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Infections by multidrug-resistant pathogens are steadily increasing worldwide. A considerable proportion of neonatal intensive care admissions have a bacterial infection with multidrug-resistant bacteria during their hospital stay. In this work, we report draft genome sequences of 70 selected isolates from high-risk neonates in the Northeast of Mexico.

Natural products from Actinomycetota have served as inspiration for many clinically relevant therapeutics. Despite early triumphs in natural product discovery, the rate of unearthing new compounds has decreased, necessitating inventive approaches. One promising strategy is to explore environments where survival is challenging. These harsh environments are hypothesized to lead to bacteria developing chemical adaptations (e.g. natural products) to enable their survival. This investigation focuses on ore-forming environments, particularly fluoride mines, which typically have extreme pH, salinity and nutrient scarcity. Herein, we have utilized metagenomics, metabolomics and evolutionary genome mining to dissect the biodiversity and metabolism in these harsh environments. This work has unveiled the promising biosynthetic potential of these bacteria and has demonstrated their ability to produce bioactive secondary metabolites. This research constitutes a pioneering endeavour in bioprospection within fluoride mining regions, providing insights into uncharted microbial ecosystems and their previously unexplored natural products.

Poly-hydroxybutyrate (PHB) is an environmentally friendly alternative for conventional fossil fuel-based plastics that is produced by various microorganisms. Large-scale PHB production is challenging due to the comparatively higher biomanufacturing costs. A PHB overproducer is the haloalkaliphilic bacterium Halomonas campaniensis, which has low nutritional requirements and can grow in cultures with high salt concentrations, rendering it resistant to contamination. Despite its virtues, the metabolic capabilities of H. campaniensis as well as the limitations hindering higher PHB production remain poorly studied. To address this limitation, we present HaloGEM, the first high-quality genome-scale metabolic network reconstruction, which encompasses 888 genes, 1528 reactions (1257 gene-associated), and 1274 metabolites. HaloGEM not only displays excellent agreement with previous growth data and experiments from this study, but it also revealed nitrogen as a limiting nutrient when growing aerobically under high salt concentrations using glucose as carbon source. Among different nitrogen source mixtures for optimal growth, HaloGEM predicted glutamate and arginine as a promising mixture producing increases of 54.2% and 153.4% in the biomass yield and PHB titer, respectively. Furthermore, the model was used to predict genetic interventions for increasing PHB yield, which were consistent with the rationale of previously reported strategies. Overall, the presented reconstruction advances our understanding of the metabolic capabilities of H. campaniensis for rationally engineering this next-generation industrial biotechnology platform. KEY POINTS: A comprehensive genome-scale metabolic reconstruction of H. campaniensis was developed. Experiments and simulations predict N limitation in minimal media under aerobiosis. In silico media design increased experimental biomass yield and PHB titer., (© 2024. The Author(s).)

Mycolicibacterium fortuitum , a fast-growing nontuberculous mycobacterium, is a significant pathogen in healthcare-associated infections, encompassing skin, soft tissue, and pulmonary diseases. In this study, we present draft genome sequences from 12 M . fortuitum strains isolated from sputum samples from patients diagnosed with pulmonary infections in Mexico., Competing Interests: The authors declare no conflict of interest.

We report the draft genomes of four Kluyveromyces marxianus isolates obtained from the elaboration process of henequen ( Agave fourcroydes ) mezcal, a Mexican alcoholic beverage. The average nucleotide identity analysis revealed that isolates derived from agave plants are distinct from those from other environments, including agave fermentations., Competing Interests: The authors declare no conflict of interest.

Genetic variation in tuberculosis is influenced by the host environment, patients with comorbidity, and tuberculosis-type 2 diabetes mellitus (TB-T2DM) and implies a higher risk of treatment failure and development of drug resistance. Considering the above, this study aimed to evaluate the influence of T2DM on the dynamic of polymorphisms related to antibiotic resistance in TB. Fifty individuals with TB-T2DM and TB were initially characterized, and serial isolates of 29 of these individuals were recovered on day 0 (diagnosis), 30, and 60. Genomes were sequenced, variants related to phylogeny and drug resistance analyzed, and mutation rates calculated and compared between groups. Lineage X was predominant. At day 0 (collection), almost all isolates from the TB group were sensitive, apart from four isolates from the TB-T2DM group showing the mutation katG S315T, from which one isolate had the mutations rpoB S450L, gyrA A90G, and gyrA D94G. This pattern was observed in a second isolate at day 30. The results provide a first overview of the dynamics of mutations in resistance genes from individuals with TB-T2DM, describing an early development of resistance to isoniazid and a rapid evolution of resistance to other drugs. Although preliminary, these results help to explain the increased risk of drug resistance in individuals with TB and T2DM.

microbeMASST, a taxonomically informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbe-derived metabolites and relative producers without a priori knowledge will vastly enhance the understanding of microorganisms' role in ecology and human health., (© 2024. The Author(s).)

