GenoVi, an open-source automated circular genome visualizer for bacteria and archaea.

The increase in microbial sequenced genomes from pure cultures and metagenomic samples reflects the current attainability of whole-genome and shotgun sequencing methods. However, software for genome visualization still lacks automation, integration of different analyses, and customizable options for non-experienced users. In this study, we introduce GenoVi, a Python command-line tool able to create custom circular genome representations for the analysis and visualization of microbial genomes and sequence elements. It is designed to work with complete or draft genomes, featuring customizable options including 25 different built-in color palettes (including 5 color-blind safe palettes), text formatting options, and automatic scaling for complete genomes or sequence elements with more than one replicon/sequence. Using a Genbank format file as the input file or multiple files within a directory, GenoVi (i) visualizes genomic features from the GenBank annotation file, (ii) integrates a Cluster of Orthologs Group (COG) categories analysis using DeepNOG, (iii) automatically scales the visualization of each replicon of complete genomes or multiple sequence elements, (iv) and generates COG histograms, COG frequency heatmaps and output tables including general stats of each replicon or contig processed. GenoVi's potential was assessed by analyzing single and multiple genomes of Bacteria and Archaea. Paraburkholderia genomes were analyzed to obtain a fast classification of replicons in large multipartite genomes. GenoVi works as an easy-to-use command-line tool and provides customizable options to automatically generate genomic maps for scientific publications, educational resources, and outreach activities. GenoVi is freely available and can be downloaded from https://github.com/robotoD/GenoVi.

Exploring the biosynthetic gene clusters in Brevibacterium: a comparative genomic analysis of diversity and distribution.

Exploring Brevibacterium strains from various ecosystems may lead to the discovery of new antibiotic-producing strains. Brevibacterium sp. H-BE7, a strain isolated from marine sediments from Northern Patagonia, Chile, had its genome sequenced to study the biosynthetic potential to produce novel natural products within the Brevibacterium genus. The genome sequences of 98 Brevibacterium strains, including strain H-BE7, were selected for a genomic analysis. A phylogenomic cladogram was generated, which divided the Brevibacterium strains into four major clades. A total of 25 strains are potentially unique new species according to Average Nucleotide Identity (ANIb) values. These strains were isolated from various environments, emphasizing the importance of exploring diverse ecosystems to discover the full diversity of Brevibacterium. Pangenome analysis of Brevibacterium strains revealed that only 2.5% of gene clusters are included within the core genome, and most gene clusters occur either as singletons or as cloud genes present in less than ten strains. Brevibacterium strains from various phylogenomic clades exhibit diverse BGCs. Specific groups of BGCs show clade-specific distribution patterns, such as siderophore BGCs and carotenoid-related BGCs. A group of clade IV-A Brevibacterium strains possess a clade-specific Polyketide synthase (PKS) BGCs that connects with phenazine-related BGCs. Within the PKS BGC, five genes, including the biosynthetic PKS gene, participate in the mevalonate pathway and exhibit similarities with the phenazine A BGC. However, additional core biosynthetic phenazine genes were exclusively discovered in nine Brevibacterium strains, primarily isolated from cheese. Evaluating the antibacterial activity of strain H-BE7, it exhibited antimicrobial activity against Salmonella enterica and Listeria monocytogenes. Chemical dereplication identified bioactive compounds, such as 1-methoxyphenazine in the crude extracts of strain H-BE7, which could be responsible of the observed antibacterial activity. While strain H-BE7 lacks the core phenazine biosynthetic genes, it produces 1-methoxyphenazine, indicating the presence of an unknown biosynthetic pathway for this compound. This suggests the existence of alternative biosynthetic pathways or promiscuous enzymes within H-BE7's genome.

Nasotracheal enterococcal carriage and resistomes: detection of optrA-, poxtA- and cfrD-carrying strains in migratory birds, livestock, pets, and in-contact humans in Spain.

