Journal of Microbiology : Journal of Microbiology

Streptomyces are a crucial source of bioactive secondary metabolites with significant clinical applications. Recent studies of bacterial and metagenome-assembled genomes have revealed that Streptomyces harbors a substantial number of uncharacterized silent secondary metabolite biosynthetic gene clusters (BGCs). These BGCs represent a vast diversity of biosynthetic pathways for natural product synthesis, indicating significant untapped potential for discovering new metabolites. To exploit this potential, genome mining using comprehensive strategies that leverage extensive genomic databases can be conducted. By linking BGCs to their encoded products and integrating genetic manipulation techniques, researchers can greatly enhance the identification of new secondary metabolites with therapeutic relevance. In this context, we present a step-by-step guide for using the antiSMASH pipeline to identify secondary metabolite-coding BGCs within the complete genome of a novel Streptomyces strain. This protocol also outlines gene manipulation methods that can be applied to Streptomyces to activate cryptic clusters of interest and validate the functions of biosynthetic genes. By following these guidelines, researchers can pave the way for discovering and characterizing valuable natural products.

Protein solubility is a critical factor in the production of recombinant proteins, which are widely used in various industries, including pharmaceuticals, diagnostics, and biotechnology. Predicting protein solubility remains a challenging task due to the complexity of protein structures and the multitude of factors influencing solubility. Recent advances in computational methods, particularly those based on machine learning, have provided powerful tools for predicting protein solubility, thereby reducing the need for extensive experimental trials. This review provides an overview of current computational approaches to predict protein solubility. We discuss the datasets, features, and algorithms employed in these models. The review aims to bridge the gap between computational predictions and experimental validations, fostering the development of more accurate and reliable solubility prediction models that can significantly enhance recombinant protein production.

CRISPR-Cas technologies have emerged as powerful and versatile tools in gene therapy. In addition to the widely used SpCas9 system, alternative platforms including modified amino acid sequences, size-optimized variants, and other Cas enzymes from diverse bacterial species have been developed to apply this technology in various genetic contexts. In addition, base editors and prime editors for precise gene editing, the Cas13 system targeting RNA, and CRISPRa/i systems have enabled diverse and adaptable approaches for genome and RNA editing, as well as for regulating gene expression. Typically, CRISPR-Cas components are transported to the target in the form of DNA, RNA, or ribonucleoprotein complexes using various delivery methods, such as electroporation, adeno-associated viruses, and lipid nanoparticles. To amplify therapeutic efficiency, continued developments in targeted delivery technologies are required, with increased safety and stability of therapeutic biomolecules. CRISPR-based therapeutics hold an inexhaustible potential for the treatment of many diseases, including rare congenital diseases, by making permanent corrections at the genomic DNA level. In this review, we present various CRISPR-based tools, their delivery systems, and clinical progress in the CRISPR-Cas technology, highlighting its innovative prospects for gene therapy.

Dengue, caused by four serotypes of dengue viruses (DENV-1 to DENV-4), is the most prevalent and widely mosquito-borne viral disease affecting humans. Dengue virus (DENV) infection has been reported in over 100 countries, and approximately half of the world's population is now at risk. The paucity of universally licensed DENV vaccines highlights the urgent need to address this public health concern. Action and attention to antibody-dependent enhancement increase the difficulty of vaccine development. With the worsening dengue fever epidemic, Dengvaxia^® (CYD-TDV) and Qdenga^® (TAK-003) have been approved for use in specific populations in affected areas. However, these vaccines do not provide a balanced immune response to all four DENV serotypes and the vaccination cannot cover all populations. There is still a need to develop a safe, broad-spectrum, and effective vaccine to address the increasing number of dengue cases worldwide. This review provides an overview of the existing DENV vaccines, as well as potential candidates for future studies on DENV vaccine development, and discusses the challenges and possible solutions in the field.

