Enzyme-Specific Coupling of Oxygen and Nitrogen Isotope Fractionation of the Nap and Nar Nitrate Reductases.

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.

Novel Metal Cation Resistance Systems from Mutant Fitness Analysis of Denitrifying Pseudomonas stutzeri.

UNLABELLED: 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+) and Zn(2+) resistance systems in Pseudomonas stutzeri strain RCH2 are compared with those experimentally implicated in Cu(2+) and Zn(2+) resistance, as determined by using a DNA-barcoded transposon mutant library. Mutant fitness data obtained under denitrifying conditions are combined with regulon predictions to yield a much more comprehensive picture of Cu(2+) and Zn(2+) resistance in strain RCH2. The results not only considerably expand what is known about well-established metal ion exporters (CzcCBA, CzcD, and CusCBA) and their accessory proteins (CzcI and CusF), they also reveal that isolates with mutations in some predicted Cu(2+) resistance systems do not show decreased fitness relative to the wild type when exposed to Cu(2+) In addition, new genes are identified that have no known connection to Zn(2+) (corB, corC, Psest_3226, Psest_3322, and Psest_0618) or Cu(2+) resistance (Mrp antiporter subunit gene, Psest_2850, and Psest_0584) but are crucial for resistance to these metal cations. Growth of individual deletion mutants lacking corB, corC, Psest_3226, or Psest_3322 confirmed the observed Zn-dependent phenotypes. Notably, to our knowledge, this is the first time a bacterial homolog of TMEM165, a human gene responsible for a congenital glycosylation disorder, has been deleted and the resulting strain characterized. Finally, the fitness values indicate Cu(2+)- and Zn(2+)-based inhibition of nitrite reductase and interference with molybdenum cofactor biosynthesis for nitrate reductase. These results extend the current understanding of Cu(2+) and Zn(2+) efflux and resistance and their effects on denitrifying metabolism. IMPORTANCE: In 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.

Production of diverse brGDGTs by Acidobacterium Solibacter usitatus in response to temperature, pH, and O2 provides a culturing perspective on brGDGT proxies and biosynthesis.

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.

Alterations in the ribosomal protein bL12 of E. coli affecting the initiation, elongation and termination of protein synthesis.

In bacteria, ribosomal protein bL12 forms the prominent stalk structure on the ribosome and binds to multiple, distinct translational GTPase factors during the sequential steps of translation. Using a genetic selection in E. coli for altered readthrough of UGA stop codons, we have isolated seven different mutations affecting the C-terminal domain of the protein that forms the interaction surface with translation factors. Analysis of these altered proteins, along with four additional alterations previously shown to affect IF2-ribosome interactions, indicates that multiple steps of translation are affected, consistent with bL12's interaction with multiple factors. Surprisingly, deletion of the release factor GTPase, RF3, has relatively little effect on bL12-promoted stop codon readthrough, suggesting that other steps in termination are also influenced by bL12.

Mechanisms of Chromium and Uranium Toxicity in Pseudomonas stutzeri RCH2 Grown under Anaerobic Nitrate-Reducing Conditions.

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][Formula: see text]) or uranyl oxycation (U[VI][Formula: see text]). 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.