This study determined the carriage rates and antimicrobial resistance (AMR) genes of enterococci from nasotracheal samples of three healthy animal species and in-contact humans. Nasal samples were collected from 27 dog-owning households (34 dogs, 41 humans) and 4 pig-farms (40 pigs, 10 pig-farmers), and they were processed for enterococci recovery (MALDI-TOF-MS identification). Also, a collection of 144 enterococci previously recovered of tracheal/nasal samples from 87 white stork nestlings were characterized. The AMR phenotypes were determined in all enterococci and AMR genes were studied by PCR/sequencing. MultiLocus-Sequence-Typing was performed for selected isolates. About 72.5% and 60% of the pigs and pig-farmers, and 29.4% and 4.9%, of healthy dogs and owners were enterococci nasal carriers, respectively. In storks, 43.5% of tracheal and 69.2% of nasal samples had enterococci carriages. Enterococci carrying multidrug-resistance phenotype was identified in 72.5%/40.0%/50.0%/23.5%/1.1% of pigs/pig-farmers/dogs/dogs' owners/storks, respectively. Of special relevance was the detection of linezolid-resistant enterococci (LRE) in (a) 33.3% of pigs (E. faecalis-carrying optrA and/or cfrD of ST59, ST330 or ST474 lineages; E. casseliflavus-carrying optrA and cfrD); (b) 10% of pig farmers (E. faecalis-ST330-carrying optrA); (c) 2.9% of dogs (E. faecalis-ST585-carrying optrA); and (d) 1.7% of storks (E. faecium-ST1736-carrying poxtA). The fexA gene was found in all optrA-positive E. faecalis and E. casseliflavus isolates, while fexB was detected in the poxtA-positive E. faecium isolate. The enterococci diversity and AMR rates from the four hosts reflect differences in antimicrobial selection pressure. The detection of LRE carrying acquired and transferable genes in all the hosts emphasizes the need to monitor LRE using a One-Health approach.

Spiractinospora alimapuensis gen. nov., sp. nov., isolated from marine sediment of Valparaíso Bay (Chile) and proposal for reclassification of two species of the genus Nocardiopsis.

An alkaliphilic actinobacterium, designated VN6-2T, was isolated from marine sediment collected from Valparaíso Bay, Chile. Strain VN6-2T formed yellowish-white branched substrate mycelium without fragmentation. Aerial mycelium was well developed, forming wavy or spiral spore chains. Strain VN6-2T exhibited a 16S rRNA gene sequence similarity of 93.9 % to Salinactinospora qingdaonensis CXB832T, 93.7 % to Murinocardiopsis flavida 14-Be-013T, and 93.7 % to Lipingzhangella halophila 14-Be-013T. Genome sequencing revealed a genome size of 5.9 Mb and an in silico G+C content of 69.3 mol%. Both of the phylogenetic analyses based on 16S rRNA gene sequences and the up-to-date bacterial core gene sequences revealed that strain VN6-2T formed a distinct monophyletic clade within the family Nocardiopsaceae. Chemotaxonomic assessment of strain VN6-2T showed that the major fatty acids were iso-C16 : 0, anteiso-C17 : 0 and 10-methyl-C18 : 0, and the predominant respiratory quinones were MK-9, MK-9(H2) and MK-9(H4). Whole-cell hydrolysates contained meso-diaminopimelic acid as the cell-wall diamino acid, and ribose and xylose as the diagnostic sugars. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, aminophospholipids, glycolipid and phospholipid. Based on the results of this polyphasic study, a novel genus, Spiractinospora gen. nov., is proposed within the family Nocardiopsaceae and the type species Spiractinospora alimapuensis gen. nov., sp. nov. The type strain is VN6-2T (CECT 30026T, CCUG 66258T). On the basis of the phylogenetic results herein, we also propose that Nocardiopsis arvandica and Nocardiopsis litoralis are later heterotypic synonyms of Nocardiopsis sinuspersici and Nocardiopsis kunsanensis, respectively, for which emended descriptions are given.

An Integrative Bioinformatic Analysis for Keratinase Detection in Marine-Derived Streptomyces.

Keratinases present promising biotechnological applications, due to their ability to degrade keratin. Streptomyces appears as one of the main sources of these enzymes, but complete genome sequences of keratinolytic bacteria are still limited. This article reports the complete genomes of three marine-derived streptomycetes that show different levels of feather keratin degradation, with high (strain G11C), low (strain CHD11), and no (strain Vc74B-19) keratinolytic activity. A multi-step bioinformatics approach is described to explore genes encoding putative keratinases in these genomes. Despite their differential keratinolytic activity, multiplatform annotation reveals similar quantities of ORFs encoding putative proteases in strains G11C, CHD11, and Vc74B-19. Comparative genomics classified these putative proteases into 140 orthologous groups and 17 unassigned orthogroup peptidases belonging to strain G11C. Similarity network analysis revealed three network communities of putative peptidases related to known keratinases of the peptidase families S01, S08, and M04. When combined with the prediction of cellular localization and phylogenetic reconstruction, seven putative keratinases from the highly keratinolytic strain Streptomyces sp. G11C are identified. To our knowledge, this is the first multi-step bioinformatics analysis that complements comparative genomics with phylogeny and cellular localization prediction, for the prediction of genes encoding putative keratinases in streptomycetes.

