicum occurs when growth slows down.     

Phototrophic growth on n-fatty acids by species of the genus Rhodocyclus sensu Imhoff et al.

Abstract Type strains of Rhodocyclus purpureus, R. gelatinosus and R. tenuis along with three local isolates of R. gelatinosus were tested for growth in the light on n -fatty acids ranging in chain length from C₅ (valerate) to C₂₂ (docosanoate).
R. purpureus , the type species of the genus, was anomalous in its limited ability to grow on n -fatty acids; no fatty acids of chain length greater than C₉ (nonanoate) were utilized. R. gelatinosus and R. tenuis , on the other hand, utilized all fatty acids in the range C₅ to C₁₈ inclusive. R. gelatinosus showed some restricted ability to use C₂₀ (eicosanoate) and C₂₂ (docosanoate) fatty acids.     

Carbon isotope ratios of plants and occurrences of C4 species under different soil moisture regimes in arid region of Northwest China

Carbon isotope ratio of leaf dry matter, δ ¹³C, was measured on species occurring within Baiyin desert community, consisting of valley, slope and ridge microhabitats, and within Shandan desert community, consisting of Gobi desert and seasonal flooded creek microhabitats, in Northwest China. δ ¹³C of C₃ species increased with a decrease in soil water availability, suggesting that water-use efficiency (WUE) increased with decreasing soil moisture, whereas for all C₄ species, δ ¹³C tended to decrease with decreasing soil water availability, suggesting that WUE also increased with decreasing soil moisture. Above results indicated that water-use pattern was conservative under drought for C₄ and C₃ plants. In this present study, C₄ species' occurrences within different microhabitats were investigated and C₄ plants were observed to be absent and/or scarce within relatively lower soil moisture microhabitats, whereas they occurred and/or even had a high abundance within relatively higher soil moisture microhabitats, suggesting limited moisture available was a key factor of limiting C₄ distribution in arid region of Northwest China.     

Biophysical properties of membrane lipids of anammox bacteria: I. Ladderane phospholipids form highly organized fluid membranes

Anammox bacteria that are capable of anaerobically oxidizing ammonium (anammox) with nitrite to nitrogen gas produce unique membrane phospholipids that comprise hydrocarbon chains with three or five linearly condensed cyclobutane rings. To gain insight into the biophysical properties of these ‘ladderane’ lipids, we have isolated a ladderane phosphatidylcholine and a mixed ladderane phosphatidylethanolamine/phosphatidylglycerol lipid fraction and reconstituted these lipids in different membrane environments. Langmuir monolayer experiments demonstrated that the purified ladderane phospholipids form fluid films with a relatively high lipid packing density. Fluid-like behavior was also observed for ladderane lipids in bilayer systems as monitored by cryo-electron microscopy on large unilamellar vesicles (LUVs) and epi-fluorescence microscopy on giant unilamellar vesicles (GUVs). Analysis of the LUVs by fluorescence depolarization revealed a relatively high acyl chain ordering in the hydrophobic region of the ladderane phospholipids. Micropipette aspiration experiments were applied to study the mechanical properties of ladderane containing lipid bilayers and showed a relatively high apparent area compressibility modulus for ladderane containing GUVs, thereby confirming the fluid and acyl chain ordered characteristics of these lipids. The biophysical findings in this study support the previous postulation that dense membranes in anammox cells protect these microbes against the highly toxic and volatile anammox metabolites.