• Title/Summary/Keyword: That-Complementation

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Molecular Characterization of Regulatory Genes Associated with Biofilm Variation in a Staphylococcus aureus Strain

  • Kim, Jong-Hyun;Kim, Cheorl-Ho;Hacker, Jorg;Ziebuhr, Wilma;Lee, Bok-Kwon;Cho, Seung-Hak
    • Journal of Microbiology and Biotechnology
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    • v.18 no.1
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    • pp.28-34
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    • 2008
  • Biofilm formation in association with the intercellular adhesion (icaADBC) gene cluster is a serious problem in nosocomial infections of Staphylococcus aureus. In all 112 S. aureus strains tested, the ica genes were present, and none of these strains formed biofilms. The biofilm formation is known to be changeable by environmental factors. We have found about 30% of phase variation in these strains with treatment of tetracycline, pristinamycin, and natrium chloride. However, this phenotype disappeared without these substances. Therefore, we have constructed stable biofilm-producing variants through a passage culture method. To explain the mechanism of this variation, nucleotide changes of ica genes were tested in strain S. aureus 483 and the biofilm-producing variants. No differences of DNA sequence in ica genes were found between the strains. Additionally, molecular analysis of three regulatory genes, the accessory gene regulator (agr) and the staphylococcal accessory regulator (sarA), and in addition, alternative transcription factor ${\sigma}^B$ (sigB), was performed. The data of Northern blot and complementation showed that SigB plays an important role for this biofilm variation in S. aureus 483 and the biofilm-producing variants. Sequence analysis of the sigB operon indicated three point mutations in the rsbU gene, especially in the stop codon, and two point mutations in the rsbW gene. This study shows that this variation of biofilm formation in S. aureus is deduced by the role of sigB, not agr and sarA.

Vegetative Compatibility Grouping and Pathogenicity of Colletotrichum gloeosporioides Isolates from Different Host Plants

  • Ahn, Il-Pyung;Kim, Soonok;Im, Kyung-Hwan;Lee, Yong-Hwan
    • The Plant Pathology Journal
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    • v.19 no.6
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    • pp.269-273
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    • 2003
  • A total of 57 isolates of Colletotrichum gloeosporioides were recovered from diseased tissues of Hall's crab apple (Malus haliana), 3 cultivars of edible apple (M. pumila var. dulcissima), red pepper (Capsicum annum), and grapevine (Vitis vinifera) fruits. All isolates showed strong virulence on their own host plants. Isolates from edible apple exhibited high level of cultivar specificity in pathogenicity tests. Ten isolates from apple cultivar 'Fuji' were virulent on 'Jonathan' and 'Rall's Genet'. However, 12 isolates from 'Jonathan' and 'Rall's Genet' were not virulent on 'Fuji'. Among the 24 isolates from red pepper, only seven and two isolates were infective on edible apple and grapevine fruits, respectively. All six isolates from grapevine were only virulent on their own host. These isolates were grouped into five vegetative compatibility groups (VCGs), A, B, C, D, and E, by demonstrating heterokaryosis through complementation using nitrate-nonutilizing (nit) mutants. Among them, isolates belong to VCG-A and VCG-D accounted for 24 and 17 isolates; those in VCG-A exhibited wide host range involving Hall's crab apple, all three edible apple cultivars, and red pepper. On the other hand, isolates of VCG-D and VCG-E showed limited host range specific to red pepper and grapevine, respectively. Taken together, the data suggest that among C. gloeosporioides isolates, the concepts of pathotype and/or forma specialis may exist, and that three is a relationship between host specificity and VCG grouping among C. gloeosporioides isolates.