Structural and mechanistic basis for the inhibition of Escherichia coli RNA polymerase by T7 Gp2.

The T7 phage-encoded small protein Gp2 is a non-DNA-binding transcription factor that interacts with the jaw domain of the Escherichia coli (Ec) RNA polymerase (RNAp) ß' subunit and inhibits transcriptionally proficient promoter-complex (RPo) formation. Here, we describe the high-resolution solution structure of the Gp2-Ec ß' jaw domain complex and show that Gp2 and DNA compete for binding to the ß' jaw domain. We reveal that efficient inhibition of RPo formation by Gp2 requires the amino-terminal σ(70) domain region 1.1 (R1.1), and that Gp2 antagonizes the obligatory movement of R1.1 during RPo formation. We demonstrate that Gp2 inhibits RPo formation not just by steric occlusion of the RNAp-DNA interaction but also through long-range antagonistic effects on RNAp-promoter interactions around the RNAp active center that likely occur due to repositioning of R1.1 by Gp2. The inhibition of Ec RNAp by Gp2 thus defines a previously uncharacterized mechanism by which bacterial transcription is regulated by a viral factor.

Enzyme Bioprospection of Marine-Derived Actinobacteria from the Chilean Coast and New Insight in the Mechanism of Keratin Degradation in Streptomyces sp. G11C.

Marine actinobacteria are viewed as a promising source of enzymes with potential technological applications. They contribute to the turnover of complex biopolymers, such as pectin, lignocellulose, chitin, and keratin, being able to secrete a wide variety of extracellular enzymes. Among these, keratinases are a valuable alternative for recycling keratin-rich waste, which is generated in large quantities by the poultry industry. In this work, we explored the biocatalytic potential of 75 marine-derived actinobacterial strains, focusing mainly on the search for keratinases. A major part of the strains secreted industrially important enzymes, such as proteases, lipases, cellulases, amylases, and keratinases. Among these, we identified two streptomycete strains that presented great potential for recycling keratin wastes-Streptomyces sp. CHA1 and Streptomyces sp. G11C. Substrate concentration, incubation temperature, and, to a lesser extent, inoculum size were found to be important parameters that influenced the production of keratinolytic enzymes in both strains. In addition, proteomic analysis of culture broths from Streptomyces sp. G11C on turkey feathers showed a high abundance and diversity of peptidases, belonging mainly to the serine and metallo-superfamilies. Two proteases from families S08 and M06 were highly expressed. These results contributed to elucidate the mechanism of keratin degradation mediated by streptomycetes.

Corynebacterium alimapuense sp. nov., an obligate marine actinomycete isolated from sediment of Valparaíso bay, Chile.

A novel Gram-positive, non-motile, non-spore-forming and aerobic bacterium, designated strain VA37-3T, was isolated from a marine sediment sample collected at 19.2 m water depth from Valparaíso bay, Chile. Strain VA37-3T exhibits 97.6 % 16S rRNA gene sequence similarity to Corynebacterium marinum D7015T, 96.4 % to Corynebacterium humireducens MFC-5T and 96 % to Corynebacterium testudinoris M935/96/4T; and a rpoB gene sequence similarity of 85.1 % to Corynebacterium pollutisoli VMS11T, both analyses suggesting that strain VA37-3T represents a novel species of Corynebacterium. Physiological testing indicated that strain VA37-3T requires artificial sea water or sodium-supplemented media for growth, representing the first obligate marine actinomycete of the genus Corynebacterium. The genome of the proposed new species, along with the type strains of its most closely related species were sequenced and characterized. In silico genome-based similarity analyses revealed an ANIb of 72.8 % (C. marinum D7015T), ANIm of 85.0 % (Corynebacterium mustelae DSM 45274T), tetra of 0.90 (Corynebacterium callunae DSM 20147T) and ggdc of 24.7 % (Corynebacterium kutscheri DSM 20755T) when compared with the closest related strains. The genomic DNA G+C content of strain VA37-3T was 57.0 %. Chemotaxonomic assessment of strain VN6-2T showed the major fatty acids were C18 : 1ω9c and C16 : 0. Menaquinones predominantly consisted of MK-8(II-H2). Polar lipids consisted of diphosphatidylglycerol, glycolipids, phosphatidylglycerol, phosphoglycolipid and phosphatidylinositol. Mycolic acids also were present. Overall, the results from phylogenetic, phenotypic and genomic analyses confirmed that strain VA37-3T represents a novel species of the genus Corynebacterium, for which the name Corynebacterium alimapuense sp. nov. is proposed, with VA37-3T as the type strain (=CCUG 69366T=NCIMB 15118T).

