• Title, Summary, Keyword: ERM

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In vitro activity comparison of Erm proteins from Firmicutes and Actinobacteria (Firmicutes와 Actinobacteria에 속하는 세균들의 Erm 단백질 in vitro 활성 비교)

  • Jin, Hyung Jong
    • Korean Journal of Microbiology
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    • v.52 no.3
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    • pp.269-277
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    • 2016
  • Erm proteins methylate the specific adenine residue ($A_{2058}$, E. coli numbering) on 23S rRNA to confer the $MLS_B$ (macrolidelincosamide-streptogramin B) antibiotic resistance on a variety of microorganisms ranging from antibiotic producers to pathogens. When phylogenetic tree is constructed, two main clusters come out forming each cluster of Actinobacteria and Firmicutes. Two representative Erm proteins from each cluster were selected and their in vitro methylation activities were compared. ErmS and ErmE from Actinobacteria cluster exhibited much higher activities than ErmB and ErmC' from Firmicutes: 9 fold difference when ErmC' and ErmE were compared and 13 fold between ErmS and ErmB. Most of the difference was observed and presumed to be caused by N-terminal and C-terminal extra region from ErmS and ErmE, respectively because NT59TE in which N-terminal end 59 amino acids was truncated from wild type ErmS exhibited only 22.5% of wild type ErmS activity. Meanwhile, even NT59TE showed three and 2.2 times more activity when it was compared to ErmB and C, respectively, suggesting core region from antibiotic producers contains extra structure enabling higher activity. This is suggested to be possible through the extra region of 197RWS199 (from both ErmS and ErmE), 261GVGGSLY267 (from ErmS), and 261GVGGNIQ267 (from ErmE) and 291SVV293 (from ErmS) and 291GAV293 (from ErmE) by multiple sequence alignment.

Molecular Analysis of Spontaneous Mutations in erm(A) and erm(C) Selected In vitro as a Constitutive MLS$_B$ Resistant Staphylococci (MLS$_B$계 항생물질 유도 내성 세균에서 In vitro로 선발된 지속성 내성형 erm(A)와 erm(C)의 분자적 특성 규명)

  • Yoon, Eun-Jeong;Jin, Sung-Hye;Choi, Eung-Chil;Shim, Mi-Ja
    • YAKHAK HOEJI
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    • v.51 no.2
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    • pp.108-114
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    • 2007
  • The predominant Macrolides-Lincosamide-Streptogramin B (MLS$_B$) antibiotics resistance genes in staphylococci are erm(A) and erm(C). There is the phenomenon that the ratio of constitutively MLS$_B$ antibiotics resistance (cMLS) in erm(A) is much higher than in erm(C). Thus, we confirmed that the difference of the mutation ratio between erm(A) and erm(C) makes the phenomenon. We examined 8 staphylococci carrying inducibly expressed (iMLS) erm(A) or erm(C) genes. After overnight incubation in the presence of the non-inducer MLS$_B$ antibiotics, spontaneous mutants constitutively expressed MLS$_B$ resistance were selected. Against our expectation, the mutation ratio of erm(A) was lower than erm(C). Therefore, possibilities of other factors determining the ratio of cMLS phenotype might be concerned. All the mutants showed sequence alterations in translational attenuator and all the alterations seemed to give rise to change the second structure of mRNA to express constitutively. For erm(A), 4 different types of sequence deletions ranging from 72 bp to 122 bp and 3 different types of duplications ranging 24 bp to 93 bp were detected. Also, there were 9 different types of duplications ranging 15bp to 154bp in erm(C).

