Publications

4477

AbstractIn 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, non-phosphorus 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 bacteriumCandidatusPelagibacter sp. HTCC7211, a member of the SAR11 clade. Our results showed that GTcpis 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 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 modelling and mutational analyses revealed binding sites for the sugar donor and the diacylglycerol lipid acceptor, which provided insights into the retaining mechanism of GTcpwith its GT-B fold. A phylogenetic analysis showed that GTcpand its homologs form a group in the GT4 glycosyltransferase family. These results not only provide new insights into the glycoglycerolipid synthesis mechanism in lipid remodelling, but also describe an efficient enzymatic tool for future synthesis of bioactive molecules.ImportanceThe 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 substitute phospholipids with non-phosphorus 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 bacteriumCandidatusPelagibacter sp. HTCC7211. GTcpand its homologs form a group in the GT4 glycosyltransferase family, and can synthesize neutral glycolipids (MGlc-DAG and MGal-DAG) and an acidic glycolipid (MGlcA-DAG). We also uncover 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 remodelling.

SummaryMicrobes are phylogenetically and metabolically diverse. Yet capturing this diversity, assigning functions to host organisms and exploring the biosynthetic potential in natural environments remains challenging. We reconstructed >25,000 draft genomes, including from >2,500 uncharacterized species, from globally-distributed ocean microbial communities, and combined them with ∼10,000 genomes from cultivated and single cells. Mining this resource revealed ∼40,000 putative biosynthetic gene clusters (BGCs), many from unknown phylogenetic groups. Among these, we discoveredCandidatusEudoremicrobiaceae as one of the most biosynthetically diverse microbes detected to date. Discrete transcriptional states structuring natural populations were associated with a potentially niche-partitioning role for BGC products. Together with the characterization of the first Eudoremicrobiaceae natural product, this study demonstrates how microbiomics enables prospecting for candidate bioactive compounds in underexplored microbes and environments.

AbstractMitochondria are specialized eukaryotic organelles that have a dedicated function in oxygen respiration and energy production. They evolved about 2 billion years ago from a free-living bacterial ancestor (probably an alphaproteobacterium), in a process known as endosymbiosis1,2. Many unicellular eukaryotes have since adapted to life in anoxic habitats and their mitochondria have undergone further reductive evolution3. As a result, obligate anaerobic eukaryotes with mitochondrial remnants derive their energy mostly from fermentation4. Here we describe ‘Candidatus Azoamicus ciliaticola’, which is an obligate endosymbiont of an anaerobic ciliate and has a dedicated role in respiration and providing energy for its eukaryotic host. ‘Candidatus A. ciliaticola’ contains a highly reduced 0.29-Mb genome that encodes core genes for central information processing, the electron transport chain, a truncated tricarboxylic acid cycle, ATP generation and iron–sulfur cluster biosynthesis. The genome encodes a respiratory denitrification pathway instead of aerobic terminal oxidases, which enables its host to breathe nitrate instead of oxygen. ‘Candidatus A. ciliaticola’ and its ciliate host represent an example of a symbiosis that is based on the transfer of energy in the form of ATP, rather than nutrition. This discovery raises the possibility that eukaryotes with mitochondrial remnants may secondarily acquire energy-providing endosymbionts to complement or replace functions of their mitochondria.

“Candidatus Liberibacter asiaticus” (CLas) and Spiroplasma citri are phloem-limited bacteria that infect citrus and are transmitted by insect vectors. S. citri causes citrus stubborn disease (CSD) and is vectored by the beet leafhopper in California. CLas is associated with the devastating citrus disease, Huanglongbing (HLB), and is vectored by the Asian citrus psyllid. CLas is a regulatory pathogen spreading in citrus on residential properties in southern California and is an imminent threat to spread to commercial citrus plantings. CSD is endemic in California and has symptoms in citrus that can be easily confused with HLB. Therefore, the objective of this study was to develop a multiplex qPCR and duplex droplet digital PCR (ddPCR) assay for simultaneous detection of CLas and S. citri to be used where both pathogens can co-exist. The multiplex qPCR assay was designed to detect multicopy genes of CLas—RNR (5 copies) and S. citri–SPV1 ORF1 (13 copies), respectively, and citrus cytochrome oxidase (COX) as internal positive control. Absolute quantitation of these pathogens was achieved by duplex ddPCR as a supplement for marginal qPCR results. Duplex ddPCR allowed higher sensitivity than qPCR for detection of CLas and S. citri. ddPCR showed higher tolerance to inhibitors and yielded highly reproducible results. The multiplex qPCR assay has the benefit of testing both pathogens at reduced cost and can serve to augment the official regulatory protocol for CLas detection in California. Moreover, the ddPCR provided unambiguous absolute detection of CLas and S. citri at very low concentrations without any standards for pathogen titer.