The increasing environmental concerns regarding conventional plastics have led to a growing demand for sustainable alternatives, such as biodegradable plastics. Yeast cell factories, specifically Saccharomyces cerevisiae and Yarrowia lipolytica, have emerged as promising platforms for bioplastic production due to their scalability, robustness, and ease of manipulation. This review highlights synthetic biology approaches aimed at developing yeast cell factories to produce key biodegradable plastics, including polylactic acid (PLA), polyhydroxyalkanoates (PHAs), and poly (butylene adipate-co-terephthalate) (PBAT). We explore recent advancements in engineered yeast strains that utilize various synthetic biology strategies, such as the incorporation of new genetic elements at the gene, pathway, and cellular system levels. The combined efforts of metabolic engineering, protein engineering, and adaptive evolution have enhanced strain efficiency and maximized product yields. Additionally, this review addresses the importance of integrating computational tools and machine learning into the Design-Build-Test-Learn cycle for strain development. This integration aims to facilitate strain development while minimizing effort and maximizing performance. However, challenges remain in improving strain robustness and scaling up industrial production processes. By combining advanced synthetic biology techniques with computational approaches, yeast cell factories hold significant potential for the sustainable and scalable production of bioplastics, thus contributing to a greener bioeconomy.

Strains Mo2-6^T, S9, KG4-3^T, and 50Mo3-2, identified as coagulase-negative, Gram-stain-positive, halotolerant, non-motile coccoid bacteria, were isolated from traditional Korean soybean foods. Strains Mo2-6^T and S9 were both catalase- and oxidase-negative, whereas KG4-3^T and 50Mo3-2 were catalase-positive but oxidase-negative. The optimal growth conditions for Mo2-6^T and S9 were 30°C, 2% NaCl, and pH 7.0, while KG4-3^T and 50Mo3-2 grew best at 35°C, 2% NaCl, and pH 7.0. All strains contained menaquinone-7 as the predominant isoprenoid quinone, with anteiso-C_15:0 and iso-C_15:0 as the major cellular fatty acids (> 10%). Additionally, anteiso-C_13:0 was a major fatty acid in strain KG4-3^T. The DNA G + C contents of strains Mo2-6^T, S9, KG4-3^T, and 50Mo3-2 were 33.4%, 33.3%, 32.5%, and 32.7%, respectively. Phylogenetic analyses based on the 16S rRNA gene and whole-genome sequences revealed that strains Mo2-6^T and S9, as well as KG4-3^T and 50Mo3-2, formed distinct lineages within the genus Staphylococcus. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses confirmed that strains Mo2-6^T and S9, as well as KG4-3^T and 50Mo3-2, belonged to the same species. Meanwhile, dDDH and ANI values between strains Mo2-6^T and KG4-3^T, as well as comparisons with other Staphylococcus type strains, were below the species delineation thresholds, indicating they represent novel species. Based on phenotypic, chemotaxonomic, and molecular data, we propose strain Mo2-6^T as the type strain of Staphylococcus parequorum sp. nov. (=KACC 23685^T =JCM 37038^T) and strain KG4-3^T as the type strain of Staphylococcus halotolerans sp. nov. (=KACC 23684^T =JCM 37037^T).

CRISPR-Cas9-based gene editing enables precise genetic modifications. However, its application to human cytomegalovirus (HCMV) remains challenging due to the large size of the viral genome and the essential roles of key regulatory genes. Here, we establish an optimized CRISPR-Cas9 system for precise labeling and functional analysis of HCMV immediate early (IE) genes. By integrating a multifunctional cassette encoding an auxin-inducible degron (AID), a self-cleaving peptide (P2A), and GFP into the viral genome via homology-directed repair (HDR), we achieved efficient knock-ins without reliance on bacterial artificial chromosome (BAC) cloning, a labor-intensive and time-consuming approach. We optimized delivery strategies, donor template designs, and component ratios to enhance HDR efficiency, significantly improving knock-in success rates. This system enables real-time fluorescent tracking and inducible protein degradation, allowing temporal control of essential viral proteins through auxin-mediated depletion. Our approach provides a powerful tool for dissecting the dynamic roles of viral proteins throughout the HCMV life cycle, facilitating a deeper understanding of viral pathogenesis and potential therapeutic targets.

The widespread use of antibiotics in aquaculture has led to the emergence of multidrug-resistant pathogens and environmental concerns, highlighting the need for sustainable, eco-friendly alternatives. In this study, we isolated and characterized three novel bacteriophages from aquaculture effluents in Korean shrimp farms that target the key Vibrio pathogens, Vibrio harveyi, and Vibrio parahaemolyticus. Bacteriophages were isolated through environmental enrichment and serial purification using double-layer agar assays. Transmission electron microscopy revealed that the phages infecting V. harveyi, designated as vB_VhaS-MS01 and vB_VhaS-MS03, exhibited typical Siphoviridae morphology with long contractile tails and icosahedral heads, whereas the phage isolated from V. parahaemolyticus (vB_VpaP-MS02) displayed Podoviridae characteristics with an icosahedral head and short tail.