Biodiversity of Actinobacteria from the South Pacific and the Assessment of Streptomyces Chemical Diversity with Metabolic Profiling.

Recently, bioprospecting in underexplored habitats has gained enhanced focus, since new taxa of marine actinobacteria can be found, and thus possible new metabolites. Actinobacteria are in the foreground due to their versatile production of secondary metabolites that present various biological activities, such as antibacterials, antitumorals and antifungals. Chilean marine ecosystems remain largely unexplored and may represent an important source for the discovery of bioactive compounds. Various culture conditions to enrich the growth of this phylum were used and 232 bacterial strains were isolated. Comparative analysis of the 16S rRNA gene sequences led to identifying genetic affiliations of 32 genera, belonging to 20 families. This study shows a remarkable culturable diversity of actinobacteria, associated to marine environments along Chile. Furthermore, 30 streptomycete strains were studied to establish their antibacterial activities against five model strains, Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica, Escherichia coli and Pseudomonas aeruginosa, demonstrating abilities to inhibit bacterial growth of Gram-positive bacteria. To gain insight into their metabolic profiles, crude extracts were submitted to liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis to assess the selection of streptomycete strains with potentials of producing novel bioactive metabolites. The combined approach allowed for the identification of three streptomycete strains to pursue further investigations. Our Chilean marine actinobacterial culture collection represents an important resource for the bioprospection of novel marine actinomycetes and its metabolites, evidencing their potential as producers of natural bioproducts.

Acute Suppurative Thyroiditis in Childhood: An Atypical Presentation.

Neck stiffness is a common clinical sign in children presenting to the emergency department that may indicate a wide variety of diagnoses. Acute suppurative thyroiditis (AST) is an infection of the thyroid gland caused by a bacterium, virus, or, less commonly, fungus. It presents as an acute or subacute development of an anterior cervical mass, with associated inflammatory signs. The pressure upon neck muscles may be reflected as a limitation of cervical mobility. AST is often preceded by an upper respiratory tract infection, and pyriform sinus fistula is the most common predisposing factor. It is particularly uncommon in the pediatric age, with limited cases reported in the literature. Therefore, a heightened suspicion is required for proper diagnosis and timely intervention, due to its high mortality. Prompt treatment with broad-spectrum parenteral antibiotic therapy and drainage is mandatory to prevent the numerous complications associated, namely, mediastinitis and sepsis. We report the case of a two-year-old female child admitted with a two-week history of intermittent high-grade fever and sore throat, followed by prostration and limitation in neck movement on admission. Examination revealed neck stiffness with positive Kernig and Brudzinski signs. The laboratory tests showed elevated inflammatory parameters. Cranial computed tomography (CT) scan and lumbar puncture were normal. On day 2 after admission, an anterior cervical mass with slight signs of fluctuation was detected. Ultrasound was compatible with a hemorrhagic/overinfected thyroid nodule, and the patient was started on broad-spectrum antibiotics. Due to clinical worsening, a cervical CT scan was performed, which documented a thyroid abscess with extension into the retropharyngeal space. She underwent surgical drainage, and Streptococcus anginosus and mixed anaerobes were isolated, sensitive to ongoing antibiotherapy. On multidisciplinary follow-up, an esophageal barium study, laryngoscopy, and cervical magnetic resonance imaging (MRI) were performed, revealing no anatomical defects. AST is a rare disease in children, but potentially fatal, so its early recognition and treatment are essential. We aim to draw attention to this disease and its differential diagnosis to reduce the associated morbimortality.

