Journal of Microbiology

Journal Articles

Rab27b regulates extracellular vesicle production in cells infected with Kaposi’s sarcoma–associated herpesvirus to promote cell survival and persistent infection: Hyungtaek Jeon , Su-Kyung Kang , Myung-Ju Lee , Changhoon Park , Seung-Min Yoo , Yun Hee Kang , Myung-Shin Lee; J. Microbiol. 2021;59(5):522-529. Published online April 20, 2021; DOI: https://doi.org/10.1007/s12275-021-1108-6

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Abstract: Extracellular vesicles (EVs) play a crucial role in cell-to-cell communication. EVs and viruses share several properties related to their structure and the biogenesis machinery in cells. EVs from virus-infected cells play a key role in virus spread and suppression using various loading molecules, such as viral proteins, host proteins, and microRNAs. However, it remains unclear how and why viruses regulate EV production inside host cells. The purpose of this study is to investigate the molecular mechanisms underlying EV production and their roles in Kaposi’s sarcoma-associated herpesvirus (KSHV)-infected cells. Here, we found that KSHV induced EV production in human endothelial cells via Rab- 27b upregulation. The suppression of Rab27b expression in KSHV-infected cells enhanced cell death by increasing autophagic flux and autolysosome formation. Our results indicate that Rab27b regulates EV biogenesis to promote cell survival and persistent viral infection during KSHV infection, thereby providing novel insights into the crucial role of Rab- 27b in the KSHV life cycle.

An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus: Guomin Han , Qian Shao , Cuiping Li , Kai Zhao , Li Jiang , Jun Fan , Haiyang Jiang , Fang Tao; J. Microbiol. 2018;56(5):356-364. Published online May 2, 2018; DOI: https://doi.org/10.1007/s12275-018-7349-3

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Abstract: Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ~ 60 positive transformants per 106 conidia using our protocol. A small-scale insertional mutant library (~ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.

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

Identification of seven novel virulence genes from Xanthomonas citri subsp. citri by Tn5-based random mutagenesis: Xue Song , Jing Guo , Wen-xiu Ma , Zhi-yuan Ji , Li-fang Zou , Gong-you Chen , Hua-song Zou; J. Microbiol. 2015;53(5):330-336. Published online May 3, 2015; DOI: https://doi.org/10.1007/s12275-015-4589-3

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Abstract: To identify novel virulence genes, a mutant library of Xanthomonas citri subsp. citri 29-1 was produced using EZ-Tn5 transposon and the mutants were inoculated into susceptible grapefruit. Forty mutants with altered virulence phenotypes were identified. Nine of the mutants showed a complete loss of citrus canker induction, and the other 31 mutants resulted in attenuated canker symptoms. Southern blot analysis revealed that each of the mutants carried a single copy of Tn5. The flanking sequence was identified by plasmid rescue and 18 different ORFs were identified in the genome sequence. Of these 18 ORFs, seven had not been previously associated with the virulence of X. citri subsp. citri and were therefore confirmed by complementation analysis. Real-time PCR analysis showed that the seven genes were upregulated when the bacteria were grown in citrus plants, suggesting that the expression of these genes was essential for canker development.

A Potent Brucella abortus 2308 Δery Live Vaccine Allows for the Differentiation between Natural and Vaccinated Infection: Junbo Zhang , Shuanghong Yin , Fei Guo , Ren Meng , Chuangfu Chen , Hui Zhang , Zhiqiang Li , Qiang Fu , Huijun Shi , Shengwei Hu , Wei Ni , Tiansen Li , Ke Zhang; J. Microbiol. 2014;52(8):681-688. Published online July 4, 2014; DOI: https://doi.org/10.1007/s12275-014-3689-9

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16 Citations

Abstract: Brucellosis is a globally distributed zoonotic disease that causes animal and human diseases. However, the current Brucella abortus vaccines (S19 and RB51) are deficient; they can cause abortion in pregnant animals. Moreover, when the vaccine S19 is used, tests cannot differentiate natural from vaccinated infection. Therefore, a safer and more potent vaccine is needed. A Brucella abortus 2308 ery promoter mutant (Δery) was constructed to overcome these drawbacks. The growth of the Δery mutant was significantly attenuated in macrophages and mice and induced high protective immunity in mice. Moreover, Δery induced an anti-Brucellaspecific IgG (immunoglobulin G) response and stimulated the expression of interferon-gamma (INF-γ) and interleukin-4 (IL-4). Furthermore, the expression of EryA antigen allowed for the serological differentiation between natural and vaccinated infection in mice. These results indicate that the Δery mutant is a potential attenuated live vaccine candidate against virulent Brucella abortus 2308 (S2308) infection.

