Journal of Microbiology

Volume 60(10); October 2022

Review

[Minireview]Biodegradation of plastics: mining of plastic-degrading microorganisms and enzymes using metagenomics approaches: Dae-Wi Kim , Jae-Hyung Ahn , Chang-Jun Cha; J. Microbiol. 2022;60(10):969-976. Published online September 27, 2022; DOI: https://doi.org/10.1007/s12275-022-2313-7

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Abstract: Plastic pollution exacerbated by the excessive use of synthetic plastics and its recalcitrance has been recognized among the most pressing global threats. Microbial degradation of plastics has gained attention as a possible eco-friendly countermeasure, as several studies have shown microbial metabolic capabilities as potential degraders of various synthetic plastics. However, still defined biochemical mechanisms of biodegradation for the most plastics remain elusive, because the widely used culture-dependent approach can access only a very limited amount of the metabolic potential in each microbiome. A culture-independent approach, including metagenomics, is becoming increasingly important in the mining of novel plastic-degrading enzymes, considering its more expanded coverage on the microbial metabolism in microbiomes. Here, we described the advantages and drawbacks associated with four different metagenomics approaches (microbial community analysis, functional metagenomics, targeted gene sequencing, and whole metagenome sequencing) for the mining of plastic-degrading microorganisms and enzymes from the plastisphere. Among these approaches, whole metagenome sequencing has been recognized among the most powerful tools that allow researchers access to the entire metabolic potential of a microbiome. Accordingly, we suggest strategies that will help to identify plastisphere-enriched sequences as de novo plastic-degrading enzymes using the whole metagenome sequencing approach. We anticipate that new strategies for metagenomics approaches will continue to be developed and facilitate to identify novel plastic-degrading microorganisms and enzymes from microbiomes.

Journal Articles

Phenotypic and genomic characteristics of Brevibacterium zhoupengii sp. nov., a novel halotolerant actinomycete isolated from bat feces: Yuyuan Huang , Lingzhi Dong , Jian Gong , Jing Yang , Shan Lu , Xin-He Lai , Dong Jin , Qianni Huang , Ji Pu , Liyun Liu , Jianguo Xu; J. Microbiol. 2022;60(10):977-985. Published online August 19, 2022; DOI: https://doi.org/10.1007/s12275-022-2134-8

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Abstract: Two strictly aerobic, Gram-staining-positive, non-spore-forming, regular rod-shaped (approximately 0.7 × 1.9 mm) bacteria (HY170T and HY001) were isolated from bat feces collected from Chongzuo city, Guangxi province (22°20􍿁54􍿂N, 106°49􍿁20􍿂E, July 2011) and Chuxiong Yi Autonomous Prefecture, Yunnan province (25°09􍿁10􍿂N, 102°04􍿁39􍿂E, October 2013) of South China, respectively. Optimal growth is obtained at 25–28°C (range, 4–32°C) on BHI-5% sheep blood plate with pH 7.5 (range, 5.0–10.0) in the presence of 0.5– 1.0% NaCl (w/v) (range, 0–15% NaCl [w/v]). The phylogenetic and phylogenomic trees based respectively on the 16S rRNA gene and 845 core gene sequences revealed that the two strains formed a distinct lineage within the genus Brevibacterium, most closely related to B. aurantiacum NCDO 739T (16S rRNA similarity, both 98.5%; dDDH, 46.7–46.8%; ANI, 91.9–92.1%). Strain HY170T contained MK-8(H2), diphosphatidylglycerol (DPG) and phosphatidylglycerol (PG), galactose and ribose as the predominant menaquinone, major polar lipids, and main sugars in the cell wall teichoic acids, respectively. The meso-diaminopimelic acid (meso-DAP) was the diagnostic diamino acid of the peptidoglycan found in strain HY170T. Anteiso-C15:0 and anteiso-C17:0 were the major fatty acids (> 10%) of strains HY170T and HY001, with anteiso-C17:1A predominant in strain HY170T but absent in strain HY001. Mining the genomes revealed the presence of secondary metabolite biosynthesis gene clusters encoding for non-alpha poly-amino acids (NAPAA), ectoine, siderophore, and terpene. Based on results from the phylogenetic, chemotaxonomic and phenotypic analyses, the two strains could be classified as a novel species of the genus Brevibacterium, for which the name Brevibacterium zhoupengii sp. nov. is proposed (type strain HY170T = CGMCC 1.18600T = JCM 34230T).