A Challenging Case of Recurrent Fever.

Syndrome of undifferentiated recurrent fever (SURF) includes heterogeneous episodes of systemic inflammation without documented infection, without response to antibiotherapy, and characterized by a paucity of specific clinical or molecular criteria. Colchicine is an effective treatment with an impact on morbimortality. We describe a case of a previously healthy one-year-old male, with consanguineous ancestry, admitted four times due to recurrent fever, associated with nonspecific symptoms and an increase of inflammatory markers in a sepsis-like pattern. No consistent infection was documented, and there was no response to broad-spectrum antibiotics. The evolution revealed corticosteroid dependency. The autoinflammatory syndrome-targeted next-generation sequencing (NGS) gene panel didn't detect relevant pathogenic variants. SURF was postulated as a diagnosis of exclusion, and the effectiveness of colchicine supports an autoinflammatory etiology. We aimed to draw attention to recurrent fevers associated with autoinflammatory disorders due to their challenging diagnosis. Improved understanding of immune pathways and advances in genetic testing will enable greater accuracy in the approach.

Mimicking Nonribosomal Peptides from the Marine Actinomycete Streptomyces sp. H-KF8 Leads to Antimicrobial Peptides.

Microorganisms within the marine environment have been shown to be very effective sources of naturally produced antimicrobial peptides (AMPs). Several nonribosomal peptides were identified based on genome mining predictions of Streptomyces sp. H-KF8, a marine Actinomycetota isolated from a remote Northern Chilean Patagonian fjord. Based on these predictions, a series of eight peptides, including cyclic peptides, were designed and chemically synthesized. Six of these peptides showed antimicrobial activity. Mode of action studies suggest that two of these peptides potentially act on the cell membrane via a novel mechanism allowing the passage of small ions, resulting in the dissipation of the membrane potential. This study shows that though structurally similar peptides, determined by NMR spectroscopy, the incorporation of small sequence mutations results in a dramatic influence on their bioactivity including mode of action. The qualified hit sequence can serve as a basis for more potent AMPs in future studies.

Unveiling metabolo-genomic insights of potent antitumoral and antibiotic activity in Streptomyces sp. VB1 from Valparaíso Bay.

Streptomyces sp. VB1, an actinomycete isolated from marine sediments in Valparaíso Bay, Chile, synthesizes antimicrobial and antiproliferative compounds. This study presents comprehensive metabolomics and comparative genomics analyses of strain VB1. LC-HRMS dereplication and Molecular Networking analysis of crude extracts identified antibiotics such as globomycin and daunorubicin, along with known and potentially novel members of the arylomycin family. These compounds exhibit activity against a range of clinically relevant bacterial and cancer cell lines. Phylogenomic analysis underscores the uniqueness of strain VB1, suggesting it represents a novel taxon. Such uniqueness is further supported by its Biosynthetic Novelty Index (BiNI) and BiG-SCAPE analysis of Gene Cluster Families (GCFs). Notably, two Biosynthetic Gene Clusters (BGCs) were found to be unique to VB1 compared to closely related strains: BGC #15, which encodes potentially novel anthracycline compounds with cancer cell growth inhibition properties, and BGC #28, which features a non-canonical configuration combining arylomycin, globomycin, and siamycin BGCs. This supercluster, the first described to consist of more than two adjacent and functional BGCs, co-produces at least three antimicrobial compounds from different antibiotic families. These findings highlight Streptomyces sp. VB1's potential for discovering new bioactive molecules, positioning it as a promising candidate for further research.

Statistical analysis of feature-based molecular networking results from non-targeted metabolomics data.

