The oral bacterial community in Melanophryniscus admirabilis (Admirable Red-Belly Toads): implications for conservation

Melanophryniscus admirabilis (admirable red-belly toad) is a microendemic and critically endangered species found exclusively along 700 m of the Forqueta River, in a fragment of the Atlantic Forest of southern Brazil. One of the greatest concerns regarding the conservation of this species is the extensive use of pesticides in areas surrounding their natural habitat. In recent years, the adaptation and persistence of animal species in human-impacted environments have been associated with microbiota. Therefore, the present study aimed to characterize the oral bacterial community of wild M. admirabilis and to address the question of how this community might contribute to this toad’s adaptation in the anthropogenic environment as well as its general metabolic capabilities. A total of 11 oral samples collected from wild M. admirabilis were characterized and analyzed via high-throughput sequencing. Fragments of the 16S rRNA variable region 4 (V4) were amplified, and sequencing was conducted using an Ion Personal Genome Machine (PGM) System with 316 chips. A total of 181,350 sequences were obtained, resulting in 16 phyla, 34 classes, 39 orders, and 77 families. Proteobacteria dominated (53%) the oral microbiota of toads, followed by Firmicutes (18%), Bacteroidetes (17%), and Actinobacteria (5%). No significant differences in microbial community profile from among the samples were reported, which suggests that the low dietary diversity observed in this population may directly influence the bacterial composition. Inferences of microbiome function were performed using PICRUSt2 software. Important pathways (e.g., xenobiotic degradation pathways for pesticides and aromatic phenolic compounds) were detected, which suggests that the bacterial communities may serve important roles in M. admirabilis health and survival in the anthropogenic environment. Overall, our results have important implications for the conservation and management of this microendemic and critically endangered specie

Effect of live Eimeria vaccination or salinomycin on growth and immune status in broiler chickens receiving in-feed inclusion of gelatin and vitamin E

This experiment determined if 2% of gelatin, to improve the levels of proline and glycine in the diet, and 70 mg/kg of vitamin E supplementation would relieve the impaired performance of male Cobb broilers vaccinated for coccidiosis. Half of the chicks were vaccinated via water (live oocysts), while the other half received medication (salinomycin) in the feed until 35 d of age. The effects of coccidiosis vaccine on performance and mRNA levels of genes involved in mucin synthesis, cytokines, trefoil family factor-2 (TFF2), and metabolic processes (CD36) in the jejunum of broilers were measured. Vaccination negatively affected performance in the first 21 d; however, the inclusion of gelatin and vitamin E reduced this negative response. Additionally, supplementation with these nutrients led to an improvement in broilers receiving the coccidiostat (P < 0.05). From 21 to 35 d, birds treated with gelatin and coccidiosis vaccine experienced better body weight gain than birds without gelatin and vitamin E (P < 0.05). Vaccinated chickens had decreased body weight and decreased anti-inflammatory cytokine expression. Fur- thermore, they had increased inflammatory cytokine expression, mucin 2 expression, and TFF2 compared to salinomycin-fed broilers (P < 0.05). Transcripts for IL- 1B, IFN-y, MUC2, TFF2 were decreased while mRNAs for IL-4 and IL-10 increased in salinomycin-fed broilers compared to vaccinated broilers (P < 0.05). In conclusion, broilers vaccinated against coccidiosis increase their pro-inflammatory immune status and mucin expression compared to broilers receiving salinomycin. These events may contribute to lower performance in vaccinated broiler chicks. Moreover, vitamin E and gelatin can minimize the vaccine’s negative immune effects and promote better performance

Selection of Reference Genes for Transcriptional Analysis of Edible Tubers of Potato (Solanum tuberosum L.)

Potato (Solanum tuberosum) yield has increased dramatically over the last 50 years and this has been achieved by a combination of improved agronomy and biotechnology efforts. Gene studies are taking place to improve new qualities and develop new cultivars. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a bench-marking analytical tool for gene expression analysis, but its accuracy is highly dependent on a reliable normalization strategy of an invariant reference genes. For this reason, the goal of this work was to select and validate reference genes for transcriptional analysis of edible tubers of potato. To do so, RT-qPCR primers were designed for ten genes with relatively stable expression in potato tubers as observed in RNA-Seq experiments. Primers were designed across exon boundaries to avoid genomic DNA contamination. Differences were observed in the ranking of candidate genes identified by geNorm, NormFinder and BestKeeper algorithms. The ranks determined by geNorm and NormFinder were very similar and for all samples the most stable candidates were C2, exocyst complex component sec3 (SEC3) and ATCUL3/ ATCUL3A/CUL3/CUL3A (CUL3A). According to BestKeeper, the importin alpha and ubiquitin-associated/ts-n genes were the most stable. Three genes were selected as reference genes for potato edible tubers in RT-qPCR studies. The first one, called C2, was selected in common by NormFinder and geNorm, the second one is SEC3, selected by NormFinder, and the third one is CUL3A, selected by geNorm. Appropriate reference genes identified in this work will help to improve the accuracy of gene expression quantification analyses by taking into account differences that may be observed in RNA quality or reverse transcription efficiency across the samples

