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.

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.

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.

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.

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.

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.

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.

Corrigendum: A Genetic System for Methanocaldococcus jannaschii: An Evolutionary Deeply Rooted Hyperthermophilic Methanarchaeon.

[This corrects the article DOI: 10.3389/fmicb.2019.01256.].

Whole genome sequence analysis reveals the broad distribution of the RtxA type 1 secretion system and four novel putative type 1 secretion systems throughout the Legionella genus.

Type 1 secretion systems (T1SSs) are broadly distributed among bacteria and translocate effectors with diverse function across the bacterial cell membrane. Legionella pneumophila, the species most commonly associated with Legionellosis, encodes a T1SS at the lssXYZABD locus which is responsible for the secretion of the virulence factor RtxA. Many investigations have failed to detect lssD, the gene encoding the membrane fusion protein of the RtxA T1SS, in non-pneumophila Legionella, which has led to the assumption that this system is a virulence factor exclusively possessed by L. pneumophila. Here we discovered RtxA and its associated T1SS in a novel Legionella taurinensis strain, leading us to question whether this system may be more widespread than previously thought. Through a bioinformatic analysis of publicly available data, we classified and determined the distribution of four T1SSs including the RtxA T1SS and four novel T1SSs among diverse Legionella spp. The ABC transporter of the novel Legionella T1SS Legionella repeat protein secretion system shares structural similarity to those of diverse T1SS families, including the alkaline protease T1SS in Pseudomonas aeruginosa. The Legionella bacteriocin (1-3) secretion systems T1SSs are novel putative bacteriocin transporting T1SSs as their ABC transporters include C-39 peptidase domains in their N-terminal regions, with LB2SS and LB3SS likely constituting a nitrile hydratase leader peptide transport T1SSs. The LB1SS is more closely related to the colicin V T1SS in Escherichia coli. Of 45 Legionella spp. whole genomes examined, 19 (42%) were determined to possess lssB and lssD homologs. Of these 19, only 7 (37%) are known pathogens. There was no difference in the proportions of disease associated and non-disease associated species that possessed the RtxA T1SS (p = 0.4), contrary to the current consensus regarding the RtxA T1SS. These results draw into question the nature of RtxA and its T1SS as a singular virulence factor. Future studies should investigate mechanistic explanations for the association of RtxA with virulence.

A Genetic System for Methanocaldococcus jannaschii: An Evolutionary Deeply Rooted Hyperthermophilic Methanarchaeon.

Phylogenetically deeply rooted methanogens belonging to the genus of Methanocaldococcus living in deep-sea hydrothermal vents derive energy exclusively from hydrogenotrophic methanogenesis, one of the oldest respiratory metabolisms on Earth. These hyperthermophilic, autotrophic archaea synthesize their biomolecules from inorganic substrates and perform high temperature biocatalysis producing methane, a valuable fuel and potent greenhouse gas. The information processing and stress response systems of archaea are highly homologous to those of the eukaryotes. For this broad relevance, Methanocaldococcus jannaschii, the first hyperthermophilic chemolithotrophic organism that was isolated from a deep-sea hydrothermal vent, was also the first archaeon and third organism for which the whole genome sequence was determined. The research that followed uncovered numerous novel information in multiple fields, including those described above. M. jannaschii was found to carry ancient redox control systems, precursors of dissimilatory sulfate reduction enzymes, and a eukaryotic-like protein translocation system. It provided a platform for structural genomics and tools for incorporating unnatural amino acids into proteins. However, the assignments of in vivo relevance to these findings or interrogations of unknown aspects of M. jannaschii through genetic manipulations remained out of reach, as the organism was genetically intractable. This report presents tools and methods that remove this block. It is now possible to knockout or modify a gene in M. jannaschii and genetically fuse a gene with an affinity tag sequence, thereby allowing facile isolation of a protein with M. jannaschii-specific attributes. These tools have helped to genetically validate the role of a novel coenzyme F420-dependent sulfite reductase in conferring resistance to sulfite in M. jannaschii and to demonstrate that the organism possesses a deazaflavin-dependent system for neutralizing oxygen.

Comparative Genomics and Proteomic Analysis of Assimilatory Sulfate Reduction Pathways in Anaerobic Methanotrophic Archaea.

