Research Articles (Biochemistry, Genetics and Microbiology (BGM))

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Bacterial Isolation from natural grassland on nitrogen-free agar yields many strains without nitrogenase
(MDPI, 2025-01) Koirala, Amrit; Alshibli, Nabilah Ali; Das, Bikram K.; Brözel, Volker Siegfried
Nitrogen inputs for sustainable crop production for a growing population require the enhancement of biological nitrogen fixation. Efforts to increase biological nitrogen fixation include bioprospecting for more effective nitrogen-fixing bacteria. As bacterial nitrogenases are extremely sensitive to oxygen, most primary isolation methods rely on the use of semisolid agar or broth to limit oxygen exposure. Without physical separation, only the most competitive strains are obtained. The distance between strains provided by plating on solid media in reduced oxygen environments has been found to increase the diversity of culturable potential diazotrophic bacteria. To obtain diverse nitrogen-fixing isolates from natural grasslands, we plated soil suspensions from 27 samples onto solid nitrogen-free agar and incubated them under atmospheric and oxygen-reducing conditions. Putative nitrogen fixers were confirmed by subculturing in liquid nitrogen-free media and PCR amplification of the nifH genes. Streaking of the 432 isolates on nitrogen-rich R2A revealed many cocultures. In most cases, only one community member then grew on NFA, indicating the coexistence of nonfixers in coculture with fixers when growing under nitrogen-limited conditions. To exclude isolates able to scavenge residual nitrogen, such as that from vitamins, we used a stringent nitrogen-free medium containing only 6.42 μmol/L total nitrogen and recultured them in a nitrogen-depleted atmosphere. Surprisingly, PCR amplification of nifH using various primer pairs yielded amplicons from only 17% of the 442 isolates. The majority of the nifH PCR-negative isolates were Bacillus and Streptomyces. It is unclear whether these isolates have highly effective uptake systems or nitrogen reduction systems that are not closely aligned with known nitrogenase families. We advise caution in determining the nitrogen fixation ability of plants from growth on nitrogen-free media, even where the total nitrogen is very limited.
Associating serum testosterone levels with African ancestral prostate cancer health disparities
(Nature Research, 2025-04) Lebelo, Maphuti Tebogo; Mmekwa, Naledi; Louw, Melanie; Jaratlerdsiri, Weerachai; Mutambirwa, Shingai B.A.; Loda, Massimo; Hayes, Vanessa M.; Bornman, Maria S. (Riana); riana.bornman@up.ac.za
Serum testosterone levels decrease in the aging male, while the risk for prostate cancer (PCa) increases concomitantly. Higher levels in younger men have been linked with racially driven PCa disparities, with African men disproportionately impacted. In turn, higher levels of serum lipids have been associated with aggressive disease, while racial disparity between serum testosterone, cholesterol and cancer mortality has been suggested. Having previously reported a 2.1-fold increased age-adjusted risk for aggressive PCa in Black South African over Black American men, we determined the serum testosterone and associated lipid levels in 250 Black South African men either with or without clinicopathologically diagnosed disease. Observing no associations with serum lipid levels, Black South Africans presented with testosterone levels between 1.24 (< 60 years) and 1.3-fold (≥ 60 years) greater than Black Americans. Notably, a rapid drop in total-, bioavailable- and free testosterone levels in men 65 years or older was significantly associated with PCa risk (P = 0.0057, 0.009 and 0.005, respectively), while irrespective of age, further associated with advanced disease (P = 0.004, 0.0012 and 0.0036, respectively). These preliminary data provide insights into the potential role of androgens in driving PCa health disparities, with important consequence for tailoring treatment for Black men.
