Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease ...spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 min. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, RT-PCR, using 177 nasopharyngeal RNA samples. For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH.
Abstract
Organohalide-respiring bacteria are key organisms for the bioremediation of soils and aquifers contaminated with halogenated organic compounds. The major players in this process are ...respiratory reductive dehalogenases, corrinoid enzymes that use organohalides as substrates and contribute to energy conservation. Here, we present the structure of a menaquinol:organohalide oxidoreductase obtained by cryo-EM. The membrane-bound protein was isolated from
Desulfitobacterium hafniense
strain TCE1 as a PceA
2
B
2
complex catalysing the dechlorination of tetrachloroethene. Two catalytic PceA subunits are anchored to the membrane by two small integral membrane PceB subunits. The structure reveals two menaquinone molecules bound at the interface of the two different subunits, which are the starting point of a chain of redox cofactors for electron transfer to the active site. In this work, the structure elucidates how energy is conserved during organohalide respiration in menaquinone-dependent organohalide-respiring bacteria.
Protein film electrochemistry is a technique in which an enzyme is immobilized on an electrode in a configuration that allows following the changes in turnover frequency as a response to changes in ...the experimental conditions. Insights into the reactivity of the enzyme can be obtained by quantitatively modeling such responses. As a consequence, the more the technique allows flexibility in changing conditions, the more useful it becomes. The most commonly used setup, based on the rotating disc electrode, allows easy stepwise increases in the concentration of nongaseous substrates, or exposure to constant concentration of dissolved gas, but does not permit to easily decrease the concentration of nongaseous substrates, or to change the concentration of dissolved gas in a stepwise fashion. To overcome the limitation by mass transport of the substrate toward the electrode when working with fast enzymes, we have designed another kind of electrochemical cell based on the wall-tube electrode (WTE). We demonstrate here that by using a system combining two syringe pumps, a commercial mixer, and the WTE, it is possible to change the concentration of species in a stepwise fashion in all directions, opening new possibilities to study redox enzymes. As a proof of concept, this device was applied to the study of the electrochemical response of the cytochrome c nitrite reductase of Desulfovibrio desulfuricans.
Electroactive bacteria combine the oxidation of carbon substrates with an extracellular electron transfer (EET) process that discharges electrons to an electron acceptor outside the cell. This ...process involves electron transfer through consecutive redox proteins that efficiently connect the inner membrane to the cell exterior. In this study, we isolated and characterized the quinone‐interacting membrane cytochrome c ImcH from Geobacter sulfurreducens, which is involved in the EET process to high redox potential acceptors. Spectroscopic and electrochemical studies show that ImcH hemes have low midpoint redox potentials, ranging from −150 to −358 mV, and connect the oxidation of the quinol‐pool to EET, transferring electrons to the highly abundant periplasmic cytochrome PpcA with higher affinity than to its homologues. Despite the larger number of hemes and transmembrane helices, the ImcH structural model has similarities with the NapC/NirT/NrfH superfamily, namely the presence of a quinone‐binding site on the P‐side of the membrane. In addition, the first heme, likely involved on the quinol oxidation, has apparently an unusual His/Gln coordination. Our work suggests that ImcH is electroneutral and transfers electrons and protons to the same side of the membrane, contributing to the maintenance of a proton motive force and playing a central role in recycling the menaquinone pool.
In prokaryotes, the proton or sodium motive force required for ATP synthesis is produced by respiratory complexes that present an ion-pumping mechanism or are involved in redox loops performed by ...membrane proteins that usually have substrate and quinone-binding sites on opposite sides of the membrane. Some respiratory complexes include a dimeric redox module composed of a quinone-interacting membrane protein of the NrfD family and an iron‑sulfur protein of the NrfC family. The QrcABCD complex of sulfate reducers, which includes the QrcCD module homologous to NrfCD, was recently shown to perform electrogenic quinone reduction providing the first conclusive evidence for energy conservation among this family. Similar redox modules are present in multiple respiratory complexes, which can be associated with electroneutral, energy-driven or electrogenic reactions. This work discusses the presence of the NrfCD/PsrBC dimeric redox module in different bioenergetics contexts and its role in prokaryotic energy conservation mechanisms.