Narrow-spectrum antibiotics are of great interest given their ability to spare the microbiome and decrease widespread antibiotic resistance compared to broad-spectrum antibiotics. Herein, we screened an in-house library of Actinobacteria strains for selective activity against Acinetobacter baumannii and successfully identified Streptomyces sp. CS-62 as a producer of a natural product with this valuable activity. Analysis of the cultures via high-resolution mass spectrometry and tandem mass spectrometry, followed by comparison with molecules in the Natural Product Atlas and the Global Natural Products Social Molecular Networking platform, suggested a novel natural product. Genome mining analysis initially supported the production of a novel kirromycin derivative. Isolation and structure elucidation via mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses revealed that the active natural product was the known natural product factumycin, exposing omissions and errors in the consulted databases. While public databases are generally very useful for avoiding rediscovery of known molecules, rediscovery remains a problem due to public databases either being incomplete or having errors that result in failed dereplication. Overall, the work describes the ongoing problem of dereplication and the continued need for public database curation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Introduction: Tuberculosis (TB) is among the deadliest diseases worldwide, and its impact is mainly due to the continuous emergence of resistant isolates during treatment due to the laborious process of resistance diagnosis, nonadherence to treatment and circulation of previously resistant isolates of Mycobacterium tuberculosis. In this study, we evaluated the performance and functionalities of web-based tools, including Mykrobe, TB-profiler, PhyResSE, KvarQ, and SAM-TB, for detecting resistance in 88 Ecuadorian isolates of Mycobacterium tuberculosis drug susceptibility tested previously. Statistical analysis was used to determine the correlation between genomic and phenotypic analysis. Our results showed that with the exception of KvarQ, all tools had the highest correlation with the conventional drug susceptibility test (DST) for global resistance detection (98% agreement and 0.941 Cohen's kappa), while SAM-TB, PhyResSE, TB-profiler and Mykrobe had better correlations with DST for first-line drug analysis individually. We also identified that in our study, only 50% of mutations characterized by the web-based tools in the rpoB, katG, embB, pncA, gyrA and rrs regions were canonical and included in the World Health Organization (WHO) catalogue. Our findings suggest that SAM-TB, PhyResSE, TB-profiler and Mykrobe were efficient in determining canonical resistance-related mutations, but more analysis is needed to improve second-line detection. Improving surveillance programs using whole-genome sequencing tools for first-line drugs, MDR-TB and XDR-TB is essential to understand the molecular epidemiology of TB in Ecuador., Importance: Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis, most commonly affects the lungs and is often spread through the air when infected people cough, sneeze, or spit. However, despite the existence of effective drug treatment, patient adherence, long duration of treatment, and late diagnosis have reduced the effectiveness of therapy and increased drug resistance. The increase in resistant cases, added to the impact of the COVID-19 pandemic, has highlighted the importance of implementing efficient and timely diagnostic methodologies worldwide. The significance of our research is in evaluating and identifying a more efficient and user-friendly web-based tool to characterize resistance in Mycobacterium tuberculosis by whole-genome sequencing, which will allow more routine application to improve TB strain surveillance programs locally., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Morey-León et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Oligosaccharides are significant in mammalian milk, where they serve as prebiotics that promote the growth of beneficial gut bacteria in infants. Comprehensive research of milk oligosaccharides requires precise and validated analytical methods for compositional studies. To address this need, the focus of our study was to develop and validate an analytical method using UPLC-MS/MS to quantify seven specific oligosaccharides found in mammalian milk. The developed and optimized method has adequate linearity, accuracy, and precision parameters. The detection (LOD) and quantification (LOQ) limits for the seven compounds ranged from 0.0018 to 0.0030 μg/mL and 0.0054-0.0063 μg/mL, respectively. The sample preparation method yielded recovery rates above 90.5 %. Furthermore, no significant matrix effect was observed. The validated method was successfully applied to human, goat, and bovine milk samples, demonstrating its proficiency in identifying variances in the concentration of oligosaccharides across different mammals. This versatile method will allow future research about factors affecting oligosaccharide composition., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rodriguez-Sanchez, Gónzalez-Salazar, Rodriguez-Orduña, Cumsille, Undabarrena, Camara, Sélem-Mojica and Licona-Cassani.)