Effects of exogenous application of methyl jasmonate and salicylic acid on the physiological and molecular response of ‘Dusa’ avocado to Rosellinia necatrix
(American Phytopathological Society, 2024-07) Moreno-Pérez, Ana; Martínez-Ferri, Elsa; Van den Berg, Noelani; Pliego, Clara
Methyl jasmonate (MeJA) and salicylic acid (SA) are important in mediating plant responses to abiotic and biotic stresses. MeJA and SA can act as elicitors by triggering plant defense responses similar to those induced by pathogens and may even provide long-term protection against them. Thus, exogenous application of MeJA and SA could protect susceptible avocado plants against white root rot (WRR) disease caused by the necrotrophic fungus Rosellinia necatrix, one of the main diseases affecting avocado orchards. This work evaluates the effects of MeJA or SA on the physiological and molecular response of susceptible ‘Dusa’ avocado rootstock and their ability to provide some protection against WRR. The application of MeJA and SA in avocado increased photoprotective mechanisms (nonphotochemical chlorophyll fluorescence quenching) and upregulated the glutathione S-transferase, suggesting the triggering of mechanisms closely related to oxidative stress relief and reactive oxygen species scavenging. In contrast to SA, MeJA’s effects were more pronounced at the morphoanatomical level, including functional traits such as high leaf mass area, high stomatal density, and high root/shoot ratio, closely related to strategies to cope with water scarcity and WRR disease. Moreover, MeJA upregulated a greater number of defense-related genes than SA, including a glu protease inhibitor, a key gene in avocado defense against R. necatrix. The overall effects of MeJA increased ‘Dusa’ avocado tolerance to R. necatrix by inducing a primed state that delayed WRR disease symptoms. These findings point toward the use of MeJA application as an environmentally friendly strategy to mitigate the impact of this disease on susceptible avocado orchards.
Comparative transcriptomics in ferns reveals key innovations and divergent evolution of the secondary cell walls
(Nature Research, 2025) Ali, Zahin; Tan, Qiao Wen; Lim, Peng Ken; Chen, Hengchi; Pfeifer, Lukas; Julca, Irene; Lee, Jia Min; Classen, Birgit; De Vries, Sophie; De Vries, Jan; Vinter, Fanny; Alvarado, Camille; Layens, Amandine; Mizrachi, Eshchar; Motawie, Mohammed Saddik; Joergensen, Bodil; Ulvskov, Peter; Van de Peer, Yves; Ho, Boon Chuan; Sibout, Richard; Mutwil, Marek
Ferns are essential for understanding plant evolution; however, their large and intricate genomes have kept their genetic landscape largely unexplored, with only a few genomes sequenced and limited transcriptomic data available. To bridge this gap, we generated extensive RNA-sequencing data across various organs from 22 representative fern species, resulting in high-quality transcriptome assemblies. These data enabled us to construct a time-calibrated phylogeny for ferns, encompassing all major clades, which revealed numerous instances of whole-genome duplication. We highlighted the distinctiveness of fern genetics, discovering that half of the identified gene families are unique to ferns. Our exploration of fern cell walls through biochemical and immunological analyses uncovered the presence of the lignin syringyl unit, along with evidence of its independent evolution in ferns. Additionally, the identification of an unusual sugar in fern cell walls suggests a divergent evolutionary trajectory in cell wall biochemistry, probably influenced by gene duplication and sub-functionalization. To facilitate further research, we have developed an online database that includes preloaded genomic and transcriptomic data for ferns and other land plants. We used this database to demonstrate the independent evolution of lignocellulosic gene modules in ferns. Our findings provide a comprehensive framework illustrating the unique evolutionary journey ferns have undertaken since diverging from the last common ancestor of euphyllophytes more than 360 million years ago.