•The NrfD family comprises integral membrane proteins that are present in many prokaryotic respiratory complexes•NrfD-like proteins associate with NrfC-like FeS proteins to form a dimeric redox module involved in quinone redox chemistry•We discuss the role of this widespread bacterial redox module in multiple bioenergetic contexts
Nitric oxide reductase (NOR) is a membrane bound enzyme involved in the metabolic denitrification pathway, reducing nitric oxide (NO) to nitrous oxide (N2O), subsequently promoting the formation of ...the NN bond. Three types of bacterial NOR are known, namely cNOR, qNOR and qCuNOR, that differ on the physiological electron donor. cNOR has been purified as a two subunit complex, the NorC, anchored to the cytoplasmic membrane, with a low-spin heme c, and the NorB subunit showing high structural homology with the HCuO subunit I, comprising a bis-histidine low-spin heme b and a binuclear iron centre. The binuclear iron centre is the catalytic site and it is formed by a heme b3 coupled to a non-heme iron (FeB) through a μ-oxo bridge. The catalytic mechanism is still under discussion and three hypotheses have been proposed: the trans-mechanism, the cis-FeB and the cis-heme b3 mechanisms. In the present work, the Pseudomonas nautica cNOR electrochemical behaviour was studied by cyclic voltammetry (CV), using a pyrolytic graphite electrode modified with the immobilised protein. The protein redox centres were observed and the formal redox potentials were determined. The binuclear iron centre presents the lowest redox potential value, and discrimination between the heme b3 and FeB redox processes was attained. Also, the number of electrons involved and correspondent surface electronic transfer rate constants were estimated. The pH dependence of the observed redox processes was determined and some new insights on the NOR catalytic mechanism are discussed.
► This work reports the cNOR characterization attained via direct electron transfer. ► Metal centre midpoint potentials and number of involved electrons were estimated. ► Redox centres' pH dependence was evaluated. ► Non-heme iron FeB process occurs at lower potential value than previously reported. ► Electrochemical results of heme b3 on turnover conditions point to cis-FeB mechanism.
The bioenergetics of anaerobic metabolism frequently relies on redox loops performed by membrane complexes with substrate- and quinone-binding sites on opposite sides of the membrane. However, in ...sulfate respiration (a key process in the biogeochemical sulfur cycle), the substrate- and quinone-binding sites of the QrcABCD complex are periplasmic, and their role in energy conservation has not been elucidated. Here we show that the QrcABCD complex of Desulfovibrio vulgaris is electrogenic, as protons and electrons required for quinone reduction are extracted from opposite sides of the membrane, with a H
/e
ratio of 1. Although the complex does not act as a H
-pump, QrcD may include a conserved proton channel leading from the N-side to the P-side menaquinone pocket. Our work provides evidence of how energy is conserved during dissimilatory sulfate reduction, and suggests mechanisms behind the functions of related bacterial respiratory complexes in other bioenergetic contexts.
Ferritins are ubiquitous and can be found in practically all organisms that utilize Fe. They are composed of 24 subunits forming a hollow sphere with an inner cavity of ∼80 Å in diameter. The main ...function of ferritin is to oxidize the cytotoxic Fe2+ ions and store the oxidized Fe in the inner cavity. It has been established that the initial step of rapid oxidation of Fe2+ (ferroxidation) by H-type ferritins, found in vertebrates, occurs at a diiron binding center, termed the ferroxidase center. In bacterial ferritins, however, X-ray crystallographic evidence and amino acid sequence analysis revealed a trinuclear Fe binding center comprising a binuclear Fe binding center (sites A and B), homologous to the ferroxidase center of H-type ferritin, and an adjacent mononuclear Fe binding site (site C). In an effort to obtain further evidence supporting the presence of a trinuclear Fe binding center in bacterial ferritins and to gain information on the states of the iron bound to the trinuclear center, bacterial ferritin from Desulfovibrio vulgaris (DvFtn) and its E130A variant was loaded with substoichiometric amounts of Fe2+, and the products were characterized by Mössbauer and EPR spectroscopy. Four distinct Fe species were identified: a paramagnetic diferrous species, a diamagnetic diferrous species, a mixed valence Fe2+Fe3+ species, and a mononuclear Fe2+ species. The latter three species were detected in the wild-type DvFtn, while the paramagnetic diferrous species was detected in the E130A variant. These observations can be rationally explained by the presence of a trinuclear Fe binding center, and the four Fe species can be properly assigned to the three Fe binding sites. Further, our spectroscopic data suggest that (1) the fully occupied trinuclear center supports an all ferrous state, (2) sites B and C are bridged by a μ-OH group forming a diiron subcenter within the trinuclear center, and (3) this subcenter can afford both a mixed valence Fe2+Fe3+ state and a diferrous state. Mechanistic insights provided by these new findings are discussed and a minimal mechanistic scheme involving O–O bond cleavage is proposed.
