Elucidation of structure-function relationships in Methanocaldococcus jannaschii RNase P, a multi-subunit catalytic ribonucleoprotein.

RNase P is a ribonucleoprotein (RNP) that catalyzes removal of the 5' leader from precursor tRNAs in all domains of life. A recent cryo-EM study of Methanocaldococcus jannaschii (Mja) RNase P produced a model at 4.6-Å resolution in a dimeric configuration, with each holoenzyme monomer containing one RNase P RNA (RPR) and one copy each of five RNase P proteins (RPPs; POP5, RPP30, RPP21, RPP29, L7Ae). Here, we used native mass spectrometry (MS), mass photometry (MP), and biochemical experiments that (i) validate the oligomeric state of the Mja RNase P holoenzyme in vitro, (ii) find a different stoichiometry for each holoenzyme monomer with up to two copies of L7Ae, and (iii) assess whether both L7Ae copies are necessary for optimal cleavage activity. By mutating all kink-turns in the RPR, we made the discovery that abolishing the canonical L7Ae-RPR interactions was not detrimental for RNase P assembly and function due to the redundancy provided by protein-protein interactions between L7Ae and other RPPs. Our results provide new insights into the architecture and evolution of RNase P, and highlight the utility of native MS and MP in integrated structural biology approaches that seek to augment the information obtained from low/medium-resolution cryo-EM models.

A Reduced F420-Dependent Nitrite Reductase in an Anaerobic Methanotrophic Archaeon.

Anaerobic methanotrophic archaea (ANME), which oxidize methane in marine sediments through syntrophic associations with sulfate-reducing bacteria, carry homologs of coenzyme F420-dependent sulfite reductase (Fsr) of Methanocaldococcus jannaschii, a hyperthermophilic methanogen from deep-sea hydrothermal vents. M. jannaschii Fsr (MjFsr) and ANME-Fsr belong to two phylogenetically distinct groups, FsrI and FsrII, respectively. MjFsrI reduces sulfite to sulfide with reduced F420 (F420H2), protecting methyl coenzyme M reductase (Mcr), an essential enzyme for methanogens, from sulfite inhibition. However, the function of FsrIIs in ANME, which also rely on Mcr and live in sulfidic environments, is unknown. We have determined the catalytic properties of FsrII from a member of ANME-2c. Since ANME remain to be isolated, we expressed ANME2c-FsrII in a closely related methanogen, Methanosarcina acetivorans. Purified recombinant FsrII contained siroheme, indicating that the methanogen, which lacks a native sulfite reductase, produced this coenzyme. Unexpectedly, FsrII could not reduce sulfite or thiosulfate with F420H2. Instead, it acted as an F420H2-dependent nitrite reductase (FNiR) with physiologically relevant Km values (nitrite, 5 µM; F420H2, 14 µM). From kinetic, thermodynamic, and structural analyses, we hypothesize that in FNiR, F420H2-derived electrons are delivered at the oxyanion reduction site at a redox potential that is suitable for reducing nitrite (E0' [standard potential], +440 mV) but not sulfite (E0', -116 mV). These findings and the known nitrite sensitivity of Mcr suggest that FNiR may protect nondenitrifying ANME from nitrite toxicity. Remarkably, by reorganizing the reductant processing system, Fsr transforms two analogous oxyanions in two distinct archaeal lineages with different physiologies and ecologies. IMPORTANCE Coenzyme F420-dependent sulfite reductase (Fsr) protects methanogenic archaea inhabiting deep-sea hydrothermal vents from the inactivation of methyl coenzyme M reductase (Mcr), one of their essential energy production enzymes. Anaerobic methanotrophic archaea (ANME) that oxidize methane and rely on Mcr, carry Fsr homologs that form a distinct clade. We show that a member of this clade from ANME-2c functions as F420-dependent nitrite reductase (FNiR) and lacks Fsr activity. This specialization arose from a distinct feature of the reductant processing system and not the substrate recognition element. We hypothesize FNiR may protect ANME Mcr from inactivation by nitrite. This is an example of functional specialization within a protein family that is induced by changes in electron transfer modules to fit an ecological need.