Functional Analysis of pilQ Gene in Xanthomanas oryzae pv. oryzae, Bacterial Blight Pathogen of Rice: Seon-Hwa Lim , Byoung-Ho So , Ji-Chun Wang , Eun-Seong Song , Young-Jin Park , Byoung-Moo Lee , Hee-Wan Kang; J. Microbiol. 2008;46(2):214-220. Published online June 11, 2008; DOI: https://doi.org/10.1007/s12275-007-0173-9

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27 Citations

Abstract: Bacterial blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most devastating bacterial disease in rice. A virulence-attenuated mutant strain HNU89K9 of X. oryzae pv. oryzae (KACC10331), with a transposon insertion in the pilQ gene was used for this study. The pilQ was involved in the gene cluster pilMNOPQ of the Xoo genome. Growth rate of the pilQ mutant was similar to that of wild-type. At level of amino acids, PilQ of Xoo showed that a high sequence identities more than 94% and 70% to Xanthomonas species and to Xyllela fastidiosa, respectively but a low sequence homology less than 30% to other bacterial species. The twitching motility forming a marginal fringe on PSA media was observed on colony of the wild-type strain KACC10331, but not in mutant HNU89K9. Wild-type Xoo cells formed a biofilm on the surface of the PVC plastic test tube, while the mutant strain HNU89K9 did not form a biofilm. The results suggest that the pilQ gene of X. oryzae pv. oryzae plays a critical role in pathogenicity, twitching motility, and biofilm formation.

Strain Improvement of Candida tropicalis for the Production of Xylitol:Biochemical and Physiological Characterization of Wild-type and Mutant Strain CT-OMV5: Ravella Sreenivas Rao , Cherukuri Pavana Jyothi , Reddy Shetty Prakasham , Chaganti Subba Rao , Ponnupalli Nageshwara Sarma , Linga Venkateswar Rao; J. Microbiol. 2006;44(1):113-120.; DOI: https://doi.org/2328 [pii]

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Abstract: Candida tropicalis was treated with ultraviolet (UV) rays, and the mutants obtained were screened for xylitol production. One of the mutants, the UV1 produced 0.81g of xylitol per gram of xylose. This was further mutated with N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), and the mutants obtained were screened for xylitol production. One of the mutants (CT-OMV5) produced 0.85g/g of xylitol from xylose. Xylitol production improved to 0.87 g/g of xylose with this strain when the production medium was supplemented with urea. The CT-OMV5 mutant strain differs by 12 tests when compared to the wild-type Candida tropicalis strain. The XR activity was higher in mutant CT-OMV5. The distinct difference between the mutant and wild-type strain is the presence of numerous chlamydospores in the mutant. In this investigation, we have demonstrated that mutagenesis was successful in generating a superior xylitol-producing strain, CT-OMV5, and uncovered distinctive biochemical and physiological characteristics of the wild-type and mutant strain, CT-OMV5.

Screening of Growth- or Development-related Genes by Using Genomic Library with Inducible Promoter in Aspergillus nidulans: Bang-Yong Lee , Sang-Yong Han , Han Gil Choi , Jee Hyun Kim , Kap-Hoon Han , Dong-Min Han; J. Microbiol. 2005;43(6):523-528.; DOI: https://doi.org/2295 [pii]

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Abstract: Using the genomic library constructed at the downstream of the niiA promoter, which induces the over-expression of an inserted DNA fragment, we have attempted to screen the genes affecting growth or development by over-expression. The wild-type strain was transformed using the AMA-niiA(p) library and cultured on 1.2 M sorbitol media, in which asexual sporulation is induced, but sexual development is repressed. Over 100,000 strains transformed to pyrG+ were analyzed with regard to any changes in phenotype. Consequently, seven strains were isolated for further analyses. These strains were designated NOT [niiA(p) over-expression transformants] stains. Four of the strains were of the inducible type, and the remaining strains were of the multi-copy suppression type. Two of the inducible-type strains, NOT1 and NOT40, harbored genes which had been inserted in reverse direction, suggesting that the mutant phenotypes had been derived from an excess amount of anti-sense mRNA. Domain analyses of the deduced polypeptides from the DNA fragments rescued from the transformants revealed that NOT1, NOT40 and NOT6 harbored a LisH motif, a forkhead domain, and a Zn(II)2Cys6 binuclear zinc cluster, respectively.