Feature-based molecular networking (FBMN) is a popular analysis approach for liquid chromatography-tandem mass spectrometry-based non-targeted metabolomics data. While processing liquid chromatography-tandem mass spectrometry data through FBMN is fairly streamlined, downstream data handling and statistical interrogation are often a key bottleneck. Especially users new to statistical analysis struggle to effectively handle and analyze complex data matrices. Here we provide a comprehensive guide for the statistical analysis of FBMN results, focusing on the downstream analysis of the FBMN output table. We explain the data structure and principles of data cleanup and normalization, as well as uni- and multivariate statistical analysis of FBMN results. We provide explanations and code in two scripting languages (R and Python) as well as the QIIME2 framework for all protocol steps, from data clean-up to statistical analysis. All code is shared in the form of Jupyter Notebooks ( https://github.com/Functional-Metabolomics-Lab/FBMN-STATS ). Additionally, the protocol is accompanied by a web application with a graphical user interface ( https://fbmn-statsguide.gnps2.org/ ) to lower the barrier of entry for new users and for educational purposes. Finally, we also show users how to integrate their statistical results into the molecular network using the Cytoscape visualization tool. Throughout the protocol, we use a previously published environmental metabolomics dataset for demonstration purposes. Together, the protocol, code and web application provide a complete guide and toolbox for FBMN data integration, cleanup and advanced statistical analysis, enabling new users to uncover molecular insights from their non-targeted metabolomics data. Our protocol is tailored for the seamless analysis of FBMN results from Global Natural Products Social Molecular Networking and can be easily adapted to other mass spectrometry feature detection, annotation and networking tools.

T7 phage protein Gp2 inhibits the Escherichia coli RNA polymerase by antagonizing stable DNA strand separation near the transcription start site.

Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the host RNA polymerase (RNAP)--a multi-subunit enzyme responsible for gene transcription--by a small ( approximately 7 kDa) phage-encoded protein called Gp2. Gp2 is also a potent inhibitor of E. coli RNAP in vitro. Here we describe the first atomic resolution structure of Gp2, which reveals a distinct run of surface-exposed negatively charged amino acid residues on one side of the molecule. Our comprehensive mutagenesis data reveal that two conserved arginine residues located on the opposite side of Gp2 are important for binding to and inhibition of RNAP. Based on a structural model of the Gp2-RNAP complex, we propose that inhibition of transcription by Gp2 involves prevention of RNAP-promoter DNA interactions required for stable DNA strand separation and maintenance of the "transcription bubble" near the transcription start site, an obligatory step in the formation of a transcriptionally competent promoter complex.

Analog neuromorphic circuit for spontaneous Ca2+ oscillations.

This study proposes an innovative analog neuromorphic circuit design to mimic spontaneous Ca2+ oscillations observed in astrocytes. Unlike traditional models, this approach does not rely on synaptic stimulation, suggesting that astrocytes may play a key role in generating neuronal activity. The circuit is built using transistor differential pairs to approximate the nonlinear sigmoidal biological functions, and its performance is validated through simulation and compared against mathematical models using phase diagram analysis. Results indicate a good fit between the circuit and the mathematical model. Finally, the circuit's ability to simulate the release of glutamate and ATP through spontaneous oscillations is demonstrated.

Glutaric Acidemia Type 1: Diagnosis, Clinical features, and Outcome in a Portuguese Cohort.

INTRODUCTION: Glutaric acidemia type 1 (GA1) is a rare autosomal recessive disorder characterized by a deficiency of glutaryl-CoA dehydrogenase, resulting in the accumulation of glutaric acid (GA), 3-hydroxyglutaric acid, and glutarylcarnitine, especially in the brain. GA1-affected children are clinically characterized by macrocephaly. Neurological abnormalities usually appear between 6 and 18 months of age, often triggered by a catabolic event. On the other hand, several biochemically affected individuals may remain asymptomatic or experience an insidious onset of mild neurological abnormalities. METHODS: Retrospective study of GA1 patients followed at a Portuguese Hereditary Metabolic Disease Center, to characterize the phenotypic and genotypic variations associated with GA1. Therefore, we analyzed the clinical, neuroradiological, biochemical, and genetic information from 14 patients. RESULTS: 14 patients (four months-27 years old) were identified in the last 26 years, 9 were male, 1 was from a consanguineous family. 11 were diagnosed by newborn screening (NBS), and 3 identified following clinical symptoms (later diagnosed, LD). There were 3 phenotypic presentations: 6 asymptomatic, 3 with a motor disability after encephalopathic crisis (EC), and 5 with insidious onset. Acute EC occurred in 1/3 of the LD patients and in 2/11 NBS-identified patients. About urinary GA concentrations: 5 were low excretors (LE), 9 were high excretors (HE). All LE showed symptoms, and 2 had EC. Concerning HE, 3 showed symptoms and 1 had EC. GCDH analysis showed: 6 compound heterozygotes and 8 homozygotes. most frequent variant was c.1204C>T (p.R402W). All of them received appropriate therapy from the time of diagnosis, with a mean age of 23.3 months in LD patients and 13.3 days in NBS-identified patients. CONCLUSION: The outcomes were different between the two groups: all the LD patients presented motor dysfunction however in the NBS-identified patients only 5 developed this symptom. Patients identified by NBS had better outcomes showing that NBS enables an early diagnosis, and treatment, and consequently improves the clinical outcomes for these patients. No correlation was observed with clinical phenotype between LE and HE, as both groups can suffer the most severe neurological manifestations. These conclusions are in agreement with previous cohorts described in the literature.

