Developing a genetic approach to target cyanobacterial producers of heterocyte glycolipids in the environment

Heterocytous cyanobacteria are important players in the carbon and nitrogen cycle. They can fix dinitrogen by using heterocytes, specialized cells containing the oxygen-sensitive nitrogenase enzyme surrounded by a thick polysaccharide and glycolipid layer which prevents oxygen diffusion and nitrogenase inactivation. Heterocyte glycolipids can be used to detect the presence of heterocytous cyanobacteria in present-day and past environments, providing insight into the functioning of the studied ecosystems. However, due to their good preservation throughout time, heterocyte glycolipids are not ideal to detect and study living communities, instead methods based on DNA are preferred. Currently cyanobacteria can be detected using untargeted genomic approaches such as metagenomics, or they can be specifically targeted by, for example, the use of primers that preferentially amplify their 16S rRNA gene or their nifH gene in the case of nitrogen fixing cyanobacteria. However, since not all cyanobacterial nitrogen fixers are heterocytous, there is currently no fast gene-based method to specifically detect and distinguish heterocytous cyanobacteria. Here, we developed a PCR-based method to specifically detect heterocytous cyanobacteria by designing primers targeting the gene (hglT) encoding the enzyme responsible for the last step in the biosynthesis of heterocyte glycolipid (i.e., a glycosyltransferase). We designed several primer sets using the publicly available sequences of 23 heterocytous cyanobacteria, after testing them on DNA extracts of 21 heterocyte-forming and 7 non-heterocyte forming freshwater cyanobacteria. The best primer set was chosen and successfully used to confirm the presence of heterocytous cyanobacteria in a marine environmental sample

Molecular analysis of intact preen waxes of Calidris canutus (Aves:Scolopacidae) by gas chromatography/mass spectrometry

The intact preen wax esters of the red knot Calidris canutus were studied with gas chromatography/mass spectrometry (GC/MS) and GC/MS/MS. In this latter technique, transitions from the molecular ion to fragment ions representing the fatty acid moiety of the wax esters were measured, providing additional resolution to the analysis of wax esters. The C-21-C-32 wax esters are composed of complex mixtures of hundreds of individual isomers. The odd carbon-numbered wax esters are predominantly composed of even carbon-numbered n-alcohols (C-14, C-16, and C-18) esterified predominantly with odd carbon-numbered 2-methyl fatty acids (C-7, C-9, C-11, and C-13), resulting in relatively simple distributions. The even carbon-numbered wax esters show a far more complex distribution due to a number of factors: (i) Their n-alcohol moieties are not dominated by even carbon-numbered n-alcohols esterified with odd carbon-numbered 2-methyl fatty acids, but odd and even carbon-numbered n-alcohols participate in approximately equal amounts; (ii) odd carbon-numbered methyl-branched alcohols participate abundantly in these wax ester clusters; and (iii) with increasing molecular weight, various isomers of the 2,6-, 2,8-, and 2,10-dimethyl branched fatty acids also participate in the even carbon-numbered wax esters. The data demonstrate that there is a clear biosynthetic control on the wax ester composition although the reasons for the complex chemistry of the waxes are not yet understood

Rapid sulfurisation of highly branched isoprenoid (HBI) alkenes in sulfidic Holocene sediments from Ellis Fjord, Antarctica

Author Posting. © Elsevier B.V., 2007. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Organic Geochemistry 38 (2007): 128-139, doi:10.1016/j.orggeochem.2006.08.003.Samples of particulate organic matter from the water column and anoxic Holocene sediment layers from the Small Meromictic Basin (SMB) in Ellis Fjord (eastern Antarctica) were analyzed to study the early incorporation of reduced inorganic sulfur species into highly branched isoprenoid (HBI) alkenes. HBIs were not detected in the water column samples from austral winter, whereas compounds containing the C25 HBI skeleton were abundant in all analyzed Holocene sediment layers. The structure of the C25:2 HBI alkene together with its enriched stable carbon isotopic composition suggest that the HBI alkene is produced by a diatom or diatoms probably belonging to the Navicula genus present in the sea-ice which covers the area most of the year. Within just 500 years of deposition, all of the HBI alkene was sulfurised. A mixture of products was formed, including components tentatively identified as a C25 HBI thiane and three S-containing dimers composed of two C25:1 HBI skeletons linked together by a sulfide bond. Most of the HBI alkene, however, was converted to polar S-containing compounds. The observed reaction rate for sulfurisation the C25:2 HBI alkene is the highest observed so far in natural systems. Sterols and other lipids known to be prone to sulfurisation were only minimally sulfurised under these depositional conditions. The reason for this is presently unclear.Funding for the collection of the sediment and water samples (by MJLC and CW) was provided by ASAC grant 1166 to JKV. This work was further supported by a grant from the Netherlands Organization for Scientific Research (NWO; Netherlands Antarctic Research Proposals 851.20.006 to JSSD)