Genomics has significantly revolutionized pathogen surveillance, particularly in epidemiological studies, the detection of drug-resistant strains, and disease control. Despite its potential, the representation of Latin American countries in the genomic catalogues of Mycobacterium tuberculosis (Mtb), the bacteria responsible for Tuberculosis (TB), remains limited. In this study, we present a whole genome sequencing (WGS)-based analysis of 85 Mtb clinical strains from 17 Mexican states, providing insights into local adaptations and drug resistance signatures in the region. Our results reveal that the Euro-American lineage (L4) accounts for 94% of our dataset, showing 4.1.2.1 (Haarlem, n = 32), and 4.1.1.3 (X-type, n = 34) sublineages as the most prevalent. We report the presence of the 4.1.1.3 sublineage, which is endemic to Mexico, in six additional locations beyond previous reports. Phenotypic drug resistance tests showed that 34 out of 85 Mtb samples were resistant, exhibiting a variety of resistance profiles to the first-line antibiotics tested. We observed high levels of discrepancy between phenotype and genotype associated with drug resistance in our dataset, including pyrazinamide-monoresistant Mtb strains lacking canonical variants of drug resistance. Expanding the Latin American Mtb genome databases will enhance our understanding of TB epidemiology and potentially provide new avenues for controlling the disease in the region., Competing Interests: The authors declare no conflict of interest., (Copyright: © 2023 Mejía-Ponce et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Small peptide aldehydes (SPAs) with protease inhibitory activity are naturally occurring compounds shown to be synthesized by non-ribosomal peptide synthetases (NRPS). SPAs are widely used in biotechnology and have been utilized as therapeutic agents. They are also physiologically relevant and have been postulated to regulate the development of their producing microorganisms. Previously, we identified an NRPS-like biosynthetic gene cluster (BGC) in Streptomyces lividans 66 that lacked a condensation (C) domain but included a tRNA-utilizing enzyme (tRUE) belonging to the leucyl/phenylalanyl (L/F) transferase family. This system was predicted to direct the synthesis of a novel SPA, which we named livipeptin. Using evolutionary genome mining approaches, here, we confirm the presence of L/F transferase tRUEs within the genomes of diverse Streptomyces and related organisms, including fusions with the anticipated C-minus NRPS-like protein. We then demonstrate genetic functional cooperation between the identified L/F-transferase divergent tRUE homolog with the C-minus NRPS, leading to the synthesis of a metabolic fraction with protease inhibitory activity. Semisynthetic assays in the presence of RNAse revealed that the productive interaction between the tRUE and the C-minus NRPS enzymes is indeed tRNA dependent. We expect our findings to boost the discovery of SPAs, as well as the development of protease-mediated biotechnologies, by exploiting the uncovered genetic basis for synthesizing putative acetyl-leu/phe-arginine protease inhibitors. Furthermore, these results will facilitate the purification and structural elucidation of livipeptin, which has proven difficult to chemically characterize., Significance: The discovery of natural products biosynthetic genes marks a significant advancement in our understanding of these metabolites, for example of their evolution, activity, and biosynthesis, but also opens biotechnological opportunities and knowledge to advance genome mining approaches. We made this possible by uncovering a new biosynthetic pathway in Streptomyces lividans 66 shown to direct the synthesis of a strong protease inhibitor, termed livipeptin, following unprecedented biosynthetic rules and genes. Thus, by shedding light on the genetic mechanisms predicted to govern the production of acetyl-leu/phe-arginine protease inhibitors, including the elusive livipeptin, this study enables novel protease-mediated biotechnologies as well as approaches for discovering protease inhibitors from genome data., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Calculations predict that testing of 5 000-10 000 molecules and >1 billion US dollars (£0.8 billion, £1=$1.2) are required for one single drug to come to the market. A solution to this problem is to establish more efficient protocols that reduce the high rate of re-isolation and continuous rediscovery of natural products during early stages of the drug development process. The study of 'rare actinobacteria' has emerged as a possible approach for increasing the discovery rate of drug leads from natural sources. Here, we define a simple genomic metric, defined as biosynthetic novelty index (BiNI), that can be used to rapidly rank strains according to the novelty of the subset of encoding biosynthetic clusters. By comparing a subset of high-quality genomes from strains of different taxonomic and ecological backgrounds, we used the BiNI score to support the notion that rare actinobacteria encode more biosynthetic gene cluster (BGC) novelty. In addition, we present the isolation and genomic characterization, focused on specialized metabolites and phenotypic screening, of two isolates belonging to genera Lentzea and Actinokineospora from a highly oligotrophic environment. Our results show that both strains harbour a unique subset of BGCs compared to other members of the genera Lentzea and Actinokineospora . These BGCs are responsible for potent antimicrobial and cytotoxic bioactivity. The experimental data and analysis presented in this study contribute to the knowledge of genome mining analysis in rare actinobacteria and, most importantly, can serve to direct sampling efforts to accelerate early stages of the drug discovery pipeline.

Background: Breastmilk is a dynamic fluid whose initial function is to provide the most adapted nutrition to the neonate. Additional attributes have been recently ascribed to breastmilk, with the evidence of a specific microbiota and the presence of various components of the immune system, such as cytokines and leukocytes. The composition of breastmilk varies through time, according to the health status of mother and child, and altogether contributes to the future health of the infant. Obesity is a rising condition worldwide that creates a state of systemic, chronic inflammation including leukocytosis. Here, we asked whether colostrum, the milk produced within the first 48 h post-partum, would contain a distinct leukocyte composition depending on the body mass index (BMI) of the mother., Methods: We collected peripheral blood and colostrum paired samples from obese (BMI > 30) and lean (BMI < 25) mothers within 48 h post-partum and applied a panel of 6 antibodies plus a viability marker to characterize 10 major leukocyte subpopulations using flow cytometry., Results: The size, internal complexity, and surface expression of CD45 and CD16 of multiple leukocyte subpopulations were selectively regulated between blood and colostrum irrespective of the study groups, suggesting a generalized cell-specific phenotype alteration. In obesity, the colostrum B lymphocyte compartment was significantly reduced, and CD16 + blood monocytes had an increased CD16 expression compared to the lean group., Conclusions: This is the first characterization of major leukocyte subsets in colostrum of mothers suffering from obesity and the first report of colostrum leukocyte subpopulations in Latin America. We evidence various significant alterations of most leukocyte populations between blood and colostrum and demonstrate a decreased colostrum B lymphocyte fraction in obesity. This pioneering study is a stepping stone to further investigate active immunity in human breastmilk., (© 2022. The Author(s).)

Streptomyces sp. strain FB2 is an actinomycete isolated from rice rhizosphere. A whole-genome assembly of the strain FB2 comprised 7,727,571 bp (number of contigs, 55; GC content, 71.96%). In total, 17 biosynthetic gene clusters (BGCs), including nonribosomal peptides, polyketides, terpenes, and ribosomally synthesized and posttranslationally modified peptides, were predicted.

Genes related to DNA damage repair in Mycobacterium tuberculosis are critical for survival and genomic diversification. The aim of this study is to compare the presence of SNPs in genes related to DNA damage repair in sensitive and drug-resistant M. tuberculosis genomes isolated from patients with and without type 2 diabetes mellitus (T2DM). We collected 399 M. tuberculosis L4 genomes from several public repositories; 224 genomes belonging to hosts without T2DM, of which 123 (54.9%) had drug sensitive tuberculosis (TB) and 101 (45.1%) had drug resistance (DR)-TB; and 175 genomes from individuals with T2DM, of which 100 (57.1%) had drug sensitive TB and 75 (42.9%) had DR-TB. The presence of SNPs in the coding regions of 65 genes related to DNA damage repair was analyzed and compared with the resistance profile and the presence/absence of T2DM in the host. The results show the phylogenetic relationships of some SNPS and L4 sub-lineages, as well as differences in the distribution of SNPs present in DNA damage repair-related genes related to the resistance profile of the infecting strain and the presence of T2DM in the host. Given these differences, it was possible to generate two discriminant functions to distinguish between drug sensitive and drug resistant genomes, as well as patients with or without T2DM.