Whole-genome sequencing produced complete, circularized genomes of 81,710 bp for vB_VhaS-MS01, 81,874 bp for vB_VhaS-MS03, and 76,865 bp for vB_VpaP-MS02, each showing a modular genome organization typical of Caudoviricetes. Genomic and phylogenetic analyses based on the terminase large subunit gene revealed that although vB_VhaS-MS01 and vB_VhaS-MS03 were closely related, vB_VpaP-MS02 exhibited a distinct genomic architecture that reflects its unique morphology and host specificity. Collectively, these comparative analyses demonstrated that all three phages possess genetic sequences markedly different from those of previously reported bacteriophages, thereby establishing their novelty. One-step growth and multiplicity of infection (MOI) experiments demonstrated significant differences in replication kinetics, such as burst size and lytic efficiency, among the phages, with vB_VhaS-MS03 maintaining the most effective bacterial control, even at an MOI of 0.01. Additionally, host range assays showed that vB_VhaS-MS03 possessed a broader spectrum of activity, supporting its potential use as a stand-alone agent or key component of phage cocktails. These findings highlight the potential of region-specific phage therapy as a targeted and sustainable alternative to antibiotics for controlling Vibrio infections in aquaculture.

Condensin plays a central role in mitotic chromosome organization and segregation by mediating long-range chromatin interactions. However, the extent to which cellular metabolic status influences condensin function remains unclear. To gain insights into the relationship of metal ion homeostasis and the function of condensin, we conducted genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) using Schizosaccharomyces pombe under iron- or zinc-deficient conditions. Under iron- or zinc-deficient conditions, ChIP-seq results revealed a selective reduction in condensin binding at high-affinity target loci, particularly genes regulated by Ace2 and Ams2, while cohesin binding remained largely unaffected. Hi-C analysis showed that iron depletion weakened chromatin interactions at these condensin targets and centromeres, without disrupting global genome architecture. DNA fluorescence in situ hybridization (FISH) confirmed that iron deficiency impaired long-range associations between centromeres and Ace2 target loci at the single-cell level. Notably, iron deficiency led to chromosome segregation defects during mitosis, suggesting that diminished condensin occupancy compromised genome stability. These changes occurred without significant alterations in condensin protein levels or global transcription, indicating a direct effect of metal ion availability on condensin activity. Collectively, our findings revealed a previously unrecognized regulatory axis in which cellular metal ion homeostasis modulated condensin-dependent chromatin organization and mitotic chromosome segregation, offering new insights into the integration of metabolic state with genome maintenance.

Extracellular vesicles derived from probiotics have received considerable attention for their pivotal role in bacterial‒host communication. These nanosized, bilayer-encapsulated vesicles carry diverse bioactive molecules, such as proteins, lipids, nucleic acids, and metabolites. Currently, ample evidence has emerged that probiotic extracellular vesicles may modulate several processes of host physiological hemostasis and offer therapeutic benefits. This review examines the biogenesis, composition, and immunomodulatory functions of probiotic-derived extracellular vesicles in probiotic–host interactions, highlighting the therapeutic potential of probiotic extracellular vesicles in the diagnosis and treatment of conditions such as cancer and inflammatory bowel disease. We further summarize the techniques for the separation and purification of extracellular vesicles, providing a methodological foundation for future research and applications. Although the field of probiotic extracellular vesicle research is still in its infancy, the prospects for their application in the biomedical field are broad, potentially emerging as a novel therapeutic approach.