Phylogenetic classification of natural product biosynthetic gene clusters based on regulatory mechanisms.

The natural products (NPs) biosynthetic gene clusters (BGCs) represent the adapting biochemical toolkit for microorganisms to thrive different microenvironments. Despite their high diversity, particularly at the genomic level, detecting them in a shake-flask is challenging and remains the primary obstacle limiting our access to valuable chemicals. Studying the molecular mechanisms that regulate BGC expression is crucial to design of artificial conditions that derive on their expression. Here, we propose a phylogenetic analysis of regulatory elements linked to biosynthesis gene clusters, to classify BGCs to regulatory mechanisms based on protein domain information. We utilized Hidden Markov Models from the Pfam database to retrieve regulatory elements, such as histidine kinases and transcription factors, from BGCs in the MIBiG database, focusing on actinobacterial strains from three distinct environments: oligotrophic basins, rainforests, and marine environments. Despite the environmental variations, our isolated microorganisms share similar regulatory mechanisms, suggesting the potential to activate new BGCs using activators known to affect previously characterized BGCs.

Activity map of the Escherichia coli RNA polymerase bridge helix.

Transcription, the synthesis of RNA from a DNA template, is performed by multisubunit RNA polymerases (RNAPs) in all cellular organisms. The bridge helix (BH) is a distinct feature of all multisubunit RNAPs and makes direct interactions with several active site-associated mobile features implicated in the nucleotide addition cycle and RNA and DNA binding. Because the BH has been captured in both kinked and straight conformations in different crystals structures of RNAP, recently supported by molecular dynamics studies, it has been proposed that cycling between these conformations is an integral part of the nucleotide addition cycle. To further evaluate the role of the BH, we conducted systematic alanine scanning mutagenesis of the Escherichia coli RNAP BH to determine its contributions to activities required for transcription. Combining our data with an atomic model of E. coli RNAP, we suggest that alterations in the interactions between the BH and (i) the trigger loop, (ii) fork loop 2, and (iii) switch 2 can help explain the observed changes in RNAP functionality associated with some of the BH variants. Additionally, we show that extensive defects in E. coli RNAP functionality depend upon a single previously not studied lysine residue (Lys-781) that is strictly conserved in all bacteria. It appears that direct interactions made by the BH with other conserved features of RNAP are lost in some of the E. coli alanine substitution variants, which we infer results in conformational changes in RNAP that modify RNAP functionality.

Characterizing the microbial colonization of a dolostone quarry: implications for stone biodeterioration and response to biocide treatments.

This study examines the microbial colonization of three fronts of an abandoned dolostone quarry (Redueña, Madrid, Spain) exposed to atmospheric conditions for different time periods since Roman times to the present. Through scanning electron microscopy in backscattered electron mode (SEM-BSE), endolithic colonization was predominantly detected in the most recently exposed front, while in the longer exposed quarry fronts, epilithic forms of growth were most often observed. These observations were confirmed by denaturing gradient gel electrophoresis (DGGE) analysis. Based on the distribution pattern of microbial colonization in the different quarry fronts, we then established a sequence of colonization events that took place over this long time frame. Bioalteration processes related to this sequential colonization were also identified. Characterizing these sequential processes can be useful for interpreting biodeterioration processes in historic dolostone monuments, especially those affecting constructions in the area of the Redueña stone quarry. In a second experimental stage, different biocide treatments were tested on this quarry rock to find the best way to avoid the microbial colonization effects identified. Through combined SEM-BSE/DGGE analysis, the efficacy of several biocides against the microorganisms inhabiting the dolostones was assessed after 4 and 16 months treatment. In general, all treatments were effective at reducing around 80% of the lichen cover, although effects on endolithic lithobiontic communities were dependent on how well the rock surface had been mechanically cleaned prior to treatment and gradually disappeared over time.