Isolation and Genetic Mapping of Paraquat-Resistant Sporulating Mutants of Streptomyces Coelicolor: Chung, Hye Jung , Kim, Eun Ja , Park, Uhn Mee , Roe, Jung Hye; J. Microbiol. 1995;33(3):215-221.

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Abstract: S. coelicolor A3(2) cells were treated with various redox-cycling agents on nutrient agar plates and examined for their effect on the growth and differentiation. When treated with plumbagin, severe effect on cell viability was observed at concentrations above 250 uM. However, the surviving colonies differentiated normally. When treated with 100 uM paraquat, growth rate was decreased and morphological differentiation was inhibited, while the survival rate was maintained at about 100% even at 5 mM paraquat. Menadione or lawsone did not cause any visible changes at concentrations up to 1 mM. The effect of paraquat was also observed when it was added to nutrient agar plate before spore inoculation. Paraquat had also observed when it was added to nutrient agar plate before spore inoculation. Paraquat had no effect on colonies growing on R2YE agar plates. Among the components of R2YE medium selectively added to nutrient agar medium, CaCl₂was found to have some protective function from the inhibitory effect of paraquat. As a first step to study the mechanism of the inhibitory effect of paraquat on differentiation, resistant mutants which sporulate well in the presence of paraquat were screened following UV mutagenesis. Three paraquat-resistant mutants were isolated with a frequency of 3 × 10^-5. Their mutation sites were determined by genetic crossings. All three mutations were mapped to a single locus near argA at about 1 o'clock on the genetic map of S. coelicolor A3(2).

cloning of Gene Encoding for Siderophore biosynthesis in Fluorescent Pseudomonas sp.: Koh, Han Cheol , Ha, Sung Cheol , Na, Jung A , Kim, Ho Sang , Yeo, Myeong Gu , Lee, Jung Sup , Kim, Sung Jun , Park, yeal; J. Microbiol. 1995;33(1):28-33.

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Abstract: Pseudomonas sp. strain PY002, isolated from soil, was mutagenized with a transposon Tn5(21). To screening of siderophore biosunthesis defective mutant, 138 kanamycin resistant mutants were tested of growth on MKB medium supplemented with iron chelator(bipydidyl and EDDHA) and in vitro antibiosis. Among 138 mutants, 32 mutants do not excreted a siderophore and lose their antibiotic activity. So, these mutants were designated Flu^-Sid^-. A gene bank of DNA from Pseudomonas sp. strain PY002 was constructed using the broad-host range conjugative cosmid pLAFR3. The recombinant cosmids contained insert DNA averaging 21 kb in length and the frequence of transduction into E. coli HB101 per 1㎍ of insert DNA was 9 × 10³. Nonfluorescent mutants were complemented by mating the gene bank en masse and identifying the 108 fluorescent transconjugants. Restriction enzyme analysis of these complemented transconjugants revealed three different types and they were named pCOM61, pCOM91 and pCOM97. Sizes of their insert DNA were 30kb, 26kb and 28kb, respectively.

Isolation of Glucose Utilizing Mutant of Alcaligenes eutrophus, its Substrate Selectivity, and Accumulation of Poly-β-hydroxybutyrate: Kim, Hye Yeon , Park, Jin Seo , Shin, Hyun Dong , Lee, yong Hyun; J. Microbiol. 1995;33(1):51-58.

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Abstract: A glucose utilizing mutant was selected from parent strain Alcaligenes eutrophus H16, and named as Glu-9. The mechanisms of glucose utilization of the mutant Glu-9 was investigated by measuring the D-[1-¹⁴C] glucose transport activity and the activities of key enzymes related to glucose and fructose uptake via facilitated diffusion. The uptaken glucose seems to activate key enzymes related to glucose matabolism. The selectivity between glucose and fructose of mutant Glu-9 was also analyzed by measuring glucose transport activity and enzyme activities under the various cultivation conditions using different carbon sources. Mutant Alcaligenes eutrophus Glu-9 preferentially consumed fructose from mixed substrates of glucose and fructose due to the inhibition of fructose to glucose transport activity. The characteristics of cell growth and PHB accumulation of Alcaligenes eutrophus Glu-9 were examined under various cultural conditions. Mutant strain Glu-9 showed tolerance in high concentration of glucose and increased yield of PHB production.

