Journal of Microbiology

Volume 58(11); November 2020

Journal Articles

[PROTOCOL] High-throughput cultivation based on dilution-to-extinction with catalase supplementation and a case study of cultivating acI bacteria from Lake Soyang: Suhyun Kim , Miri S. Park , Jaeho Song , Ilnam Kang , Jang-Cheon Cho; J. Microbiol. 2020;58(11):893-905. Published online October 30, 2020; DOI: https://doi.org/10.1007/s12275-020-0452-2

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Abstract: Multi-omics approaches, including metagenomics and single- cell amplified genomics, have revolutionized our understanding of the hidden diversity and function of microbes in nature. Even in the omics age, cultivation is an essential discipline in microbial ecology since microbial cultures are necessary to assess the validity of an in silico prediction about the microbial metabolism and to isolate viruses infecting bacteria and archaea. However, the ecophysiological characteristics of predominant freshwater bacterial lineages remain largely unknown due to the scarcity of cultured representatives. In an ongoing effort to cultivate the uncultured majority of freshwater bacteria, the most abundant freshwater Actinobacteria acI clade has recently been cultivated from Lake Soyang through catalase-supplemented high-throughput cultivation based on dilution-to-extinction. This method involves physical isolation of target microbes from mixed populations, culture media simulating natural habitats, and removal of toxic compounds. In this protocol, we describe detailed procedures for isolating freshwater oligotrophic microbes, as well as the essence of the dilution-to-extinction culturing. As a case study employing the catalase-supplemented dilution-to-extinction protocol, we also report a cultivation trial using a water sample collected from Lake Soyang. Of the 480 cultivation wells inoculated with a single lake-water sample, 75 new acI strains belonging to 8 acI tribes (acI-A1, A2, A4, A5, A6, A7, B1, B4, C1, and C2) were cultivated, and each representative strain per subclade could be revived from glycerol stocks. These cultivation results demonstrate that the protocol described in this study is efficient in isolating freshwater bacterioplankton harboring streamlined genomes.

Influence of dragon bamboo with different planting patterns on microbial community and physicochemical property of soil on sunny and shady slopes: Weiyi Liu , Fang Wang , Yanmei Sun , Lei Yang , Huihai Chen , Weijie Liu , Bin Zhu , Chaomao Hui , Shiwei Wang; J. Microbiol. 2020;58(11):906-914. Published online October 30, 2020; DOI: https://doi.org/10.1007/s12275-020-0082-8

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Abstract: Dragon bamboo (Dendrocalamus giganteus) is a giant sympodial bamboo species widely distributed in Asia. However, it remains unclear how dragon bamboo and soil microbes interact to affect soil properties. In this study, we investigated the planting patterns (semi-natural and artificial) on different slopes (sunny and shady) to determine the effects on soil properties and microbial community. The results showed that the soil in which dragon bamboo was grown was acidic, with a pH value of ~5. Also, the soil organic matter content, nitrogen hydrolysate concentration, total nitrogen, available potassium, and total potassium of the dragon bamboo seminatural forest significantly improved, especially on the sunny slope. In contrast, the available phosphorus level was higher in the artificial bamboo forest, probably owing to the phosphate fertilizer application. The bacterial and fungal diversity and the bacterial abundance were all higher on the sunny slope of the semi-natural forest than those in the other samples. The microbial operational taxonomic units (OTUs) shared between the shady and sunny slopes accounted for 47.8–62.2%, but the core OTUs of all samples were only 24.4– 30.4% of each sample, suggesting that the slope type had a significant effect on the microbial community. Some acidophilic microbes, such as Acidobacteria groups, Streptomyces and Mortierella, became dominant in dragon bamboo forest soil. A PICRUSt analysis of the bacterial functional groups revealed that post-translational modification, cell division, and coenzyme transport and metabolism were abundant in the semi-natural forest. However, some microorganisms with strong stress resistance might be activated in the artificial forest. Taken together, these results illustrated the influence of dragon bamboo growth on soil physicochemical property and microbial community, which might help understand the growth status of dragon bamboo under different planting patterns.

