Branched glycerol dialkyl glycerol tetraethers (brGDGTs) are ubiquitous and well preserved sedimentary biomarkers. These compounds serve as important palaeoenvironmental indicators due to strong empirical correlations between brGDGT distributions and temperature and pH in modern environments. However, the mechanistic link between temperature, pH, and brGDGT production has been impossible to ascertain thus far due to the absence of a clear biological source for brGDGTs. Here, we report that oxygen limitation triggers brGDGT production in at least one cultured species of Acidobacteria and confirm for the first time the biosynthesis of three structural varieties of brGDGTs, including an uncharacterised isomer of brGDGT Ic. This discovery helps explain why brGDGT producers have been so difficult to identify and provides a pathway towards uncovering the genetic basis and biological function of brGDGTs, which will lead to a more comprehensive understanding of their palaeoenvironmental significance. If the oxygen effects observed here apply more broadly, the empirical calibrations for brGDGT-based temperature and pH reconstructions may currently be missing the effects of oxygen as a relevant and possibly dominant control in the environmental distributions of brGDGTs.

Dissimilatory nitrate reduction (DNR) to nitrite is the first step in denitrification, the main process through which bioavailable nitrogen is removed from ecosystems. DNR is catalyzed by both cytosolic (Nar) and periplasmic (Nap) nitrate reductases and fractionates the stable isotopes of nitrogen (14N, 15N) and oxygen (16O, 18O), which is reflected in residual environmental nitrate pools. Data on the relationship between the pattern in oxygen vs nitrogen isotope fractionation (18ε/15ε) suggests that systematic differences exist between marine and terrestrial ecosystems that are not fully understood. We examined the 18ε/15ε of nitrate-reducing microorganisms that encode Nar, Nap, or both enzymes, as well as gene deletion mutants of Nar and Nap to test the hypothesis that enzymatic differences alone could explain the environmental observations. We find that the distribution of 18ε/15ε fractionation ratios of all examined nitrate reductases forms two distinct peaks centered around an 18ε/15ε proportionality of 0.55 (Nap) and 0.91 (Nar), with the notable exception of the Bacillus Nar reductases, which cluster isotopically with the Nap reductases. Our findings may explain differences in 18ε/15ε fractionation between marine and terrestrial systems and challenge current knowledge about Nar 18ε/15ε signatures.

Metal ion transport systems have been studied extensively, but the specificity of a given transporter is often unclear from amino acid sequence data alone. In this study, predicted Cu 2؉ IMPORTANCEIn this study, genome-wide mutant fitness data in P. stutzeri RCH2 combined with regulon predictions identify several proteins of unknown function that are involved in resisting zinc and copper toxicity. For zinc, these include a member of the UPF0016 protein family that was previously implicated in Ca 2؉ /H ؉ antiport and a human congenital glycosylation disorder, CorB and CorC, which were previously linked to Mg 2؉ transport, and Psest_3322 and Psest_0618, two proteins with no characterized homologs. Experiments using mutants lacking Psest_3226, Psest_3322, corB, corC, or czcI verified their proposed functions, which will enable future studies of these little-characterized zinc resistance determinants. Likewise, Psest_2850, annotated as an ion antiporter subunit, and the conserved hypothetical protein Psest_0584 are implicated in copper resistance. Physiological connections between previous studies and phenotypes presented here are discussed. Functional and mechanistic understanding of transport proteins improves the understanding of systems in which members of the same protein family, including those in humans, can have different functions.T he responses of microorganisms to metal toxicity have been well studied (1). In brief, metal ions can be toxic by binding to essential proteins or other molecules, causing them to become nonfunctional or function incorrectly. This occurs, for example, where the toxic metal ion binds to a binding site that requires a different metal ion to function. Toxic metal ions can also catalyze reactions that are detrimental to the cell, such as hydroxyl radical generation catalyzed by free iron (Fe 3ϩ/2ϩ ) (2) or iron-sulfur cluster degradation by copper (3).Understanding modes of toxicity and metal resistance systems is important, as the information gained from bacteria can be applied to human physiology and medicine. For example, Wilson's disease and Menkes disease are two human diseases caused by mutations in copper transporters (4). Likewise, acrodermatitis enteropathica, while less well-studied at a mechanistic level, is caused by a mutation of the SLC39A4 zinc transporter gene (5). Wilson's disease results in copper accumulation, which is known to cause tissue damage due to the generation of reactive oxygen species (4). Similarly, copper and zinc are known to be involved in neurodegenerative diseases, such as Alzheimer's and Parkinson's, again with a connection to oxidative stress, although their precise roles are still unclear (6). In addition, copper has long been used as