Chloroplast HCF101 is a scaffold protein for [4Fe-4S] cluster assembly

Oxygen-evolving chloroplasts possess their own iron-sulfur cluster assembly proteins including members of the SUF (sulfur mobilization) and the NFU family. Recently, the chloroplast protein HCF101 (high chlorophyll fluorescence 101) has been shown to be essential for the accumulation of the membrane complex Photosystem I and the soluble ferredoxin-thioredoxin reductases, both containing [4Fe-4S] clusters. The protein belongs to the FSC-NTPase ([4Fe-4S]-cluster-containing P-loop NTPase) superfamily, several members of which play a crucial role in Fe/S cluster biosynthesis. Although the C-terminal ISC-binding site, conserved in other members of the FSC-NTPase family, is not present in chloroplast HCF101 homologues using Mössbauer and EPR spectroscopy, we provide evidence that HCF101 binds a [4Fe-4S] cluster. 55Fe incorporation studies of mitochondrially targeted HCF101 in Saccharomyces cerevisiae confirmed the assembly of an Fe/S cluster in HCF101 in an Nfs1-dependent manner. Site-directed mutagenesis identified three HCF101-specific cysteine residues required for assembly and/or stability of the cluster. We further demonstrate that the reconstituted cluster is transiently bound and can be transferred from HCF101 to a [4Fe-4S] apoprotein. Together, our findings suggest that HCF101 may serve as a chloroplast scaffold protein that specifically assembles [4Fe-4S] clusters and transfers them to the chloroplast membrane and soluble target proteins

In silico pathway reconstruction: Iron-sulfur cluster biogenesis in Saccharomyces cerevisiae

BACKGROUND: Current advances in genomics, proteomics and other areas of molecular biology make the identification and reconstruction of novel pathways an emerging area of great interest. One such class of pathways is involved in the biogenesis of Iron-Sulfur Clusters (ISC). RESULTS: Our goal is the development of a new approach based on the use and combination of mathematical, theoretical and computational methods to identify the topology of a target network. In this approach, mathematical models play a central role for the evaluation of the alternative network structures that arise from literature data-mining, phylogenetic profiling, structural methods, and human curation. As a test case, we reconstruct the topology of the reaction and regulatory network for the mitochondrial ISC biogenesis pathway in S. cerevisiae. Predictions regarding how proteins act in ISC biogenesis are validated by comparison with published experimental results. For example, the predicted role of Arh1 and Yah1 and some of the interactions we predict for Grx5 both matches experimental evidence. A putative role for frataxin in directly regulating mitochondrial iron import is discarded from our analysis, which agrees with also published experimental results. Additionally, we propose a number of experiments for testing other predictions and further improve the identification of the network structure. CONCLUSION: We propose and apply an iterative in silico procedure for predictive reconstruction of the network topology of metabolic pathways. The procedure combines structural bioinformatics tools and mathematical modeling techniques that allow the reconstruction of biochemical networks. Using the Iron Sulfur cluster biogenesis in S. cerevisiae as a test case we indicate how this procedure can be used to analyze and validate the network model against experimental results. Critical evaluation of the obtained results through this procedure allows devising new wet lab experiments to confirm its predictions or provide alternative explanations for further improving the models

Radical SAM enzyme QueE defines a new minimal core fold and metal-dependent mechanism