Resistance to Macrolide, Lincosamide and Streptogramin Antibiotics in Staphylococci Isolated in Istanbul, Turkey

  • Aktas, Zerrin;Aridogan, Aslihan;Kayacan, Cigdem Bal;Aydin, Derya
    • Journal of Microbiology
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    • v.45 no.4
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    • pp.286-290
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    • 2007
  • The purpose of this study was to investigate the prevalence and genetic mechanisms of erythromycin resistance in staphylococci. A total of 102 erythromycin resistant non-duplicate clinical isolates of staphylococci [78. coagulase negative stapylococci (CNS), 24 Staphylococcus aureus] were collected between October 2003 and August 2004 in Istanbul Faculty of Medicine in Turkey. The majority of the isolates were from blood and urine specimens. Antimicrobial susceptibilities were determined by the agar dilution procedure and the resistance phenotypes by the double disk induction test. A multiplex PCR was performed, using primers specific for erm(A), erm(B), erm(C), and msrA genes.. Among the 78 CNS isolates, 57.8% expressed the $MLS_{B}-constitutive$, 20.6% the $MLS_{B}-inducible$, and 21.6% the $MS_B$ phenotypes. By PCR, 78.2% of these isolates harbored the erm(C) gene, 8.9% erm(A), 6.4% erm(B), and 11.5% msrA genes. In S. aureus, the constitutive $MLS_B$ (58.3 %) was more common than the inducible phenotype (20.8%). erm(A) was detected in 50% and erm(C) in 62.5% of the isolates, while 37.5% contained both erm(A) and erm(C). erm(C)-associated macrolide resistance was the most prevalent in CNS, while ermC) and erm(A, C) was the most prevalent in S. aureus.

Site-directed Mutagenesis Analysis Elucidates the Role of 223/227 Arginine in 23S rRNA Methylation, Which Is in 'Target Adenine Binding Loop' Region of ErmSF (위치 지정 치환 변이를 이용한 ErmSF의 '타깃 Adenine Binding Loop'을 형성하는 부위에 존재하는 223/227 Arginine 잔기의 23S rRNA Methylation 활성에서의 역할 규명)

  • Jin, Hyung-Jong
    • Korean Journal of Microbiology
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    • v.48 no.2
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    • pp.79-86
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    • 2012
  • ErmSF is one of the Erm family proteins which catalyze S-adenosyl-$_L$-methionine dependent modification of a specific adenine residue (A2058, E. coli numbering) in bacterial 23S rRNA, thereby conferring resistance to clinically important macrolide, lincosamide and streptogramin B ($MLS_B$) antibiotics. $^{222}FXPXPXVXS^{230}$ (ErmSF numbering) sequence appears to be a consensus sequence among the Erm family. This sequence was supposed to be involved in direct interaction with the target adenine from the structural studies of Erm protein ErmC'. But in DNA methyltarnsferase M. Taq I, this interaction have been identified biochemically and from the complex structure with substrate. Arginine 223 and 227 in this sequence are not conserved among Erm proteins, but because of the basic nature of residues, it was expected to interact with RNA substrates. Two amino acid residues were replaced with Ala by site-directed mutagenesis. Two mutant proteins still maintained its activity in vivo and resistant to the antibiotic erythromycin. Compared to the wild-type ErmSF, R223A and R227A proteins retained about 50% and 88% of activity in vitro, respectively. Even though those arginine residues are not essential in the catalytic step, with their positive charge they may play an important role for RNA binding.

Deletion of N-terminal End Region of ErmSF Leads to an Amino Acid Having Important Role in Methyl Transfer Reaction (ErmSF에서 특이적으로 발견되는 N-terminal End Region의 점차적인 제거에 의한 활성에 중요한 아미노산의 규명)

  • Lee Hak Jin;Jin Hyung Jong
    • Korean Journal of Microbiology
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    • v.40 no.4
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    • pp.257-262
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    • 2004
  • ErmSF is one of the ERM proteins which transfer the methyl group to A2058 in 23S rRNA to confer the resis­tance to MLS (macrolide-lincosamide-streptogramin B) antibiotics on microorganism. Unlike other ERM pro­teins, ErmSF contains long N-terminal end region (NTER), of which $25\%$ is composed of arginine that is known to interact with RNA well. Gradual deletion of NTER leaded us to the point where mutant protein lost much of activity in vivo. Overexpressed and purified mutant protein showed much reduced activity in vitro: $2\%$ activity relative to that of wild type protein. This fact suggests that this amino acid interact with RNA close to meth­ylatable adenine to locate it at an active site properly.

