Journal of Microbiology

Research Article

Single nucleotide genome recognition and selective bacterial lysis using synthetic phages loaded with CRISPR-Cas12f1-truncated sgRNA: Ho Joung Lee, Song Hee Jeong, Sang Jun Lee; J. Microbiol. 2025;63(2):e2501012. Published online February 27, 2025; DOI: https://doi.org/10.71150/jm.2501012

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Abstract PDF Supplementary Material: Phage specificity primarily relies on host cell-surface receptors. However, integrating cas genes and guide RNAs into phage genomes could enhance their target specificity and regulatory effects. In this study, we developed a CRISPR-Cas12f1 system-equipped bacteriophage λ model capable of detecting Escherichia coli target genes. We demonstrated that synthetic λ phages carrying Cas12f1-sgRNA can effectively prevent lysogen formation. Furthermore, we showcased that truncating the 3'-end of sgRNA enables precise identification of single-nucleotide variations in the host genome. Moreover, infecting E. coli strains carrying various stx2 gene subtypes encoding Shiga toxin with bacteriophages harboring Cas12f1 and truncated sgRNAs resulted in the targeted elimination of strains with matching subtype genes. These findings underscore the ability of phages equipped with the CRISPR-Cas12f1 system to precisely control microbial hosts by recognizing genomic sequences with high resolution.

Journal Articles

H-NS is a Transcriptional Repressor of the CRISPR-Cas System in Acinetobacter baumannii ATCC 19606: Kyeongmin Kim, Md Maidul Islam, Seunghyeok Bang, Jeongah Kim, Chung-Young Lee, Je Chul Lee, Minsang Shin; J. Microbiol. 2024;62(11):999-1012. Published online November 11, 2024; DOI: https://doi.org/10.1007/s12275-024-00182-5

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Abstract: Acinetobacter baumannii is a multidrug-resistant opportunistic pathogen primarily associated with hospital-acquired infections. The bacterium can gain multidrug resistance through several mechanisms, including horizontal gene transfer. A CRISPR-Cas system including several Cas genes could restrict the horizontal gene transfer. However, the molecular mechanism of CRISPR- Cas transcriptional regulation remains unclear. We identified a type I-F CRISPR-Cas system in A. baumannii ATCC 19606^T standard strain based on sequence analysis. We focused on the transcriptional regulation of Cas3, a key protein of the CRISPR-Cas system. We performed a DNA affinity chromatography-pulldown assay to identify transcriptional regulators of the Cas3 promoter. We identified several putative transcriptional factors, such as H-NS, integration host factor, and HU, that can bind to the promoter region of Cas3. We characterized AbH-NS using size exclusion chromatography and cross-linking experiments and demonstrated that the Cas3 promoter can be regulated by AbH-NS in a concentration-dependent manner via an in vitro transcription assay. CRISPR-Cas expression levels in wild-type and hns mutant strains in the early stationary phase were examined by qPCR and β-galactosidase assay. We found that H-NS can act as a repressor of Cas3. Our transformation efficiency results indicated that the hns mutation decreased the transformation efficiency, while the Cas3 mutation increased it. We report the existence and characterization of the CRISPR-Cas system in A. baumannii 19606^T and demonstrate that AbH-NS is a transcriptional repressor of CRISPR-Cas-related genes in A. baumannii.

Mammaliicoccus sciuri's Pan-Immune System and the Dynamics of Horizontal Gene Transfer Among Staphylococcaceae: a One-Health CRISPR Tale: Allan de Carvalho, Marcia Giambiagi-deMarval, Ciro César Rossi; J. Microbiol. 2024;62(9):775-784. Published online July 22, 2024; DOI: https://doi.org/10.1007/s12275-024-00156-7

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Abstract: Recently emancipated from the Staphylococcus genus due to genomic differences, Mammaliicoccus sciuri, previously classified as an occasional pathogen, emerges as a significant player in the landscape of resistance gene dissemination among Staphylococcaceae. Despite its classification, its role remained enigmatic. In this study, we delved into the genomic repertoire of M. sciuri to unravel its contribution to resistance and virulence gene transfer in the context of One Health. Through comprehensive analysis of publicly available genomes, we unveiled a diverse pan-immune system adept at defending against exogenous genetic elements, yet concurrently fostering horizontal gene transfer (HGT). Specifically, exploration of CRISPR-Cas systems, with spacer sequences as molecular signatures, elucidated a global dissemination pattern spanning environmental, animal, and human hosts. Notably, we identified the integration of CRISPR-Cas systems within SCCmecs (Staphylococcal Cassette Chromosome mec), harboring key genes associated with pathogenicity and resistance, especially the methicillin resistance gene mecA, suggesting a strategic adaptation to outcompete other mobile genetic elements. Our findings underscored M. sciuri's active engagement in HGT dynamics and evolutionary trajectories within Staphylococcaceae, emphasizing its central role in shaping microbial communities and highlighting the significance of understanding its implications in the One Health framework, an interdisciplinary approach that recognizes the interconnectedness of human, animal, and environmental health to address global health challenges.