Plasmids encode and can mobilize onion pathogenicity in Pantoea agglomerans
(Oxford University Press, 2025-03) Shin, Gi Yoon; Asselin, Jo Ann; Smith, Amy; Aegerter, Brenna; Coutinho, Teresa A.; Zhao, Mei; Dutta, Bhabesh; Mazzone, Jennie; Neupane, Ram; Gugino, Beth; Hoepting, Christy; Khanal, Manzeal; Malla, Subas; Nischwitz, Claudia; Sidhu, Jaspreet; Burke, Antoinette Machado; Davey, Jane; Uchanski, Mark; Derie, Michael L.; Du Toit, Lindsey J.; Stresow-Cortez, Stephen; Bonasera, Jean M.; Stodghill, Paul; Kvitko, Brian
Pantoea agglomerans is one of four Pantoea species reported in the USA to cause bacterial rot of onion bulbs. However, not all P. agglomerans strains are pathogenic to onion. We characterized onion-associated strains of P. agglomerans to elucidate the genetic and genomic signatures of onion-pathogenic P. agglomerans. We collected >300 P. agglomerans strains associated with symptomatic onion plants and bulbs from public culture collections, research laboratories, and a multi-year survey in 11 states in the USA. Combining the 87 genome assemblies with 100 high-quality, public P. agglomerans genome assemblies we identified two well-supported P. agglomerans phylogroups. Strains causing severe symptoms on onion were only identified in Phylogroup II and encoded the HiVir pantaphos biosynthetic cluster, supporting the role of HiVir as a pathogenicity factor. The P. agglomerans HiVir cluster was encoded in two distinct plasmid contexts: (i) as an accessory gene cluster on a conserved P. agglomerans plasmid (pAggl), or (ii) on a mosaic cluster of plasmids common among onion strains (pOnion). Analysis of closed genomes revealed that the pOnion plasmids harbored alt genes conferring tolerance to Allium thiosulfinate defensive chemistry and many harbored cop genes conferring resistance to copper. We demonstrated that the pOnion plasmid pCB1C can act as a natively mobilizable pathogenicity plasmid that transforms P. agglomerans Phylogroup I strains, including environmental strains, into virulent pathogens of onion. This work indicates a central role for plasmids and plasmid ecology in mediating P. agglomerans interactions with onion plants, with potential implications for onion bacterial disease management.
Self-growth suppression in Bradyrhizobium diazoefficiens is caused by a diffusible antagonist
(Oxford University Press, 2025-03) Sandhu, Armaan Kaur; Fischer, Brady R.; Subramanian, Senthil; Hoppe, Adam D.; Brözel, Volker Siegfried
Microbes in soil navigate interactions by recognizing kin, forming social groups, exhibiting antagonistic behavior, and engaging in competitive kin rivalry. Here, we investigated a novel phenomenon of self-growth suppression (sibling rivalry) observed in Bradyrhizobium diazoefficiens USDA 110. Swimming colonies of USDA 110 developed a distinct demarcation line and inter-colony zone when inoculated adjacent to each other. In addition to self, USDA 110 suppressed growth of other Bradyrhizobium strains and several other soil bacteria. We demonstrated that the phenomenon of sibling rivalry is due to growth suppression but not cell death. The cells in the inter-colony zone were culturable but had reduced respiratory activity, ATP levels, and motility. The observed growth suppression was due to the presence of a diffusible effector compound. This effector was labile, preventing extraction, and identification, but it is unlikely a protein or a strong acid or base. This counterintuitive phenomenon of self-growth suppression suggests a strategic adaptation for conserving energy and resources in competitive soil environments. Bradyrhizobium’s utilization of antagonism including self-growth suppression likely provides a competitive advantage for long-term success in soil ecosystems.
Bradyrhizobium diazoefficiens cultures display phenotypic heterogeneity
(Oxford University Press, 2025-03) Sarao, Sukhvir K.; Sandhu, Armaan K.; Hanson, Ryan L.; Govil, Tanvi; Brözel, Volker Siegfried
Bacteria growing in liquid culture are assumed to be homogenous in phenotype. Characterization of individual cells shows that some clonal cultures contain more than one phenotype. Bacteria appear to employ bet hedging where various phenotypes help the species survive in diverse niches in soil and rhizosphere environments. We asked whether the agriculturally significant bacterium Bradyrhizobium diazoefficiens USDA 110, which fixes nitrogen with soybean plants, displays phenotypic heterogeneity when grown under laboratory conditions. We observed differential binding of sugar-specific lectins in isogenic populations, revealing differential surface properties. We employed Percoll™ density gradient centrifugation to separate clonal populations of exponential and stationary phase B. diazoefficiens into four fractions and characterized their phenotype by proteomics. Specific phenotypes were then characterized in detail. Fractions varied by cell size, polyhydroxyalkanoate content, lectin binding profile, growth rate, cellular adenosine triphosphate, chemotaxis, and respiration activity. Phenotypes were not heritable because the specific buoyant densities of fractions equilibrated within 10 generations. We propose that heterogeneity helps slow growing B. diazoefficiens proliferate and maintain populations in the different environments in soil and the rhizosphere.
