Journal of Microbiology

Volume 62(1); January 2024

Journal Articles

[Protocol] Use of Cas9 Targeting and Red Recombination for Designer Phage Engineering: Shin-Yae Choi , Danitza Xiomara Romero-Calle , Han-Gyu Cho , Hee-Won Bae , You-Hee Cho; J. Microbiol. 2024;62(1):1-10. Published online February 1, 2024; DOI: https://doi.org/10.1007/s12275-024-00107-2

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Abstract: Bacteriophages (phages) are natural antibiotics and biological nanoparticles, whose application is significantly boosted by recent advances of synthetic biology tools. Designer phages are synthetic phages created by genome engineering in a way to increase the benefits or decrease the drawbacks of natural phages. Here we report the development of a straightforward genome engineering method to efficiently obtain engineered phages in a model bacterial pathogen, Pseudomonas aeruginosa. This was achieved by eliminating the wild type phages based on the Streptococcus pyogenes Cas9 (SpCas9) and facilitating the recombinant generation based on the Red recombination system of the coliphage λ (λRed). The producer (PD) cells of P. aeruginosa strain PAO1 was created by miniTn7-based chromosomal integration of the genes for SpCas9 and λRed under an inducible promoter. To validate the efficiency of the recombinant generation, we created the fluorescent phages from a temperate phage MP29. A plasmid bearing the single guide RNA (sgRNA) gene for selectively targeting the wild type gp35 gene and the editing template for tagging the Gp35 with superfolder green fluorescent protein (sfGFP) was introduced into the PD cells by electroporation. We found that the targeting efficiency was affected by the position and number of sgRNA. The fluorescent phage particles were efficiently recovered from the culture of the PD cells expressing dual sgRNA molecules. This protocol can be used to create designer phages in P. aeruginosa for both application and research purposes.

Flavivirga spongiicola sp. nov. and Flavivirga abyssicola sp. nov., Isolated from Marine Environments: Sung-Hyun Yang , Mi-Jeong Park , Hyun-Myung Oh , Yeong-Jun Park , Kae Kyoung Kwon; J. Microbiol. 2024;62(1):11-19. Published online February 6, 2024; DOI: https://doi.org/10.1007/s12275-023-00102-z

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Abstract: Two novel Gram-stain-negative, strictly-aerobic, rod-shaped (1.2 ± 3.4 μm × 0.3 ± 0.7 μm), and non-motile marine bacterial species, designated MEBiC05379T and MEBiC07777T, were isolated from a marine sponge Pseudaxinella sp. in Gangneung City and deep-sea sediments of the Ulleung basin in the East Sea of Korea, respectively. The 16S rRNA gene sequence analysis revealed high levels of similarities between these strains and members of the genus Flavivirga (97.0–98.4% sequence identities). Both novel strains revealed as mesophilic, neutrophilic in pH and slightly halophilic. Similar to those of other Flavivirga members, the primary cellular fatty acids of both strains were iso-C15:0, iso-C15:1 G, iso-C15:03-OH, and iso-C17:0 3-OH, with MEBiC05379T and MEBiC07777T containing relatively higher proportions of C12: 0 and summed feature 3 ( C16:1ω7c and/or C16: 1ω6c). In both taxa, the major isoprenoid quinone was MK-6. The DNA G + C contents of MEBiC05379T and MEBiC07777T genomes were 32.62 and 32.46 mol%, respectively. Compared to other members of Flavivirga, both strains exhibited similar DNA G + C ratio and fatty acids pattern, yet enzyme expression and carbon sources utilization pattern were different. Genomes of the genus Flavivirga showed enzyme preferences to fucoidan and sulfated galactans. Considering the monophyly rule, AAI values delineate the genus Flavivirga from adjacent genera calculated to be 76.0–78.7%. Based on the phenotypic, genomic and biochemical data, strains for MEBiC05379T and MEBiC07777T thus represent two novel species in the genus Flavivirga, for which the names Flavivirga spongiicola sp. nov. ( MEBiC05379T [= KCTC 92527 T = JCM 16662 T]), and Flavivirga abyssicola sp. nov. ( MEBiC07777T [= KCTC 92563 T = JCM 36477 T]) are proposed.