Microbial co-occurrence network in the rhizosphere microbiome: its association with physicochemical properties and soybean yield at a regional scale: Sarbjeet Niraula , Meaghan Rose , Woo-Suk Chang; J. Microbiol. 2022;60(10):986-997. Published online September 27, 2022; DOI: https://doi.org/10.1007/s12275-022-2363-x

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Abstract: Microbial communities in the rhizosphere play a crucial role in determining plant growth and crop yield. A few studies have been performed to evaluate the diversity and co-occurrence patterns of rhizosphere microbiomes in soybean (Glycine max) at a regional scale. Here, we used a culture-independent
method
to compare the bacterial communities of the soybean rhizosphere between Nebraska (NE), a high-yield state, and Oklahoma (OK), a low-yield state. It is well known that the rhizosphere microbiome is a subset of microbes that ultimately get colonized by microbial communities from the surrounding bulk soil. Therefore, we hypothesized that differences in the soybean yield are attributed to the variations in the rhizosphere microbes at taxonomic, functional, and community levels. In addition, soil physicochemical properties were also evaluated from each sampling site for comparative study. Our result showed that distinct clusters were formed between NE and OK in terms of their soil physicochemical property. Among 3 primary nutrients (i.e., nitrogen, phosphorus, and potassium), potassium is more positively correlated with the high-yield state NE samples. We also attempted to identify keystone communities that significantly affected the soybean yield using co-occurrence network patterns. Network analysis revealed that communities formed distinct clusters in which members of modules having significantly positive correlations with the soybean yield were more abundant in NE than OK. In addition, we identified the most influential bacteria for the soybean yield in the identified modules. For instance, included are class Anaerolineae, family Micromonosporaceae, genus Plantomyces, and genus Nitrospira in the most complex module (ME9) and genus Rhizobium in ME23. This research would help to further identify a way to increase soybean yield in low-yield states in the U.S. as well as worldwide by reconstructing the microbial communities in the rhizosphere.

Enzyme activity of Aspergillus section Nigri strains isolated from the Korean fermentation starter, nuruk: Eunji Jeong , Jeong-Ah Seo; J. Microbiol. 2022;60(10):998-1006. Published online August 19, 2022; DOI: https://doi.org/10.1007/s12275-022-2071-6

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Abstract: Aspergillus section Nigri is a fungus used industrially because of its ability to produce enzymes such as cellulolytic, amylolytic and proteolytic enzymes. In this study, we obtained twentyeight strains of Aspergillus section Nigri from the traditional Korean fermentation starter, nuruk, which is known as a mixed culture of enzymatic filamentous fungi and yeasts. All strains were identified as Aspergillus section Nigri through combined phylogenetic analysis using partial β-tubulin and calmodulin gene sequences. The cellulase, amylase and protease activities of Korean strains were measured and compared with ten reference strains of Aspergillus niger. Most Korean strains showed higher cellulolytic activity than reference strains, and Aspergillus neoniger KCN5 showed the highest β-glucosidase activity. Two-thirds of the Korean strains showed similar levels of α- and glucoamylase activity as the reference strains. The protease activity of Aspergillus section Nigri strains was the highest at pH 3.0, and A. niger KSJ2 showed the highest acidic protease activity. By comparing ten reference strains and twenty-eight Korean strains, our results suggested useful Aspergillus section Nigri strains from nuruk with high enzyme activity, such as KCN5 and KSJ2, and their potential for industrial applications as enzyme producers.