DPANN symbiont of Haloferax volcanii accelerates xylan degradation by the non-host haloarchaeon Halorhabdus sp.
(Elsevier, 2025-02-21) Reva, Oleg N.; La Cono, Violetta; Marturano, Laura; Crisafi, Francesca; Smedile, Francesco; Mudaliyar, Manasi; Ghosal, Debnath; Selivanova, Elena A.; Ignatenko, Marina E.; Ferrer, Manuel; Fernandez-Lopez, Laura; Krupovic, Mart; Yakimov, Michail M.; oleg.reva@up.ac.za
This study examines a natural consortium of halophilic archaea, comprising xylan-degrading Halorhabdus sp. SVX81, consortium cohabitant Haloferax volcanii SVX82 (formerly H. lucentense SVX82), and its DPANN ectosymbiont Ca. Nanohalococcus occultus SVXNc. Transcriptomics and targeted metabolomics demonstrated that the tripartite consortium outperformed individual and the Halorhabdus sp. SVX81 with H. volcanii SVX82 bipartite cultures in xylan degradation, exhibiting a division of labor: the DPANN symbiont processed glycolysis products, while other members performed xylan depolymerization and biosynthesis of essential compounds. Electron microscopy and cryo-electron tomography revealed the formation of heterocellular biofilms interlinked by DPANN cells. The findings demonstrated that DPANN symbionts can interact directly with other members of microbial communities, which are not their primary hosts, influencing their gene expression. However, DPANN proliferation requires their primary host presence. The study highlights the collective contribution of consortium members to xylan degradation and their potential for biotechnological applications in the management of hypersaline environments.
IMA GENOME-F20 a draft genome assembly of Agroathelia rolfsii, Ceratobasidium papillatum, Pyrenopeziza brassicae, Neopestalotiopsis macadamiae, Sphaerellopsis filum and genomic resources for Colletotrichum spaethianum and Colletotrichum fructicola
(Pensoft Publishers, 2025-02) D’Angelo, Davide; Sorrentino, Roberto; Nkomo, Tiphany; Zhou, Xianzhi; Vaghefi, Niloofar; Sonnekus, Byron; Bose, Tanay; Cerrato, Domenico; Cozzolino, Loredana; Creux, Nicky; D'Agostino, Nunzio; Fourie, Gerda; Fusco, Giovanna; Hammerbacher, Almuth; Idnurm, Alexander; Kiss, Levente; Hu, Yanping; Hu, Hongli; Lahoz, Ernesto; Risteski, Jason; Steenkamp, Emma Theodora; Viscardi, Maurizio; Van der Nest, Magrieta Aletta; Wu, Yuan; Yu, Hao; Zhou, Jianjin; Dewage, Chinthani S. Karandeni; Kotta-Loizou, Loly I.; Stotz, Henrik U.; Fitt, Bruce D.L.; Huang, Yong-Ju; Wingfield, Brenda D.; brenda.wingfield@up.ac.za
Profiling the diversity of the village chicken faecal microbiota using 16S rRNA gene and metagenomic sequencing data to reveal patterns of gut microbiome signatures
(Frontiers Media, 2025-02) Nene, Mxolisi; Kunene, Nokuthula W.; Pierneef, Rian; Hadebe, Khanyisile
INTRODUCTION: The production environment of extensively raised village chickens necessitates their adaptability to low-resource systems. The gut microbiome plays a critical role in supporting this adaptability by influencing health and productivity. This study aimed to investigate the diversity and functional capacities of the faecal microbiome in village chickens from Limpopo and KwaZulu-Natal provinces of South Africa. METHODS: Using a combination of 16S rRNA gene sequencing and shotgun metagenomic sequencing technologies, we analysed 98 16S rRNA and 72 metagenomic datasets. Taxonomic profiles and functional gene annotations were derived, focusing on microbial diversity, antibiotic resistance genes (ARGs), and potential zoonotic pathogens. RESULTS: Taxonomic