Occurrence of four waterborne viruses at five typical raw water resources in the Republic of Korea during August 2013 to February 2019: Young Yil Bahk , Min-Ho Kim , Tong-Soo Kim , Sang Jung Park , Jeong-Myeong Kim , Ok-Jae Rhee , Sang-Seob Lee; J. Microbiol. 2020;58(11):915-925. Published online October 30, 2020; DOI: https://doi.org/10.1007/s12275-020-0231-0

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Abstract: Waterborne diseases have critical public health issues and socioeconomic relevancy worldwide. Various viral pathogens are ordinarily associated with waterborne diseases. Six-yearsurveillance (a total of 20 times) of norovirus, hepatitis A virus, group C rotavirus, and enterovirus was conducted at five raw water sampling sites including two lakes (Lakes Soyang and Juam), Hyundo region of Geum River in Daejeon City, and Guui region of Han River in Seoul Metropolitan City and Moolgeum region of Nakdong River in Gimhae City which are located near two water intake plants. In this study, we routinely investigated virus contamination in water samples through reverse transcription polymerase chain reaction (RTPCR) and integrated cell culture RT-PCR with high sensitivity and specificity. A total 100 samples were tested. Most of the targeted viruses were found in 32% of the samples and at least one of the indicator bacteria was detected in 65% of these occurrences. Among all the detected viruses, enterovirus was the most prevalent with a detection frequency of 12% and 2.71 MPN/10 L on average, while hepatitis A virus was the least prevalent with a detection frequency of 4%. Nearly all of the analyzed viruses (except for group C rotavirus) were present in samples from Han River (the Guui region), Geum River (the Hyundo region), Lake Juam, and Nakdong River (the Moolgeum region), while group C rotavirus was detected in those from the Guui region. During the six-year sampling period, the targeted waterborne viruses in water samples exhibited seasonal patterns in their occurrence that were different from the indicator bacteria levels in the water samples. The fact that they were detected in the five representative Korean water environments makes it necessary to establish the chemical and biological analysis systems for waterborne viruses and sophisticated management systems.

The effects of cigarettes and alcohol on intestinal microbiota in healthy men: Renbin Lin , Yawen Zhang , Luyi Chen , Yadong Qi , Jiamin He , Mengjia Hu , Ying Zhang , Lina Fan , Tao Yang , Lan Wang , Misi Si , Shujie Chen; J. Microbiol. 2020;58(11):926-937. Published online October 30, 2020; DOI: https://doi.org/10.1007/s12275-020-0006-7

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Abstract: Human intestinal microbiota is affected by the exogenous microenvironment. This study aimed to determine the effects of cigarettes and alcohol on the gut microbiota of healthy men. In total, 116 healthy male subjects were enrolled and divided into four groups: non-smoking and non-drinking (Group A), smoking only (Group B), drinking only (Group C), and smoking and drinking combined (Group D). Fecal samples were collected and sequenced using 16S rRNA to analyze the microbial composition. Short-chain fatty acid (SCFAs) levels in feces were determined by gas chromatography. We found that cigarette and alcohol consumptions can alter overall composition of gut microbiota in healthy men. The relative abundances of phylum Bacteroidetes and Firmicutes and more than 40 genera were changed with cigarette and alcohol consumptions. SCFAs decreased with smoking and alcohol consumption. Multivariate analysis indicated that when compared with group A, group B/C/D had higher Bacteroides, and lower Phascolarctobacterium, Ruminococcaceae_ UCG-002, Ruminococcaceae_UCG-003, and Ruminiclostridium_ 9 regardless of BMI and age. Additionally, the abundance of Bacteroides was positively correlated with the smoking pack-year (r = 0.207, p < 0.05), the abundance of predicted pathway of bacterial toxins (r = 0.3672, p < 0.001) and the level of carcinoembryonic antigen in host (r = 0.318, p < 0.01). Group D shared similar microbial construction with group B, but exerted differences far from group C with lower abundance of Haemophilus. These results demonstrated that cigarette and alcohol consumption separately affected the intestinal microbiota and function in healthy men; furthermore, the co-occurrence of cigarette and alcohol didn’t exacerbate the dysbiosis and cigarette played the predominated role on the alteration.

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.

