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All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Evolutionary Diversification of Host-Targeted Bartonella Effectors Proteins Derived from a Conserved FicTA Toxin-Antitoxin Module

Proteins containing a FIC domain catalyze AMPylation and other post-translational modifications (PTMs). In bacteria, they are typically part of FicTA toxin-antitoxin modules that control conserved biochemical processes such as topoisomerase activity, but they have also repeatedly diversified into host-targeted virulence factors. Among these, Bartonella effector proteins (Beps) comprise a particularly diverse ensemble of FIC domains that subvert various host cellular functions. However, no comprehensive comparative analysis has been performed to infer molecular mechanisms underlying the biochemical and functional diversification of FIC domains in the vast Bep family. Here, we used X-ray crystallography, structural modelling, and phylogenetic analyses to unravel the expansion and diversification of Bep repertoires that evolved in parallel in three Bartonella lineages from a single ancestral FicTA toxin-antitoxin module. Our analysis is based on 99 non-redundant Bep sequences and nine crystal structures. Inferred from the conservation of the FIC signature motif that comprises the catalytic histidine and residues involved in substrate binding, about half of them represent AMP transferases. A quarter of Beps show a glutamate in a strategic position in the putative substrate binding pocket that would interfere with triphosphate-nucleotide binding but may allow binding of an AMPylated target for deAMPylation or another substrate to catalyze a distinct PTM. The β-hairpin flap that registers the modifiable target segment to the active site exhibits remarkable structural variability. The corresponding sequences form few well-defined groups that may recognize distinct target proteins. The binding of Beps to promiscuous FicA antitoxins is well conserved, indicating a role of the antitoxin to inhibit enzymatic activity or to serve as a chaperone for the FIC domain before translocation of the Bep into host cells. Taken together, our analysis indicates a remarkable functional plasticity of Beps that is mostly brought about by structural changes in the substrate pocket and the target dock. These findings may guide future structure–function analyses of the highly versatile FIC domains.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Structural analysis of extracellular ATP-independent chaperones of streptococcal species and protein substrate interactions

ABSTRACT During infection, bacterial pathogens rely on secreted virulence factors to manipulate the host cell. However, in gram-positive bacteria, the molecular mechanisms underlying the folding and activity of these virulence factors after membrane translocation are not clear. Here, we solved the protein structures of two secreted parvulin and two secreted cyclophilin-like peptidyl-prolyl isomerase (PPIase) ATP-independent chaperones found in gram-positive streptococcal species. The extracellular parvulin-type PPIase, PrsA inStreptococcus pneumoniaeandStreptococcus mutansmaintain dimeric crystal structures reminiscent of folding catalysts that consist of two domains, a PPIase and foldase domain. Structural comparison of the two cyclophilin-like extracellular chaperones fromS. pneumoniaeandStreptococcus pyogeneswith other cyclophilins demonstrates that this group of cyclophilin-like chaperones has novel structural appendages formed by 9- and 24-residue insertions. Furthermore, we demonstrate that deletion ofprsAandslrAgenes impairs the secretion of the cholesterol-dependent pore-forming toxin, pneumolysin inS. pneumoniae. Using protein pull-down and biophysical assays, we demonstrate a direct interaction between PrsA and SlrA with Ply. Then, we developed chaperone-assisted folding assays that show that theS. pneumoniaePrsA and SlrA extracellular chaperones accelerate pneumolysin folding. In addition, we demonstrate that SlrA and, for the first time,S. pyogenes PpiA exhibit PPIase activity and can bind the immunosuppressive drug, cyclosporine A. Altogether, these findings suggest a mechanistic role for streptococcal PPIase chaperones in the activity and folding of secreted virulence factors such as pneumolysin. IMPORTANCE Streptococcal species are a leading cause of lower respiratory infections that annually affect millions of people worldwide. During infection, streptococcal species secrete a medley of virulence factors that allow the bacteria to colonize and translocate to deeper tissues. In many gram-positive bacteria, virulence factors are secreted from the cytosol across the bacterial membrane in an unfolded state. The bacterial membrane-cell wall interface is exposed to the potentially harsh extracellular environment, making it difficult for native virulence factors to fold before being released into the host. ATP-independent PPIase-type chaperones, PrsA and SlrA, are thought to facilitate folding and stabilization of several unfolded proteins to promote the colonization and spread of streptococci. Here, we present crystal structures of the molecular chaperones of PrsA and SlrA homologs from streptococcal species. We provide evidence that theStreptococcus pyogenesSlrA homolog, PpiA, has PPIase activity and binds to cyclosporine A. In addition, we show thatStreptococcus pneumoniaePrsA and SlrA directly interact and fold the cholesterol-dependent pore-forming toxin and critical virulence determinant, pneumolysin.

