Journal of Microbiology

Review

Expanding the genetic code: In vivo approaches for incorporating non-proteinogenic monomers: Dongheon Lee, Suk Min Yun, Jong-il Choi; J. Microbiol. 2025;63(3):e2501005. Published online March 28, 2025; DOI: https://doi.org/10.71150/jm.2501005

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The application of genetic code expansion has enabled the incorporation of non-canonical amino acids (ncAAs) into proteins, introducing novel functional groups and significantly broadening the scope of protein engineering. Over the past decade, this approach has extended beyond ncAAs to include non-proteinogenic monomers (npMs), such as β-amino acids and hydroxy acids. In vivo incorporation of these monomers requires maintaining orthogonality between endogenous and engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pairs while optimizing the use of the translational machinery. This review introduces the fundamental principles of genetic code expansion and highlights the development of orthogonal aaRS/tRNA pairs and ribosomal engineering to incorporate npMs. Despite these advancements, challenges remain in engineering aaRS/tRNA pairs to accommodate npMs, especially monomers that differ significantly from L-α-amino acids due to their incompatibility with existing translational machinery. This review also introduces recent methodologies that allow aaRSs to recognize and aminoacylate npMs without reliance on the ribosomal translation system, thereby unlocking new possibilities for synthesizing biopolymers with chemically diverse monomers.

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Advancing microbial engineering through synthetic biology
Ki Jun Jeong
Journal of Microbiology.2025; 63(3): e2503100. CrossRef

Journal Articles

Iron interferes with quorum sensing-mediated cooperation in Pseudomonas aeruginosa by affecting the expression of ppyR and mexT, in addition to rhlR: Feng Sun , Na Li , Lijia Wang , Huajun Feng , Dongsheng Shen , Meizhen Wang; J. Microbiol. 2020;58(11):938-944. Published online October 30, 2020; DOI: https://doi.org/10.1007/s12275-020-0264-4

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Abstract

The stabilization of quorum sensing (QS) is vital for bacterial survival in various environments. Although the mechanisms of QS stabilization in certain conditions have been well studied, the impact of environmental factors has received much less attention. In this study, we show that the supplementation of 25 μM iron in competition experiments and 50 μM in evolution experiments to casein growth cultures significantly increased the possibility of population collapse by affecting elastase production. However, the expression of lasI and lasR remained constant regardless of iron concentration and hence this effect was not through interference with the LasIR circuit, which mainly regulates the secretion of elastase in Pseudomonas aeruginosa. However, the expression of rhlR was significantly inhibited by iron treatment, which could affect the production of elastase. Further, based on both reverse transcription quantitative polymerase chain reaction and gene knock-out assays, we show that iron inhibits the transcription of ppyR and enhances the expression of mexT, both of which decrease elastase production and correspondingly interfere with QS stabilization. Our findings show that environmental factors can affect the genes of QS circuits, interfering with QS stabilization. These findings are not only beneficial in understanding the mechanistic effect of iron on QS stabilization, but also demonstrate the complexity of QS stabilization by linking non-QS-related genes with QS traits.

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Effect of LuxS/AI-2-mediated quorum sensing system on bacteriocin production of Lactobacillus plantarum NMD-17
Li-Li Man, Dian-Jun Xiang
Folia Microbiologica.2023; 68(6): 855. CrossRef
PtsN in Pseudomonas aeruginosa Is Phosphorylated by Redundant Upstream Proteins and Impacts Virulence-Related Genes
Simon A. M. Underhill, Somalisa Pan, Mary Erdmann, Matthew T. Cabeen, Joseph Bondy-Denomy
Journal of Bacteriology.2023;[Epub] CrossRef
The immune responses to different Uropathogens call individual interventions for bladder infection
Linlong Li, Yangyang Li, Jiali Yang, Xiang Xie, Huan Chen
Frontiers in Immunology.2022;[Epub] CrossRef
Design of Polymeric Thin Films to Direct Microbial Biofilm Growth, Virulence, and Metabolism
Trevor Franklin, Yinan Wu, Jiayan Lang, Sijin Li, Rong Yang
Biomacromolecules.2021; 22(12): 4933. CrossRef