Review

Apoptotic Factors, CaNma111 and CaYbh3, Function in Candida albicans Filamentation by Regulating the Hyphal Suppressors, Nrg1 and Tup1: Suyoung Kim , Se Hyeon Kim , Eunjoong Kweon , Jinmi Kim; J. Microbiol. 2023;61(4):403-409. Published online March 27, 2023; DOI: https://doi.org/10.1007/s12275-023-00034-8

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Abstract: The morphological switch from the yeast to hyphal form is a key virulence attribute of the opportunistic fungal pathogen, Candida albicans. Our recent report showed that deletion of the newly identified apoptotic factor, CaNma111 or CaYbh3, leads to hyperfilamentation and increased virulence in a mouse infection model. CaNma111 and CaYbh3 are homologs of the pro-apoptotic protease, HtrA2/Omi, and BH3-only protein, respectively. In this study, we examined the effects of CaNMA111 and CaYBH3 deletion mutations on the expression levels of the hypha-specific transcr!ption factors, Cph1 (a hyphal activator), Nrg1 (a hyphal repressor), and Tup1 (a hyphal repressor). The protein levels of Nrg1 were decreased in Caybh3/Caybh3 cells while those of Tup1 were decreased in both Canma111/Canma111 and Caybh3/Caybh3 cells. These effects on Nrg1 and Tup1 proteins were retained during serum-induced filamentation and appear to explain the hyperfilamentation phenotypes of the CaNMA111 and CaYBH3 deletion mutants. Treatment with the apoptosis-inducing dose of farnesol decreased the Nrg1 protein levels in the wild-type strain and more evidently in Canma111/Canma111 and Caybh3/Caybh3 mutant strains. Together, our results suggest that CaNma111 and CaYbh3 are key regulators of Nrg1 and Tup1 protein levels in C. albicans.

Journal Articles

Gut microbiota metabolic characteristics in coronary artery disease patients with hyperhomocysteine: Ran Tian , Hong-Hong Liu , Si-Qin Feng , Yi-Fei Wang , Yi-Yang Wang , Yu-Xiong Chen , Hui Wang , Shu-Yang Zhang; J. Microbiol. 2022;60(4):419-428. Published online March 4, 2022; DOI: https://doi.org/10.1007/s12275-022-1451-2

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Abstract

Hyperhomocysteine (HHcy) is known as a risk factor for coronary artery disease (CAD). Despite the knowledge that gut microbiota related metabolism pathway shares metabolites with that of Hcy, little has been shown concerning the association between HHcy and gut microbiota. To explore their relationship in the context of CAD, 105 patients and 14 healthy controls were recruited from one single medical center located in Beijing, China. Their serum and fecal samples were collected, with multi-omics analyses performed via LC/MS/ MS and 16S rRNA gene V3-V4 region sequencing, respectively. Participants from the prospective cohort were divided into CAD, CAD & HHcy and healthy controls (HC) groups based on the diagnosis and serum Hcy concentration. The
results
revealed significant different metabolic signatures between CAD and CAD & HHcy groups. CAD patients with HHcy suffered a heavier atherosclerotic burden compared to CAD patients, and the difference was closely associated to betaine-homocysteine S-methyltransferase (BHMT)-related metabolites and trimethylamine N-oxide (TMAO)-related metabolites. Dimethylglycine (DMG) exhibited a strong positive correlation with serum total Hcy (tHcy), and TMAO and trimethylysine (TML) were associated with heavier atherosclerotic burden. Multiple other metabolites were also identified to be related to distinct cardiovascular risk factors. Additionally, Clostridium cluster IV and Butyricimonas were enriched in CAD patients with elevated tHcy. Our study suggested that CAD patients with elevated tHcy were correlated with higher atherosclerotic burden, and the impaired Hcy metabolism and cardiovascular risk were closely associated with BHMT-related metabolites, TMAO-related metabolites and impaired gut microbiota homeostasis.

Citations

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Melatonin attenuates microbiota dysbiosis of jejunum in short-term sleep deprived mice: Ting Gao , Zixu Wang , Jing Cao , Yulan Dong , Yaoxing Chen; J. Microbiol. 2020;58(7):588-597. Published online May 18, 2020; DOI: https://doi.org/10.1007/s12275-020-0094-4

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Abstract

Our study demonstrated that sleep deprivation resulted in homeostasis disorder of colon. Our study goes deeper into the positive effects of melatonin on small intestinal microbiota disorder caused by sleep deprivation. We successfully established a multiplatform 72 h sleep deprivation mouse model with or without melatonin supplementation, and analyzed the change of small intestinal microbiota using high-throughput sequencing of the 16S rRNA. We found melatonin supplementation suppressed the decrease of plasma melatonin level in sleep deprivation mice. Meanwhile, melatonin supplementation improved significantly the reduction in OTU numbers and the diversity and richness of jejunal microbiota and the abundance of Bacteroidaeae and Prevotellaceae, as well as an increase in the Firmicutes-to-Bacteroidetes ratio and the content of Moraxellaceae and Aeromonadaceae in the jejunum of sleep deprived-mice. Moreover, melatonin supplementation reversed the change of metabolic pathway in sleep deprived-mice, including metabolism, signal transduction mechanisms and transcription etc, which were related to intestinal health. Furthermore, melatonin supplementation inverted the sleep deprivation-induced a decline of anti-inflammatory cytokines (IL-22) and an increase of the ROS and proinflammatory cytokines (IL-17) in jejunum. These findings suggested that melatonin, similar to a probiotics agent, can reverse sleep deprivation-induced small intestinal microbiota disorder by suppressing oxidative stress and inflammation response.

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Review

REVIEW] When a Virus is not a Parasite: The Beneficial Effects of Prophages: Joseph Bondy-Denomy , Alan R. Davidson; J. Microbiol. 2014;52(3):235-242. Published online March 1, 2014; DOI: https://doi.org/10.1007/s12275-014-4083-3

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Abstract

Most organisms on the planet have viruses that infect them. Viral infection may lead to cell death, or to a symbiotic relationship where the genomes of both virus and host replicate together. In the symbiotic state, both virus and cell potentially experience increased fitness as a result of the other. The viruses that infect bacteria, called bacteriophages (or phages), well exemplify the symbiotic relationships that can develop between viruses and their host. In this review, we will discuss the many ways that prophages, which are phage genomes integrated into the genomes of their hosts, influence bacterial behavior and virulence.

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