Microbiology↗
DOE OSTI2022

An Abundant and Diverse New Family of Electron Bifurcating Enzymes With a Non-canonical Catalytic Mechanism

Microorganisms utilize electron bifurcating enzymes in metabolic pathways to carry out thermodynamically unfavorable reactions. Bifurcating FeFe-hydrogenases (HydABC) reversibly oxidize NADH (E′∼−280 mV, under physiological conditions) and reduce protons to H 2 gas (E°′−414 mV) by coupling this endergonic reaction to the exergonic reduction of protons by reduced ferredoxin (Fd) (E′∼−500 mV). We show here that HydABC homologs are surprisingly ubiquitous in the microbial world and are represented by 57 phylogenetically distinct clades but only about half are FeFe-hydrogenases. The others have replaced the hydrogenase domain with another oxidoreductase domain or they contain additional subunits, both of which enable various third reactions to be reversibly coupled to NAD + and Fd reduction. We hypothesize that all of these enzymes carry out electron bifurcation and that their third substrates can include hydrogen peroxide, pyruvate, carbon monoxide, aldehydes, aryl-CoA thioesters, NADP + , cofactor F 420 , formate, and quinones, as well as many yet to be discovered. Some of the enzymes are proposed to be integral membrane-bound proton-translocating complexes. These different functionalities are associated with phylogenetically distinct clades and in many cases with specific microbial phyla. We propose that this new and abundant class of electron bifurcating enzyme be referred to as the Bfu family whose defining feature is a conserved bifurcating BfuBC core. This core contains FMN and six iron sulfur clusters and it interacts directly with ferredoxin (Fd) and NAD(H). Electrons to or from the third substrate are fed into the BfuBC core via BfuA. The other three known families of electron bifurcating enzyme (abbreviated as Nfn, EtfAB, and HdrA) contain a special FAD that bifurcates electrons to high and low potential pathways. The Bfu family are proposed to use a different electron bifurcation mechanism that involves a combination of FMN and three adjacent iron sulfur clusters, including a novel [2Fe-2S] cluster with pentacoordinate and partial non-Cys coordination. The absolute conservation of the redox cofactors of BfuBC in all members of the Bfu enzyme family indicate they have the same non-canonical mechanism to bifurcate electrons. A hypothetical catalytic mechanism is proposed as a basis for future spectroscopic analyses of Bfu family members.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Bactericidal effectors of the Stenotrophomonas maltophilia type IV secretion system: functional definition of the nuclease TfdA and structural determination of TfcB