The effects of deletion of cellobiohydrolase genes on carbon source-dependent growth and enzymatic lignocellulose hydrolysis in Trichoderma reesei: Meibin Ren , Yifan Wang , Guoxin Liu , Bin Zuo , Yuancheng Zhang , Yunhe Wang , Weifeng Liu , Xiangmei Liu , Yaohua Zhong; J. Microbiol. 2020;58(8):687-695. Published online June 10, 2020; DOI: https://doi.org/10.1007/s12275-020-9630-5

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Abstract

The saprophytic fungus Trichoderma reesei has long been used as a model to study microbial degradation of lignocellulosic biomass. The major cellulolytic enzymes of T. reesei are the cellobiohydrolases CBH1 and CBH2, which constitute more than 70% of total proteins secreted by the fungus. However, their physiological functions and effects on enzymatic hydrolysis of cellulose substrates are not sufficiently elucidated. Here, the cellobiohydrolase-encoding genes cbh1 and cbh2 were deleted, individually or combinatively, by using an auxotrophic marker-recycling technique in T. reesei. When cultured on media with different soluble carbon sources, all three deletion strains (Δcbh1, Δcbh2, and Δcbh1Δcbh2) exhibited no dramatic variation in morphological phenotypes, but their growth rates increased apparently when cultured on soluble cellulase-inducing carbon sources. In addition, Δcbh1 showed dramatically reduced growth and Δcbh1Δcbh2 could hardly grew on microcrystalline cellulose (MCC), whereas all strains grew equally on sodium carboxymethyl cellulose (CMC-Na), suggesting that the influence of the CBHs on growth was carbon source-dependent. Moreover, five representative cellulose substrates were used to analyse the influence of the absence of CBHs on saccharification efficiency. CBH1 deficiency significantly affected the enzymatic hydrolysis rates of various cellulose substrates, where acid pre-treated corn stover (PCS) was influenced the least. CBH2 deficiency reduced the hydrolysis of MCC, PCS, and acid pre-treated and delignified corncob but improved the hydrolysis ability of filter paper. These results demonstrate the specific contributions of CBHs to the hydrolysis of different types of biomass, which could facilitate the development of tailor-made strains with highly efficient hydrolysis enzymes for certain biomass types in the biofuel industry.

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An efficient CRISPR/Cas9 genome editing system based on a multiple sgRNA processing platform in Trichoderma reesei for strain improvement and enzyme production
Jiaxin Zhang, Kehang Li, Yu Sun, Cheng Yao, Weifeng Liu, Hong Liu, Yaohua Zhong
Biotechnology for Biofuels and Bioproducts.2024;[Epub] CrossRef
Transcriptome-wide analysis of a superior xylan degrading isolate Penicillium oxalicum 5–18 revealed active lignocellulosic degrading genes
Shuang Hu, Pei Han, Bao-Teng Wang, Long Jin, Hong-Hua Ruan, Feng-Jie Jin
Archives of Microbiology.2024;[Epub] CrossRef
Engineering the secretome of Aspergillus niger for cellooligosaccharides production from plant biomass
Fernanda Lopes de Figueiredo, Fabiano Jares Contesini, César Rafael Fanchini Terrasan, Jaqueline Aline Gerhardt, Ana Beatriz Corrêa, Everton Paschoal Antoniel, Natália Sayuri Wassano, Lucas Levassor, Sarita Cândida Rabelo, Telma Teixeira Franco, Uffe Hasb
Microbial Cell Factories.2024;[Epub] CrossRef
Constitutive overexpression of cellobiohydrolase 2 in Trichoderma reesei reveals its ability to initiate cellulose degradation
Yubo Wang, Meibin Ren, Yifan Wang, Lu Wang, Hong Liu, Mei Shi, Yaohua Zhong
Engineering Microbiology.2023; 3(1): 100059. CrossRef
Inducer-free recombinant protein production in Trichoderma reesei: secretory production of endogenous enzymes and heterologous nanobodies using glucose as the sole carbon source
Toshiharu Arai, Mayumi Wada, Hiroki Nishiguchi, Yasushi Takimura, Jun Ishii
Microbial Cell Factories.2023;[Epub] CrossRef
The Influence of Trctf1 Gene Knockout by CRISPR–Cas9 on Cellulase Synthesis by Trichoderma reesei with Various Soluble Inducers
Yudian Chen, Yushan Gao, Zancheng Wang, Nian Peng, Xiaoqin Ran, Tingting Chen, Lulu Liu, Yonghao Li
Fermentation.2023; 9(8): 746. CrossRef
The effect of cellobiohydrolase 1 gene knockout for composition and hydrolytic activity of the enzyme complex secreted by filamentous fungus Penicillium verruculosum
Valeriy Yu. Kislitsin, Andrey M. Chulkin, Ivan N. Zorov, Yuri А. Denisenko, Arkadiy P. Sinitsyn, Alexandra M. Rozhkova
Bioresource Technology Reports.2022; 18: 101023. CrossRef
Deciphering the efficient cellulose degradation by the thermophilic fungus Myceliophthora thermophila focused on the synergistic action of glycoside hydrolases and lytic polysaccharide monooxygenases
Xing Qin, Jiahuan Zou, Kun Yang, Jinyang Li, Xiaolu Wang, Tao Tu, Yuan Wang, Bin Yao, Huoqing Huang, Huiying Luo
Bioresource Technology.2022; 364: 128027. CrossRef