Development of TaqMan Probe-Based Real-Time PCR Method for erm(A), erm(B), and erm(C), Rapid Detection of Macrolide-Lincosamide-Streptogramin B Resistance Genes, from Clinical Isolates

  • Jung, Jae-Hyuk;Yoon, Eun-Jeong;Choi, Eung-Chil;Choi, Sung-Sook
    • Journal of Microbiology and Biotechnology
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    • v.19 no.11
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    • pp.1464-1469
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    • 2009
  • To achieve more accurate and rapid detection of macrolide-lincosamide-streptogramin B resistance genes, erm(A), erm(B), and erm(C), we developed a TaqMan probe-based real-time PCR (Q-PCR) method and compared it with conventional PCR (C-PCR), which is the most widely using erm gene identification method. The detection limit of Q-PCR was 5 fg of genomic DNA or 5-8 CFU of bacterial cells of Staphylococcus aureus. The utilization of Q-PCR might shorten the time to erm detection from 3-4 h to about 50 min. These data indicated that Q-PCR assay appears to be not only highly sensitive and specific, but also the most rapid diagnostic method. Therefore, the appropriate application of the Q-PCR assay will permit rapid and accurate identification of erm genes from clinical and other samples.

Establishment of Hertwig's Epithelial Root Sheath/Epithelial Rests of Malassez Cell Line from Human Periodontium

  • Nam, Hyun;Kim, Ji-Hye;Kim, Jae-Won;Seo, Byoung-Moo;Park, Joo-Cheol;Kim, Jung-Wook;Lee, Gene
    • Molecules and Cells
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    • v.37 no.7
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    • pp.562-567
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    • 2014
  • Human Hertwig's epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cells are epithelial remnants of teeth residing in the periodontium. Although the functional roles of HERS/ERM cells have yet to be elucidated, they are a unique epithelial cell population in adult teeth and are reported to have stem cell characteristics. Therefore, HERS/ERM cells might play a role as an epithelial component for the repair or regeneration of dental hard tissues; however, they are very rare population in periodontium and the primary isolation of them is considered to be difficult. To overcome these problems, we immortalized primary HERS/ERM cells isolated from human periodontium using SV40 large T antigen (SV40 LT) and performed a characterization of the immortalized cell line. Primary HERS/ERM cells could not be maintained for more than 6 passages; however, immortalized HERS/ERM cells were maintained for more than 20 passages. There were no differences in the morphological and immunophenotypic characteristics of HERS/ERM cells and immortalized HERS/ERM cells. The expression of epithelial stem cell and embryonic stem cell markers was maintained in immortalized HERS/ERM cells. Moreover, immortalized HERS/ERM cells could acquire mesenchymal phenotypes through the epithelial-mesenchymal transition via TGF-${\beta}1$. In conclusion, we established an immortalized human HERS/ERM cell line with SV40 LT and expect this cell line to contribute to the understanding of the functional roles of HERS/ERM cells and the tissue engineering of teeth.

Investigation on Inhibitory Effect of ErmSF N-Terminal End Region Peptide on ErmSF Methyltansferase Activity In Vivo Through Development of Co-Expression System of Two Different Proteins in One Cell (서로 다른 두 단백질의 세포 내 동시 발현 체계의 개발을 통한 ErmSF에서 특이적으로 발견되는 N-Terminal End Region (NTER)을 포함하는 펩타이드의 생체내에서의 ErmSF 활성 억제 효과 검색)