Marine organisms often form symbiotic relationships with various microorganisms to adapt and thrive in harsh environments. These symbiotic microbes contribute to host survival by providing nutrition, modulating the hosts’ immune system, and supporting overall physiological stability. Advances in high-throughput sequencing technologies have enabled a deeper understanding of the structure and function of symbiotic microbial communities, as well as host-microbe interactions. Notably, symbiotic bacteria associated with marine invertebrates such as corals and sponges are recognized as a potential source of useful bioactive compounds, including antibiotics and enzymes. However, obtaining high-quality microbial DNA from host tissues still remains a technical challenge due to the presence of unknown substances. This study focuses on optimizing sample preparation and DNA extraction procedures and additional purification to improve the recovery of microbial DNA while minimizing host DNA contamination. Comparison between several methods was conducted using sponge samples to evaluate DNA quality and microbial recovery. A sample designated as 2110BU-001 was collected from the east coast of the Republic of Korea and used for culture-independent microbial cell isolation. Total bacterial DNA was extracted by using a manual Phenol-Chloroform protocol and three commercial kits. DNA extracted using the standard manual method showed both the highest yield and the largest fragment size. However, PCR (Polymerase chain reaction) test showed that quality of manually extracted DNA was not enough for sequencing. Therefore, the quality of DNA was improved through additional purification steps. Briefly, host eukaryotic cells were removed by mechanical process and almost only bacterial DNA was successfully obtained by combination of manual extraction method and further purification processes. The established protocol was successfully introduced to extraction of metagenomic DNA from mussel and jellyfish microbiomes, indicating that it can be widely applied to various marine organisms.

The escalating antibiotic resistance crisis poses a significant challenge to global public health, threatening the efficacy of current treatments and driving the emergence of multidrug-resistant pathogens. Among the various factors associated with bacterial antibiotic resistance, small regulatory RNAs (sRNAs) have emerged as pivotal post-transcriptional regulators which orchestrate bacterial adaptation to antibiotic pressure via diverse mechanisms. This review consolidates the current knowledge on sRNA-mediated mechanisms, focusing on drug uptake, drug efflux systems, lipopolysaccharides, cell wall modification, biofilm formation, and mutagenesis. Recent advances in transcriptomics and functional analyses have revealed novel sRNAs and their regulatory networks, expanding our understanding of resistance mechanisms. These findings highlight the potential of targeting sRNA-mediated pathways as an innovative therapeutic strategy to combat antibiotic resistance, and offer promising avenues for managing challenging bacterial infections.

Existing microbial engineering strategies—encompassing metabolic engineering, systems biology, and systems metabolic engineering—have significantly enhanced the potential of microbial cell factories as sustainable alternatives to the petrochemical industry by optimizing metabolic pathways. Recently, systems metabolic engineering, which integrates tools from synthetic biology, enzyme engineering, omics technology, and evolutionary engineering, has been successfully developed. By leveraging modern engineering strategies within the Design-Build-Test-Learn (DBTL) cycle framework, these advancements have revolutionized the biosynthesis of valuable compounds. This review highlights recent progress in the metabolic engineering of Corynebacterium glutamicum, a versatile microbial platform, achieved through various approaches from traditional metabolic engineering to advanced systems metabolic engineering, all within the DBTL cycle. A particular focus is placed C5 platform chemicals derived from L-lysine, one of the key amino acid production pathways of C. glutamicum. The development of DBTL cycle-based metabolic engineering strategies for this process is discussed.

Akkermansia muciniphila (AKK, A. muciniphila) fortifies the intestinal barrier, inhibits the colonization of pathogenic bacteria, and protects the host’s health. Nevertheless, the existing literature offers inadequate evidence to ascertain whether A. muciniphila can effectively treat Candida albicans (C. albicans) infections in vitro, and the underlying mechanisms remain ambiguous. This study, animal models were established through gavage with clinical isolates of C. albicans to induce gastrointestinal tract colonization and subsequent translocation infection. The models were subsequently administered A. muciniphila. We examined the analysis of 16S rRNA gene sequencing, metabolomics of colonic contents, and transcriptomics of colonic tissue. The intestinal barrier, inflammatory responses, and immune cell infiltration are analyzed. This study revealed that A. muciniphila markedly mitigated C. albicans translocation infection and modified the intestinal microbial community structure and metabolic attributes in model mice. After administering A. muciniphila to the translocation infection group, there was a notable increase in the prevalence of bacteria that produce short-chain fatty acids, including Eubacterium_F. Moreover, there was a significant increase in the levels of specific pathogens, including Faecalibaculum, Turicibacter, and Turicimonas. The study demonstrated that A. muciniphila treatment can improve the composition of intestinal microbiota and metabolites, augment the tight junctions of colonic tissue and diminish systemic inflammatory response. This presents an innovative therapeutic approach for the potential treatment of intestinal C. albicans infection using A. muciniphila.