Catechol-containing compounds are a broad class of protein aggregation inhibitors: Redox state is a key determinant of the inhibitory activities.

A range of neurodegenerative and related aging diseases, such as Alzheimer's disease and type 2 diabetes, are linked to toxic protein aggregation. Yet the mechanisms of protein aggregation inhibition by small molecule inhibitors remain poorly understood, in part because most protein targets of aggregation assembly are partially unfolded or intrinsically disordered, which hinders detailed structural characterization of protein-inhibitor complexes and structural-based inhibitor design. Herein we employed a parallel small molecule library-screening approach to identify inhibitors against three prototype amyloidogenic proteins in neurodegeneration and related proteinopathies: amylin, Aß and tau. One remarkable class of inhibitors identified from these screens against different amyloidogenic proteins was catechol-containing compounds and redox-related quinones/anthraquinones. Secondary assays validated most of the identified inhibitors. In vivo efficacy evaluation of a selected catechol-containing compound, rosmarinic acid, demonstrated its strong mitigating effects of amylin amyloid deposition and related diabetic pathology in transgenic HIP rats. Further systematic investigation of selected class of inhibitors under aerobic and anaerobic conditions revealed that the redox state of the broad class of catechol-containing compounds is a key determinant of the amyloid inhibitor activities. The molecular insights we gained not only explain why a large number of catechol-containing polyphenolic natural compounds, often enriched in healthy diet, have anti-neurodegeneration and anti-aging activities, but also could guide the rational design of therapeutic or nutraceutical strategies to target a broad range of neurodegenerative and related aging diseases.

Coenzyme F420-Dependent Glucose-6-Phosphate Dehydrogenase-Coupled Polyglutamylation of Coenzyme F420 in Mycobacteria.

Coenzyme F420 plays a key role in the redox metabolisms of various archaea and bacteria, including Mycobacterium tuberculosis In M. tuberculosis, F420-dependent reactions have been linked to several virulence factors. F420 carries multiple glutamate residues in the side chain, forming F420-n species (n, number of glutamate residues), and the length of this side chain impacts cellular physiology. M. tuberculosis strains with F420 species carrying shorter side chains exhibit resistance to delamanid and pretomanid, two new tuberculosis (TB) drugs. Thus, the process of polyglutamylation of F420 is of great interest. It has been known from genetic analysis that in mycobacteria an F420-0 γ-glutamyl ligase (FbiB) introduces up to seven glutamate residues into F420 However, purified FbiB of M. tuberculosis (MtbFbiB) is either inefficient or incapable of incorporating more than two glutamates. We found that, in vitro, MtbFbiB synthesized side chains containing up to seven glutamate residues if F420 was presented to the enzyme in a two-electron reduced state (F420H2). Our genetic analysis in Mycobacterium bovis BCG and Mycobacterium smegmatis and an analysis of literature data on M. tuberculosis revealed that in these mycobacteria the polyglutamylation process requires the assistance of F420-dependent glucose-6-phosphate dehydrogenase (Fgd) which reduces F420 to F420H2 We hypothesize that, starting with F420-0H2, the amino-terminal domain of FbiB builds F420-2H2, which is then transferred to the carboxy-terminal domain for further glutamylation; F420-2H2 modifies the carboxy-terminal domain structurally to accommodate longer glutamyl chains. This system is analogous to folylpolyglutamate synthase, which introduces more than one glutamate residue into folate only after this vitamin is reduced to tetrahydrofolate.IMPORTANCE Coenzyme F420-dependent reactions of Mycobacterium tuberculosis, which causes tuberculosis, potentially contributes to the virulence of this bacterium. The coenzyme carries a glutamic acid-derived tail, the length of which influences the metabolism of M. tuberculosis Mutations that eliminate the production of F420 with longer tails make M. tuberculosis resistant to two new tuberculosis drugs. This report describes that the synthesis of longer glutamyl tails of F420 requires concerted actions of two enzymes, one of which reduces the coenzyme prior to the action of the other, which catalyzes polyglutamylation. This knowledge will help to develop more effective tuberculosis (TB) drugs. Remarkably, the introduction of multiple glutamate residues into the sidechain of folate (vitamin B9) requires similar concerted actions, where one enzyme reduces the vitamin to tetrahydrofolate and the other catalyzes polyglutamylation; folate is required for DNA and amino acid synthesis. Thus, the reported research has also revealed a key similarity between two important cellular systems.