Cloning and Nucleotide Sequence Analysis of the rpoH Gene from Methylovorus sp. Strain SS1 DSM11726 (Methylovorus sp. Strain SS1 DSM11726으로부터 rpoH 유전자의 클로닝과 염기서열 분석)

  • Eom, Chi-Yong;Song, Seung-Eun;Park, Mi-Hwa;Kim, Young-Min
    • Microbiology and Biotechnology Letters
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    • v.35 no.3
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    • pp.177-183
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    • 2007
  • Using complementation of RpoH deficient E. coli strain A7448, the rpoH gene encoding heat shock sigma factor 32 (${\sigma}^{32}$) from Methylovorus sp. strain SS1 DSM11726 was cloned and sequenced. Sequence analysis of a stretch of 1,796-bp revealed existence of an open reading frame encoding a polypeptide of 284 amino acid (32,006 dalton). Deduced amino acid sequence of the Methylovorus sp. strain SS1 RpoH showed that 59.6%, 39.1% and 51.4% identities with those of Nitrosomonas europaea (${\beta}$-proteobacteria), Agrobacterium tumefaciens ($\alpha$-proteobacteria) and E. coli (${\gamma}$-proteobacteria). The expression level of the functional ortholog of RpoH of Methylovorus sp. strain SS1 was increased transiently after heat induction, further indicating that it functions as a heat shock sigma factor.

The Effects of Fashion Involvement on Self-Satisfaction by Conspicuous Consumption Tendency Groups (과시소비성향집단별 패션관여가 자기만족도에 미치는 영향)

  • Park, Hyun-Ju;Park, Sook-Hyun
    • Journal of the Korean Society of Clothing and Textiles
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    • v.36 no.11
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    • pp.1162-1173
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    • 2012
  • This study classifies conspicuous consumption groups and the difference of fashion involvement and selfsatisfaction by each group. It also examined the effect of fashion involvement on self-satisfaction by each group. A questionnaire method was used for the study method and the subjects of the study were females in their 20s-50s. A total of 580 sets of questionnaires were distributed and 554 sets were used for the final analysis. Data were analyzed by factor analysis, t-test, ANOVA, factor analysis, cluster analysis, Cronbach's alpha coefficients, and multiple regression analysis. The results of this study were as follows: First, this study classified 4 groups of active conspicuous consumption, the group of passive conspicuous consumption, the group of syntonic conspicuous consumption and the group pursuing individuality & frugal consumption. Second, as a result of the examination of the impact of fashion involvement for each group with a propensity for conspicuous consumption on their self-satisfaction, it was found that the sex appeal of fashion involvement had no significant impact on the economic satisfaction in the group of active conspicuous consumption, and had no significant impact on all elements of self-satisfaction in the group of passive conspicuous consumption. It was also found that social symbolism had a negative impact on satisfaction with looks in the group of syntonic conspicuous consumption, and the physical complementation and directions of looks had a negative impact on satisfaction with living, the social symbolism on satisfaction with looks and the syntone on satisfaction with looks in the group of pursuing individuality & frugal consumption.

Cloning and Expression of the Gene Encoding Glucose Permease of the Phosphotransferase System from Brevibacterium flavum in Escherichia coli

  • Kwon, Il;Lee, Kyu-Nam;Lee, Jung-Kee;Pan, Jae-Gu;Oh, Tae-Kwang;Lee, Hyung-Hoan;Yoon, Ki-Hong
    • Journal of Microbiology and Biotechnology
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    • v.5 no.4
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    • pp.188-193
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    • 1995
  • A Brevibacterium flavum gene coding for glucose permease of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was cloned by complementing the Escherichia coli ZSCl13 mutations affecting a ptsG gene with the B. flavum genomic library. From the E. coli clone grown as red colony on a MacConkey plate supplemented with glucose as an additional carbon source, a recombinant plasmid was isolated and named pBFT93. The plasmid pBFT93 was identified as carrying a 3.6-kb fragment of B. flavum chromosomal DNA which enables the E. coli transformant to use glucose or man nose as a sole carbon source in an M9 minimal medium. The non-metabolizable sugar analogues, 2-deoxy-D-glucose (2-DG) and methyl-$\alpha$-D-glucopyranoside (MeGlc) affected the growth of ZSCl13 cells carrying the plasmid pBFT93 on minimal medium supplemented with non-PTS carbohydrate, glycerol, as a sole cabon source, while the analogues did not repress the growth of ZSCl13 cells without pBFT93. It was also found that both $2-deoxy-D-[U-^{14}C]glucose{\;}and{\;}methyl-{\alpha}-D-[U-^{14}C]glucopyranoside$ could be effectively transported into ZSCl13 cells transformed with plasmid pBFT93. Several in vivo complementation studies suggested that the B. flavum DNA in pBFT93 encodes a glucose permease specific for glucose and mannose.