Snake venoms are rich sources of proteins with potential biotechnological and pharmaceutical applications. Among them, metalloproteases (MPs) and phospholipases A2 (PLA 2 ) are the most abundant. Their isolation involves a multistep chromatographic approach, which has proven to be effective, however implies high operating costs and long processing times. In this study, a cost-effective and simple method based on aqueous two-phase systems (ATPS) was developed to recover MPs and PLA 2 from Crotalus molossus nigrescens venom. A system with PEG 400 g mol -1 , volume ratio (V R ) 1, tie line length (TLL) 25% w/w and pH 7 showed the best performance for PLA 2 recovery. In systems with PEG 400 g mol -1 , V R 1, TLL 15% w/w, pH 7 and 1 and 3% w/w of NaCl, selective recovery of MP subtype P-III was achieved; whereas, in a system with PEG 400 g mol -1 , V R 1, TLL 25% w/w and pH 8.5, MP subtypes P-I and P-III were recovered. Due to their low costs, ethanol-salt systems were also evaluated, however, failed to differentially partition PLA 2 and MPs. The use of ATPS could contribute to the simplification and cost reduction of protein isolation processes from snake venoms and other toxin fluids, as well as potentially aid their biochemical, proteomic and biological analyses., (© 2021. The Author(s).)

Given its biocompatibility, rheological, and physiological properties, hyaluronic acid (HA) has become a biomaterial of increasing interest with multiple applications in medicine and cosmetics. In recent decades, microbial fermentations have become an important source for the industrial production of HA. However, due to its final applications, microbial HA must undergo critical and long purification processes to ensure clinical and cosmetic grade purity. Aqueous two-phase systems (ATPS) have proven to be an efficient technique for the primary recovery of high-value biomolecules. Nevertheless, their implementation in HA downstream processing has been practically unexplored. In this work, polyethylene glycol (PEG)-citrate ATPS were used for the first time for the primary recovery of HA produced with an engineered strain of Streptococcus equi subsp. zooepidemicus. The effects of PEG molecular weight (MW), tie-line length (TLL), volume ratio (V R ), and sample load on HA recovery and purity were studied with a clarified fermentation broth as feed material. HA was recovered in the salt-rich bottom phase, and its recovery increased when a PEG MW of 8000 g mol -1 was used. Lower V R values (0.38) favoured HA recovery, whereas purity was enhanced by a high V R (3.50). Meanwhile, sample load had a negative impact on both recovery and purity. The ATPS with the best performance was PEG 8000 g mol -1 , TLL 43% (w/w), and V R 3.50, showing 79.4% HA recovery and 74.5% purity. This study demonstrated for the first time the potential of PEG-citrate ATPS as an effective primary recovery strategy for the downstream process of microbial HA., (© 2021. The Author(s).)

Hyaluronic acid (HA) is a biopolymer with a wide range of applications, mainly in the cosmetic and pharmaceutical sectors. Typical industrial-scale production utilizes organisms that generate HA during their developmental cycle, such as Streptococcus equi sub. zooepidemicus . However, a significant disadvantage of using Streptococcus equi sub. zooepidemicus is that it is a zoonotic pathogen, which use at industrial scale can create several risks. This creates opportunities for heterologous, or recombinant, production of HA. At an industrial scale, the recovery and purification of HA follow a series of precipitation and filtration steps. Current recombinant approaches are developing promising alternatives, although their industrial implementation has yet to be adequately assessed. The present study aims to create a theoretical framework to forecast the advantages and disadvantages of endogenous and recombinant strains in production with the same downstream strategy. The analyses included a selection of the best cost-related recombinant and endogenous production strategies, followed by a sensitivity analysis of different production variables in order to identify the three most critical parameters. Then, all variables were analyzed by varying them simultaneously and employing multiple linear regression. Results indicate that, regardless of HA source, production titer, recovery yield and bioreactor scale are the parameters that affect production costs the most. Current results indicate that recombinant production needs to improve current titer at least 2-fold in order to compete with costs of endogenous production. This study serves as a platform to inform decision-making for future developments and improvements in the recombinant production of HA., Competing Interests: Authors LM-G, MG-G, and MB-M was employed by Biomentum SAPI de CV. The remaining authors declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Torres-Acosta, Castaneda-Aponte, Mora-Galvez, Gil-Garzon, Banda-Magaña, Marcellin, Mayolo-Deloisa and Licona-Cassani.)

Ulcerative colitis (UC) is a frequent type of inflammatory bowel disease, characterized by periods of remission and exacerbation. Gut dysbiosis may influence pathophysiology and clinical response in UC. The purpose of this study was to evaluate whether gut microbiota is related to the active and remission phases of pancolitis in patients with UC as well as in healthy participants. Fecal samples were obtained from 18 patients with UC and clinical-endoscopic evidenced pancolitis (active phase n = 9 and remission phase n = 9), as well as 15 healthy participants. After fecal DNA extraction, the 16S rRNA gene was amplified and sequenced (Illumina MiSeq), operational taxonomic units were analyzed with the QIIME software. Gut microbiota composition revealed a higher abundance of the phyla Proteobacteria and Fusobacteria in active pancolitis, as compared with remission and healthy participants. Likewise, a marked abundance of the genus Bilophila and Fusobacteria were present in active pancolitis, whereas a higher abundance of Faecalibacterium characterized both remission and healthy participants. LEfSe analysis showed that the genus Roseburia and Faecalibacterium were enriched in remission pancolitis, and genera Bilophila and Fusobacterium were enriched in active pancolitis. The relative abundance of Fecalibacterium and Roseburia showed a higher correlation with fecal calprotectin, while Bilophila and Fusobacterium showed AUCs (area under the curve) of 0.917 and 0.988 for active vs. remission pancolitis. The results of our study highlight the relation of gut dysbiosis with clinically relevant phases of pancolitis in patients with UC. Particularly, Fecalibacterium, Roseburia, Bilophila, and Fusobacterium were identified as genera highly related to the different clinical phases of pancolitis., (© 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Tetanus is a fatal disease caused by Clostridium tetani infections. To prevent infections, a toxoid vaccine, developed almost a century ago, is routinely used in humans and animals. The vaccine is listed in the World Health Organisation list of Essential Medicines and can be produced and administered very cheaply in the developing world for less than one US Dollar per dose. Recent developments in both analytical tools and frameworks for systems biology provide industry with an opportunity to gain a deeper understanding of the parameters that determine C. tetani virulence and physiological behaviour in bioreactors. Here, we compared a traditional fermentation process with a fermentation medium supplemented with five heavily consumed amino acids. The experiment demonstrated that amino acid catabolism plays a key role in the virulence of C. tetani. The addition of the five amino acids favoured growth, decreased toxin production and changed C. tetani morphology. Using time-course transcriptomics, we created a "fermentation map", which shows that the tetanus toxin transcriptional regulator BotR, P21 and the tetanus toxin gene was downregulated. Moreover, this in-depth analysis revealed potential genes that might be involved in C. tetani virulence regulation. We observed differential expression of genes related to cell separation, surface/cell adhesion, pyrimidine biosynthesis and salvage, flagellar motility, and prophage genes. Overall, the fermentation map shows that, mediated by free amino acid concentrations, virulence in C. tetani is regulated at the transcriptional level and affects a plethora of metabolic functions.

