Co-Fermentation and Genomic Insights into Lactic Acid Bacteria for Enhanced Propionic Acid Production Using a Non-GMO Approach

Propionic acid (PA) is an important organic acid with applications in food preservation, feed additives, and bio-based chemical production. While industrial PA is mostly derived from petrochemical processes, sustainable microbial alternatives are gaining attention. In this study, we explored a co-fermentation strategy using lactic acid bacteria (LAB) with complementary metabolic capabilities to enhance PA biosynthesis via the 1,2-propanediol (PDO) pathway. Genome-based screening identified a metabolic division between strains capable of producing PDO (e.g., Carnobacterium maltaromaticum IBB3447) and those converting PDO to PA (e.g., Levilactobacillus brevis IBB3735). Notably, we discovered that C. maltaromaticum IBB3447 is capable of PDO 24 biosynthesis, a function previously undescribed in this species. Phenotypic assays confirmed glycerol metabolism and acid tolerance among strains. In co-culture fermentation trials, the highest PA concentration (6.87 mM) was achieved using simultaneous fermentation in a fructose–sorbitol–glucose (FRC-SOR-GLC) medium, accompanied by prior PDO accumulation (up to 13.13 mM). No single strain produced PA independently, confirming that metabolic cooperation is required. These findings reveal a novel LAB-based bioprocess for sustainable PA and PDO production, using cross-feeding interactions and the valorization of industrial waste streams. The study supports future optimization and scale-up for circular bioeconomy applications

Streptococcus anginosus orchestrates antibacterial potential of NETs facilitating survival of accompanying pathogens

Streptococcus anginosus is considered an emerging opportunistic pathogen causing life-threatening infections, including abscesses and empyema. Noticeably, clinical data revealed that S. anginosus also constitutes an important component of polymicrobial infections. Here, we showed for the first time that S. anginosus inactivates the antibacterial potential of neutrophil extracellular traps (NETs). The process is determined by a cell wall-anchored nuclease referred to as SanA, which high expression dominates in clinical strains isolated from severe infections. Nuclease activity protects S. anginosus against the antibacterial activity of NETs, supporting at the same time the survival of coexisting highly pathogenic species of Enterobacteriales. Obtained data suggest that SanA nuclease should be recognized as a critical S. anginosus virulence factor determining severe monospecies purulent infections but also shielding other pathogens promoting the development of polymicrobial infections

The TRAPPC8/TRS85 subunit of the Arabidopsis TRAPPIII tethering complex regulates endoplasmic reticulum function and autophagy

Transport protein particle (TRAPP) tethering complexes are known for their function as Rab GTPase exchange factors. Two versions of the complex are considered functionally separate: TRAPPII, an activator of the Rab11 family (RabA in plants) GTPases that function in post-Golgi sorting, and TRAPPIII, activating Rab1 family (RabD in plants) members that regulate endoplasmic reticulum (ER)-to-Golgi trafficking and autophagy. In Arabidopsis (Arabidopsis ahaliana), the TRAPPIII complex has been identified and its subunit composition established, but little is known about its functions. Here, we found that binary subunit interactions of the plant TRAPPIII complex are analogous to those of metazoan TRAPPIII, with the 2 large subunits TRAPPC8 and TRAPPC11 linking the TRAPP core and the small C12 to C13 dimer. To gain insight into the functions of TRAPPIII in plants, we characterized 2 A. thaliana trappc8 mutants. These mutants display abnormalities in plant morphology, particularly in flower and seed development. They also exhibit autophagic defects, a constitutive ER stress response, and elevated levels of the ER lipid dolichol (Dol), which is an indispensable cofactor in protein glycosylation. These results indicate that plant TRAPPC8 is involved in multiple cellular trafficking events and suggest a link between ER stress responses and Dol levels