A Novel F420-dependent Thioredoxin Reductase Gated by Low Potential FAD: A TOOL FOR REDOX REGULATION IN AN ANAEROBE.

A recent report suggested that the thioredoxin-dependent metabolic regulation, which is widespread in all domains of life, existed in methanogenic archaea about 3.5 billion years ago. We now show that the respective electron delivery enzyme (thioredoxin reductase, TrxR), although structurally similar to flavin-containing NADPH-dependent TrxRs (NTR), lacked an NADPH-binding site and was dependent on reduced coenzyme F420 (F420H2), a stronger reductant with a mid-point redox potential (E'0) of -360 mV; E'0 of NAD(P)H is -320 mV. Because F420 is a deazaflavin, this enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). It transferred electrons from F420H2 to thioredoxin via protein-bound flavin; Km values for thioredoxin and F420H2 were 6.3 and 28.6 µm, respectively. The E'0 of DFTR-bound flavin was approximately -389 mV, making electron transfer from NAD(P)H or F420H2 to flavin endergonic. However, under high partial pressures of hydrogen prevailing on early Earth and present day deep-sea volcanoes, the potential for the F420/F420H2 pair could be as low as -425 mV, making DFTR efficient. The presence of DFTR exclusively in ancient methanogens and mostly in the early Earth environment of deep-sea volcanoes and DFTR's characteristics suggest that the enzyme developed on early Earth and gave rise to NTR. A phylogenetic analysis revealed six more novel-type TrxR groups and suggested that the broader flavin-containing disulfide oxidoreductase family is more diverse than previously considered. The unprecedented structural similarities between an F420-dependent enzyme (DFTR) and an NADPH-dependent enzyme (NTR) brought new thoughts to investigations on F420 systems involved in microbial pathogenesis and antibiotic production.

Proline utilization system is required for infection by the pathogenic α-proteobacterium Brucella abortus.

Proline utilization (Put) systems have been described in a number of bacteria; however, the importance and functionality of the Put system in the intracellular pathogen Brucellaabortus has not been explored. Generally, bacterial Put systems are composed of the bifunctional enzyme proline dehydrogenase PutA and its transcriptional activator PutR. Here, we demonstrate that the genes putA (bab2_0518) and putR (bab2_0517) are critical for the chronic infection of mice by B. abortus, but putA and putR are not required for the survival and replication of the bacteria in naive macrophages. Additionally, in vitro experiments revealed that putR is necessary for the ability of the bacteria to withstand oxidative stress, as a ΔputR deletion strain is hypersensitive to hydrogen peroxide exposure. Quantitative reverse transcription-PCR and putA-lacZ transcriptional reporter studies revealed that PutR acts as a transcriptional activator of putA in Brucella, and electrophoretic mobility shift assays confirmed that PutR binds directly to the putA promoter region. Biochemical analyses demonstrated that a purified recombinant B. abortus PutA protein possesses quintessential proline dehydrogenase activity, as PutA is capable of catalysing the conversion of proline to glutamate. Altogether, these data are the first to reveal that the Put system plays a significant role in the ability of B. abortus to replicate and survive within its host, as well as to describe the genetic regulation and biochemical activity of the Put system in Brucella.

Thioredoxin targets fundamental processes in a methane-producing archaeon, Methanocaldococcus jannaschii.