Background: One of the main functions of diet is to nurture the gut microbiota and this relationship affects the health of the host. However, different analysis strategies can generate different views on the relative abundance of each microbial taxon, which can affect our conclusions about the significance of diet to gut health in lean and obese subjects. Here we explored the impact of using different analysis strategies to study the gut microbiota in a context of diet, health and obesity., Methods: Over 15 million 16S rRNA gene sequences from published studies involving dietary interventions in obese laboratory rodents were analyzed. Three strategies were used to assign the 16S sequences to Operational Taxonomic Units (OTUs) based on the GreenGenes reference OTU sequence files clustered at 97% and 99% similarity., Results: Different strategies to select OTUs influenced the relative abundance of all bacterial taxa, but the magnitude of this phenomenon showed a strong study effect. Different taxa showed up to 20% difference in relative abundance within the same study, depending on the analysis strategy. Very few OTUs were shared among the samples. ANOSIM test on unweighted UniFrac distances showed that study, sequencing technique, animal model, and dietary treatment (in that order) were the most important factors explaining the differences in bacterial communities. Except for obesity status, the contribution of diet and other factors to explain the variability in bacterial communities was lower when using weighted UniFrac distances. Predicted functional profile and high-level phenotypes of the microbiota showed that each study was associated with unique features and patterns., Conclusions: The results confirm previous findings showing a strong study effect on gut microbial composition and raise concerns about the impact of analysis strategies on the membership and composition of the gut microbiota. This study may be helpful to guide future research aiming to investigate the relationship between diet, health, and the gut microbiota., Competing Interests: Jose F. Garcia-Mazcorro is an employee of MNA de Mexico, a company of Animal Nutrition., (©2020 Garcia-Mazcorro et al.)

Chronic wounds are a major health problem that cause millions of dollars in expenses every year. Among all the treatments used, active wound treatments such as enzymatic treatments represent a cheaper and specific option with a fast growth category in the market. In particular, bacterial and plant proteases have been employed due to their homology to human proteases, which drive the normal wound healing process. However, the use of these proteases has demonstrated results with low reproducibility. Therefore, alternative sources of proteases such as snake venom have been proposed. Here, we performed a functional mining of proteases from rattlesnakes ( Crotalus ornatus , C. molossus nigrescens , C. scutulatus, and C. atrox ) due to their high protease predominance and similarity to native proteases. To characterize Crotalus spp. Proteases, we performed different protease assays to measure and confirm the presence of metalloproteases and serine proteases, such as the universal protease assay and zymography, using several substrates such as gelatin, casein, hemoglobin, L-TAME, fibrinogen, and fibrin. We found that all our venom extracts degraded casein, gelatin, L-TAME, fibrinogen, and fibrin, but not hemoglobin. Crotalus ornatus and C. m. nigrescens extracts were the most proteolytic venoms among the samples. Particularly, C. ornatus predominantly possessed low molecular weight proteases (P-I metalloproteases). Our results demonstrated the presence of metalloproteases capable of degrading gelatin (a collagen derivative) and fibrin clots, whereas serine proteases were capable of degrading fibrinogen-generating fibrin clots, mimicking thrombin activity. Moreover, we demonstrated that Crotalus spp. are a valuable source of proteases that can aid chronic wound-healing treatments.

Skin wounds have been extensively studied as their healing represents a critical step towards achieving homeostasis following a traumatic event. Dependent on the severity of the damage, wounds are categorized as either acute or chronic. To date, chronic wounds have the highest economic impact as long term increases wound care costs. Chronic wounds affect 6.5 million patients in the United States with an annual estimated expense of $25 billion for the health care system. Among wound treatment categories, active wound care represents the fastest-growing category due to its specific actions and lower costs. Within this category, proteases from various sources have been used as successful agents in debridement wound care. The wound healing process is predominantly mediated by matrix metalloproteinases (MMPs) that, when dysregulated, result in defective wound healing. Therapeutic activity has been described for animal secretions including fish epithelial mucus, maggot secretory products and snake venom, which contain secreted proteases (SPs). No further alternatives for use, sources or types of proteases used for wound healing have been found in the literature to date. Through the present review, the context of enzymatic wound care alternatives will be discussed. In addition, substrate homology of SPs and human MMPs will be compared and contrasted. The purpose of these discussions is to identify and propose the stages of wound healing in which SPs may be used as therapeutic agents to improve the wound healing process., (Copyright: © Isabela Avila-Rodríguez et al.)