Genomic and physiological analysis of C50 carotenoid-producing novel Halorubrum ruber sp. nov.: Chi Young Hwang , Eui-Sang Cho , Won Jong Rhee , Eunjung Kim , Myung-Ji Seo; J. Microbiol. 2022;60(10):1007-1020. Published online August 26, 2022; DOI: https://doi.org/10.1007/s12275-022-2173-1

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Abstract: A novel haloarchaeal species designated as MBLA0099T was isolated from seawater near Yeongheung Island. Cells were Gram-negative, non-motile, red-pigmented, and rod-shaped. They grew at 10–45°C, within pH 5.5–9.0, and between 7.5% and 30% NaCl concentrations. Cells were able to grow without Mg2+ and were lysed in distilled water. The size of the whole-genome and G + C content of DNA was 3.02 Mb and 68.9 mol%, respectively. Phylogenetic analysis shows that the strain MBLA0099T belongs to the genus Halorubrum. The average nucleotide and amino acid identity, and in silico DNA-DNA hybridization values were below the species delineation threshold. Pan-genomic analysis revealed that 3.2% of all genes present in strain MBLA0099T were unique to the strain. The red carotenoid produced by strain MBLA0099T was subjected to spectrometric and chromatographic analyses and confirmed to be bacterioruberin as C50 carotenoid. Mevalonic acid, terpenoid backbone, and carotenoid biosynthesis pathway were annotated for strain MBLA0099T. The C50 carotenoid production by strain MBLA0099T was also enhanced under various stress conditions including relatively netural pH, high oxidative and salinity conditions. Additionally, the strain MBLA0099T-derived bacterioruberin showed the antioxidant activity with EC50 value of 12.29 μg/ml, based on the evaluation of DPPH free radical scavenging activity. The present study would be the first report on the identification of C50 carotenoid from the strain MBLA0099T representing a novel species of the genus Halorubrum, for which the name Halorubrum ruber sp. nov. is proposed. The typestrain used was MBLA0099T (= KCTC 4296T = JCM 34701T).

Observational Study

Early gut microbiota in very low and extremely low birth weight preterm infants with feeding intolerance: a prospective case-control study: Ling Liu , Dang Ao , Xiangsheng Cai , Peiyi Huang , Nali Cai , Shaozhu Lin , Benqing Wu; J. Microbiol. 2022;60(10):1021-1031. Published online August 19, 2022; DOI: https://doi.org/10.1007/s12275-022-2180-2

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Abstract: The potential role of the gut microbiota in the pathogenesis of feeding intolerance (FI) remains unclear. Understanding the role of the gut microbiota could provide a new avenue for microbiota-targeted therapeutics. This study aimed to explore the associations between aberrant gut microbiota and FI in very low or extremely low birth weight (VLBW/ELBW) preterm infants. In this observational case-control study, VLBW/ ELBW infants were divided into two groups: FI group and feeding tolerance (FT) group. 16S rRNA gene sequencing was performed to analyze the gut microbial diversity and composition of the infants. The differences in the gut microbiota of the two groups were compared. In total, 165 stool samples were obtained from 44 infants, among which, 31 developed FI and 13 served as controls. Alpha diversity was the highest in the meconium samples of the two groups. LEfSe analysis revealed that the abundances of Peptostreptococcaceae, Clostridiales and Clostridia in the FT group were significantly higher than in the FI group. At the phylum level, the FI group was dominated by Proteobacteria, and the FT group was dominated by Firmicutes. The meconium samples of the FI group had higher proportions of γ-proteobacteria and Escherichia-Shigella and a lower proportion of Bacteroides compared with the FT group. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that aberrant gut bacteria in the FI group were strongly associated with dysregulation of C5-Brancheddibasic- acid-metabolism, protein kinases, and sporulation. These findings reveal candidate microbial markers to prevent FI. Increased relative abundances of γ-proteobacteria and Escherichia-Shigella and decreased abundance of Bacteroides in meconium were associated with an increased risk of FI, while Peptostreptococcaceae, Clostridiales and Clostridia reduced the risk of FI in VLBW/ELBW infants.