analysis showed that the predominant phyla in both provinces were Firmicutes, Bacteroidota, Proteobacteria, and Actinobacteria. At the genus level, Escherichia and Shigella were prevalent, with Escherichia coli and Shigella dysenteriae identified as major contributors to the gut microbiome. ARGs were identified, with MarA, PmrF, and AcrE detected in KwaZulu-Natal, and cpxA, mdtG, and TolA in Limpopo. These genes primarily mediate antibiotic efflux and alteration. DISCUSSION: The detection of zoonotic bacteria such as Escherichia coli and Streptococcus spp. highlights potential health risks to humans through the food chain, emphasizing the importance of improved household hygiene practices. This study underscores the role of the gut microbiome in village chicken health and adaptability, linking microbial diversity to production efficiency in lowresource settings. Targeted interventions and further research are crucial for mitigating zoonotic risks and enhancing sustainability in village chicken farming.
Gene transfer between fungal species triggers repeated coffee wilt disease outbreaks
(Public Library of Science, 2024-12-06) Wingfield, Brenda D.; Wingfield, Michael J.; brenda.wingfield@fabi.up.ac.za
Two outbreaks of coffee wilt disease have devastated African coffee production. A PLOS Biology study suggests that horizontal gene transfer via large Starship transposons between 2 fungal species played a key role in the repeated emergence of the disease.
Genomic diversity and antimicrobial susceptibility of invasive Neisseria meningitidis in South Africa, 2016–2021
(Oxford University Press, 2024-12-15) Mikhari, Rito L.; Meiring, Susan; De Gouveia, Linda; Chan, Wai Yin; Jolley, Keith A.; Van Tyne, Daria; Harrison, Lee H.; Marjuki, Henju; Ismail, Arshad; Quan, Vanessa; Cohen, Cheryl; Walaza, sibongile; Von Gottberg, Anne; Du Plessis, Mignon
Please read abstract in the article.
Growth rate extremes of a Sciaenid in an ocean-warming hotspot
(Elsevier, 2025-05) Jagger, Charmaine E.; Potts, Warren M.; Pringle, Brett A.; Bordbar, M. Hadi; Henriques, Romina; McKeown, Niall J.; Wilhelm, Margit R.
Argyrosomus inodorus is a cool water Sciaenid, important to the recreational and commercial coastal fishery in Namibia, the northern Benguela. Given recent findings on the northern Benguela as an ocean-warming hotspot and A. inodorus being more sensitive to warm temperatures than its congeneric A. coronus, it is important to study long-term growth responses of this Sciaenid. We developed a 34-year otolith biochronology for A. inodorus. The linear mixed-effects models fitted to A. inodorus’ otolith biochronology indicated that the best linear unbiased predictor of growth was significantly positively correlated with mean sea surface temperatures (SSTs) in September of the year of formation and significantly negatively correlated with mean summer SSTs (November y-1), in the central Namibian area (20–24◦S, 12–14◦E). Thus, faster annual growth was observed during warmer winter/spring temperatures (up to 16℃) and slower growth was noted during warmer summer temperatures (> 16℃). These results indicate that the species has a narrow range of thermal plasticity, and highlight the recent reductions in growth of this species in response to rapid thermal change observed in the ocean warming hotspot. Our findings suggest that A. inodorus would not be able to adapt readily with future climate warming predicted to happen and already happening along the northernmost part of the Namibian coast.