ABSTRACT Stenotrophomonas maltophilia expresses a type IV protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria and does so partly by secreting the effector TfcB. Here, we report the structure of TfcB, comprising an N-terminal domain similar to the catalytic domain of glycosyl hydrolase (GH-19) chitinases and a C-terminal domain for recognition and translocation by the T4SS. Utilizing a two-hybrid assay to measure effector interactions with the T4SS coupling protein VirD4, we documented the existence of five more T4SS substrates. One of these was protein 20845, an annotated nuclease. A S. maltophilia mutant lacking the gene for 20845 was impaired for killing Escherichia coli , Klebsiella pneumoniae , and Pseudomonas aeruginosa . Moreover, the cloned 20845 gene conferred robust toxicity, with the recombinant E. coli being rescued when 20845 was co-expressed with its cognate immunity protein. The 20845 effector was an 899 amino-acid protein, comprised of a GHH-nuclease domain in its N-terminus, a large central region of indeterminant function, and a C-terminus for secretion. Engineered variants of the 20845 gene that had mutations in the predicted catalytic site did not impede E. coli , indicating that the antibacterial effect of 20845 involves its nuclease activity. Using flow cytometry with DNA staining, we determined that 20845, but not its mutant variants, confers a loss in DNA content of target bacteria. Database searches revealed that uncharacterized homologs of 20845 occur within a range of bacteria. These data indicate that the S. maltophilia T4SS promotes interbacterial competition through the action of multiple toxic effectors, including a potent, novel DNase. IMPORTANCE Stenotrophomonas maltophilia is a multi-drug-resistant, Gram-negative bacterium that is an emerging pathogen of humans. Patients with cystic fibrosis are particularly susceptible to S. maltophilia infection. In hospital water systems and various types of infections, S. maltophilia co-exists with other bacteria, including other pathogens such as Pseudomonas aeruginosa . We previously demonstrated that S. maltophilia has a functional VirB/D4 type VI protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria. Since most work on antibacterial systems involves the type VI secretion system, this observation remains noteworthy. Moreover, S. maltophilia currently stands alone as a model for a human pathogen expressing an antibacterial T4SS. Using biochemical, genetic, and cell biological approaches, we now report both the discovery of a novel antibacterial nuclease (TfdA) and the first structural determination of a bactericidal T4SS effector (TfcB).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Storage and persistence of organic carbon in the upper three meters of soil under arable and native prairie land use

Aims: Land use change from native grasslands to arable lands globally impacts soil ecosystem functions, including the storage of soil organic carbon (SOC). Understanding the factors affecting SOC changes in topsoil and subsoil due to land use is crucial for effective mitigation strategies. We determined SOC storage and persistence as affected by land use change from native prairies to arable lands. Methods: Here we examined SOC stocks, soil δ 13 C and Δ 14 C signatures, microbial communities (bacteria and fungi), and soil mineral characteristics under native prairies and long-term arable lands (i.e., > 40 years) down to 3 m in the U.S. Midwest. Results: Native prairie soils had higher SOC stocks in the A horizon and 0–50 cm depth increment than arable soils. For both land use types, the δ 13 C and Δ 14 C values significantly decreased with depth, with the latter pointing towards highly stabilized SOC, especially in the B- and C-horizons. Analysis of the microbial communities indicated that the diversity of bacteria and fungi decreased with increasing soil depth. The content of oxalate soluble Al appeared to be the single most important predictor of SOC across horizons and land use types. Conclusion: Our data suggest that most SOC gains and losses and transformation and translocation processes seem to be restricted to the uppermost 50 cm. Increasing SOC retention in the A and B horizons within the 0–50 cm depth would enhance organic material serving as substrate and nutrients for microbes and plants (A horizon) and facilitate long-term SOC storage in the subsoil (B horizon).

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Structure and mechanism of human vesicular polyamine transporter

Polyamines play essential roles in gene expression and modulate neuronal transmission in mammals. Vesicular polyamine transporters (VPAT) from the SLC18 family exploit the transmembrane H + gradient to translocate polyamines into secretory vesicles, enabling the quantal release of polyamine neuromodulators and underpinning learning and memory formation. Here, we report the cryo-electron microscopy structures of human VPAT in complex with spermine, spermidine, H + , or tetrabenazine, elucidating discrete lumen-facing states of the antiporter and pivotal interactions between VPAT and its substrate or inhibitor. Leveraging structure-inspired mutagenesis studies and protein structure prediction, we deduce an unforeseen mechanism whereby the polyamine and H + compete for multiple acidic protein residues both directly and indirectly, and rationalize how the antidopaminergic therapeutic tetrabenazine impedes vesicular transport of polyamines. This study unravels the mechanism of an H + -coupled polyamine antiporter, reveals mechanistic diversity between VPAT and other SLC18 antiporters, and raises new prospects for combating human disorders of polyamine homeostasis.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1994