Preliminary study on microeukaryotic community analysis using NGS technology to determine postmortem submersion interval (PMSI) in the drowned pig: Cheol-ho Hyun , Heesoo Kim , Seongho Ryu , Won Kim; J. Microbiol. 2019;57(11):1003-1011. Published online September 25, 2019; DOI: https://doi.org/10.1007/s12275-019-9198-0

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Abstract

While several methods for determining postmortem submersion interval (PMSI) in drowning cases have been suggested, the estimation of PMSI remains difficult. Next-generation sequencing (NGS) technology enables simultaneous identification of multiple taxa from environmental samples. Although NGS has been applied to estimate time since death, this application has been mainly focused on terrestrial cases. As a case study, we investigated microeukaryotic biodiversity and community structures in submerged car bonnet and drowned pig using NGS technology. NGS analysis showed that the microeukaryotic biodiversity in pig carcass was relevantly lower than that in car bonnet. NGS results also revealed that water molds and algae were related to decomposition. Relative abundances of Filobasidium, Achlya, Saprolegnia, Hydrodicton, Lobosphaera, and Scenedesmus varied with decomposition period. This data indicated that these taxa might be useful as good indicators to estimate PMSI. This study showed microeukaryotic community analysis using NGS technology may help solve drowning cases in forensic investigation.

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A review of human decomposition in marine environments
Britny A. Martlin, Gail S. Anderson, Lynne S. Bell
Canadian Society of Forensic Science Journal.2023; 56(2): 92. CrossRef
Determination of the corpse’s stay in the water duration according to maceration degree of its skin
A.Yu. Vavilov, A.A. Khalikov, I.A. Rykunov, K.O. Kuznetsov, R.H. Sagidullin
Sudebno-meditsinskaya ekspertiza.2023; 66(3): 64. CrossRef
Predicting the Postmortem Interval Based on Gravesoil Microbiome Data and a Random Forest Model
Chunhong Cui, Yang Song, Dongmei Mao, Yajun Cao, Bowen Qiu, Peng Gui, Hui Wang, Xingchun Zhao, Zhi Huang, Liqiong Sun, Zengtao Zhong
Microorganisms.2022; 11(1): 56. CrossRef
Bacterial Succession in Microbial Biofilm as a Potential Indicator for Postmortem Submersion Interval Estimation
Finkelbergs Dmitrijs, Juanjuan Guo, Yecao Huang, Yafei Liu, Xinyue Fang, Kankan Jiang, Lagabaiyila Zha, Jifeng Cai, Xiaoliang Fu
Frontiers in Microbiology.2022;[Epub] CrossRef