Expanding the toolkit for ploidy manipulation in Chlamydomonas reinhardtii
(Wiley, 2025) Van de Vloet, Antoine; Prost-Boxoen, Lucas; Bafort, Quinten; Paing, Yunn Thet; Casteleyn, Griet; Jomat, Lucile; Lemaire, Stephane D.; De Clerck, Olivier; Van de Peer, Yves
Whole-genome duplications, widely observed in plant lineages, have significant evolutionary and ecological impacts. Yet, our current understanding of the direct implications of ploidy shifts on short- and long-term plant evolution remains fragmentary, necessitating further investigations across multiple ploidy levels. Chlamydomonas reinhardtii is a valuable model organism with profound potential to study the impact of ploidy increase on the longer term in a laboratory environment. This is partly due to the ability to increase the ploidy level. We developed a strategy to engineer ploidy in C. reinhardtii using noninterfering, antibiotic, selectable markers. This approach allows us to induce higher ploidy levels in C. reinhardtii and is applicable to field isolates, which expands beyond specific auxotroph laboratory strains and broadens the genetic diversity of parental haploid strains that can be crossed. We implement flow cytometry for precise measurement of the genome size of strains of different ploidy. We demonstrate the creation of diploids, triploids, and tetraploids by engineering North American field isolates, broadening the application of synthetic biology principles in C. reinhardtii. However, our newly formed triploids and tetraploids show signs of rapid aneuploidization. Our study greatly facilitates the application of C. reinhardtii to study polyploidy, in both fundamental and applied settings.
Asymmetric genome merging leads to gene expression novelty through nucleo-cytoplasmic disruptions and transcriptomic shock in Chlamydomonas triploids
(Wiley, 2025-01) Prost-Boxoen, Lucas; Bafort, Quinten; Van de Vloet, Antoine; Almeida-Silva, Fabricio; Paing, Yunn Thet; Casteleyn, Griet; D'hondt, Sofie; De Clerck, Olivier; Van de Peer, Yves
Genome merging is a common phenomenon causing a wide range of consequences on phenotype, adaptation, and gene expression, yet its broader implications are not well-understood. Two consequences of genome merging on gene expression remain particularly poorly understood: dosage effects and evolution of expression. We employed Chlamydomonas reinhardtii as a model to investigate the effects of asymmetric genome merging by crossing a diploid with a haploid strain to create a novel triploid line. Five independent clonal lineages derived from this triploid line were evolved for 425 asexual generations in a laboratory natural selection experiment. Utilizing fitness assays, flow cytometry, and RNA-Seq, we assessed the immediate consequences of genome merging and subsequent evolution. Our findings reveal substantial alterations in genome size, gene expression, protein homeostasis, and cytonuclear stoichiometry. Gene expression exhibited expression-level dominance and transgressivity (i.e. expression level higher or lower than either parent). Ongoing expression-level dominance and a pattern of ‘functional dominance’ from the haploid parent was observed. Despite major genomic and nucleo-cytoplasmic disruptions, enhanced fitness was detected in the triploid strain. By comparing gene expression across generations, our results indicate that proteostasis restoration is a critical component of rapid adaptation following genome merging in Chlamydomonas reinhardtii and possibly other systems.