Thioredoxin (Trx), a small redox protein, controls multiple processes in eukaryotes and bacteria by changing the thiol redox status of selected proteins. The function of Trx in archaea is, however, unexplored. To help fill this gap, we have investigated this aspect in methanarchaea--strict anaerobes that produce methane, a fuel and greenhouse gas. Bioinformatic analyses suggested that Trx is nearly universal in methanogens. Ancient methanogens that produce methane almost exclusively from H2 plus CO2 carried approximately two Trx homologs, whereas nutritionally versatile members possessed four to eight. Due to its simplicity, we studied the Trx system of Methanocaldococcus jannaschii--a deeply rooted hyperthermophilic methanogen growing only on H2 plus CO2. The organism carried two Trx homologs, canonical Trx1 that reduced insulin and accepted electrons from Escherichia coli thioredoxin reductase and atypical Trx2. Proteomic analyses with air-oxidized extracts treated with reduced Trx1 revealed 152 potential targets representing a range of processes--including methanogenesis, biosynthesis, transcription, translation, and oxidative response. In enzyme assays, Trx1 activated two selected targets following partial deactivation by O2, validating proteomics observations: methylenetetrahydromethanopterin dehydrogenase, a methanogenesis enzyme, and sulfite reductase, a detoxification enzyme. The results suggest that Trx assists methanogens in combating oxidative stress and synchronizing metabolic activities with availability of reductant, making it a critical factor in the global carbon cycle and methane emission. Because methanogenesis developed before the oxygenation of Earth, it seems possible that Trx functioned originally in metabolic regulation independently of O2, thus raising the question whether a complex biological system of this type evolved at least 2.5 billion years ago.

F420H2 Is Required for Phthiocerol Dimycocerosate Synthesis in Mycobacteria.

UNLABELLED: Phthiocerol dimycocerosates (PDIM) are a group of cell surface-associated apolar lipids of Mycobacterium tuberculosis and closely related mycobacteria, such as Mycobacterium bovis and Mycobacterium leprae A characteristic methoxy group of these lipids is generated from the methylation of a hydroxyl group of the direct precursors, the phthiotriols. The precursors arise from the reduction of phthiodiolones, the keto intermediates, by a ketoreductase. The putative phthiodiolone ketoreductase (PKR) is encoded by Rv2951c in M. tuberculosis and BCG_2972c in M. bovis BCG, and these open reading frames (ORFs) encode identical amino acid sequences. We investigated the cofactor requirement of the BCG_2972c protein. A comparative analysis based on the crystallographic structures of similar enzymes identified structural elements for binding of coenzyme F420 and hydrophobic phthiodiolones in PKR. Coenzyme F420 is a deazaflavin coenzyme that serves several key functions in pathogenic and nonpathogenic mycobacteria. We found that an M. bovis BCG mutant lacking F420-dependent glucose-6-phosphate dehydrogenase (Fgd), which generates F420H2 (glucose-6-phosphate + F420 → 6-phosphogluconate + F420H2), was devoid of phthiocerols and accumulated phthiodiolones. When the mutant was provided with F420H2, a broken-cell slurry of the mutant converted accumulated phthiodiolones to phthiocerols; F420H2 was generated in situ from F420 and glucose-6-phosphate by the action of Fgd. Thus, the reaction mixture was competent in reducing phthiodiolones to phthiotriols (phthiodiolones + F420H2 → phthiotriols + F420), which were then methylated to phthiocerols. These results established the mycobacterial phthiodiolone ketoreductase as an F420H2-dependent enzyme (fPKR). A phylogenetic analysis of close homologs of fPKR revealed potential F420-dependent lipid-modifying enzymes in a broad range of mycobacteria. IMPORTANCE: Mycobacterium tuberculosis is the causative agent of tuberculosis, and phthiocerol dimycocerosates (PDIM) protect this pathogen from the early innate immune response of an infected host. Thus, the PDIM synthesis system is a potential target for the development of effective treatments for tuberculosis. The current study shows that a PDIM synthesis enzyme is dependent on the coenzyme F420 F420 is universally present in mycobacteria and absent in humans. This finding expands the number of experimentally validated F420-dependent enzymes in M. tuberculosis to six, each of which helps the pathogen to evade killing by the host immune system, and one of which activates an antituberculosis drug, PA-824. This work also has relevance to leprosy, since similar waxy lipids are found in Mycobacterium leprae.