Middle East respiratory syndrome coronavirus-encoded ORF8b strongly antagonizes IFN-β promoter activation: its implication for vaccine design: Jeong Yoon Lee , Sojung Bae , Jinjong Myoung; J. Microbiol. 2019;57(9):803-811. Published online August 27, 2019; DOI: https://doi.org/10.1007/s12275-019-9272-7

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Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) is a causative agent of severe-to-fatal pneumonia especially in patients with pre-existing conditions, such as smoking and chronic obstructive pulmonary disease (COPD). MERS-CoV transmission continues to be reported in the Saudi Arabian Peninsula since its discovery in 2012. However, it has rarely been epidemic outside the area except one large outbreak in South Korea in May 2015. The genome of the epidemic MERS-CoV isolated from a Korean patient revealed its homology to previously reported strains. MERS-CoV encodes 5 accessory proteins and generally, they do not participate in the genome transcription and replication but rather are involved in viral evasion of the host innate immune responses. Here we report that ORF8b, an accessory protein of MERSCoV, strongly inhibits both MDA5- and RIG-I-mediated activation of interferon beta promoter activity while downstream signaling molecules were left largely unaffected. Of note, MDA5 protein levels were significantly down-regulated by ORF8b and co-expression of ORF4a and ORF4b. These novel findings will facilitate elucidation of mechanisms of virus-encoded evasion strategies, thus helping design rationale antiviral countermeasures against deadly MERS-CoV infection.

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Review

REVIEW] The role of laboratory diagnostics in emerging viral infections: the example of the Middle East respiratory syndrome epidemic: Jasper F. W. Chan , Siddharth Sridhar , Cyril C. Y. Yip , Susanna K. P. Lau , Patrick C. Y. Woo; J. Microbiol. 2017;55(3):172-182. Published online February 28, 2017; DOI: https://doi.org/10.1007/s12275-017-7026-y

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Abstract

Rapidly emerging infectious disease outbreaks place a great strain on laboratories to develop and implement sensitive and specific diagnostic tests for patient management and infection control in a timely manner. Furthermore, laboratories also play a role in real-time zoonotic, environmental, and epidemiological investigations to identify the ultimate source of the epidemic, facilitating measures to eventually control the outbreak. Each assay modality has unique pros and cons; therefore, incorporation of a battery of tests using traditional culture-based, molecular and serological diagnostics into diagnostic algorithms is often required. As such, laboratories face challenges in assay development, test evaluation, and subsequent quality assurance. In this review, we describe the different testing modalities available for the ongoing Middle East respiratory syndrome (MERS) epidemic including cell culture, nucleic acid amplification, antigen detection, and antibody detection assays. Applications of such tests in both acute clinical and epidemiological investigation settings are highlighted. Using the MERS epidemic as an example, we illustrate the various challenges faced by laboratories in test development and implementation in the setting of a rapidly emerging infectious disease. Future directions in the diagnosis of MERS and other emerging infectious disease investigations are also highlighted.

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Development and validation of different indirect ELISAs for MERS-CoV serological testing
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Research Support, Non-U.S. Gov't

Variations in 16S rRNA-based Microbiome Profiling between Pyrosequencing Runs and between Pyrosequencing Facilities: Minseok Kim , Zhongtang Yu; J. Microbiol. 2014;52(5):355-365. Published online April 11, 2014; DOI: https://doi.org/10.1007/s12275-014-3443-3

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Abstract

Pyrosequencing of 16S rRNA gene amplicons on the 454 FLX Titanium platform has been widely used to analyze microbiomes in various environments. However, different results may stem from variations among sequencing runs or among sequencing facilities. This study aimed to evaluate these variations between different pyrosequencing runs by sequencing 16S rRNA gene amplicon libraries generated from three sets of rumen samples twice each on the 454 FLX Titanium system at two independent sequencing facilities. Similar relative abundances were found for predominant taxa represented by large numbers of sequence reads but not for minor taxa represented by small numbers of sequence reads. The two sequencing facilities revealed different bac-terial profiles with respect to both predominant taxa and minor taxa, including the most predominant genus Prevo-tella, the family Lachnospiraceae, and the phylum Proteo-bacteria. Differences in primers used to generate amplicon libraries may be a major source of variations in microbiome profiling. Because different primers and regions of 16S rRNA genes are often used by different researchers, significant variations likely exist among studies. Quantitative interpre-tation for relative abundance of taxa, especially minor taxa, from prevalence of sequence reads and comparisons of re-sults from different studies should be done with caution.