Chromosome-level assemblies for the pine pitch canker pathogen Fusarium circinatum
(MDPI, 2024-01) De Vos, Lieschen; Van der Nest, Magrieta Aletta; Santana, Quentin C.; Van Wyk, Stephanie; Leeuwendaal, Kyle; Wingfield, Brenda D.; Steenkamp, Emma Theodora; emma.steenkamp@fabi.up.ac.za
The pine pitch canker pathogen, Fusarium circinatum, is globally regarded as one of the most important threats to commercial pine-based forestry. Although genome sequences of this fungus are available, these remain highly fragmented or structurally ill-defined. Our overall goal was to provide high-quality assemblies for two notable strains of F. circinatum, and to characterize these in terms of coding content, repetitiveness and the position of telomeres and centromeres. For this purpose, we used Oxford Nanopore Technologies MinION long-read sequences, as well as Illumina short sequence reads. By leveraging the genomic synteny inherent to F. circinatum and its close relatives, these sequence reads were assembled to chromosome level, where contiguous sequences mostly spanned from telomere to telomere. Comparative analyses unveiled remarkable variability in the twelfth and smallest chromosome, which is known to be dispensable. It presented a striking length polymorphism, with one strain lacking substantial portions from the chromosome’s distal and proximal regions. These regions, characterized by a lower gene density, G+C content and an increased prevalence of repetitive elements, contrast starkly with the syntenic segments of the chromosome, as well as with the core chromosomes. We propose that these unusual regions might have arisen or expanded due to the presence of transposable elements. A comparison of the overall chromosome structure revealed that centromeric elements often underpin intrachromosomal differences between F. circinatum strains, especially at chromosomal breakpoints. This suggests a potential role for centromeres in shaping the chromosomal architecture of F. circinatum and its relatives. The publicly available genome data generated here, together with the detailed metadata provided, represent essential resources for future studies of this important plant pathogen.
Sex pheromone receptor Ste2 orchestrates chemotropic growth towards pine root extracts in the pitch canker pathogen Fusarium circinatum
(MDPI, 2024-05) Ramaswe, J.B.; Steenkamp, Emma Theodora; De Vos, Lieschen; Fru, Felix Fon; Adegeye, O.O.; Wingfield, Brenda D.; emma.steenkamp@fabi.up.ac.za
In ascomycetous fungi, sexual mate recognition requires interaction of the Ste2 receptor protein produced by one partner with the α-factor peptide pheromone produced by the other partner. In some fungi, Ste2 is further needed for chemotropism towards plant roots to allow for subsequent infection and colonization. Here, we investigated whether this is also true for the pine pitch canker fungus, Fusarium circinatum, which is a devastating pathogen of pine globally. Ste2 knockout mutants were generated for two opposite mating-type isolates, after which all strains were subjected to chemotropism assays involving exudates from pine seedling roots and synthetic α-factor pheromone, as well as a range of other compounds for comparison. Our data show that Ste2 is not required for chemotropism towards any of these other compounds, but, in both wild-type strains, Ste2 deletion resulted in the loss of chemotropism towards pine root exudate. Also, irrespective of mating type, both wild-type strains displayed positive chemotropism towards α-factor pheromone, which was substantially reduced in the deletion mutants and not the complementation mutants. Taken together, these findings suggest that Ste2 likely has a key role during the infection of pine roots in production nurseries. Our study also provides a strong foundation for exploring the role of self-produced and mate-produced α-factor pheromone in the growth and overall biology of the pitch canker pathogen.
Inconsistencies within the proposed framework for stabilizing fungal nomenclature risk further confusion
(American Society for Microbiology, 2024-04) Kidd, Sarah E.; Hagen, Ferry; Halliday, Catriona L.; Abdolrasouli, Alireza; Boekhout, Teun; Crous, Pedro W.; Ellis, David H.; Elvy, Juliet; Forrest, Graeme N.; Groenewald, Marizeth; Hahn, Rosane C.; Houbraken, Jos; Rodrigues, Anderson M.; Scott, James; Sorrell, Tania C.; Summerbell, Richard C.; Tsui, Clement K.M.; Yurkov, Andrey M.; Chen, Sharon C.-A.
We read with interest the recent publication by de Hoog and colleagues in the Journal of Clinical Microbiology (1) and support the goal to stabilize fungal nomenclature. Although we recognize the importance of stable naming, we offeroffercomments on the need for clarity around the concepts introduced, consistency of the recommended nomenclature, process issues regarding the endorsement by professional groups and societies and the proposed oversight committee (working group), and the relationship between the proposed database and existing, officially recognized nomenclatural repositories.
FEP augmentation as a means to solve data paucity problems for machine learning in chemical biology
(American Chemical Society, 2024-04-23) Burger, Pieter B.; Hu, Xiaohu; Balabin, Ilya; Muller, Morne; Stanley, Megan; Joubert, Fourie; Kaiser, Thomas M.