Reclassification of Desulfurococcus mobilis as a synonym of Desulfurococcus mucosus, Desulfurococcus fermentans and Desulfurococcus kamchatkensis as synonyms of Desulfurococcus amylolyticus, and emendation of the D. mucosus and D. amylolyticus species descriptions.

Representatives of the crenarchaeal genus Desulfurococcus are strictly anaerobic hyperthermophiles with an organotrophic type of metabolism. Since 1982, five Desulfurococcus species names have been validly published: Desulfurococcus mucosus, D. mobilis, D. amylolyticus, D. fermentans and D. kamchatkensis. Recently, the genomic sequences of all five species became available, promoting the refinement of their taxonomic status. Analysis of full-length high-quality 16S rRNA gene sequences shows that the sequences of D. mobilis and D. mucosus are 100 % identical and differ by 2.2 % from those of D. amylolyticus, D. fermentans and D. kamchatkensis. The latter three sequences differ from each other by 0.1-0.3 % (99.9 % similarity in the D amylolyticus-D. kamchatkensis pair and 99.7 % in the pairs involving D. fermentans). In silico prediction of DNA-DNA hybridization (DDH) values by comparison of genomes using ggdc 2.0 blast+ at http://ggdc.dsmz.de/ produced results that correlated with the 16S rRNA gene sequence similarity values. In the D. mucosus-D. mobilis and D. amylolyticus-D. kamchatkensis pairs, the predicted DDH values were 99 and 92 %, respectively, much higher than the recommended 70 % species-delimiting DDH value. Between members of different pairs, these values were no higher than 20 %. For D. fermentans, its predicted DDH values were around 70 % with D. amylolyticus and D. kamchatkensis and no higher than 20 % with D. mobilis and D. mucosus. These results indicated that D. mobilis should be reclassified as a synonym of D. mucosus, whereas D. kamchatkensis and D. fermentans should be reclassified as synonyms of D. amylolyticus.

Ferredoxin:thioredoxin reductase (FTR) links the regulation of oxygenic photosynthesis to deeply rooted bacteria.

Uncovered in studies on photosynthesis 35 years ago, redox regulation has been extended to all types of living cells. We understand a great deal about the occurrence, function, and mechanism of action of this mode of regulation, but we know little about its origin and its evolution. To help fill this gap, we have taken advantage of available genome sequences that make it possible to trace the phylogenetic roots of members of the system that was originally described for chloroplasts-ferredoxin, ferredoxin:thioredoxin reductase (FTR), and thioredoxin as well as target enzymes. The results suggest that: (1) the catalytic subunit, FTRc, originated in deeply rooted microaerophilic, chemoautotrophic bacteria where it appears to function in regulating CO(2) fixation by the reverse citric acid cycle; (2) FTRc was incorporated into oxygenic photosynthetic organisms without significant structural change except for addition of a variable subunit (FTRv) seemingly to protect the Fe-S cluster against oxygen; (3) new Trxs and target enzymes were systematically added as evolution proceeded from bacteria through the different types of oxygenic photosynthetic organisms; (4) an oxygenic type of regulation preceded classical light-dark regulation in the regulation of enzymes of CO(2) fixation by the Calvin-Benson cycle; (5) FTR is not universally present in oxygenic photosynthetic organisms, and in certain early representatives is seemingly functionally replaced by NADP-thioredoxin reductase; and (6) FTRc underwent structural diversification to meet the ecological needs of a variety of bacteria and archaea.

Ribosomal protein L7Ae is a subunit of archaeal RNase P.

To the mounting evidence of nonribosomal functions for ribosomal proteins, we now add L7Ae as a subunit of archaeal RNase P, a ribonucleoprotein (RNP) that catalyzes 5'-maturation of precursor tRNAs (pre-tRNAs). We first demonstrate that L7Ae coelutes with partially purified Methanococcus maripaludis (Mma) RNase P activity. After establishing in vitro reconstitution of the single RNA with four previously known protein subunits (POP5, RPP21, RPP29, and RPP30), we show that addition of L7Ae to this RNase P complex increases the optimal reaction temperature and k(cat)/K(m) (by approximately 360-fold) for pre-tRNA cleavage to those observed with partially purified native Mma RNase P. We identify in the Mma RNase P RNA a putative kink-turn (K-turn), the structural motif recognized by L7Ae. The large stimulatory effect of Mma L7Ae on RNase P activity decreases to

Nitrite reductase activity in F420-dependent sulphite reductase (Fsr) from Methanocaldococcus jannaschii.

