Journal of Microbiology

Volume 60(12); December 2022

Journal Articles

Construction of high-density transposon mutant library of Staphylococcus aureus using bacteriophage ϕ11: Wonsik Lee; J. Microbiol. 2022;60(12):1123-1129. Published online November 24, 2022; DOI: https://doi.org/10.1007/s12275-022-2476-2

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Abstract: Transposon mutant libraries are an important resource to study bacterial metabolism and pathogenesis. The fitness analysis of mutants in the libraries under various growth conditions provides important clues to study the physiology and biogenesis of structural components of a bacterial cell. A transposon library in conjunction with next-generation sequencing techniques, collectively named transposon sequencing (Tnseq), enables high-throughput genome profiling and synthetic lethality analysis. Tn-seq has also been used to identify essential genes and to study the mode of action of antibacterials. To construct a high-density transposon mutant library, an efficient delivery system for transposition in a model bacterium is essential. Here, I describe a detailed protocol for generating a high-density phage-based transposon mutant library in a Staphylococcus aureus strain, and this protocol is readily applicable to other S. aureus strains including USA300 and MW2.

Microbial metabolic responses and CO2 emissions differentiated by soil water content variation in subarctic tundra soils: Dockyu Kim , Namyi Chae , Mincheol Kim , Sungjin Nam , Tai Kyoung Kim , Ki-Tea Park , Bang Yong Lee , Eungbin Kim , Hyoungseok Lee; J. Microbiol. 2022;60(12):1130-1138. Published online November 24, 2022; DOI: https://doi.org/10.1007/s12275-022-2378-3

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Abstract: Recent rapid air temperature increases across the northernlatitude tundra have prolonged permafrost thawing and snow melting periods, resulting in increased soil temperature (Ts) and volumetric soil water content (SWC). Under prolonged soil warming at 8°C, Alaskan tundra soils were incubated in a microcosm system and examined for the SWC differential influence on the microbial decomposition activity of large molecular weight (MW) humic substances (HS). When one microcosm soil (AKC1-1) was incubated at a constant SWC of 41% for 90 days (T = 90) and then SWC was gradually decreased from 41% to 29% for another T = 90, the initial HS was partly depolymerized. In contrast, in AKC1-2 incubated at a gradually decreasing SWC from the initial 32% to 10% for T = 90 and then increasing to 27% for another T = 90, HS depolymerization was undetected. Overall, the microbial communities in AKC1-1 could maintain metabolic activity at sufficient and constant SWC during the initial T = 90 incubation. In contrast, AKC1-2 microbes may have been damaged by drought stress during the drying SWC regimen, possibly resulting in the loss of HS decomposition activity, which did not recover even after re-wetting to an optimal SWC range (20–40%). After T = 90, the CO2 production in both treatments was attributed to the increased decomposition of small-MW organic compounds (including aerobic HS-degradative products) within an optimal SWC range. We expect this study to provide new insights into the early effects of warming- and topography-induced SWC variations on the microbial contribution to CO2 emissions via HS decomposition in northern-latitude tundra soil.

Negative regulation of the acsA1 gene encoding the major acetyl-CoA synthetase by cAMP receptor protein in Mycobacterium smegmatis: Eon-Min Ko , Yuna Oh , Jeong-Il Oh; J. Microbiol. 2022;60(12):1139-1152. Published online October 24, 2022; DOI: https://doi.org/10.1007/s12275-022-2347-x

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Abstract: Acetyl-CoA synthetase (ACS) is the enzyme that irreversibly catalyzes the synthesis of acetyl-CoA from acetate, CoA-SH, and ATP via acetyl-AMP as an intermediate. In this study, we demonstrated that AcsA1 (MSMEG_6179) is the predominantly expressed ACS among four ACSs (MSMEG_6179, MSMEG_0718, MSMEG_3986, and MSMEG_5650) found in Mycobacterium smegmatis and that a deletion mutation of acsA1 in M. smegmatis led to its compromised growth on acetate as the sole carbon source. Expression of acsA1 was demonstrated to be induced during growth on acetate as the sole carbon source. The acsA1 gene was shown to be negatively regulated by Crp1 (MSMEG_6189) that is the major cAMP receptor protein (CRP) in M. smegmatis. Using DNase I footprinting analysis and site-directed mutagenesis, a CRPbinding site (GGTGA-N6-TCACA) was identified in the upstream regulatory region of acsA1, which is important for repression of acsA1 expression. We also demonstrated that inhibition of the respiratory electron transport chain by inactivation of the major terminal oxidase, aa3 cytochrome c oxidase, led to a decrease in acsA1 expression probably through the activation of CRP. In conclusion, AcsA1 is the major ACS in M. smegmatis and its gene is under the negative regulation of Crp1, which contributes to some extent to the induction of acsA1 expression under acetate conditions. The growth of M. smegmatis is severely impaired on acetate as the sole carbon source under respiration-inhibitory conditions.