Significance Symbiotic dinitrogen (N 2 )-fixing trees fulfill a critical function in tropical forests by bringing in new nitrogen, yet it remains unclear how they overcome constraints by highly weathered, nutrient-poor tropical soils. We advance forest biogeochemistry and microbial ecology with the discovery from field trials in Panama that fast-growing N 2 -fixing trees in tropical forests exhibit accelerated mineral weathering and distinctive soil metagenomes that improve their access to inorganic nutrients in nutrient-poor soils. Furthermore, we show that N 2 -fixing trees exert similar effects on non-N 2 –fixing trees nearby thus having previously overlooked community-wide effects on tropical forest nutrient cycling. These results offer insights into the role of N 2 -fixing trees and their associated microbiomes in safeguarding the function of tropical forests within the global biosphere.

Peptidoglycan (PG) is essential for bacterial survival and maintaining cell shape. The rod-shaped model bacterium Escherichia coli has a set of seven endopeptidases that remodel the PG during cell growth. The gamma proteobacterium Candidatus Thiosymbion oneisti is also rod-shaped and attaches to the cuticle of its nematode host by one pole. It widens and divides by longitudinal fission using the canonical proteins MreB and FtsZ. The PG layer of Ca. T. oneisti has an unusually high peptide cross-linkage of 67% but relatively short glycan chains with an average length of 12 disaccharides. Curiously, it has only two predicted endopeptidases, MepA and PBP4. Cellular localization of symbiont PBP4 by fluorescently labeled antibodies reveals its polar localization and its accumulation at the constriction sites, suggesting that PBP4 is involved in PG biosynthesis during septum formation. Isolated symbiont PBP4 protein shows a different selectivity for β-lactams compared to its homologue from E. coli. Bocillin-FL binding by PBP4 is activated by some β-lactams, suggesting the presence of an allosteric binding site. Overall, our data point to a role of PBP4 in PG cleavage during the longitudinal cell division and to a PG that might have been adapted to the symbiotic lifestyle.

Abstract BackgroundArchaea were originally discovered in extreme environments, and thrive in many extreme habitats including soda lakes with high pH and salinity. Characteristic and diverse archaeal community played a significant role in biogeochemical cycles; however, the archaeal community and their functions are still less-studied in the intricate sediment of soda lakes. ResultsIn this article, the archaeal community of the deep sediment (40-50 cm depth) of five artificially-separated ponds with a salinity range from 7.0% to 33.0% in a soda saline lake was systematically surveyed using culture-independent metagenomics combined with the next-generation sequencing of the archaeal 16S rRNA amplicons. Nine archaeal phyla were detected, which accounted for 2.2% to 35.73% of microbial community in the five deep sediments. Besides the well-known class Halobacteria, one novel archaeal order (Candidatus Natranaeroarchaeales) of the class Thermoplasmata was even more abundant than Halobacteria in some deep sediment samples. Of 69 dereplicated archaeal metagenome-assembled genomes (MAGs), 30 MAGs belonged to Ca. Natranaeroarchaeales. Different genera of the Ca. Natranaeroarchaeales preferred to inhabit in the different salinities, and the divergent halophilic adaptation strategies (salt-out or salt-in) suggested the fast evolution adaptation within this lineage. Most high-quality MAGs had the genes of Wood-Ljungdahl pathway, organic acid fermentation and sulfur respiration, suggesting the putative functions in carbon fixation and sulfur reduction. Interestingly, heterodisulfide reductase and F420-non-reducing hydrogenase complex HdrABC-MvhADG were widely distributed in Ca. Natranaeroarchaeales and may play the core roles in energy metabolism from hydrogen. The regeneration of CoM-S-S-CoB was coupled to succinate or 2-oxoglutarate production in Ca. Natranaeroarchaeales instead of methanogenesis in the close related Methanomassiliicoccales. It suggested that methyl-coenzyme M reductase in Methanomassiliicoccales may be laterally transferred from other methanogens. ConclusionA novel archaeal order Ca. Natranaeroarchaeales of Thermoplasmata was found by culture-independent approaches. This order was the most abundant archaeal lineage in the deep sediment of soda lakes, with the characteristic environmental adaptation and biogeochemical potentials in carbon fixation and sulfur reduction. The difference in fermentation products coupled to energy metabolism between methanogens and Ca. Natranaeroarchaeales provided additional insights into the origination of methanogenesis in Thermoplasmata from the energy metabolism perspective.