Methanocaldococcus jannaschii (Mj), a hyperthermophilic and evolutionarily deeply rooted methanogenic archaeon from a deep-sea hydrothermal vent, produces F420-dependent sulphite reductase (Fsr) in response to exposure to sulphite. This enzyme allows Mj to detoxify sulphite, a potent inhibitor of methyl coenzyme-M reductase (Mcr), by reducing it to sulphide with reduced coenzyme F420 (F420H2) as an electron donor; Mcr is essential for energy production for a methanogen. Fsr allows Mj to utilize sulphite as a sulphur source. Nitrite is another potent inhibitor of Mcr and is toxic to methanogens. It is reduced by most sulphite reductases. In this study, we report that MjFsr reduced nitrite to ammonia with F420H2 with physiologically relevant K m values (nitrite, 8.9 µM; F420H2, 9.7 µM). The enzyme also reduced hydroxylamine with a K m value of 112.4 µM, indicating that it was an intermediate in the reduction of nitrite to ammonia. These results open the possibility that Mj could use nitrite as a nitrogen source if it is provided at a low concentration of the type that occurs in its habitat.

Evolving understanding of rumen methanogen ecophysiology.

Production of methane by methanogenic archaea, or methanogens, in the rumen of ruminants is a thermodynamic necessity for microbial conversion of feed to volatile fatty acids, which are essential nutrients for the animals. On the other hand, methane is a greenhouse gas and its production causes energy loss for the animal. Accordingly, there are ongoing efforts toward developing effective strategies for mitigating methane emissions from ruminant livestock that require a detailed understanding of the diversity and ecophysiology of rumen methanogens. Rumen methanogens evolved from free-living autotrophic ancestors through genome streamlining involving gene loss and acquisition. The process yielded an oligotrophic lifestyle, and metabolically efficient and ecologically adapted descendants. This specialization poses serious challenges to the efforts of obtaining axenic cultures of rumen methanogens, and consequently, the information on their physiological properties remains in most part inferred from those of their non-rumen representatives. This review presents the current knowledge of rumen methanogens and their metabolic contributions to enteric methane production. It also identifies the respective critical gaps that need to be filled for aiding the efforts to mitigate methane emission from livestock operations and at the same time increasing the productivity in this critical agriculture sector.

Nitrogen transformation processes catalyzed by manure microbiomes in earthen pit and concrete storages on commercial dairy farms.

Storing manure is an essential aspect of nutrient management on dairy farms. It presents the opportunity to use manure efficiently as a fertilizer in crop and pasture production. Typically, the manure storages are constructed as earthen, concrete, or steel-based structures. However, storing manure can potentially emit aerial pollutants to the atmosphere, including nitrogen and greenhouse gases, through microbial and physicochemical processes. We have characterized the composition of the microbiome in two manure storage structures, a clay-lined earthen pit and an aboveground concrete storage tank, on commercial dairy farms, to discern the nitrogen transformation processes, and thereby, inform the development of mitigation practices to preserve the value of manure. First, we analyzed the 16S rRNA-V4 amplicons generated from manure samples collected from several locations and depths (0.3, 1.2, and 2.1-2.75 m below the surface) of the storages, identifying a set of Amplicon Sequence Variant (ASVs) and quantifying their abundances. Then, we inferred the respective metabolic capabilities. These results showed that the manure microbiome composition was more complex and exhibited more location-to-location variation in the earthen pit than in the concrete tank. Further, the inlet and a location with hard surface crust in the earthen pit had unique consortia. The microbiomes in both storages had the potential to generate ammonia but lacked the organisms for oxidizing it to gaseous compounds. However, the microbial conversion of nitrate to gaseous N2, NO, and N2O via denitrification and to stable ammonia via dissimilatory nitrite reduction seemed possible; minor quantities of nitrate was present in manure, potentially originating from oxidative processes occurring on the barn floor. The nitrate-transformation linked ASVs were more prevalent at the near-surface locations and all depths of the inlet. Anammox bacteria and archaeal or bacterial autotrophic nitrifiers were not detected in either storage. Hydrogenotrophic Methanocorpusculum species were the primary methanogens or methane producers, exhibiting higher abundance in the earthen pit. These findings suggested that microbial activities were not the main drivers for nitrogen loss from manure storage, and commonly reported losses are associated with the physicochemical processes. Finally, the microbiomes of stored manure had the potential to emit greenhouse gases such as NO, N2O, and methane.

