Journal of Microbiology

Volume 57(8); August 2019

Review

MINIREVIEW] Development of bacteria as diagnostics and therapeutics by genetic engineering: Daejin Lim , Miryoung Song; J. Microbiol. 2019;57(8):637-643. Published online May 11, 2019; DOI: https://doi.org/10.1007/s12275-019-9105-8

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18 Citations

Abstract: Bacteria sense and respond to the environment, communicate, and continuously interact with their surroundings, including host bodies. For more than a century, engineers have been trying to harness the natural ability of bacteria as live biotherapeutics for the treatment of diseases. Recent advances in synthetic biology facilitate the enlargement of the repertoire of genetic parts, tools, and devices that serve as a framework for biotherapy. This review describes bacterial species developed for specific diseases shown in in vitro studies and clinical stages. Here, we focus on drug delivery by programing bacteria and discuss the challenges for safety and improvement.

Journal Articles

PROTOCOL] Applications of different solvents and conditions for differential extraction of lipopolysaccharide in Gram-negative bacteria: Mai Phuong Nguyen , Le Viet Ha Tran , Hyun Namgoong , Yong-Hak Kim; J. Microbiol. 2019;57(8):644-654. Published online May 23, 2019; DOI: https://doi.org/10.1007/s12275-019-9116-5

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Abstract: Lipopolysaccharide (LPS) is one of the major components in the outer membrane of Gram-negative bacteria. However, its heterogeneity and variability in different bacteria and differentiation conditions make it difficult to extract all of the structural variants. We designed a solution to improve quality and biological activity of LPS extracted from various bacteria with different types of LPS, as compared to conventional
methods
. We introduced a quality index as a simple measure of LPS purity in terms of a degree of polysaccharide content detected by absorbance at 204 nm. Further experiments using gel electrophoresis, endotoxin test, and macrophage activation test were performed to evaluate the performance and reliability of a proposed ‘T-sol’ method and the biological effectiveness and character of the LPS products. We presented that the T-sol method had differential effects on extraction of a RAW 264.7 cell-activating LPS, which was effective in the macrophage activation with similar effects in stimulating the production of TNF-alpha. In conclusion, the T-sol method provides a simple way to improve quality and biological activity of LPS with high yield.

Fulvimarina endophytica sp. nov., a novel endophytic bacterium isolated from bark of Sonneratia caseolaris: Li Tuo , Xiao-Rui Yan; J. Microbiol. 2019;57(8):655-660. Published online June 11, 2019; DOI: https://doi.org/10.1007/s12275-019-8627-4

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Abstract: A Gram-negative, aerobic, short-rod-shaped, motile (with a terminal flagellum), non-spore-forming bacterium, designated strain 85T, was isolated from a surface-sterilized bark of Sonneratia caseolaris collected from Qinzhou in Guangxi, China and was analyzed using a polyphasic approach to determine its taxonomic position. Strain 85T grew optimally in the presence of 1–2% (w/v) NaCl at 30°C and pH 6.0–7.0. Phylogenetic analysis based on 16S rRNA gene sequence suggested that strain 85T belonged to the genus Fulvimarina and shared the highest 16S rRNA gene sequence similarity with Fulvimarina pelagi HTCC2506T (96.16%). The cell-wall peptidoglycan contained meso-diaminopimelic acid and ubiquinone Q-10 was the predominant respiratory lipoquinone. The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unidentified amino lipid, three unidentified phospholipids and six unidentified lipids. The major fatty acid was C18:1 ω7c. The DNA G+C content of strain 85T was 65.4 mol%, and the average nucleotide identity and estimated DDH values between strain 85T and the type strain of Fulvimarina pelagi HTCC2506T were 77.3% and 21.7%, respectively. Based on the phylogenetic, phenotypic, and chemotaxonomic analyses, strain 85T should be considered as a novel species of the genus Fulvimarina with the proposed name Fulvimarina endophytica sp. nov., and its type strain is 85T (= KCTC 62717T = CGMCC 1.13665T).

Sulfitobacter profundi sp. nov., isolated from deep seawater: Jaeho Song , Hye-Jin Jang , Yochan Joung , Ilnam Kang , Jang-Cheon Cho; J. Microbiol. 2019;57(8):661-667. Published online April 22, 2019; DOI: https://doi.org/10.1007/s12275-019-9150-3

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9 Citations

Abstract: A Gram-stain-negative, rod-shaped, obligately aerobic, chemoheterotrophic bacterium which is motile by means of a single polar flagellum, designated SAORIC-263T, was isolated from deep seawater of the Pacific Ocean. Phylogenetic analyses based on 16S rRNA gene sequences and genomebased phylogeny revealed that strain SAORIC-263T belonged to the genus Sulfitobacter and shared 96.1–99.9% 16S rRNA gene sequence similarities with Sulfitobacter species. Wholegenome sequencing of strain SAORIC-263T revealed a genome size of 3.9􍾘Mbp and DNA G+C content of 61.3 mol%. The SAORIC-263T genome shared an average nucleotide identity and digital DNA-DNA hybridization of 79.1–88.5% and 18.9–35.0%, respectively, with other Sulfitobacter genomes. The SAORIC-263T genome contained the genes related to benzoate degradation, which are frequently found in deep-sea metagenome. The strain contained summed feature 8 (C18:1 ω7c), C18:1 ω7c 11-methyl, and C16:0 as the predominant cellular fatty acids as well as ubiquinone-10 (Q-10) as the major respiratory quinone. The major polar lipids of the strain were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, and aminolipid. On the basis of taxonomic data obtained in this study, it is suggested that strain SAORIC-263T represents a novel species of the genus Sulfitobacter, for which the name Sulfitobacter profundi sp. nov. is proposed. The type strain is SAORIC-263T (= KACC 21183T = NBRC 113428T).