ATP Synthesis in the Extremely Halophilic Bacteria

The proton-translocating ATPases are multimeric enzymes that carry out a multitude of essential functions. Their origin and evolution represent a seminal event in the early evolution of life. Amino acid sequences of the two largest subunits from archaeal ATPases (A-ATPases), vacuolar ATPases (V-ATPases), and FOF1-ATP syntheses (FATPases) suggest these ATPases evolved from an ancestral vacuolar-like ATP syntheses. A necessary consequence of this notion is that the A-ATPases are ATP syntheses. With the possible exception of the A-ATPase from Halobacterium salinarium. no A-ATPase has been demonstrated to synthesize ATP. The evidence for this case is dubious since ATP synthesis occurs only when conditions are distinctively unphysiological. We demonstrated that ATP synthesis in H.saccharovorum is inconsistent with the operation of an A-type ATPase. In order to determine if this phenomenon was unique to H. saccharovorum, ATP synthesis was examined in various extremely halophilic bacteria with the goal of ascertaining if it resembled what occurred in a. saccharovorum, or was consistent with the operation of an A-type ATPase. A-, V-, and F-type ATPases respond singularly to certain inhibitors. Therefore, the effect of these inhibitors on ATP synthesis in several extreme halophiles was determined. Inhibitors that either blocked or collapsed proton-gradients inhibited the steady state synthesis of ATP thus verifying that synthesis took place at the expense of a proton gradient. Azide, an inhibitor of F-ATPases inhibited ATP synthesis. Since the arginine-dependent synthesis of ATP, which occurs by way of substrate-level phosphorylation, was unaffected by azide, it was unlikely that azide acted as an "uncoupler." N -ethylmaleimide and nitrate, which inhibit V- and A-ATPases, either did not inhibit ATP synthesis or resulted in higher steady-state levels of ATP. These results suggest there are two types of proton-motive ATPases in the extreme halophiles (and presumably in other Archaea). One, the V-like enzyme which, provides protons that are subsequently used for solute translocation. The other ATPase is the familiar and ubiquitous F-ATPase that functions as a reversible proton pump and is the ATP Synthase in the extreme halophiles. Thus, while the suggested evolution of the proton -translocating ATPases accounts for the relationship among these ATPases, this scheme does not account for the presence of F-ATPases in the Archaea. Discounting lateral gene transfer, perhaps an F-type ATPase evolved before the eucaryal-archaeal and bacterial bifurcation. The presence of V-type ATPases in the Bacterial Domain is consistent with this suggestion. Finally, it is of interest to note that if an F-type ATPase appeared before the bifurcation, an endosymbiotic event need not be invoked to explain the presence of F-ATPases in the Eucarya.

Hochstein, Lawrence I.↗
DOE OSTI2022

Uncovering translation roadblocks during the development of a synthetic tRNA

Ribosomes are remarkable in their malleability to accept diverse aminoacyl-tRNA substrates from both the same organism and other organisms or domains of life. This is a critical feature of the ribosome that allows the use of orthogonal translation systems for genetic code expansion. Optimization of these orthogonal translation systems generally involves focusing on the compatibility of the tRNA, aminoacyl-tRNA synthetase, and a non-canonical amino acid with each other. As we expand the diversity of tRNAs used to include non-canonical structures, the question arises as to the tRNA suitability on the ribosome. Specifically, we investigated the ribosomal translation of allo-tRNA UTu1 , a uniquely shaped (9/3) tRNA exploited for site-specific selenocysteine insertion, using single-molecule fluorescence. With this technique we identified ribosomal disassembly occurring from translocation of allo-tRNA UTu1 from the A to the P site. Using cryo-EM to capture the tRNA on the ribosome, we pinpointed a distinct tertiary interaction preventing fluid translocation. Through a single nucleotide mutation, we disrupted this tertiary interaction and relieved the translation roadblock. With the continued diversification of genetic code expansion, our work highlights a targeted approach to optimize translation by distinct tRNAs as they move through the ribosome.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Genetically manipulated chloroplast stromal phosphate levels alter photosynthetic efficiency