Functional analysis of ascP in Aeromonas veronii TH0426 reveals a key role in the regulation of virulence: Yongchao Guan , Meng Zhang , Yingda Wang , Zhongzhuo Liu , Zelin Zhao , Hong Wang , Dingjie An , Aidong Qian , Yuanhuan Kang , Wuwen Sun , Xiaofeng Shan; J. Microbiol. 2022;60(12):1153-1161. Published online November 10, 2022; DOI: https://doi.org/10.1007/s12275-022-2373-8

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Abstract: Aeromonas veronii is a pathogen which can induce diseases in humans, animals and aquatic organisms, but its pathogenic mechanism and virulence factors are still elusive. In this study, we successfully constructed a mutant strain (ΔascP) by homologous recombination. The results showed that the deletion of the ascP gene significantly down-regulated the expression of associated effector proteins in A. veronii compared to its wild type. The adhesive and invasive abilities of ΔascP to EPC cells were 0.82-fold lower in contrast to the wild strain. The toxicity of ΔascP to cells was decreased by about 2.91-fold (1 h) and 1.74-fold (2 h). Furthermore, the LD50 of the mutant strain of crucian carp was reduced by 19.94-fold, and the virulence was considerably attenuated. In contrast to the wild strain, the ΔascP content in the liver and spleen was considerably lower. The titers of serum cytokines (IL-8, TNF-α, and IL-1β) in crucian carp after the infection of the ΔascP strain were considerably lower in contrast to the wild strain. Hence, the ascP gene is essential for the etiopathogenesis of A. veronii TH0426.

Transcript-specific selective translation by specialized ribosomes bearing genome-encoded heterogeneous rRNAs in V. vulnificus CMCP6: Younkyung Choi , Minju Joo , Wooseok Song , Minho Lee , Hana Hyeon , Hyun-Lee Kim , Ji-Hyun Yeom , Kangseok Lee , Eunkyoung Shin; J. Microbiol. 2022;60(12):1162-1167. Published online November 24, 2022; DOI: https://doi.org/10.1007/s12275-022-2437-9

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Abstract: Ribosomes composed of genome-encoded heterogeneous rRNAs are implicated in the rapid adaptation of bacterial cells to environmental changes. A previous study showed that ribosomes bearing the most heterogeneous rRNAs expressed from the rrnI operon (I-ribosomes) are implicated in the preferential translation of a subset of mRNAs, including hspA and tpiA, in Vibrio vulnificus CMCP6. In this study, we show that HspA nascent peptides were predominantly bound to I-ribosomes. Specifically, I-ribosomes were enriched more than two-fold in ribosomes that were pulled down by immunoprecipitation of HspA peptides compared with the proportion of I-ribosomes in crude ribosomes and ribosomes pulled down by immunoprecipitation of RNA polymerase subunit ß peptides in the wild-type (WT) and rrnI-completed strains. Other methods that utilized the incorporation of an affinity tag in 23S rRNA or chimeric rRNA tethering 16S and 23S rRNAs, which generated specialized functional ribosomes in Escherichia coli, did not result in functional I-ribosomes in V. vulnificus CMCP6. This study provides direct evidence of the preferential translation of hspA mRNA by I-ribosomes.

Physiological roles of catalases Cat1 and Cat2 in Myxococcus xanthus: Kimura Yoshio , Yuri Yoshioka , Kie Toshikuni; J. Microbiol. 2022;60(12):1168-1177. Published online October 24, 2022; DOI: https://doi.org/10.1007/s12275-022-2277-7

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Abstract: Catalases are key antioxidant enzymes in aerobic organisms. Myxococcus xanthus expresses two monofunctional catalases, small-subunit Cat1 and large-subunit Cat2. The Km of H2O2 for recombinant Cat1 and Cat2 were 14.0 and 9.0 mM, respectively, and the catalytic efficiency of Cat2 (kcat/Km = 500 sec-1 mM-1) was 4-fold higher than that of Cat1. The activity ratio of Cat1 to Cat2 in the exponential growth phase of M. xanthus was 1 to 3–4. A Cat1-deficient strain was constructed, whereas a Cat2-deficient strain could not be produced. In H2O2-supplemented medium, the cat1 mutant exhibited marked growth retardation and a longer generation time than the wild-type (wt) strain. After 2 h of incubation in 0.5 mM H2O2-supplemented medium, the catalase activity of the wt strain significantly increased (by 64-fold), but that of the cat1 mutant strain did not. Under starvation-induced developmental conditions, catalase activity was induced by approximately 200-fold in both wt and cat1 strains, although in the mutant the activity increase as well as spore formation occurred one day later, indicating that the induction of catalase activity during starvation was due to Cat2. In wt starved cells, catalase activity was not induced by H2O2. These results suggest that Cat2 is the primary housekeeping catalase during M. xanthus growth and starvation-induced development, whereas Cat1 may have a complementary role, being responsible for the rapid degradation of H2O2 in proliferating vegetative cells subjected to oxidative stress.