Journal Articles

DNA vaccine dual-expressing viral hemorrhagic septicemia virus glycoprotein and C-C motif chemokine ligand 19 induces the expression of immune-related genes in zebrafish (Danio rerio): Jin-Young Kim , Hyoung Jun Kim , Jeong Su Park , Se Ryun Kwon; J. Microbiol. 2022;60(10):1032-1038. Published online August 1, 2022; DOI: https://doi.org/10.1007/s12275-022-2231-8

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Abstract: Glycoprotein (G protein)-based DNA vaccines are effective in protecting aquaculture fish from rhabdoviruses but the degree of immune response they elicit depends on plasmid concentration and antigen cassette. Here, we developed a DNA vaccine using the viral hemorrhagic septicemia virus G (VG) gene and chemokine (C-C motif) ligand 19 (CCL19)a.2 regulated by the CMV promoter as the molecular adjuvant. After transfection of the prepared plasmid (pVG + CCL19) into epithelioma papulosum cyprini cells, mRNA expression was confirmed through quantitative real-time polymerase chain reaction. The vaccine was intramuscularly injected into zebrafish (Danio rerio), and 28 days after immunization, viral hemorrhagic septicemia virus (105 TCID50/10 μl/fish) was intraperitoneally injected. A survival rate of 68% was observed in the pVG + CCL19 group but this was not significantly different from the survival rate of fish treated with pVG alone, that is, without the adjuvant. However, the expression of interferonand cytokine-related genes in the spleen and kidney tissues of zebrafish was significantly increased (p < 0.05) on days 1, 3, 7, and 14 after immunization. Thus, CCL19a.2 induced an initial immune response as a molecular adjuvant, which may provide initial protection against virus infection before vaccination- induced antibody formation. This study provides insights on the functions of CCL19a.2 adjuvant in DNA vaccines.

Core promoter mutation of nucleotides A1762T and G1764A of hepatitis B virus increases core promoter transactivation by hepatocyte nuclear factor 1: Mi So Seong , Hyeon Jeong Hwang , Eun Ah Jang , Jeong Ah Jang , Wah Wah Aung , Yi Yi Kyaw , JaeHun Cheong; J. Microbiol. 2022;60(10):1039-1047. Published online September 27, 2022; DOI: https://doi.org/10.1007/s12275-022-1675-1

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Abstract: Hepatitis B virus (HBV) infection highly increases the risk for liver cirrhosis and hepatocellular carcinoma (HCC). The clinical manifestation of HBV infection is determined by the mutual interplay of the viral genotype, host genetic factors, mode of transmission, adaptive mutations, and environmental factors. Core promoter activation plays a critical role in the pre-genomic RNA transcription of HBV for HBV replication. The mutations of core promoter have been implicated in HCC development. We had obtained HBV genes from Myanmar HBV infectants and identified gene variations at the core promoter region. For measuring the relative transactivation activity on core promoter, we prepared the core-promoter reporter construct. Both of A1762T and G1764A mutation were consistently found in the HBV genes with hepatocellular carcinoma. The A1762T/G1764A mutation was corresponding to K130M/V131I of HBx protein. We prepared the core promoter- luciferase reporter construct containing the double A1762T/G1764A mutation and the K130M/V131I HBx protein expression construct. The A1762T/G1764A mutation highly was responsive to core promoter transactivation by HBx, regardless of HBx mutation. The A1762T/G1764A mutation newly created hepatocyte nuclear factor 1 (HNF1) responsive element. Ectopic expression of HNF1 largely increased the HBV core promoter containing A1762T/G1764A mutation. In addition, hepatic rich fatty acid, palmitic acid and oleic acid, increased K130M/V131I HBx level by core promoter activation. These results provide biological properties and clinical significance of specific HBV core promoter mutants related with HCC development.

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