Abstract The concentration of inorganic phosphate (Pi) in the chloroplast stroma must be maintained within narrow limits to sustain photosynthesis and to direct the partitioning of fixed carbon. However, it is unknown if these limits or the underlying contributions of different chloroplastic Pi transporters vary throughout the photoperiod or between chloroplasts in different leaf tissues. To address these questions, we applied live Pi imaging to Arabidopsis (Arabidopsis thaliana) wild-type plants and 2 loss-of-function transporter mutants: triose phosphate/phosphate translocator (tpt), phosphate transporter 2;1 (pht2;1), and tpt pht2;1. Our analyses revealed that stromal Pi varies spatially and temporally, and that TPT and PHT2;1 contribute to Pi import with overlapping tissue specificities. Further, the series of progressively diminished steady-state stromal Pi levels in these mutants provided the means to examine the effects of Pi on photosynthetic efficiency without imposing nutritional deprivation. ΦPSII and nonphotochemical quenching (NPQ) correlated with stromal Pi levels. However, the proton efflux activity of the ATP synthase (gH+) and the thylakoid proton motive force (pmf) were unaltered under growth conditions, but were suppressed transiently after a dark to light transition with return to wild-type levels within 2 min. These results argue against a simple substrate-level limitation of ATP synthase by depletion of stromal Pi, favoring more integrated regulatory models, which include rapid acclimation of thylakoid ATP synthase activity to reduced Pi levels.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Mapping Hsp104 interactions using cross‐linking mass spectrometry

Molecular machines from the AAA+ (ATPases Associated with diverse cellular Activity) superfamily of protein disaggregases play important roles in protein folding, disaggregation and DNA processing. Recent cryo-EM structures of AAA+ molecular machines have uncovered nuanced changes in their conformation that underlie their specialized functions. Structural knowledge of these molecular machines in complex with substrates begins to explain their mechanism of activity. Here, we explore how cross-linking mass spectrometry (XL-MS) can be used to interpret changes in conformation induced by ATP in Hsp104 and how a substrate may interact with Hsp104. We applied a panel of cross-linking reagents to produce cross-linking maps of Hsp104 and interpret our data on previously determined X-ray and cryo-EM structures of Hsp104 from a thermophilic yeast, Calcarisporiella thermophila. We developed an analysis pipeline to differentiate between intra-subunit and inter-subunit contacts within the hexameric homo-oligomer. We identify cross-links that break the asymmetry that is present in Hsp104 in an ATP-hydrolysis competent conformation but is absent in an ATP-hydrolysis-defective mutant. Finally, we identify contacts between Hsp104 and a selected protein (proprotein convertase subtilisin/kexin type 9 PCSK9) to reveal contacts on the central channel of Hsp104 across the length of this protein indicating that we might have trapped interactions consistent with its translocation. Our simple and robust XL-MS-based experiments and methods help interpret how these molecular machines change conformation and bind to other proteins even in the context of homo-oligomeric assemblies enabling coupling state-of-the-art modeling approaches with XL-MS.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Identification of novel small molecule inhibitors of twin arginine translocation (Tat) pathway and their effect on the control of Campylobacter jejuni in chickens