Multiple mechanisms of termination modulate the dynamics of RNAPI transcription

Transcription elongation is stochastic, driven by a Brownian ratchet, making it subject to changes in velocity. On the rDNA, multiple polymerases are linked by "torsional entrainment" generated by DNA rotation. We report that release of entrainment by co-transcriptional 3' end cleavage, is permissive for relative movement between polymerases, promoting pausing and backtracking. Subsequent termination (polymerase release) is facilitated by the 5' exonuclease Rat1 (Xrn2) and backtracked transcript cleavage by the RNA polymerase I (RNAPI) subunit Rpa12. These activities are reproduced in vitro. Short nascent transcripts close to the transcriptional start site, combined with nascent transcript folding energy, similarly facilitate RNAPI pausing. Nascent, backtracked transcripts at pause sites are terminated by forward and reverse "torpedoes": Rat1 and the exosome cofactor Trf4/5-Air1/2-Mtr4 polyadenylation (TRAMP), respectively. Topoisomerase 2 localizes adjacent to RNAPI pause sites, potentially allowing continued elongation by downstream polymerases. Mathematical modeling supported substantial premature termination. These basic insights into transcription in vivo will be relevant to many systems

SorCS2 Is Important for Astrocytic Function in Neurovascular Signaling

Introduction: The receptor SorCS2 is involved in the trafficking of membrane receptors and transporters. It has been implicated in brain disorders and has previously been reported to be indispensable for ionotropic glutamatergic neurotransmission in the hippocampus. Aim: We aimed to study the role of SorCS2 in the control of astrocyte-neuron communication, critical for neurovascular coupling. Methods: Brain slices from P8 and 2-month-old wild-type and SorCS2 knockout (Sorcs2−/−) mice were immunostained for SorCS2, GFAP, AQP4, IB4, and CD31. Neurovascular coupling was assessed in vivo using laser speckle contrast imaging and ex vivo in live brain slices loaded with calcium-sensitive dye. Bulk and cell surface fraction proteomics was analyzed on freshly isolated and cultured astrocytes, respectively, and validated with Western blot and qPCR. Results: SorCS2 was strongly expressed in astrocytes, primarily in their endfeet, of P8 mice; however, it was sparsely repre-sented in 2-month-old mice. Sorcs2−/− mice demonstrated reduced neurovascular coupling associated with a reduced astrocytic calcium response to neuronal excitation. No differences in vascularization or endothelium-dependent relaxation ex vivo between the 2-month-old groups were observed. Proteomics suggested changes in glutamatergic signaling and suppressed calcium sign-aling in Sorcs2−/− brains from both P8 and 2-month-old mice. The increased abundance of glutamate metabotropic receptor 3 in Sorcs2−/− astrocytes was validated by PCR and Western blot. In cultured Sorcs2−/− astrocytes, AQP4 abundance was increased in the bulk lysate but reduced in the cell surface fraction, suggesting impaired trafficking

Symmetric adenine methylation is an essential DNA modification in the early-diverging fungus Rhizopus microsporus

The discovery of N6-methyladenine (6mA) in eukaryotic genomes, typically found in prokaryotic DNA, has revolutionized epigenetics. Here, we show that symmetric 6mA is essential in the early diverging fungus Rhizopus microsporus, as the absence of the MT-A70 complex (MTA1c) responsible for this modification results in a lethal phenotype. 6mA is present in 70% of the genes, correlating with the presence of H3K4me3 and H2A.Z in open euchromatic regions. This modification is found predominantly in nucleosome linker regions, influencing the nucleosome positioning around the transcription start sites of highly expressed genes. Controlled downregulation of MTA1c reduces symmetric 6mA sites affecting nucleosome positioning and histone modifications, leading to altered gene expression, which is likely the cause of the severe phenotypic changes observed. Our study highlights the indispensable role of the DNA 6mA in a multicellular organism and delineates the mechanisms through which this epigenetic mark regulates gene expression in a eukaryotic genome

Short coiled-coil proteins from plants and metazoans – the ‘jacks of all trades’

