• 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.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.51 no.4
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• pp.397-403
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• 2018

We determined the resistance rates of pathogenic bacteria isolated from cultured olive flounder Paralichthys olivaceus to erythromycin (Em), antibiotic typically used in aquaculture and analyzed the genotypes of resistant bacteria using polymerase chain reaction (PCR). We isolated and utilized 160 isolates of Streptococcus parauberis, 1 of S. iniae, 66 of Edwardsiella tarda, 56 of Vibrio sp. and 23 of unidentified bacteria from presumed infected olive flounder from Jeju Island from March 2016 to October 2017. Of the 306 isolated strains, Em-resistant strains included 33 of S. parauberis, 39 of E. tarda and 2 of Vibrio sp. We conducted PCR to assess the resistance determination of Em-resistant strains. Five different types of Em-resistance genes were detected in the 74 Em-resistant strains: erm (A), erm (B), erm (C), mef (A) and mef (E); erm (A) and erm (B) were detected in 1 (3%) and 24 (72.7%) S. parauberis isolates, respectively. In E. tarda, erm (B) was detected in five isolates (12.8 %) and no Em-resistance genes were detected in the two Vibrio sp. isolates.

• 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.

• 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.

• 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.

• Korean Journal of Microbiology
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• v.49 no.2
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• pp.105-111
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• 2013

The Erm family of adenine-$N^6$ methyltransferases (MTases) is responsible for the development of resistance to macrolide-lincosamide-streptogramin B antibiotics through the methylation of 23S ribosomal RNA. Recently, it has been proposed that well conserved amino acids in ErnC' located in concave cleft between N-terminal 'catalytic' domain and C-terminal 'RNA-binding' domain interacts with substrate RNA. We carried out the site-directed mutagenesis and studied the function of the ErmSF R237 mutant in vitro and in vivo. R237 amino acid residue is located in the concave cleft between two domains. Furthermore this residue is very highly conserved in almost all the Erm family. Purified mutant protein exhibited only 51% enzyme activity compared to wild-type. Escherichia coli with R237A mutant protein compared to the wild-type protein expressing E. coli did not show any difference in its MIC (minimal inhibitory concentration) suggesting that even with lowered enzyme activity, mutant protein was able to efficiently methylate 23S rRNA to confer the resistance on E. coli expressing this protein. But this observation strongly suggests that R237 of ErmSF probably interacts with substrate RNA affecting enzyme activity significantly.

• Archives of Pharmacal Research
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• v.29 no.12
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• pp.1154-1157
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• 2006

The ermK gene from Bacillus lichenformis encodes an inducible rRNA methylase that confers resistance to the macrolide-lincosamide-streptogramin B antibiotics. The ermK mRNA leader sequence has a total length of 357 nucleotides and encodes a 14-amino acid leader peptide together with its ribosome binding site. The secondary structure of ermK leader mRNA and a leader peptide sequence have been reported as the elements that control expression. In this study, the contribution of specific leader peptide amino acid residues to induction of ermK was studied using the PCR-based megaprimer mutation method. ermK methylases with altered leader peptide codons were translationally fused to E. coli ${\beta}-galactosidase$ reporter gene. The deletion of the codons for Thr-2 through Ser-4 reduced inducibility by erythromycin, whereas that for Thr-2 and His-3 was not. The replacement of the individual codons for Ser-4, Met-5 and Arg-6 with termination codon led to loss of inducibility, but stop mutation of codon Phe-9 restored inducibility by erythromycin. Collectively, these findings suggest that the codons for residue 4, 5 and 6 comprise the critical region for induction. The stop mutation at Leu-7 expressed constitutively ermK gene. Thus, ribosome stalling at codon 7 appears to be important for ermK induction.

• Proceedings of the PSK Conference
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• 2000.10a
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• pp.204.1-204.1
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• 2000

• Journal of Applied Biological Chemistry
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• v.56 no.3
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• pp.165-170
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• 2013

The SCO3388 gene from Streptomyces coelicolor is homologous to tmrB, the tunicamycin resistance gene of Bacillus subtilis. The SCO3388-inactivation strain (SY-tbl-1) was generated by replacing SCO3388 with thiostrepton resistance gene. Spores of S. coelicolor derivatives were prepared on mannitol-soy flour (MS) agar on which SY-tbl-1 displayed no significant defect in growth and development. When plated on R4 agar, spores of SYtbl-1 displayed retardation in growth and sporulation, whereas its mycelium gave rise to normal growth. Thus, SCO3388 is suggested to be involved in the dormant spore germination. Expression of SCO3388 under the ermE1 promoter restored but only partially the ability to sporulate in SY-tbl-1. Neither SY-tbl-1 nor SY-tbl-1/ermE1p-SCO3388 showed a difference in tunicamycin resistance to the wild type whereas, interestingly, the introduction of ermE1p-SCO3388 dramatically enhanced spore survival to heat and detergent treatments, suggesting that SCO3388 might play a role in the maintenance of spore cell wall integrity.

• Korean Journal of Microbiology
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• v.43 no.3
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• pp.166-172
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• 2007

ERM proteins transfer the methyl group to $A_{2058}$ in 23S rRNA, which reduces the affinity of MLS (macrolide-lincosamide-streptogramin B) antibiotics to 23S rRNA, thereby confer the antibiotic resistance on micro-organisms ranging from antibiotic producers to pathogens and are classified into monomethyltransferase and dimethyltransferase. To investigate the differences between mono- and dimethyltransferase, tirD, a representative monomethylase gene was cloned in Escherichia coli from Streptomyces fradiae which contains ermSF, dimethylase gene as well to overexpress the TlrD for the first time. T7 promoter driven expression system successfully overexpress tlrD as a insoluble aggregate at $37^{\circ}C$ accumulating to around 55% of the total cell protein but unlike ErmSF, culturing at temperature as low as $18^{\circ}C$ did not make insoluble aggregate of protein into soluble protein. Coexpression of Thioredoxin and GroESL, chaperone was not helpful in turning into soluble protein either as in case of ErmSF. These results might suggest that differences between mono- and dimethylase could be investigated on the basis of the characteristics of protein structure. However, a very small amount of soluble protein which could not be detected by SDS-PAGE conferred antibiotic resistance on E. coli as in ErmSF which was expected from the activity exerted by monmethylase in a cell.