  • Jin, Hyung-Jong
    • Korean Journal of Microbiology
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    • v.47 no.3
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    • pp.200-208
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    • 2011
  • Most problematic antibiotic resistance mechanism for MLS (macrolide-lincosamide-streptogramn B) antibiotics encountered in clinical practice is mono- or dimethylation of specific adenine residue at 2058 (E. coli coordinate) of 23S rRNA which is performed by Erm (erythromycin ribosome resistance) protein through which bacterial ribosomes reduce the affinity to the antibiotics and become resistant to them. ErmSF is one of the four gene products produced by Streptomyces fradiae to be resistant to its own antibiotic, tylosin. Unlike other Erm proteins, ErmSF harbors idiosyncratic long N-terminal end region (NTER) 25% of which is comprised of arginine well known to interact with RNA. Furthermore, NTER was found to be important because when it was truncated, most of the enzyme activity was lost. Based on these facts, capability of NTER peptide to inhibit the enzymatic activity of ErmSF was sought. For this, expression system for two different proteins to be expressed in one cell was developed. In this system, two plasmids, pET23b and pACYC184 have unique replication origins to be compatible with each other in a cell. And expression system harboring promoter, ribosome binding site and transcription termination signal is identical but disparate amount of protein could be expressed according to the copy number of each vector, 15 for pACYC and 40 for pET23b. Expression of NTER peptide in pET23b together with ErmSF in pACYC 184 in E. coli successfully gave more amounts of NTER than ErmSF but no inhibitory effects were observed suggesting that there should be dynamicity in interaction between ErmSF and rRNA rather than simple and fixed binding to each other in methylation of 23S rRNA by ErmSF.

Domain Expression of ErmSF, MLS (macrolide-lincosamide-streptogramin B) Antibiotic Resistance Factor Protein (MLS (macrolide-lincosamide-streptogramin B) 항생제 내성인자 단백질인 ErmSF의 domain발현)

  • 진형종
    • Korean Journal of Microbiology
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    • v.37 no.4
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    • pp.245-252
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    • 2001
  • Erm proteins, MLS (macrolide-lincosamide-streptogramin B) resistance factor proteins, show high degree of amino acid sequence homology and comprise of a group of structurally homologous N-methyltransferases. On the basis of the recently determined structures of ErmC` and ErmAM, ErmSF was divided into two domains, N-terminal end catalytic domain and C-terminal end substrate binding domain and attempted to overexpress catalytic domain in E. coli using various pET expression systems. Three DNA fragments were used to express the catalytic domain: DNA fragment 1 encoding Met 1 through Glu 186, DNA fragment 2 encoding Arg 60 to Glu 186 and DNA fragment 3 encoding Arg 60 through Arg 240. Among the pET expression vectors used, pET 19b successfully expressed the DNA fragment 3 and pET23b succeeded in expression of DNA fragment 1 and 2. But the overexpressed catalytic domains existed as inclusion body, a insoluble aggregate. To assist the soluble expression of ErmSF catalytic domains, Coexpression of chaperone GroESL or Thioredoxin and lowering the incubation temperature to $22^{\circ}C$ were attempted, as did in the soluble expression of the whole ErmSF protein. Both strategies did not seem to be helpful. Solubilization with guanidine-HCl and renaturation with gradual removal of denaturant and partial digestion of overexpressed whole ErmSF protein (expressed to the level of 126 mg/ι culture as a soluble protein) with proteinase K, nonspecific proteinase are under way.

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Cloning of Inducible MLS Antibiotics Resistance Genes and their Expression Control Mechanism - ermC-4, a macrolide-lincosamide-streptogramin B resistance determinant on pMB4 from Staphylococcus aureus TR-1 (MLS계 항생물질 유도내성 유전자의 크로닝과 유전자의 발현조절 기전 - Staphylococus aureus TR-1균주의 프라스미드 pMB4에 존재하는 MLS 내성 유전자 ermC-4)

  • 김수환;최응칠;김병각;심미자
    • YAKHAK HOEJI
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    • v.35 no.1
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    • pp.22-29
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    • 1991
  • pMB4 is a 2.4-kilobase plasmid of Staphylococcus aureus TR-1 that confers inducible resistance to the macrolide-lincosamide-streptogramin B(MLS) antibiotics. By subcloning studies, it was found that the MLS resistance determinant was located at 1.0Kb fragment between Sau3AI and TaqI sites. DNA sequence of the MLS resistant determinant, named ermC-4 was determined, and found to be highly homologous with that of ermC. Because the leader peptide sequence of ermC-4 was identical with that of ermC, the expression of the resistance gene is thought to be controlled by posttranscriptional attenuation in S. aureus TR-1.

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