Isolation and characterization of pre-tRNA^Val splicing Mutants of Schizosaccharomyces pombe: Hwang, Ku Chan , Kim, Dae Myung; J. Microbiol. 1997;35(4):334-340.

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Abstract: A collection of 132 temperature sensitive (ts) mutants was generated by the chemical mutagenesis of Schizosaccharomyces pombe wild type strain and screened for tRNA splicing defects on Northern blots by hybridization with an oligonucleotide that recognizes the exon of the S. pombe tRNA^Val as a probe. We identidied 6 mutants which accumulate precursor tRNA^Val. Among them, 2 mutants exhibited remarkable morphological differences compared to wild type cells. One tRNA splicing mutant showed elongated cell shape in permissive as well as non-permissive cultures. The other mutant exhibited shortened cell morphology only in nonpermissive culture. The total RNA pattern in the splicing mutants appeared to be normal. Genetic analysis of four tRNA^Val splicing mutants demonstrated that the mutation reside in different genes.

Factors affecting pheromone induction of schizosaccharomyces pomba and isolation of pheromone induction mutants: Jun, Jai Hyun , Kim, Young Min , Lee, Joo Hun , Chung, In Kwon , Kim, Dae Myung; J. Microbiol. 1998;36(3):196-202.

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Abstract: The mating pheromones of Schizosaccharomyces pombe are induced by nutritional starvation. However, this nutritional signaling pathway is largely unknown. For a complete understanding of pheromone induction, we examined the environmental factors affecting the induction afer cells were transferred to a nitrogen-starved medium. It appeared that the induction of mfm2 transcription was affected by the general environmental stress including incubation time, incubation temperature, and the growth phase of the cells. We identified 7 pheromone induction mutants by screening temperature sensitive mutant bank. Three of these mutants showed elongated cell shapes and one mutant exhibited swollen cell morphology in permissive culture, suggesting that their cell cycles were also impaired. Characterization of the pheromone induction mutants may elucidate the components required in nutritional signaling pathway leading to pheromone induction.

Isolation of the Regulator Gene Responsible for Overproduction of Catalase A in H 2 O 2 -resistant Mutant of Streptomyces coelicolor: Ji-Sook Hahn , So-Young Oh , Keith F. Chater , Jung-Hye Roe; J. Microbiol. 2000;38(1):18-23.

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Abstract: Streptomyces coelicolor produces three kinds of catalases to cope with oxidative stress and to allow nor-mal differentiation. Catalase A is the major vegetative catalase which functions in removing hydrogen peroxide generated during the process of aerobic metabolism. To understand the regulatory mechanism of response against oxidative stress, hydrogen peroxide-resistant mutant (HR40) was isolated from S. coelicolor J1501 following UV mutagenesis. The mutant overproduced catalase A more than 50-fold compared with the wild type. The mutation locus catR1 was mapped closed to the mthB2 locus by genetic crossings. An ordered cosmid library of S. coelicolor encompassing the mthB2 locus was used to isolate the regulator gene (catR) which represses catalase overproduction when introduced into HR40. A candidate catR gene was found to encode a Fur-like protein of 138 amino acids (15319 Da).

Energy Status of Neurospora crassa Mutant nap in Relation to Accumulation of Carotenoids: Tatyana A. Belozerskaya^ , Tatyana V. Potapova^† , Elena P. Isakova , Eugene I. Shurubor , Ludmila V. Savel'eva , Renata A. Zvyagilskaya; J. Microbiol. 2003;41(1):41-45.

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Abstract: N. crassa mutant strain nap showed reduced growth rate, decreased electric membrane potential, and elevated intracellular ATP content in comparison to the wild type. Blue light induced a hyperpolarization of the membrane potential in both strains. The analysis of oxidative and phosphorylation activities of mitochondria isolated from the two strains has revealed that nap utilized more efficient oxidative pathways. The higher intracellular ATP content in the nap was presumably due to impaired transport systems of the plasma membrane, and to a lesser extent to the functioning of the fully competent respiratory chain. The excess ATP possibly accounts for carotenoid accumulation in the mutant.

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