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"A La Recherche" of Functions for the Spore Protein SASP-E from Bacillus subtilis

  • Ruzal, Sandra M.;Bustos, Patricia L.;Sanchez-Rivas, Carmen
    • Journal of Microbiology and Biotechnology
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    • v.23 no.1
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    • pp.15-21
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    • 2013
  • We previously observed that Bacillus subtilis spores from sspE mutants presented a lower germination capacity in media containing high salt concentrations (0.9M NaCl). This deficiency was attributed to the absence of SASP-E (gamma-type small-acid-soluble protein), rich in osmocompatible amino acids released by degradation. Herein we observed that, in addition, this mutant spore presented a reduced capacity to use L-alanine as germinant (L-ala pathway), required longer times to germinate in calcium dipicolinate ($Ca^{2+}$-DPA), but germinated well in asparagine, glucose, fructose, and potassium chloride (AGFK pathway). Moreover, mild sonic treatment of mutant spores partially recovered their germination capacity in L-ala. Spore qualities were also altered, since sporulating colonies from the sspE mutant showed a pale brownish color, a higher adherence to agar plates, and lower autofluorescence, properties related to their spore coat content. Furthermore, biochemical analysis showed a reduced partition in hexadecane and a higher content of $Ca^{2+}$-DPA when compared with its isogenic wild-type control. Coat protein preparations showed a different electrophoretic pattern, in particular when detected with antibodies against CotG and CotE. The complementation with a wild-type sspE gene in a plasmid allowed for recovering the wild-type coat phenotype. This is the first report of a direct involvement of SASP-E in the spore coat assembly during the differentiation program of sporulation.

Increased Antibiotic Resistance of Methicillin-Resistant Staphylococcus aureus USA300 Δpsm Mutants and a Complementation Study of Δpsm Mutants Using Synthetic Phenol-Soluble Modulins

  • Song, Hun-Suk;Bhatia, Shashi Kant;Choi, Tae-Rim;Gurav, Ranjit;Kim, Hyun Joong;Lee, Sun Mi;Park, Sol Lee;Lee, Hye Soo;Joo, Hwang-Soo;Kim, Wooseong;Seo, Seung-Oh;Yang, Yung-Hun
    • Journal of Microbiology and Biotechnology
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    • v.31 no.1
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    • pp.115-122
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    • 2021
  • Phenol-soluble modulins (PSMs) are responsible for regulating biofilm formation, persister cell formation, pmtR expression, host cell lysis, and anti-bacterial effects. To determine the effect of psm deletion on methicillin-resistant Staphylococcus aureus, we investigated psm deletion mutants including Δpsmα, Δpsmβ, and Δpsmαβ. These mutants exhibited increased β-lactam antibiotic resistance to ampicillin and oxacillin that was shown to be caused by increased N-acetylmannosamine kinase (nanK) mRNA expression, which regulates persister cell formation, leading to changes in the pattern of phospholipid fatty acids resulting in increased anteiso-C15:0, and increased membrane hydrophobicity with the deletion of PSMs. When synthetic PSMs were applied to Δpsmα and Δpsmβ mutants, treatment of Δpsmα with PSMα1-4 and Δpsmβ with PSMβ1-2 restored the sensitivity to oxacillin and slightly reduced the biofilm formation. Addition of a single fragment showed that α1, α2, α3, and β2 had an inhibiting effect on biofilms in Δpsmα; however, β1 showed an enhancing effect on biofilms in Δpsmβ. This study demonstrates a possible reason for the increased antibiotic resistance in psm mutants and the effect of PSMs on biofilm formation.

Cucumber Mosaic Virus 1a Protein Interacts with the Tobacco SHE1 Transcription Factor and Partitions between the Nucleus and the Tonoplast Membrane