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Journal Article

Effect of pH on Conjugated Linoleic Acid (CLA) Formation of Linolenic Acid Biohydrogenation by Ruminal Microorganisms: Yongjae Lee; J. Microbiol. 2013;51(4):471-476. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-1070-z

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Abstract

Conventional beliefs surrounding the linolenic acid (LNA; cis-9 cis-12 cis-15 C18:3) biohydrogenation (BH) pathway propose that it converts to stearic acid (SA) without the formation of conjugated linoleic acid (CLA) as intermediate isomers. However, an advanced study (Lee and Jenkins, 2011) verified that LNA BH yields multiple CLAs. This study utilized the stable isotope tracer to investigate the BH intermediates of 13C-LNA with different pH conditions (5.5 and 6.5). The 13C enrichment was calculated as a 13C/12C ratio of labeled minus unlabeled. After 24 h, eight CLA isomers were significantly enriched on both pH treatment, this result verifies that these CLAs originated from 13C-LNA BH which supports the results of Lee and Jenkins (2011). The enrichment of cis-cis double bond CLAs (cis-9 cis-11 and cis-10 cis-12 CLA) were significantly higher at low pH conditions. Furthermore, the concentration of cis-10 cis-12 CLA at low pH was four times higher than at high pH conditions after a 3 h incubation. These differences support the LNA BH pathways partial switch under different pH conditions, with a strong influence on the cis-cis CLA at low pH. Several mono-, di-, and tri-enoic fatty acid isomers were enriched during 24 h of incubation, but the enrichment was decreased or restricted at low pH treatment. Based on these results, it is proposed that low pH conditions may cause a changed or limited capacity of the isomerization and reduction steps in BH.

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Research Support, Non-U.S. Gov'ts

NOTE] Development of Porphyromonas gingivalis-Specific Quantitative Real-Time PCR Primers Based on the Nucleotide Sequence of rpoB: Soon-Nang Park , Jae-Yoon Park , Joong-Ki Kook; J. Microbiol. 2011;49(2):315-319. Published online May 3, 2011; DOI: https://doi.org/10.1007/s12275-011-1028-y

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Abstract: Species-specific quantitative real-time PCR (qPCR) primers were developed for the detection of Porphyromonas gingivalis. These primers, Pg-F/Pg-R, were designed based on the nucleotide sequences of RNA polymerase β-subunit gene (rpoB). Species-specific amplicons were obtained from the tested P. gingivalis strains but not in any of the other strains (46 strains of 46 species). The qPCR primers could detect as little as 4 fg of P. gingivalis chromosomal DNA. These findings suggest that these qPCR primers are suitable for applications in epidemiological studies.

Isolation and Characterization of a Rhodococcus Species Strain Able to Grow on ortho- and para-Xylene: Jung Yeon Jang , Dockyu Kim , Hyun Won Bae , Ki Young Choi , Jong-Chan Chae , Gerben J. Zylstra , Young Min Kim , Eungbin Kim; J. Microbiol. 2005;43(4):325-330.; DOI: https://doi.org/2258 [pii]

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Abstract: Rhodococcus sp. strain YU6 was isolated from soil for the ability to grow on o-xylene as the sole carbon and energy source. Unlike most other o-xylene-degrading bacteria, YU6 is able to grow on p-xylene. Numerous growth substrate range experiments, in addition to the ring-cleavage enzyme assay data, suggest that YU6 initially metabolizes o- and p-xylene by direct aromatic ring oxidation. This leads to the formation of dimethylcatechols, which was further degraded largely through meta-cleavage pathway. The gene encoding meta-cleavage dioxygenase enzyme was PCR cloned from genomic YU6 DNA using previously known gene sequence data from the o-xylene-degrading Rhodococcus sp. strain DK17. Subsequent sequencing of the 918-bp PCR product revealed a 98% identity to the gene, encoding methylcatechol 2,3-dioxygenase from DK17. PFGE analysis followed by Southern hybridization with the catechol 2,3-dioxygenase gene demonstrated that the gene is located on an approximately 560-kb megaplasmid, designated pJYJ1 <br>