The published online version of this paper contains mistake. The authors first and last names have been interchanged. The correct version is given above.

An inter-kingdom beach warfare between a Streptomyces sp. and Aspergillus sp. co-isolated from shallow water beach sand, collected off Heron Island, Queensland, Australia, saw the bacteriostatic Aspergillus metabolite cyclo-(L-Phe-trans-4-hydroxy-L-Pro) (3) stimulate the Streptomyces to produce nitric oxide (NO), which in turn mediated transcriptional activation of a silent biosynthetic gene cluster (BGC) for fungistatic heronapyrrole B (1). Structure activity relationship studies, coupled with the use of NO synthase inhibitors, donors and scavangers, and both genomic and transcriptomic analyses, confirmed the extraordinary chemical cue specificity of 3, and its NO-mediated mechanism of transcriptional action. Our findings reveal the importance of inter-kingdom (fungal-bacterial) chemical communication in the regulation of silent BGCs coding for chemical defenses. We propose that the detection and characterisation of NO mediated transcriptional activation (NOMETA) of silent chemical defences in the environment, may inspire broader application in the field of microbial biodiscovery.

Streptomyces are Gram-positive bacteria that occupy diverse ecological niches including host-associations with animals and plants. Members of this genus are known for their overwhelming repertoire of natural products, which has been exploited for almost a century as a source of medicines and agrochemicals. Notwithstanding intense scientific and commercial interest in Streptomyces natural products, surprisingly little is known of the intra- and/or inter-species ecological roles played by these metabolites. In this report we describe the chemical structures, biological properties, and biosynthetic relationships between natural products produced by Streptomyces isolated from internal tissues of predatory Conus snails, collected from the Great Barrier Reef, Australia. Using chromatographic, spectroscopic and bioassays methodology, we demonstrate that Streptomyces isolated from five different Conus species produce identical chemical and antifungal profiles - comprising a suite of polycyclic tetramic acid macrolactams (PTMs). To investigate possible ecological (and evolutionary) relationships we used genome analyses to reveal a close taxonomic relationship with other sponge-derived and free-living PTM producing Streptomyces (i.e., Streptomyces albus ). In-depth phylogenomic analysis of PTM biosynthetic gene clusters indicated PTM structure diversity was governed by a small repertoire of genetic elements, including discrete gene acquisition events involving dehydrogenases. Overall, our study shows a Streptomyces - Conus ecological relationship that is concomitant with specific PTM chemical profiles. We provide an evolutionary framework to explain this relationship, driven by anti-fungal properties that protect Conus snails from fungal pathogens.

Desferrioxamines are hydroxamate siderophores widely conserved in both aquatic and soil-dwelling Actinobacteria. While the genetic and enzymatic bases of siderophore biosynthesis and their transport in model families of this phylum are well understood, evolutionary studies are lacking. Here, we perform a comprehensive desferrioxamine-centric (des genes) phylogenomic analysis, which includes the genomes of six novel strains isolated from an iron and phosphorous depleted oasis in the Chihuahuan desert of Mexico. Our analyses reveal previously unnoticed desferrioxamine evolutionary patterns, involving both biosynthetic and transport genes, likely to be related to desferrioxamines chemical diversity. The identified patterns were used to postulate experimentally testable hypotheses after phenotypic characterization, including profiling of siderophores production and growth stimulation of co-cultures under iron deficiency. Based in our results, we propose a novel des gene, which we term desG, as responsible for incorporation of phenylacetyl moieties during biosynthesis of previously reported arylated desferrioxamines. Moreover, a genomic-based classification of the siderophore-binding proteins responsible for specific and generalist siderophore assimilation is postulated. This report provides a much-needed evolutionary framework, with specific insights supported by experimental data, to direct the future ecological and functional analysis of desferrioxamines in the environment., (© FEMS 2017.)

Sphingobacterium sp. CZ-UAM was isolated from a methanotrophic consortium in mineral medium using methane as the only carbon source. A draft genome of 5.84 Mb with a 40.77% G+C content is reported here. This genome sequence will allow the investigation of potential methanotrophy in this isolated strain., (Copyright © 2017 Steffani-Vallejo et al.)

Although the specific function of SCO2127 remains elusive, it has been assumed that this hypothetical protein plays an important role in carbon catabolite regulation and therefore in antibiotic biosynthesis in Streptomyces coelicolor. To shed light on the functional relationship of SCO2127 to the biosynthesis of actinorhodin, a detailed analysis of the proteins differentially produced between the strain M145 and the Δsco2127 mutant of S. coelicolor was performed. The delayed morphological differentiation and impaired production of actinorhodin showed by the deletion strain were accompanied by increased abundance of gluconeogenic enzymes, as well as downregulation of both glycolysis and acetyl-CoA carboxylase. Repression of mycothiol biosynthetic enzymes was further observed in the absence of SCO2127, in addition to upregulation of hydroxyectoine biosynthetic enzymes and SCO0204, which controls nitrite formation. The data generated in this study reveal that the response regulator SCO0204 greatly contributes to prevent the formation of actinorhodin in the ∆sco2127 mutant, likely through the activation of some proteins associated with oxidative stress that include the nitrite producer SCO0216.