7-carboxy-7-deazaguanine synthase (QueE) catalyzes a key S-adenosyl-L-methionine (AdoMet)- and Mg[superscript 2+]-dependent radical-mediated ring contraction step, which is common to the biosynthetic pathways of all deazapurine-containing compounds. QueE is a member of the AdoMet radical superfamily, which employs the 5′-deoxyadenosyl radical from reductive cleavage of AdoMet to initiate chemistry. To provide a mechanistic rationale for this elaborate transformation, we present the crystal structure of a QueE along with structures of pre- and post-turnover states. We find that substrate binds perpendicular to the [4Fe-4S]-bound AdoMet, exposing its C6 hydrogen atom for abstraction and generating the binding site for Mg[superscript 2+], which coordinates directly to the substrate. The Burkholderia multivorans structure reported here varies from all other previously characterized members of the AdoMet radical superfamily in that it contains a hypermodified ([β [subscript 6] over α [subscript 3]]) protein core and an expanded cluster-binding motif, CX[subscript 14]CX[subscript 2]C.United States. Dept. of Energy. Office of Biological and Environmental ResearchUnited States. Dept. of Energy. Office of Basic Energy SciencesNational Center for Research Resources (U.S.) (P41RR012408)National Institute of General Medical Sciences (U.S.) (P41GM103473)National Center for Research Resources (U.S.) (5P41RR015301-10)National Institute of General Medical Sciences (U.S.) (8 P41 GM 103403-10)United States. Dept. of Energy (Contract DE-AC02-06CH11357

Metabolic reconstruction of sulfur assimilation in the extremophile Acidithiobacillus ferrooxidans based on genome analysis

BACKGROUND: Acidithiobacillus ferrooxidans is a gamma-proteobacterium that lives at pH2 and obtains energy by the oxidation of sulfur and iron. It is used in the biomining industry for the recovery of metals and is one of the causative agents of acid mine drainage. Effective tools for the study of its genetics and physiology are not in widespread use and, despite considerable effort, an understanding of its unusual physiology remains at a rudimentary level. Nearly complete genome sequences of A. ferrooxidans are available from two public sources and we have exploited this information to reconstruct aspects of its sulfur metabolism. RESULTS: Two candidate mechanisms for sulfate uptake from the environment were detected but both belong to large paralogous families of membrane transporters and their identification remains tentative. Prospective genes, pathways and regulatory mechanisms were identified that are likely to be involved in the assimilation of sulfate into cysteine and in the formation of Fe-S centers. Genes and regulatory networks were also uncovered that may link sulfur assimilation with nitrogen fixation, hydrogen utilization and sulfur reduction. Potential pathways were identified for sulfation of extracellular metabolites that may possibly be involved in cellular attachment to pyrite, sulfur and other solid substrates. CONCLUSIONS: A bioinformatic analysis of the genome sequence of A. ferrooxidans has revealed candidate genes, metabolic process and control mechanisms potentially involved in aspects of sulfur metabolism. Metabolic modeling provides an important preliminary step in understanding the unusual physiology of this extremophile especially given the severe difficulties involved in its genetic manipulation and biochemical analysis

Global diversity of enterococci and description of 18 novel species

Bacteria of the genus Enterococcus colonize the guts of diverse animals. Some species have acquired multiple antibiotic resistances on top of a high level of intrinsic resistance and have emerged as leading causes of hospital-associated infection. Although clinical isolates of enterococcal species E. faecalis and E. faecium have been studied with respect to their antibiotic resistances and infection pathogenesis, comparatively little is known about the biology of enterococci in their natural context of the guts of humans and other land animals, including arthropods and other invertebrates. Importantly, little is also known about the global pool of genes already optimized for expression in an enterococcal background with the potential to be readily acquired by hospital adapted strains of E. faecalis and E. faecium , known facile exchangers of mobile genetic elements. We therefore undertook a global study designed to reach into maximally diverse habitats, to establish a first approximation of the genetic diversity of enterococci on Earth. Presumptive enterococci from over 900 diverse specimens were initially screened by PCR using a specific reporter gene that we found to accurately reflect genomic diversity. The genomes of isolates exceeding an operationally set threshold for diversity were then sequenced in their entirety and analyzed. This provided us with data on the global occurrence of many known enterococcal species and their association with various hosts and ecologies and identified 18 novel species expanding the diversity of the genus Enterococcus by over 25%. The 18 novel enterococcal species harbor a diverse array of genes associated with toxins, detoxification, and resource acquisition that highlight the capacity of the enterococci to acquire and adapt novel functions from diverse gut environments. In addition to the discovery and characterization of new species, this expanded diversity permitted a higher resolution analysis of the phylogenetic structure of the Enterococcus genus, including identification of distinguishing features of its 4 deeply rooted clades and genes associated with range expansion such as B-vitamin biosynthesis and flagellar motility. Collectively, this work provides an unprecedentedly broad and deep view of the genus Enterococcus , along with new insights into their potential threat to human health