Development of Strain-specific PCR Primers Based on a DNA Probe Fu12 for the Identification of Fusobacterium nucleatum subsp. nucleatum ATCC 25586^T: Hwa-Sook Kim , Soo Keun Song , So Young Yoo , Dong Chun Jin , Hwan Seon Shin , Chae Kwang Lim , Myung-Soo Kim , Jin-Soo Kim , Son-Jin Choe , Joong-Ki Kook; J. Microbiol. 2005;43(4):331-336.; DOI: https://doi.org/2257 [pii]

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Abstract: The objective of this study was to assess the strain-specificity of a DNA probe, Fu12, for Fusobacterium nucleatum subsp. nucleatum ATCC 25586^T (F. nucleatum ATCC 25586^T), and to develop sets of strain-specific polymerase chain reaction (PCR) primers. Strain-specificity was tested against 16 strains of F. nucleatum and 3 strains of distinct Fusobacterium species. Southern blot hybridization revealed that the Fu12 reacted exclusively with the HindIII-digested genomic DNA of F. nucleatum ATCC 25586^T. The results of PCR revealed that three pairs of PCR primers, based on the nucleotide sequence of Fu12, generated the strain-specific amplicons from F. nucleatum ATCC 25586^T. These results suggest that the DNA probe Fu12 and the three pairs of PCR primers could be useful in the identification of F. nucleatum ATCC 25586^T, especially with regard to the determination of the authenticity of the strain.

Genetic Variation of BIV Isolates Characterized by PCR Using Degenerate Primers: Kwon, Oh Sik , Sninsky, John J.; J. Microbiol. 1995;33(3):252-259.

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Abstract: The PCR was employed to detect and characterize the bovine immunodeficiency-like virus (BIV), which is a newly recognized member of the Lentivirinae of the retroviruses. Degenerate primers representing the conserved regions in the pol genes of the Lentivirinae, were used to detect proviral DNA obtained from the bovine embryonic spleen cell cultures infected with BIV. The PCR amplified DNA fragment was molecularly cloned and sequenced. The BIV DNA fragment contained a sequence identical to that reported by Garvey et al. (Garvey et al., 1990. Virology, 175, 391-409). With the degenerate primers, peripheral blood mononuclear cells (PBMCs) of sick cattle and cells cultured with BIV were tested to determine genetic variation of BIV pol conserved sequence. We found the sequence heterogeneity within cultures and most variations occurred at the third base of codons that would not lead to amino acid substitutions. Another change was GAG (Glu) to AAG (Lys) within the BIV isolates. Interestingly, the altered sequence is also found in other lentiviruses such as HIV-2, SIV mac, CAEV and EIAV.

Evaluation of Several Parameters of in situ Polymerase Chain Reaction (ISPCR) to Reduce the Leakage of Amplificants from Cells: Jae Yung Lee , Chung-Kyoon Auh , George W. Jordan; J. Microbiol. 2002;40(1):70-76.

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Abstract: Proviral DNAs from HIV-1-infected CD4+ T cells (Molt/LAV cells) were amplified and detected in infected individual cells using polymerase chain reaction and in situ hybridization. In this in situ PCR, three parameters were considered to achieve effective amplification and retention of amplificants inside the cells by making high molecular weight PCR products intracellularly, forming agarose matrix against the cells, and maintaining the appropriate PCR temperature profile. Over the cycles of amplification, tailed primers with complementary overhanging sequences at their 5 sides manufactured high molecular weight products by using short primary products as a repeating unit. Agarose matrix could prevent the diffusion of the amplificants from the cells. Use of Thermanox coverslip inside the PCR tube offered target cells a similar temperature profile to that of conventional PCR in solution.

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