Chromium and uranium are highly toxic metals that contaminate many natural environments. We investigated their mechanisms of toxicity under anaerobic conditions using nitrate-reducing Pseudomonas stutzeri RCH2, which was originally isolated from a chromium-contaminated aquifer. A random barcode transposon site sequencing library of RCH2 was grown in the presence of the chromate oxyanion (Cr[VI]O42−) or uranyl oxycation (U[VI]O22+). Strains lacking genes required for a functional nitrate reductase had decreased fitness as both metals interacted with heme-containing enzymes required for the later steps in the denitrification pathway after nitrate is reduced to nitrite. Cr[VI]-resistance also required genes in the homologous recombination and nucleotide excision DNA repair pathways, showing that DNA is a target of Cr[VI] even under anaerobic conditions. The reduced thiol pool was also identified as a target of Cr[VI] toxicity and psest_2088, a gene of previously unknown function, was shown to have a role in the reduction of sulfite to sulfide. U[VI] resistance mechanisms involved exopolysaccharide synthesis and the universal stress protein UspA. As the first genome-wide fitness analysis of Cr[VI] and U[VI] toxicity under anaerobic conditions, this study provides new insight into the impact of Cr[VI] and U[VI] on an environmental isolate from a chromium contaminated site, as well as into the role of a ubiquitous protein, Psest_2088.

Branched glycerol dialkyl glycerol tetraethers (brGDGTs) are bacterial membrane lipids that are frequently employed as paleoenvironmental proxies because of the strong empirical correlations between their relative abundances and environmental temperature and pH. Despite the ubiquity of brGDGTs in modern and paleoenvironments, the source organisms of these enigmatic compounds have remained elusive, requiring paleoenvironmental applications to rely solely on observed environmental correlations. Previous laboratory and environmental studies have suggested that the globally abundant bacterial phylum of the Acidobacteria may be an important brGDGT producer in nature. Here, we report on experiments with a cultured Acidobacterium, Solibacter usitatus, that makes a large portion of its cellular membrane (24 ± 9% across all experiments) out of a structurally diverse set of tetraethers including the common brGDGTs Ia, IIa, IIIa, Ib, and IIb. Solibacter usitatus was grown across a range of conditions including temperatures from 15 to 30°C, pH from 5.0 to 6.5, and O2 from 1% to 21%, and demonstrated pronounced shifts in the degree of brGDGT methylation across these growth conditions. The temperature response in culture was in close agreement with trends observed in published environmental datasets, supporting a physiological basis for the empirical relationship between brGDGT methylation number and temperature. However, brGDGT methylation at lower temperatures (15 and 20°C) was modulated by culture pH with higher pH systematically increasing the degree of methylation. In contrast, pH had little effect on brGDGT cyclization, supporting the hypothesis that changes in bacterial community composition may underlie the link between cyclization number and pH observed in environmental samples. Oxygen concentration likewise affected brGDGT methylation highlighting the potential for this environmental parameter to impact paleotemperature reconstruction. Low O2 culture conditions further resulted in the production of uncommon brGDGT isomers that could be indicators of O2 limitation. Finally, the production of brGTGTs (trialkyl tetraethers) in addition to the previously discovered iso‐C15‐based mono‐ and diethers in S. usitatus suggests a potential biosynthetic pathway for brGDGTs that uses homologs of the archaeal tetraether synthase (Tes) enzyme for tetraether synthesis from diethers.