  • Yoon, Ju-Yeon;Palukaitis, Peter
    • The Plant Pathology Journal
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    • v.37 no.2
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    • pp.182-193
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    • 2021
  • The transcription factor SHE1 was identified as an interacting partner with the cucumber mosaic virus (CMV) 1a protein in the yeast two-hybrid system, by a pull-down assay, and via bimolecular fluorescent complementation. Using fluorescent-tagged proteins and confocal microscopy, the CMV 1a protein itself was found distributed predominantly between the nucleus and the tonoplast membrane, although it was also found in speckles in the cytoplasm. The SHE1 protein was localized in the nucleus, but in the presence of the CMV 1a protein was partitioned between the nucleus and the tonoplast membrane. SHE1 expression was induced by infection of tobacco with four tested viruses: CMV, tobacco mosaic virus, potato virus X and potato virus Y. Transgenic tobacco expressing the CMV 1a protein showed constitutive expression of SHE1, indicating that the CMV 1a protein may be responsible for its induction. However, previously, such plants also were shown to have less resistance to local and systemic movement of tobacco mosaic virus (TMV) expressing the green fluorescent protein, suggesting that the CMV 1a protein may act to prevent the function of the SHE1 protein. SHE1 is a member of the AP2/ERF class of transcription factors and is conserved in sequence in several Nicotiana species, although two clades of SHE1 could be discerned, including both different Nicotiana species and cultivars of tobacco, varying by the presence of particular insertions or deletions.

Identification of a Cupin Protein Gene Responsible for Pathogenicity, Phage Susceptibility and LPS Synthesis of Acidovorax citrulli

  • Rahimi-Midani, Aryan;Kim, Min-Jung;Choi, Tae-Jin
    • The Plant Pathology Journal
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    • v.37 no.6
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    • pp.555-565
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    • 2021
  • Bacteriophages infecting Acidovorax citrulli, the causal agent of bacterial fruit blotch, have been proven to be effective for the prevention and control of this disease. However, the occurrence of bacteriophage-resistant bacteria is one of hurdles in phage biocontrol and the understanding of phage resistance in this bacterium is an essential step. In this study, we aim to investigate possible phage resistance of A. citrulli and relationship between phage resistance and pathogenicity, and to isolate and characterize the genes involved in these phenomena. A phage-resistant and less-virulent mutant named as AC-17-G1 was isolated among 3,264 A. citrulli Tn5 mutants through serial spot assays and plaque assays followed by pathogenicity test using seed coating method. The mutant has the integrated Tn5 in the middle of a cupin protein gene. This mutant recovered its pathogenicity and phage sensitivity by complementation with corresponding wild-type gene. Site-directed mutation of this gene from wild-type by CRISPR/Cas9 system resulted in the loss of pathogenicity and acquisition of phage resistance. The growth of AC-17-G1 in King's B medium was much less than the wild-type, but the growth turned into normal in the medium supplemented with D-mannose 6-phosphate or D-fructose 6-phosphate indicating the cupin protein functions as a phosphomannos isomerase. Sodium dodecyl sulfa analysis of lipopolysaccharide (LPS) extracted from the mutant was smaller than that from wild-type. All these data suggest that the cupin protein is a phosphomannos isomerase involved in LPS synthesis, and LPS is an important determinant of pathogenicity and phage susceptibility of A. citrulli.

The Formative Processes and Ages of Paleo-coastal Sediments in Dangjeong-ri, Seocheon-gun in the Western Coast, South Korea (II): Complementation of Chronological Data and a Developmental Model of Coastal Geomorphology over the Past 200,000 Years (서해안 서천군 당정리 일대에 분포하는 육상 고해안 퇴적물의 형성 과정과 형성 시기(II): 추가 연대 자료 및 제4기 후기 연안 지형 발달 모델)

  • Shin, Jae-Ryul;Hong, Yeong-Min;Ryu, Hui-Gyeong;Hong, Seongchan
    • Journal of The Geomorphological Association of Korea
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    • v.28 no.3
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    • pp.51-61
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    • 2021
  • Following the previous study, we report a complementary dating data on the silty layers deposited in paleo-tidal conditions of the study area, Dangjeong-ri, Seocheon-gun and suggest coastal terrain development processes over the past 200,000 years. Based on the dating results, the silty layers distributed up to 25 m above mean sea level were deposited between 171 and 183 ka, and the gravel layer deposited in a fluvial environment of a paleo-Dangjeong stream was found to have formed between 78 and 83 ka. Considering relative altitudes of distribution, an uplift rate of the study area in the western coast is judged to be relatively 0.5~0.7 low to that of Pohang area in the eastern coast. Compared to Busan and Sacheon areas in the southern coast, it is assumed that an uplift rate of the study area shows a similar level with those during the late Quaternary.