Bacteria produce some of the most potent biomolecules known, of which many cause serious diseases such as tetanus. For prevention, billions of people and countless animals are immunised with the highly effective vaccine, industrially produced by large-scale fermentation. However, toxin production is often hampered by low yields and batch-to-batch variability. Improved productivity has been constrained by a lack of understanding of the molecular mechanisms controlling toxin production. Here we have developed a reproducible experimental framework for screening phenotypic determinants in Clostridium tetani under a process that mimics an industrial setting. We show that amino acid depletion induces production of the tetanus toxin. Using time-course transcriptomics and extracellular metabolomics to generate a 'fermentation atlas' that ascribe growth behaviour, nutrient consumption and gene expression to the fermentation phases, we found a subset of preferred amino acids. Exponential growth is characterised by the consumption of those amino acids followed by a slower exponential growth phase where peptides are consumed, and toxin is produced. The results aim at assisting in fermentation medium design towards the improvement of vaccine production yields and reproducibility. In conclusion, our work not only provides deep fermentation dynamics but represents the foundation for bioprocess design based on C. tetani physiological behaviour under industrial settings., (Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Actinomycetes populate soils and aquatic sediments that impose biotic and abiotic challenges for their survival. As a result, actinomycetes metabolism and genomes have evolved to produce an overwhelming diversity of specialized molecules. Polyketides, non-ribosomal peptides, post-translationally modified peptides, lactams, and terpenes are well-known bioactive natural products with enormous industrial potential. Accessing such biological diversity has proven difficult due to the complex regulation of cellular metabolism in actinomycetes and to the sparse knowledge of their physiology. The past decade, however, has seen the development of omics technologies that have significantly contributed to our better understanding of their biology. Key observations have contributed toward a shift in the exploitation of actinomycete's biology, such as using their full genomic potential, activating entire pathways through key metabolic elicitors and pathway engineering to improve biosynthesis. Here, we review recent efforts devoted to achieving enhanced discovery, activation, and manipulation of natural product biosynthetic pathways in model actinomycetes using genome-scale biological datasets.

Streptomyces species are native inhabitants of soil, a natural environment where nutrients can be scarce and competition fierce. They have evolved ways to metabolize unusual nutrients, such as purines and its derivatives, which are highly abundant in soil. Catabolism of these uncommon carbon and nitrogen sources needs to be tightly regulated in response to nutrient availability and environmental stimulus. Recently, the allantoin degradation pathway was characterized in Streptomyces coelicolor. However, there are questions that remained unanswered, particularly regarding pathway regulation. Here, using a combination of proteomics and genetic approaches, we identified the negative regulator of the allantoin pathway, AllR. In vitro studies confirmed that AllR binds to the promoter regions of allantoin catabolic genes and determined the AllR DNA binding motif. In addition, effector studies showed that allantoic acid, and glyoxylate, to a lesser extent, inhibit the binding of AllR to the DNA. Inactivation of AllR repressor leads to the constitutive expression of the AllR regulated genes and intriguingly impairs actinorhodin and undecylprodigiosin production. Genetics and proteomics analysis revealed that among all genes from the allantoin pathway that are upregulated in the allR mutant, the hyi gene encoding a hydroxypyruvate isomerase (Hyi) is responsible of the impairment of antibiotic production., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Actinomycetes undergo a dramatic reorganization of metabolic and cellular machinery during a brief period of growth arrest ("metabolic switch") preceding mycelia differentiation and the onset of secondary metabolite biosynthesis. This study explores the role of phosphorylation in coordinating the metabolic switch in the industrial actinomycete Saccharopolyspora erythraea. A total of 109 phosphopeptides from 88 proteins were detected across a 150-h fermentation using open-profile two-dimensional LC-MS proteomics and TiO(2) enrichment. Quantitative analysis of the phosphopeptides and their unphosphorylated cognates was possible for 20 pairs that also displayed constant total protein expression. Enzymes from central carbon metabolism such as putative acetyl-coenzyme A carboxylase, isocitrate lyase, and 2-oxoglutarate dehydrogenase changed dramatically in the degree of phosphorylation during the stationary phase, suggesting metabolic rearrangement for the reutilization of substrates and the production of polyketide precursors. In addition, an enzyme involved in cellular response to environmental stress, trypsin-like serine protease (SACE&#95;6340/NC&#95;009142&#95;6216), decreased in phosphorylation during the growth arrest stage. More important, enzymes related to the regulation of protein synthesis underwent rapid phosphorylation changes during this stage. Whereas the degree of phosphorylation of ribonuclease Rne/Rng (SACE&#95;1406/NC&#95;009142&#95;1388) increased during the metabolic switch, that of two ribosomal proteins, S6 (SACE&#95;7351/NC&#95;009142&#95;7233) and S32 (SACE&#95;6101/NC&#95;009142&#95;5981), dramatically decreased during this stage of the fermentation, supporting the hypothesis that ribosome subpopulations differentially regulate translation before and after the metabolic switch. Overall, we show the great potential of phosphoproteomic studies to explain microbial physiology and specifically provide evidence of dynamic protein phosphorylation events across the developmental cycle of actinomycetes.

The glycolytic intermediate phosphoenolpyruvate (PEP) is a precursor of several cellular components, including various aromatic compounds. Modifications to the PEP node such as PEP:sugar phosphotransferase system (PTS) or pyruvate kinase inactivation have been shown to have a positive effect on aromatics production capacity in Escherichia coli and Bacillus subtilis. In this study, pyruvate kinase and PTS-deficient B. subtilis strains were employed for the construction of derivatives lacking shikimate kinase activity that accumulate two industrially valuable chemicals, the intermediates of the common aromatic pathway, shikimic and dehydroshikimic acids. The pyruvate kinase-deficient strain (CLC6-PYKA) showed the best production parameters under resting-cell conditions. Compared to the PTS-deficient strain, the shikimic and dehydroshikimic acids specific production rates for CLC6-PYKA were 1.8- and 1.7-fold higher, respectively. A batch fermentor culture using complex media supplemented with 83 g/l of glucose was developed with strain CLC6-PYKA, where final titers of 4.67 g/l (shikimic acid) and 6.2 g/l (dehydroshikimic acid) were produced after 42 h., (© 2013 S. Karger AG, Basel.)