The molecular functions of short coiled-coil proteins remain poorly charac- terized. These proteins typically act as facilitators rather than essential components of metabolic processes, contributing to cellular homeostasis, and are aptly described as ‘jacks of all trades but masters of none’. They are found across diverse groups of organisms, including both plants and animals. LSU (RESPONSE TO LOW SULFUR) are plant proteins induced under sulfur deficiency and other environmental stresses. They par- ticipate in metabolic pathways, including sulfate assimilation, and manage oxidative stress by stabilizing and protecting antioxidative enzymes. In metazoans, SCOC (SHORT COILED-COIL) proteins regulate autophagy initiation by recruiting proteins essential for forming autophagosomes — key vesicles involved in cellular degradation. SCOC proteins also interact with factors critical for maintaining membrane dynamics and intracellular transport. Despite some functional similarities, the roles of these proteins have diverged significantly between plants and animals, reflecting organism-specific adaptations shaped by evolutionary pressures. This diver- gence underscores their adaptive versatility and highlights their potential as promising targets for future biological research

Characteristics of Two Saccharomyces cerevisiae Strains and Their Extracellular Vesicles as New Candidates for Probiotics

There is a huge disparity between the number of bacterial and yeast probiotics in favor of the former. The latest reports indicate that extracellular vehicles (EVs) play a significant role in probiotic mechanisms. In the present work, we compared the probiotic properties of Saccharomyces cerevisiae strains (WUT3 and WUT151), which have never been previously characterized in this context, with commercial probiotic yeast—Saccharomyces cerevisiae var. boulardii CNCM-745. Notably, WUT3 and WUT151 reacted more mildly to the unfavorable simulated environment of saliva, stomach, small, and large intestines. As a result, we confirmed that WUT3 and WUT151 were superior to S. boulardii in terms of probiotic properties. Then, we performed a complex analysis of their EVs, isolated by a multistep filtration process. The nanoparticle tracing analysis showed no significant difference in the diameter of the vesicles between the strains. MTT studies confirmed that EVs are not toxic against normal human colorectal cell lines CCD-18 Co and CCD 841 CoN. However, toxicity was observed against the HT-29 cancer line. By staining EVs with Nile Red, we successfully visualized EVs–cell interactions. Finally, we explored the profile of proteins transported with the EVs, identifying a significant overrepresentation of extracellular proteins. Based on comparison with other proteomic data, we selected marker proteins for S.cerevisiae EVs. This knowledge will be helpful for further studies on tracking the transfer of the protein cargo of yeast EVs to human cells using, for instance, specific antibodies to these marker proteins

Differential scanning fluorimetry followed by microscale thermophoresis and/or isothermal titration calorimetry as an efficient tool for ligand screening

Various biophysical and biochemical techniques have been developed to measure the affinity of interacting molecules. This review analyzes the combination of three methods: differential scanning fluorimetry as the initial high-throughput screening technique and microscale thermophoresis and isothermal titration calorimetry as complementary methods to quantify binding affinity. The presented work is the first to detailed compare the strengths and flaws of these three specific methods, as well as their application possibilities and complementarity. The fundamentals of these methods will be covered, including the most often-used models for characterizing observable phenomena and an emphasis on methods for analyzing data. A comprehensive review of numerous approaches to data analysis found in the literature is additionally provided, with the benefits and drawbacks of each, as well as the pitfalls and related concerns. Finally, examples of different systems will be presented, and methods used and some discrepancies in results will be described and discussed

RNA polymerase III transcription machinery and tRNA processing are regulated by the ubiquitin ligase Rsp5

Transfer RNA (tRNA) biogenesis in yeast involves synthesis of the primary transcript by RNA polymerase III (Pol III), followed by processing to remove 5′ and 3′ ends, further maturation, and export to the cytoplasm. In the present study, we found that both tRNA transcription and the initial processing of tRNA precursors are affected by the ubiquitin ligase Rsp5. We observed high levels of unprocessed primary tRNA transcripts in rsp5 mutants at elevated temperature, which were reduced upon the overexpression of RPR1, the catalytic subunit of RNase P. This observation suggests a role for Rsp5 in the maturation of 5′ ends of tRNA precursors. Under the same conditions, in vivo labeling showed that the amount of newly synthesized tRNA decreased. Furthermore, we found that Rsp5 directly interacted with the Tfc3 subunit of the TFIIIC transcription factor, which is modified by ubiquitination. The inactivation of Rsp5 catalytic activity influenced the interaction between the general Pol III factors TFIIIB and TFIIIC and decreased the recruitment of TFIIIC to tRNA genes. These findings suggest that Rsp5 ligase is implicated in the control of Pol III transcription in yeast