Complete genome sequence of Desulfurococcus fermentans, a hyperthermophilic cellulolytic crenarchaeon isolated from a freshwater hot spring in Kamchatka, Russia.

Desulfurococcus fermentans is the first known cellulolytic archaeon. This hyperthermophilic and strictly anaerobic crenarchaeon produces hydrogen from fermentation of various carbohydrates and peptides without inhibition by accumulating hydrogen. The complete genome sequence reported here suggested that D. fermentans employs membrane-bound hydrogenases and novel glycohydrolases for hydrogen production from cellulose.

Conversion of NO2 to NO by reduced coenzyme F420 protects mycobacteria from nitrosative damage.

In mycobacteria, F(420), a deazaflavin derivative, acts as a hydride transfer coenzyme for an F(420)-specific glucose-6-phosphate dehydrogenase (Fgd). Physiologically relevant reactions in the mycobacteria that use Fgd-generated reduced F(420) (F(420)H(2)) are unknown. In this work, F(420)H(2) was found to be oxidized by NO only in the presence of oxygen. Further analysis demonstrated that NO(2), produced from NO and O(2), was the oxidant. UV-visible spectroscopic and NO-sensor-based analyses proved that F(420)H(2) reduced NO(2) to NO. This reaction could serve as a defense system for Mycobacterium tuberculosis, which is more sensitive to NO(2) than NO under aerobic conditions. Activated macrophages produce NO, which in acidified phagosomes is converted to NO(2). Hence, by converting NO(2) back to NO with F(420)H(2), M. tuberculosis could decrease the effectiveness of antibacterial action of macrophages; such defense would correspond to active tuberculosis conditions where the bacterium grows aerobically. This hypothesis was consistent with the observation that a mutant strain of Mycobacterium smegmatis, a nonpathogenic relative of M. tuberculosis, which either did not produce or could not reduce F(420), was approximately 4-fold more sensitive to NO(2) than the wild-type strain. The phenomenon is reminiscent of the anticancer activity of gamma-tocopherol, which reduces NO(2) to NO and protects human cells from NO(2)-induced carcinogenesis.

Reduced protein sequence patterns in identifying key structural elements of dissimilatory sulfite reductase homologs.

Methanogenic archaea carry homologs of dissimilatory sulfite reductase (Dsr), called Dsr Like proteins (DsrLP). Dsr reduces sulfite to sulfide, a key step in an Earth's ancient metabolic process called dissimilatory sulfate reduction. The DsrLPs do not function as Dsr, and a computational approach is needed to develop hypotheses for guiding wet bench investigations on DsrLP's function. To make the computational analysis process efficient, the DsrLP amino acid sequences were transformed using only eight alphabets functionally representing twenty amino acids. The resultant reduced amino acid sequences were analyzed to identify conserved signature patterns in DsrLPs. Many of these patterns mapped on critical structural elements of Dsr and some were associated tightly with particular DsrLP groups. A search into the UniProtKB database identified several proteins carrying DsrLP's signature patterns; cysteine desulfurase, nucleosidase, and uroporphyrinogen III methylase were such matches. These outcomes provided clues to the functions of DsrLPs and highlighted the utility of the computational approach used.

Dominant remodelling of cattle rumen microbiome by Schedonorus arundinaceus (tall fescue) KY-31 carrying a fungal endophyte.