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

AbstractAnammox 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

Organohalide-respiring Desulfoluna species isolated from marine environments

The online version of this article (https://doi.org/10.1038/s41396-019-0573-y) contains supplementary material, which is available to authorized usersThe genus Desulfoluna comprises two anaerobic sulfate-reducing strains, D. spongiiphila AA1T and D. butyratoxydans MSL71T, of which only the former was shown to perform organohalide respiration (OHR). Here we isolated a third strain, designated D. spongiiphila strain DBB, from marine intertidal sediment using 1,4-dibromobenzene and sulfate as the electron acceptors and lactate as the electron donor. Each strain harbors three reductive dehalogenase gene clusters (rdhABC) and corrinoid biosynthesis genes in their genomes, and dehalogenated brominated but not chlorinated organohalogens. The Desulfoluna strains maintained OHR in the presence of 20?mM sulfate or 20?mM sulfide, which often negatively affect other organohalide-respiring bacteria. Strain DBB sustained OHR with 2\% oxygen in the gas phase, in line with its genetic potential for reactive oxygen species detoxification. Reverse transcription-quantitative PCR revealed differential induction of rdhA genes in strain DBB in response to 1,4-dibromobenzene or 2,6-dibromophenol. Proteomic analysis confirmed expression of rdhA1 with 1,4-dibromobenzene, and revealed a partially shared electron transport chain from lactate to 1,4-dibromobenzene and sulfate, which may explain accelerated OHR during concurrent sulfate reduction. Versatility in using electron donors, de novo corrinoid biosynthesis, resistance to sulfate, sulfide and oxygen, and concurrent sulfate reduction and OHR may confer an advantage to marine Desulfoluna strains.We thank Johanna Gutleben and Maryam Chaib de Mares for sediment sampling, W. Irene C. Rijpstra for fatty acid analysis, and Andreas Marquardt (Proteomics Centre of the University of Konstanz) for proteomic analyses. We acknowledge the China Scholarship Council (CSC) for the support to PP and YL. The authors thank BE-BASIC funds (grants F07.001.05 and F08.004.01) from the Dutch Ministry of Economic Affairs, ERC grant (project 323009), the Gravitation grant (project 024.002.002) and the UNLOCK project (NRGWI.obrug.2018.005) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO), and National Natural Science Foundation of China (project No.51709100) for funding.info:eu-repo/semantics/publishedVersio

2,6,10,15,19-Pentamethylicosenes in Methanolobus bombayensis, a marine methanogenic archaeon, and in Methanosarcina mazei

2,6,10,15,19-Pentamethylicosenes (PMEs) containing three to five double bonds have been found in the methanogenic archaea Methanosarcina mazei (DSM 3338), a strain isolated from sewage sludge, and in Methanolobus bombayensis (OCM 438), a non-extremophilic archaeon isolated from a marine sediment. This finding gives additional support for the use of compounds with the PME carbon skeleton as markers for methanogenic activity in marine environments. (C) 1997 Elsevier Science Ltd

Impact of Electron Acceptor Availability on Methane-Influenced Microorganisms in an Enrichment Culture Obtained From a Stratified Lake

Methanotrophs are of major importance in limiting methane emissions from lakes. They are known to preferably inhabit the oxycline of stratified water columns, often assumed due to an intolerance to atmospheric oxygen concentrations, but little is known on the response of methanotrophs to different oxygen concentrations as well as their preference for different electron acceptors. In this study, we enriched a methanotroph of theMethylobactergenus from the oxycline and the anoxic water column of a stratified lake, which was also present in the oxic water column in the winter. We tested the response of thisMethylobacter-dominated enrichment culture to different electron acceptors, i.e., oxygen, nitrate, sulfate, and humic substances, and found that, in contrast to earlier results with water column incubations, oxygen was the preferred electron acceptor, leading to methane oxidation rates of 45-72 pmol cell(-1)day(-1). Despite the general assumption of methanotrophs preferring microaerobic conditions, methane oxidation was most efficient under high oxygen concentrations (>600 mu M). Low (<30 mu M) oxygen concentrations still supported methane oxidation, but no methane oxidation was observed with trace oxygen concentrations (<9 mu M) or under anoxic conditions. Remarkably, the presence of nitrate stimulated methane oxidation rates under oxic conditions, raising the methane oxidation rates by 50% when compared to oxic incubations with ammonium. Under anoxic conditions, no net methane consumption was observed; however, methanotroph abundances were two to three times higher in incubations with nitrate and sulfate compared to anoxic incubations with ammonium as the nitrogen source. Metagenomic sequencing revealed the absence of a complete denitrification pathway in the dominant methanotrophMethylobacter, but the most abundant methylotrophMethyloteneraseemed capable of denitrification, which can possibly play a role in the enhanced methane oxidation rates under nitrate-rich conditions

HglT fragments of heterocytous cyanobacteria

Assembled hglT gene fragments obtained via PCR and direct sequencing using primer set 2 (Fw1 mix B + Rv1, Supplementary table 2) on the heterocytous cyanobacterial cultures listed in Table 2, Supplementary Table 8 and shown in Figure 5 (sequences in blue) in Pérez Gallego et al. 2023 Nucleotide sequences were obtained via Sanger sequencing and were processed using Geneious Prime (v 2023.0.4). Sequences were trimmed using an error probability limit of 0.01. Potential heterozygous bases in single reads were identified using a 50% peak similarity cutoff, peak detection height was set at 10%. When available, the consensus sequence was obtained by aligning forward and reverse reads with Geneious assembler using the highest sensitivity settings. When appropriate, sequences belonging to the pCR™4-TOPO™ vector (Invitrogen, Carlsbad, CA, USA) were identified and removed. To generate the phylogenetic trees sequences were aligned using MAFFT (v7.407) with L-INS-i iterative refinement method (Katoh and Standley, 2013) and poorly aligned regions were removed using trimAl (Capella-Gutiérrez et al., 2009). Phylogenetic trees were built using IQ-tree (v1.6.7) and its in-built nucleotide substitution model finder (Kalyaanamoorthy et al., 2017), using 1000 replicates to perform SH-like approximate likelihood ratio test (SH-aLRT) (Guindon et al., 2010) and 1000 bootstrap replicates.LV and JSSD also received funding from the Soehngen Institute for Anaerobic Microbiology (SIAM) through a Gravitation Grant (024.002.002) from the Dutch Ministry of Education, Culture, and Science (OCW)