Purines are a primary source of carbon and nitrogen in soil; however, their metabolism is poorly understood in Streptomyces. Using a combination of proteomics, metabolomics, and metabolic engineering, we characterized the allantoin pathway in Streptomyces coelicolor. When cells grew in glucose minimal medium with allantoin as the sole nitrogen source, quantitative proteomics identified 38 enzymes upregulated and 28 downregulated. This allowed identifying six new functional enzymes involved in allantoin metabolism in S. coelicolor. From those, using a combination of biochemical and genetic engineering tools, it was found that allantoinase (EC 3.5.2.5) and allantoicase (EC 3.5.3.4) are essential for allantoin metabolism in S. coelicolor. Metabolomics showed that under these growth conditions, there is a significant intracellular accumulation of urea and amino acids, which eventually results in urea and ammonium release into the culture medium. Antibiotic production of a urease mutant strain showed that the catabolism of allantoin, and the subsequent release of ammonium, inhibits antibiotic production. These observations link the antibiotic production impairment with an imbalance in nitrogen metabolism and provide the first evidence of an interaction between purine metabolism and antibiotic biosynthesis.

Background: Accurate bacterial genome annotations provide a framework to understanding cellular functions, behavior and pathogenicity and are essential for metabolic engineering. Annotations based only on in silico predictions are inaccurate, particularly for large, high G + C content genomes due to the lack of similarities in gene length and gene organization to model organisms., Results: Here we describe a 2D systems biology driven re-annotation of the Saccharopolyspora erythraea genome using proteogenomics, a genome-scale metabolic reconstruction, RNA-sequencing and small-RNA-sequencing. We observed transcription of more than 300 intergenic regions, detected 59 peptides in intergenic regions, confirmed 164 open reading frames previously annotated as hypothetical proteins and reassigned function to open reading frames using the genome-scale metabolic reconstruction. Finally, we present a novel way of mapping ribosomal binding sites across the genome by sequencing small RNAs., Conclusions: The work presented here describes a novel framework for annotation of the Saccharopolyspora erythraea genome. Based on experimental observations, the 2D annotation framework greatly reduces errors that are commonly made when annotating large-high G + C content genomes using computational prediction algorithms.

Background: Actinobacteria form a major bacterial phylum that includes numerous human pathogens. Actinobacteria are primary contributors to carbon cycling and also represent a primary source of industrial high value products such as antibiotics and biopesticides. Consistent with other members of the actinobacterial phylum, Saccharopolyspora erythraea undergo a transitional switch. This switch is characterized by numerous metabolic and morphological changes., Results: We performed RNA sequencing to analyze the transcriptional changes that occur during growth of Saccharopolyspora erythraea in batch culture. By sequencing RNA across the fermentation time course, at a mean coverage of 4000X, we found the vast majority of genes to be prominently expressed, showing that we attained close to saturating sequencing coverage of the transcriptome. During the metabolic switch, global changes in gene expression influence the metabolic machinery of Saccharopolyspora erythraea, resetting an entirely novel gene expression program. After the switch, global changes include the broad repression of half the genes regulated by complex transcriptional mechanisms. Paralogous transposon clusters, delineate these transcriptional programs. The new transcriptional program is orchestrated by a bottleneck event during which mRNA levels are severely restricted by targeted mRNA degradation., Conclusions: Our results, which attained close to saturating sequencing coverage of the transcriptome, revealed unanticipated transcriptional complexity with almost one third of transcriptional content originating from un-annotated sequences. We showed that the metabolic switch is a sophisticated mechanism of transcriptional regulation capable of resetting and re-synchronizing gene expression programs at extraordinary speed and scale.

Genome-scale metabolic reconstructions are routinely used for the analysis and design of metabolic engineering strategies for production of primary metabolites. The use of such reconstructions for metabolic engineering of antibiotic production is not common due to the lack of simple design algorithms in the absence of a cellular growth objective function. Here, we present the metabolic network reconstruction for the erythromycin producer Saccharopolyspora erythraea NRRL23338. The model was manually curated for primary and secondary metabolism pathways and consists of 1,482 reactions (2,075 genes) and 1,646 metabolites. As part of the model validation, we explored the potential benefits of supplying amino acids and identified five amino acids "compatible" with erythromycin production, whereby if glucose is supplemented with this amino acid on a carbon mole basis, the in silico model predicts that high erythromycin yield is possible without lowering biomass yield. Increased erythromycin titre was confirmed for four of the five amino acids, namely valine, isoleucine, threonine and proline. In bioreactor experiments, supplementation with 2.5 % carbon mole of valine increased the growth rate by 20 % and simultaneously the erythromycin yield on biomass by 50 %. The model presented here can be used as a framework for the future integration of high-throughput biological data sets in S. erythraea and ultimately to realise strain designs capable of increasing erythromycin production closer to the theoretical yield.

Despite the abundance of xylose in nature, the production of chemicals from C5 sugars remains challenging in metabolic engineering. By deleting xylFGH genes and using adaptive evolution, an efficient E. coli strain capable of producing D-lactate from xylose was engineered. Quantitative proteomics and genome sequencing were used to understand the new phenotype and the metabolic limitations of xylose conversion to D-lactate. Proteomics identified major changes in enzyme concentration in the glycolytic and tricarboxylic acid pathways. Whole genome sequencing of the evolved strain identified a point mutation in the gatC gene, which resulted in a change from serine to leucine at position 184 of the GatC protein. The knockout of gatC in a number of strains and the insertion of the mutation in the non-evolved strain confirmed its activity as a xylose transporter and demonstrated that the mutation is responsible for the high xylose consumption phenotype in the evolved strain. The newly found xylose transporter is a candidate for future strain engineering for converting C5-C6 syrups into valuable chemicals., (Copyright © 2012 Elsevier Inc. All rights reserved.)