Trichoderma biodiversity in major ecological systems of China: Kai Dou , Jinxin Gao , Chulong Zhang , Hetong Yang , Xiliang Jiang , Jishun Li , Yaqian Li , Wei Wang , Hongquan Xian , Shigui Li , Yan Liu , Jindong Hu , Jie Chen; J. Microbiol. 2019;57(8):668-675. Published online May 23, 2019; DOI: https://doi.org/10.1007/s12275-019-8357-7

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26 Citations

Abstract: An investigation of Trichoderma biodiversity involving a large-scale environmental gradient was conducted to understand the Trichoderma distribution in China. A total of 3,999 isolates were isolated from forestry, grassland, wetland and agriculture ecosystems, and 50 species were identified based on morphological characteristics and sequence analysis of genetic markers. Trichoderma harzianum showed the largest proportion of isolates and the most extensive distribution. Hypocrea semiorbis, T. epimyces, T. konilangbra, T. piluliferum, T. pleurotum, T. pubescens, T. strictipilis, T. hunua, T. oblongisporum and an unidentified species, Trichoderma sp. MA 3642, were first reported in China. Most Trichoderma species were distributed in Jilin and Heilongjiang Provinces in northeast China and the fewest were distributed in Qinghai Province. Based on the division of ecological and geographic factors, forestry ecosystems and low-altitude regions have the greatest species biodiversity of Trichoderma.

Isolation, cultivation, and genome analysis of proteorhodopsincontaining SAR116-clade strain Candidatus Puniceispirillum marinum IMCC1322: Junhak Lee , Kae Kyoung Kwon , Seung-Il Lim , Jaeho Song , Ah Reum Choi , Sung-Hyun Yang , Kwang-Hwan Jung , Jung-Hyun Lee , Sung Gyun Kang , Hyun-Myung Oh , Jang-Cheon Cho; J. Microbiol. 2019;57(8):676-687. Published online June 14, 2019; DOI: https://doi.org/10.1007/s12275-019-9001-2

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Abstract: Strain IMCC1322 was isolated from a surface water sample from the East Sea of Korea. Based on 16S rRNA analysis, IMCC1322 was found to belong to the OCS28 sub-clade of SAR116. The cells appeared as short vibrioids in logarithmicphase culture, and elongated spirals during incubation with mitomycin or in aged culture. Growth characteristics of strain IMCC1322 were further evaluated based on genomic information; proteorhodopsin (PR), carbon monoxide dehydrogenase, and dimethylsulfoniopropionate (DMSP)-utilizing enzymes. IMCC1322 PR was characterized as a functional retinylidene protein that acts as a light-driven proton pump in the cytoplasmic membrane. However, the PR-dependent phototrophic potential of strain IMCC1322 was only observed under CO-inhibited and nutrient-limited culture conditions. A DMSP-enhanced growth response was observed in addition to cultures grown on C1 compounds like methanol, formate, and methane sulfonate. Strain IMCC1322 cultivation analysis revealed biogeochemical processes characteristic of the SAR116 group, a dominant member of the microbial community in euphotic regions of the ocean. The polyphasic taxonomy of strain IMCC1322 is given as Candidatus Puniceispirillum marinum, and was confirmed by chemotaxonomic tests, in addition to 16S rRNA phylogeny and cultivation analyses.

Expression of sexual genes in Aspergillus fumigatus homogeneous culture produced by vegetative mass mating: Joo-Yeon Lim , Hee-Moon Park; J. Microbiol. 2019;57(8):688-693. Published online May 11, 2019; DOI: https://doi.org/10.1007/s12275-019-9094-7

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Abstract: There are presently no studies on the genes for sexual development of Aspergillus fumigatus in situ using mating culture, primarily because of challenging experimental conditions that require a significantly long period of induction and produce developmentally heterogenous culture, harboring very few sexual organs. In order to overcome these challenges, we developed an efficient and convenient procedure called ‘vegetative mass mating (VeM)’ for study at a molecular level. The VeM method enabled production of a developmentally homogenous A. fumigatus culture, harboring many sexual organs in a plate within a short period of two weeks. Feasibility of the use of VeM for functional study of genes during A. fumigatus sexual development was evaluated by analyzing the transcription pattern of genes involved in pheromone signal transduction and regulation of sexual development. Here, we present for the first time, an in situ expression pattern of sexual genes during the mating process, induced by the VeM
method
, which will enable and promote the sexual development study of A. fumigatus at the molecular level.