LAMMER Kinase Governs the Expression and Cellular Localization of Gas2, a Key Regulator of Flocculation in Schizosaccharomyces pombe: Won-Hwa Kang , Yoon-Dong Park , Joo-Yeon Lim , Hee-Moon Park; J. Microbiol. 2024;62(1):21-31. Published online January 5, 2024; DOI: https://doi.org/10.1007/s12275-023-00097-7

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Abstract: It was reported that LAMMER kinase in Schizosaccharomyces pombe plays an important role in cation-dependent and galactose-specific flocculation. Analogous to other flocculating yeasts, when cell wall extracts of the Δlkh1 strain were treated to the wild-type strain, it displayed flocculation. Gas2, a 1,3-β-glucanosyl transferase, was isolated from the EDTA-extracted cell-surface proteins in the Δlkh1 strain. While disruption of the gas2+ gene was not lethal and reduced the flocculation activity of the Δlkh1 strain, the expression of a secreted form of Gas2, in which the GPI anchor addition sequences had been removed, conferred the ability to flocculate upon the WT strain. The Gas2-mediated flocculation was strongly inhibited by galactose but not by glucose. Immunostaining analysis showed that the cell surface localization of Gas2 was crucial for the flocculation of fission yeast. In addition, we identified the regulation of mbx2+ expression by Lkh1 using RT-qPCR. Taken together, we found that Lkh1 induces asexual flocculation by regulating not only the localization of Gas2 but also the transcription of gas2+ through Mbx2.

Comparative Transcriptomic Analysis of Flagellar‑Associated Genes in Salmonella Typhimurium and Its rnc Mutant: Seungmok Han , Ji-Won Byun , Minho Lee; J. Microbiol. 2024;62(1):33-48. Published online January 5, 2024; DOI: https://doi.org/10.1007/s12275-023-00099-5

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Abstract: Salmonella enterica serovar Typhimurium (S. Typhimurium) is a globally recognized foodborne pathogen that affects both animals and humans. Endoribonucleases mediate RNA processing and degradation in the adaptation of bacteria to environmental changes and have been linked to the pathogenicity of S. Typhimurium. Not much is known about the specific regulatory mechanisms of these enzymes in S. Typhimurium, particularly in the context of environmental adaptation. Thus, this study carried out a comparative transcriptomic analysis of wild-type S. Typhimurium SL1344 and its mutant (Δrnc), which lacks the rnc gene encoding RNase III, thereby elucidating the detailed regulatory characteristics that can be attributed to the rnc gene. Global gene expression analysis revealed that the Δrnc strain exhibited 410 upregulated and 301 downregulated genes (fold-change > 1.5 and p < 0.05), as compared to the wild-type strain. Subsequent bioinformatics analysis indicated that these differentially expressed genes are involved in various physiological functions, in both the wild-type and Δrnc strains. This study provides evidence for the critical role of RNase III as a general positive regulator of flagellar-associated genes and its involvement in the pathogenicity of S. Typhimurium.

Mycobacterium tuberculosis PE_PGRS45 (Rv2615c) Promotes Recombinant Mycobacteria Intracellular Survival via Regulation of Innate Immunity, and Inhibition of Cell Apoptosis: Tao Xu , Chutong Wang , Minying Li , Jing Wei , Zixuan He , Zhongqing Qian , Xiaojing Wang , Hongtao Wang; J. Microbiol. 2024;62(1):49-62. Published online February 9, 2024; DOI: https://doi.org/10.1007/s12275-023-00101-0

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Abstract: Tuberculosis (TB), a bacterial infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis), is a significant global public health problem. Mycobacterium tuberculosis expresses a unique family of PE_PGRS proteins that have been implicated in pathogenesis. Despite numerous studies, the functions of most PE_PGRS proteins in the pathogenesis of mycobacterium infections remain unclear. PE_PGRS45 (Rv2615c) is only found in pathogenic mycobacteria. In this study, we successfully constructed a recombinant Mycobacterium smegmatis (M. smegmatis) strain which heterologously expresses the PE_PGRS45 protein. We found that overexpression of this cell wall-associated protein enhanced bacterial viability under stress in vitro and cell survival in macrophages. MS_PE_PGRS45 decreased the secretion of pro-inflammatory cytokines such as IL-1β, IL-6, IL-12p40, and TNF-α. We also found that MS_PE_PGRS45 increased the expression of the anti-inflammatory cytokine IL-10 and altered macrophage-mediated immune responses. Furthermore, PE_PGRS45 enhanced the survival rate of M. smegmatis in macrophages by inhibiting cell apoptosis. Collectively, our findings show that PE_PGRS45 is a virulent factor actively involved in the interaction with the host macrophage.

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