Sulfate is the predominant electron acceptor for anaerobic oxidation of methane (AOM) in marine sediments. This process is carried out by a syntrophic consortium of anaerobic methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB) through an energy conservation mechanism that is still poorly understood. It was previously hypothesized that ANME alone could couple methane oxidation to dissimilatory sulfate reduction, but a genetic and biochemical basis for this proposal has not been identified. Using comparative genomic and phylogenetic analyses, we found the genetic capacity in ANME and related methanogenic archaea for sulfate reduction, including sulfate adenylyltransferase, APS kinase, APS/PAPS reductase and two different sulfite reductases. Based on characterized homologs and the lack of associated energy conserving complexes, the sulfate reduction pathways in ANME are likely used for assimilation but not dissimilation of sulfate. Environmental metaproteomic analysis confirmed the expression of 6 proteins in the sulfate assimilation pathway of ANME. The highest expressed proteins related to sulfate assimilation were two sulfite reductases, namely assimilatory-type low-molecular-weight sulfite reductase (alSir) and a divergent group of coenzyme F420-dependent sulfite reductase (Group II Fsr). In methane seep sediment microcosm experiments, however, sulfite and zero-valent sulfur amendments were inhibitory to ANME-2a/2c while growth in their syntrophic SRB partner was not observed. Combined with our genomic and metaproteomic results, the passage of sulfur species by ANME as metabolic intermediates for their SRB partners is unlikely. Instead, our findings point to a possible niche for ANME to assimilate inorganic sulfur compounds more oxidized than sulfide in anoxic marine environments.

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.

Tn2008-driven carbapenem resistance in Acinetobacter baumannii isolates from a period of increased incidence of infections in a Southwest Virginia hospital (USA).

OBJECTIVES: The objectives of this study were (i) to determine the genetic basis for carbapenem resistance in multidrug-resistant (MDR) Acinetobacter baumannii strains isolated from patients affected by a sudden increase in the incidence of infections by such organisms in a tertiary care hospital in Virginia, USA, in 2009-2010 and (ii) to examine whether such strains are commonly encountered in the hospital setting. METHODS: The whole genomes of one outbreak strain as well as one carbapenem-resistant and one carbapenem-sensitive strain from sporadic infections in 2010-2012 were sequenced and analysed. Then, 5 outbreak isolates and 57 sporadic isolates (of which 39 were carbapenem-resistant) were screened by PCR for relevant DNA elements identified in the genomics investigation. RESULTS: All three strains for which whole-genome sequences were obtained carried resistance genes linked to MDR phenotypes and a ca. 111-kbp plasmid (pCMCVTAb1) without drug resistance genes. Of these, the two carbapenem-resistant strains possessed a ca. 74-kbp plasmid (pCMCVTAb2) carrying a Tn2008 transposon that provides high-level carbapenem resistance. PCR analysis showed that all of the outbreak isolates carried both plasmids and Tn2008, and of the sporadic isolates 88% carried pCMCVTAb1, 25% contained pCMCVTAb2 and 50% of the latter group carried Tn2008. CONCLUSIONS: Carbapenem resistance in outbreak strains and 12% of sporadic isolates was due to the pCMCVTAb2-borne Tn2008. This is the first report of a Tn2008-driven outbreak of carbapenem-resistant A. baumannii infections in the Commonwealth of Virginia, which followed similar cases in Pennsylvania and Ohio.

A Reexamination of Thioredoxin Reductase from Thermoplasma acidophilum, a Thermoacidophilic Euryarchaeon, Identifies It as an NADH-Dependent Enzyme.

Flavin-containing Trx reductase (TrxR) of Thermoplasma acidophilum (Ta), a thermoacidophilic facultative anaerobic archaeon, lacks the structural features for the binding of 2'-phosphate of nicotinamide adenine dinucleotide phosphate (NADPH), and this feature has justified the observed lack of activity with NADPH; NADH has also been reported to be ineffective. Our recent phylogenetic analysis identified Ta-TrxR as closely related to the NADH-dependent enzymes of Thermotoga maritima and Desulfovibrio vulgaris, both being anaerobic bacteria. This observation instigated a reexamination of the activity of the enzyme, which showed that Ta-TrxR is NADH dependent; the apparent Km for NADH was 3.1 µM, a physiologically relevant value. This finding is consistent with the observation that NADH:TrxR has thus far been found primarily in anaerobic bacteria and archaea.

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.

Complete Genome Sequence of Bordetella pertussis Pelita III, the Production Strain for an Indonesian Whole-Cell Pertussis Vaccine.

PT Bio Farma, the sole World Health Organization-approved Indonesian vaccine producer, manufactures a whole-cell whooping cough vaccine (wP) that, as part of a pentavalent diphtheria-tetanus-pertussis/hepatitis B/Haemophilus influenzae b (DTP/HB/Hib) vaccine, is used in Indonesia and many other countries. We report here the whole-genome sequence for Bordetella pertussis Pelita III, PT Bio Farma's wP production strain.