FgIlv3a is crucial in branched-chain amino acid biosynthesis, vegetative differentiation, and virulence in Fusarium graminearum: Xin Liu , Yichen Jiang , Yinghui Zhang , Mingzheng Yu , Hongjun Jiang , Jianhong Xu , Jianrong Shi; J. Microbiol. 2019;57(8):694-703. Published online May 11, 2019; DOI: https://doi.org/10.1007/s12275-019-9123-6

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13 Citations

Abstract: Dihydroxyacid dehydratase (DHAD), encoded by ILV3, catalyses the third step in the biosynthetic pathway of branchedchain amino acids (BCAAs), which include isoleucine (Ile), leucine (Leu), and valine (Val). Enzymes involved in BCAA biosynthesis exist in bacteria, plants, and fungi but not in mammals and are therefore attractive targets for antimicrobial or herbicide development. In this study, three paralogous ILV3 genes (FgILV3A, FgILV3B, and FgILV3C) were identified in the genome of Fusarium graminearum, the causal agent of Fusarium head blight (FHB). Deletion of FgILV3A alone or combined with FgILV3B or FgILV3C indicated an important role for FgILV3A in BCAA biosynthesis. FgILV3A deletion mutants lost the ability to grow on medium lacking amino acids. Exogenous supplementation of 1 mM Ile and Val rescued the auxotrophy of ΔFgIlv3A, though 5 mM was required to recover the growth defects in ΔFgIlv3AB and ΔFgIlv3AC strains, indicating that FgIlv3b and FgIlv3c exhibit redundant but accessory roles with FgIlv3a in BCAA biosynthesis. The auxotrophy of ΔFgIlv3A resulted in pleiotropic defects in aerial hyphal growth, in conidial formation and germination, and in aurofusarin accumulation. In addition, the mutants showed reduced virulence and deoxynivalenol production. Overall, our study demonstrates that FgIlv3a is crucial for BCAA biosynthesis in F. graminearum and a candidate fungicide target for FHB management.

Differential expression of the major catalase, KatA in the two wild type Pseudomonas aeruginosa strains, PAO1 and PA14: Bi-o Kim , In-Young Chung , You-Hee Cho; J. Microbiol. 2019;57(8):704-710. Published online June 11, 2019; DOI: https://doi.org/10.1007/s12275-019-9225-1

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Abstract: KatA is the major catalase required for hydrogen peroxide (H2O2) resistance and acute virulence in Pseudomonas aeruginosa PA14, whose transcription is governed by its dual promoters (katAp1 and katAp2). Here, we observed that KatA was not required for acute virulence in another wild type P. aeruginosa strain, PAO1, but that PAO1 exhibited higher KatA expression than PA14 did. This was in a good agreement with the observation that PAO1 was more resistant than PA14 to H2O2 as well as to the antibiotic peptide, polymyxin B (PMB), supposed to involve reactive oxygen species (ROS) for its antibacterial activity. The higher KatA expression in PAO1 than in PA14 was attributed to both katAp1 and katAp2 transcripts, as assessed by S1 nuclease mapping. In addition, it was confirmed that the PMB resistance is attributed to both katAp1 and katAp2 in a complementary manner in PA14 and PAO1, by exploiting the promoter mutants for each -10 box (p1m, p2m, and p1p2m). These results provide an evidence that the two widely used P. aeruginosa strains display different virulence mechanisms associated with OxyR and Anr, which need to be further characterized for better understanding of the critical virulence pathways that may differ in various P. aeruginosa strains.

Low-density lipoprotein as an opsonin promoting the phagocytosis of Pseudomonas aeruginosa by U937 cells: Yuxin Li , Zhi Liu , Jinli Yang , Ling Liu , Runlin Han; J. Microbiol. 2019;57(8):711-716. Published online May 11, 2019; DOI: https://doi.org/10.1007/s12275-019-8413-3

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Abstract: Low-density lipoprotein (LDL) was recently reported to be an opsonin, enhancing the phagocytosis of group A Streptococcus (GAS) by human monocytic leukemia U937 cells due to the binding of LDL to some GAS strains. We postulated that LDL might also promote the opsonophagocytosis of Pseudomonas aeruginosa by U937 cells since this bacterium interacts with LDL. In this study, P. aeruginosa (CMCC10104), U937 cells, and human LDL were used in phagocytosis assays to test our hypothesis. Escherichia coli strain BL21, which does not interact with LDL, was used as a negative control. Colony counting and fluorescence microscopy were used to determine the bacterial quantity in the opsonophagocytosis assays. After incubation of U937 cells and P. aeruginosa with LDL (100 μg/ml) for 15 and 30 min, phagocytosis was observed to be increased by 22.71% and 32.90%, respectively, compared to that seen in the LDL-free group. However, LDL did not increase the phagocytosis of E. coli by U937 cells. In addition, we identified CD36 as a major opsonin receptor on U937 cells, since an anti-CD36 monoclonal antibody, but not an anti- CD4 monoclonal antibody, almost completely abolished the opsonophagocytosis of P. aeruginosa by U937 cells.

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