Introduction Control ofCampylobacterfrom farm to fork is challenging due to the frequent emergence of antimicrobial-resistant isolates. Furthermore, poultry production systems are known reservoirs ofCampylobacter. The twin-arginine translocation (Tat) pathway is a crucial bacterial secretion system that allowsCampylobacterto colonize the host intestinal tract by using formate as the main source of energy. However, Tat pathway is also a major contributing factor for resistance to copper sulfate (CuSO 4 ). Methods Since mammals and chickens do not have proteins or receptors that are homologous to bacterial Tat proteins, identification of small molecule (SM) inhibitors targeting the Tat system would allow the development of safe and effective control methods to mitigateCampylobacterin infected or colonized hosts in both pre-harvest and post-harvest. In this study, we screened 11 commercial libraries (n = 50,917 SM) for increased susceptibility to CuSO 4 (1 mM) inC. jejuni81–176, a human isolate which is widely studied. Results Furthermore, we evaluated 177 SM hits (2.5 μg/mL and above) that increased the susceptibility to CuSO 4 for the inhibition of formate dehydrogenase (Fdh) activity, a Tat-dependent substrate. Eight Tat-dependent inhibitors (T1–T8) were selected for further studies. These selected eight Tat inhibitors cleared all testedCampylobacterstrains (n = 12) at >10 ng/mL in the presence of 0.5 mM CuSO 4 in vitro. These selected SMs were non-toxic to colon epithelial (Caco-2) cells when treated with 50 μg/mL for 24 h and completely cleared intracellularC. jejunicells when treated with 0.63 μg/mL of SM for 24 h in the presence of 0.5 mM of CuSO 4 . Furthermore, 3 and 5-week-old chicks treated with SM candidates for 5 days had significantly decreased cecal colonization (up to 1.2 log;p < 0.01) with minimal disruption of microbiota.In silicoanalyses predicted that T7 has better drug-like properties than T2 inhibitor and might target a key amino acid residue (glutamine 165), which is located in the hydrophobic core of TatC protein. Discussion Thus, we have identified novel SM inhibitors of the Tat pathway, which represent a potential strategy to controlC. jejunispread on farms.

Microbiology↗
DOE OSTI2026

Shrub and sedge rhizosphere communities display distinct affinities toward exudates and soil organic matter degradation: a quantitative stable isotope probing analysis

Warming temperatures are accelerating permafrost thaw and changing tundra vegetation, where woody shrubs are displacing sedges. Shrubs, such as Betula nana, and sedges, such as Eriophorum vaginatum, exhibit distinct life strategies including unique root-associated, or rhizosphere microbial communities. As permafrost thaws it unlocks previously unavailable carbon and nutrient sources resulting in deeper roots and a translocation of rhizosphere communities. Because permafrost microbial communities contain lower diversity and biomass than rhizosphere communities, the coalescence of rhizosphere and permafrost microbial communities could alter soil organic matter (SOM) degradation rates and increase greenhouse gas emissions. To identify metabolic strategies across distinct rhizosphere and permafrost microbial communities we conducted an isotope tracing incubation experiment. We inoculated thawed permafrost with shrub and sedge rhizosphere communities while adding exudates or water daily and compared this to an uninoculated control. After 46 days, we spiked samples with 18O enriched water or 13C enriched exudates and measured isotope incorporation into microbial DNA with quantitative stable isotope probing (qSIP). Our results indicate that exudate additions had little effect on uninoculated permafrost communities but the addition of exudates and rhizosphere inoculants had a compounding effect on respiration rates. We found that soils inoculated with shrub rhizosphere communities contained a mixture of exudate and SOM degraders while soils inoculated with sedge rhizosphere communities contained mainly SOM degraders. Finally, we found that individual microbial taxa exhibited maximum growth rates under specific combinations of microbial inoculant communities and exudate addition treatments. Our results reveal that microbial niches are strongly influenced by substrate preferences and community context, and suggest that a reduction in sedges and an expansion of shrubs may provide a mechanism by which permafrost carbon losses are mitigated through corresponding shifts in microbial communities and their substrate preferences.

Schaefer, Sean R [Natural Resources and Earth Syst↗