In the realm of medicinal chemistry, the primary objective is to swiftly optimize a multitude of chemical properties of a set of compounds to yield a clinical candidate poised for clinical trials. In recent years, two computational techniques, machine learning (ML) and physics-based methods, have evolved substantially and are now frequently incorporated into the medicinal chemist’s toolbox to enhance the efficiency of both hit optimization and candidate design. Both computational methods come with their own set of limitations, and they are often used independently of each other. ML’s capability to screen extensive compound libraries expediently is tempered by its reliance on quality data, which can be scarce especially during early-stage optimization. Contrarily, physics-based approaches like free energy perturbation (FEP) are frequently constrained by low throughput and high cost by comparison; however, physics-based methods are capable of making highly accurate binding affinity predictions. In this study, we harnessed the strength of FEP to overcome data paucity in ML by generating virtual activity data sets which then inform the training of algorithms. Here, we show that ML algorithms trained with an FEP-augmented data set could achieve comparable predictive accuracy to data sets trained on experimental data from biological assays. Throughout the paper, we emphasize key mechanistic considerations that must be taken into account when aiming to augment data sets and lay the groundwork for successful implementation. Ultimately, the study advocates for the synergy of physics-based methods and ML to expedite the lead optimization process. We believe that the physics-based augmentation of ML will significantly benefit drug discovery, as these techniques continue to evolve.
Plasmid DNA-based reverse genetics as a platform for manufacturing of bluetongue vaccine
(American Society for Microbiology, 2025-04) Mlingo, Tendai A.M.; Theron, Jacques; Mokoena, Nobalanda B.
Control of bluetongue disease is predominantly through vaccination with licensed inactivated or live-attenuated vaccines (LAVs). Manufacturing of LAVs in endemic countries requires formulation with a high number of serotypes for effective protection. Herein, we evaluated a plasmid DNA-based reverse genetics platform for manufacturing a multivalent vaccine. The synthetic vaccine was characterized by a common BTV1 backbone, with exchange of outer capsid proteins. Recombinant South African vaccine serotypes 1, 5, and 14 were rescued by exchanging the VP2 protein on the backbone. BTV6 rescue was achieved following the exchange of VP2 and VP5 proteins. The particle sizes were comparable to commercial vaccines of respective serotypes. BTV1, BTV5, and BTV6 had distinct growth profiles compared to commercial vaccines, while BTV14 was indistinguishable. Stability and shelf-life determination under various storage conditions showed that commercial vaccines were more stable. Formulated antigens were evaluated for vaccine safety and immunogenicity in sheep. Serotyped BTV1 monovalent vaccine was safe, as no clinical signs were observed. Neutralizing antibodies (nAbs) were induced on day 14 and peaked at 32 on day 28. The multivalent synthetic vaccine containing four serotypes elicited BTV6 nAbs from day 21 with a titer of 52, which decreased to 33 by day 42. BTV1 elicited a weak immune response with a titer of 1 on day 42. No nAbs were detected against BTV5 and BTV14. This is a first report comparing reverse genetics-derived antigens with commercial vaccines. Data generated on production yields, stability, and immunogenicity demonstrated that some serotypes can be implemented as novel synthetic vaccines using this platform. IMPORTANCE : Vaccination is the most effective control strategy for viral diseases that affect livestock. To date, only live-attenuated and inactivated vaccines have been licensed for control of bluetongue (BT). This study demonstrated the use of reverse genetics as a possible platform for BTV vaccine production. Data generated in the study contribute toward the advancement of an alternative manufacturing platform for licensing of BT vaccines. Information on production yields and stability of synthetic vaccines in comparison to the conventional products demonstrated that optimization is required for some serotypes to fully translate the reverse genetics platform for manufacturing the BTV vaccine. The study highlighted the safety and immunogenicity of vaccines manufactured using the plasmid DNA-based reverse-genetics platform.

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