Tall fescue KY-31 is an important primary forage for beef cattle. It carries a fungal endophyte that produces ergovaline, the main cause of tall fescue toxicosis that leads to major revenue loss for livestock producers. The MaxQ, an engineered cultivar, hosts an ergovaline nonproducing strain of the fungus and consequently is nontoxic. However, it is less attractive economically. It is not known how rumen microbiome processes these two forages towards nutrient generation and ergovaline transformation. We have analysed the rumen microbiome compositions of cattle that grazed MaxQ with an intervening KY-31 grazing period using the 16S rRNA-V4 element as an identifier and found that KY-31 remodelled the microbiome substantially, encompassing both cellulolytic and saccharolytic functions. The effect was not evident at the whole microbiome levels but was identified by analysing the sessile and planktonic fractions separately. A move from MaxQ to KY-31 lowered the Firmicutes abundance in the sessile fraction and increased it in planktonic part and caused an opposite effect for Bacteroidetes, although the total abundances of these dominant rumen organisms remained unchanged. The abundances of Fibrobacter , which degrades less degradable fibres, and certain cellulolytic Firmicutes such as Pseudobutyrivibrio and Butyrivibrio 2, dropped in the sessile fraction, and these losses were apparently compensated by increased occurrences of Eubacterium and specific Ruminococcaceae and Lachnospiraceae . A return to MaxQ restored the original Firmicutes and Bacteroidetes distributions. However, several KY-31 induced changes, such as the low abundance of Fibrobacter and Butyrivibrio two remained in place, and their substitutes maintained significant presence. The rumen microbiome was distinct from previously reported faecal microbiomes. In summary, KY-31 and MaxQ were digested in the cattle rumen with distinct consortia and the KY-31-specific features were dominant. The study also identified candidate ergovaline transforming bacteria. It highlighted the importance of analysing sessile and planktonic fractions separately.

Nutritional Evaluation of Black Soldier Fly Frass as an Ingredient in Florida Pompano (Trachinotus carolinus L.) Diets.

The aquaculture industry is in need of sustainable fish feed to reduce the use of expensive and environmentally invasive wild-caught fish currently fed to many carnivorous species. The black soldier fly (BSF) has become a popular sustainable alternative protein source; however, the nutritional waste byproduct of BSF, frass, has not been extensively studied as a feed replacement in carnivorous species. This study evaluates the potential of BSF frass on the growth, body composition, and intestinal microbiome of the Florida pompano, Trachinotus carolinus. Four experimental diets were formulated containing different levels of frass, replacing plant-based carbohydrate sources. As a result of this study, the frass did not improve the growth performance, resulting in a lower specific growth rate and higher feed conversion rate. While the frass diets did not alter the body composition, the visceral somatic index (VSI) significantly increased compared to the control diet and the hepatosomatic index (HIS) was lowered. The microbiome analysis showed high variation among the diets, with the control diet having the most distinct consortia, which may have been driven by the increased levels of starch compared to frass diets. This study indicates that BSF frass may not be a suitable feed replacement for carnivorous pompano; however, frass could still potentially be a replacement feed for herbivore or detritivore fish and should be further studied.

Structure of an archaeal-type phosphoenolpyruvate carboxylase sensitive to inhibition by aspartate.

The crystal structure of an archaeal-type phosphoenolpyruvate carboxylase from Clostridium perfringens has been determined based on X-ray data extending to 3 Å. The asymmetric unit of the structure includes two tetramers (each a dimer-of-dimers) of the enzyme. The precipitant, malonate, employed for the crystallization is itself a weak inhibitor of phosphoenolpyruvate carboxylase and a malonate molecule is seen in the active-site in the crystal structure. The allosteric binding sites for aspartate (an inhibitor) and glucose-6-phosphate (an activator) observed in the Escherichia coli and Zea mays phosphoenolpyruvate carboxylase structures, respectively, are not conserved in the C. perfringens structure. Aspartate inhibits the C. perfringens enzyme competitively with respect to the substrate, Mg(++.) phosphoenolpyruvate. A mechanism for inhibition is proposed based on the structure and sequence comparisons with other archaeal-type phosphoenolpyruvate carboxylases with differing sensitivity to inhibition by aspartate.