Respiratory nitric oxide reductase (NOR) was purified from membrane extract of
Pseudomonas
(
Ps.
)
nautica
cells to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein ...is a heterodimer with subunits of molecular masses of 54 and 18 kDa. The gene encoding both subunits was cloned and sequenced. The amino acid sequence shows strong homology with enzymes of the cNOR class. Iron/heme determinations show that one heme
c
is present in the small subunit (NORC) and that approximately two heme
b
and one non-heme iron are associated with the large subunit (NORB), in agreement with the available data for enzymes of the cNOR class. Mössbauer characterization of the as-purified, ascorbate-reduced and dithionite-reduced enzyme confirms the presence of three heme groups (the catalytic heme
b
3
, and the electron transfer heme
b
and heme
c
) and one redox-active non-heme Fe (Fe
B
). Consistent with results obtained for other cNORs, heme
c
and heme
b
in
Ps. nautica
cNOR were found to be low-spin while Fe
B
was found to be high-spin. Unexpectedly, as opposed to the presumed high-spin state for heme
b
3
, the Mössbauer data demonstrate unambiguously that heme
b
3
is, in fact, low-spin in both ferric and ferrous states, suggesting that heme
b
3
is six-coordinated regardless of its oxidation state. EPR spectroscopic measurements of the as-purified enzyme show resonances at the
g
~ 6 and
g
~ 2–3 regions very similar to those reported previously for other cNORs. The signals at
g
= 3.60, 2.99, 2.26 and 1.43 are attributed to the two charge-transfer low-spin ferric heme
c
and heme
b
. Previously, resonances at the g ~ 6 region were assigned to a small quantity of uncoupled high-spin Fe
III
heme
b
3
. This assignment is now questionable because heme
b
3
is low-spin. On the basis of our spectroscopic data, we argue that the
g
= 6.34 signal is likely arising from a spin-spin coupled binuclear center comprising the low-spin Fe
III
heme
b
3
and the high-spin Fe
B
III
. Activity assays performed under various reducing conditions indicate that heme
b
3
has to be reduced for the enzyme to be active. But, from an energetic point of view, the formation of a ferrous heme-NO as an initial reaction intermediate for NO reduction is disfavored because heme FeNO
7
is a stable product. We suspect that the presence of a sixth ligand in the Fe
II
-heme
b
3
may weaken its affinity for NO and thus promotes, in the first catalytic step, binding of NO at the Fe
B
II
site. The function of heme
b
3
would then be, to orient the Fe
B
-bound NO molecules for the formation of the N-N bound and to provide reducing equivalents for NO reduction.
Abstract
The gold standard for COVID-19 diagnostic testing relies on RNA extraction from naso/oropharyngeal swab followed by amplification through RT-PCR with fluorogenic probes. While the test is ...extremely sensitive and specific, its high cost and the potential discomfort associated with specimen collection made it suboptimal for public health screening purposes.
In this study, we developed an equally reliable, but cheaper and less invasive alternative test based on a one-step RT-PCR with the DNA-intercalating dye SYBR Green, which enables the detection of SARS-CoV-2 directly from saliva samples or RNA isolated from nasopharyngeal swabs. Importantly, we found that this type of testing can be fine-tuned to discriminate SARS-CoV-2 variants of concern.
The saliva RT-PCR SYBR Green test was successfully used in a mass-screening initiative targeting nearly 4500 asymptomatic children under the age of 12. Testing was performed at a reasonable cost, and in some cases, the saliva test outperformed nasopharyngeal rapid antigen tests in identifying infected children. Whole genome sequencing revealed that the antigen testing failure could not be attributed to a specific lineage of SARS-CoV-2.
Overall, this work strongly supports the view that RT-PCR saliva tests based on DNA-intercalating dyes represent a powerful strategy for community screening of SARS-CoV-2. The tests can be easily applied to other infectious agents and, therefore, constitute a powerful resource for an effective response to future pandemics.
PDF