Characteristic alterations of gut microbiota in uncontrolled gout: Asad ul-Haq , Kyung-Ann Lee , Hoonhee Seo , Sukyung Kim , Sujin Jo , Kyung Min Ko , Su-Jin Moon , Yun Sung Kim , Jung Ran Choi , Ho-Yeon Song , Hyun-Sook Kim; J. Microbiol. 2022;60(12):1178-1190. Published online November 24, 2022; DOI: https://doi.org/10.1007/s12275-022-2416-1

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Abstract: Microbiome research has been on the rise recently for a more in-depth understanding of gout. Meanwhile, there is a need to understand the gut microbiome related to uric acid-lowering drug resistance. In this study, 16S rRNA gene-based microbiota analysis was performed for a total of 65 stool samples from 17 healthy controls and 48 febuxostat-treated gout patients (including 28 controlled subjects with decreased uric acid levels and 20 uncontrolled subjects with non-reduced uric acid levels). Alpha diversity of bacterial community decreased in the healthy control, controlled, and uncontrolled groups. In the case of beta diversity, the bacterial community was significantly different among groups (healthy control, controlled, and uncontrolled groups). Taxonomic biomarker analysis revealed the increased population of g-Bifidobacterium in healthy controls and g-Prevotella in uncontrolled patients. PCR further confirmed this result at the species level. Additionally, functional metagenomics predictions led to the exploration of various functional biomarkers, including purine metabolism. The results of this study can serve as a basis for developing potential new strategies for diagnosing and treating gout from microbiome prospects.

Synthesis of pinene in the industrial strain Candida glycerinogenes by modification of its mevalonate pathway: Tengfei Ma , Hong Zong , Xinyao Lu , Bin Zhuge; J. Microbiol. 2022;60(12):1191-1200. Published online October 24, 2022; DOI: https://doi.org/10.1007/s12275-022-2344-0

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Abstract: Terpenes have many applications and are widely found in nature, but recent progress in synthetic biology has enabled the use of microorganisms as chassis cells for the synthesis of these compounds. Candida glycerinogenes (C. glycerinogenes) is an industrial strain that may be developed as a chassis for the synthesis of terpenes since it has a tolerance to hyperosmolality and high sugar, and has a complete mevalonate (MVA) pathway. However, monoterpenes such as pinene are highly toxic, and the tolerance of C. glycerinogenes to pinene was investigated. We also measured the content of mevalonate and squalene to evaluate the strength of the MVA pathway. To determine terpene synthesis capacity, a pathway for the synthesis of pinene was constructed in C. glycerinogenes. Pinene production was improved by overexpression, gene knockdown and antisense RNA inhibition. Pinene production was mainly enhanced by strengthening the upstream MVA pathway and inhibiting the production of by-products from the downstream pathway. With these strategies, yield could be increased by almost 16 times, to 6.0 mg/L. Overall, we successfully constructed a pinene synthesis pathway in C. glycerinogenes and enhanced pinene production through metabolic modification.

Coumarin-based combined computational study to design novel drugs against Candida albicans: Akhilesh Kumar Maurya , Nidhi Mishra; J. Microbiol. 2022;60(12):1201-1207. Published online November 10, 2022; DOI: https://doi.org/10.1007/s12275-022-2279-5

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Abstract: Candida species cause the most prevalent fungal illness, candidiasis. Candida albicans is known to cause bloodstream infections. This species is a commensal bacterium, but it can cause hospital–acquired diseases, particularly in COVID-19 patients with impaired immune systems. Candida infections have increased in patients with acute respiratory distress syndrome. Coumarins are both naturally occurring and synthetically produced. In this study, the biological activity of 40 coumarin derivatives was used to create a three-dimensional quantitative structure activity relationship (3D-QSAR) model. The training and test minimum inhibitory concentration values of C. albicans active compounds were split, and a regression model based on statistical data was established. This model served as a foundation for the creation of coumarin derivative QSARs. This is a unique way to create new therapeutic compounds for various ailments. We constructed novel structural coumarin derivatives using the derived QSAR model, and the models were confirmed using molecular docking and molecular dynamics simulation.

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