Similarities and differences between 6S RNAs from Bradyrhizobium japonicum and Sinorhizobium meliloti: Olga Y. Burenina , Daria A. Elkina , Anzhela Y. Migur , Tatiana S. Oretskaya , Elena Evguenieva-Hackenberg , RolK. Hartmann , Elena A. Kubareva; J. Microbiol. 2020;58(11):945-956. Published online October 30, 2020; DOI: https://doi.org/10.1007/s12275-020-0283-1

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Abstract: 6S RNA, a conserved and abundant small non-coding RNA found in most bacteria, regulates gene expression by inhibiting RNA polymerase (RNAP) holoenzyme. 6S RNAs from α-proteobacteria have been studied poorly so far. Here, we present a first in-depth analysis of 6S RNAs from two α-proteobacteria species, Bradyrhizobium japonicum and Sinorhizobium meliloti. Although both belong to the order Rhizobiales and are typical nitrogen-fixing symbionts of legumes, their 6S RNA expression profiles were found to differ: B. japonicum 6S RNA accumulated in the stationary phase, thus being reminiscent of Escherichia coli 6S RNA, whereas S. meliloti 6S RNA level peaked at the transition to the stationary phase, similarly to Rhodobacter sphaeroides 6S RNA. We demonstrated in vitro that both RNAs have hallmarks of 6S RNAs: they bind to the σ70-type RNAP holoenzyme and serve as templates for de novo transcription of so-called product RNAs (pRNAs) ranging in length from ~13 to 24 nucleotides, with further evidence of the synthesis of even longer pRNAs. Likewise, stably bound pRNAs were found to rearrange the 6S RNA structure to induce its dissociation from RNAP. Compared with B. japonicum 6S RNA, considerable conformational heterogeneity was observed for S. meliloti 6S RNA and its complexes with pRNAs, even though the two 6S RNAs share ~75% sequence identity. Overall, our findings suggest that the two rhizobial 6S RNAs have diverged with respect to their regulatory impact on gene expression throughout the bacterial life cycle.

Analyses of DNA double-strand break repair pathways in tandem arrays of HXT genes of Saccharomyces cerevisiae: Ju-Hee Choi , Ye-Seul Lim , Min-Ku Kim , Sung-Ho Bae; J. Microbiol. 2020;58(11):957-966. Published online October 30, 2020; DOI: https://doi.org/10.1007/s12275-020-0461-1

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Abstract: Eukaryotic genomes contain numerous homologous repeat sequences including redundant genes with divergent homology that can be potential recombination targets. Recombination between divergent sequences is rare but poses a substantial threat to genome stability. The hexose transporter (HXT) gene family shares high sequence similarities at both protein and DNA levels, and some members are placed close together in tandem arrays. In this study, we show that spontaneous interstitial deletions occur at significantly high rates in HXT gene clusters, resulting in chimeric HXT sequences that contain a single junction point. We also observed that DNA double-strand breaks created between HXT genes produce primarily interstitial deletions, whereas internal cleavage of the HXT gene resulted in gene conversions as well as deletion products. Interestingly, interstitial deletions were less constrained by sequence divergence than gene conversion. Moreover, recombination-defective mutations differentially affected the survival frequency. Mutations that impair single-strand annealing (SSA) pathway greatly reduced the survival frequency by 10–1,000-fold, whereas disruption of Rad51-dependent homologous recombination exhibited only modest reduction. Our results indicate that recombination in the tandemly repeated HXT genes occurs primarily via SSA pathway.

1Medical Convergence Materials Commercialization Center, Gyeongsan: JaeJin An , Eun-Mi Ha; J. Microbiol. 2020;58(11):967-977. Published online October 30, 2020; DOI: https://doi.org/10.1007/s12275-020-0375-y

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Abstract: Lactobacillus plantarum-derived metabolites (LDMs) increase drug sensitivity to 5-FU and antimetastatic effects in 5-FUresistant colorectal cancer cells (HCT-116/5FUR). In this study, we evaluated the effects of LDMs on the regulation of genes and proteins involved in HCT-116/5-FUR cell proliferation and metastasis. HCT-116/5-FUR cells showed high metastatic potential, significantly reduced tight junction (TJ) integrity, including increased migration and paracellular permeability, and upregulation of claudin-1 (CLDN-1). The genetic silencing of CLDN-1 increased the sensitivity of HCT- 116/5FUR to 5-FU and inhibited its metastatic potential by regulating the expression of epithelial-mesenchymal transition (EMT) related genes. Co-treatment of HCT-116/5FUR with LDMs and 5-FU suppressed chemoresistant and metastatic behavior by downregulating CLDN-1 expression. Finally, we designed LDMs-based therapeutic strategies to treatment for metastatic 5-FU-resistant colorectal cancer cells. These
results
suggested that LDMs and 5-FU cotreatments can synergistically target 5-FU-resistant cells, making it a candidate strategy to overcome 5-FU chemoresistance improve anticancer drug efficacy.

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