• Journal of Microbiology and Biotechnology
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• v.16 no.1
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• pp.118-125
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• 2006

The region responsible for replication of the megaplasmid pAtC58 in the nopaline-type Agrobacterium tumefaciens strain C58 was determined. A derivative ofa Co1E1 vector, pBluscript SK-, incapable of autonomous replication in Agrobacterium spp, was cloned with a 7.6-kb Bg1II-HindIII fragment from a cosmid clone of pAtC58, which contains a region adjacent to the operon for the utilization of deoxyfructosyl glutamine (DFG). The resulting plasmid conferred resistance to carbenicillin on the A. tumefaciens strain UIA5 that is a plasmidfree derivative of C58. The plasmid was stably maintained in the strain even after consecutive cultures for generations. Analysis of nested deletions of the 7.6-kb fragment showed that a 4.3-kb BglII-XhoI region sufficiently confers replication of the derivative of the ColE1 vector on UIA5. The region comprises three ORFs, which have high homologies with repA, repB, and repC of plasm ids in virulent Agrobacterium spp. including pTiC58, pTiB6S3, pTi-SAKURA, and pRiA4b as well as those of symbiotic plasmids from Rhizobium spp. Phylogenie analysis showed that rep genes in pAtC58 are more closely related to those in pRiA4 than to pTi plasmids including pTiC58, suggesting that the two inborn plasmids, pTiC58 and pAtC58, harbored in C58 evolved from distinct origins.

• The Plant Pathology Journal
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• v.37 no.3
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• pp.243-257
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• 2021

Bacillus pumilus is the causal agent of trunk bulges disease affecting rubber and rubberwood quality and yield production. In this study, B. pumilus and other closely related species were included in B. pumilus group, as they shared over 99.5% similarity from 16S rRNA analysis. Multilocus sequence analysis (MLSA) of five housekeeping genes and repetitive elements-based polymerase chain reaction (rep-PCR) using REP, ERIC, and BOX primers conducted to analyze the diversity and systematic relationships of 20 isolates of B. pumilus group from four rubber tree plantations in Peninsular Malaysia (Serdang, Tanah Merah, Baling, and Rawang). Multi rep-PCR results revealed the genetic profiling among the B. pumilus group isolates, while MLSA results showed 98-100% similarity across the 20 isolates of B. pumilus group species. These 20 isolates, formerly established as B. pumilus, were found not to be grouped with B. pumilus. However, being distributed within distinctive groups of the B. pumilus group comprising of two clusters, A and B. Cluster A contained of 17 isolates close to B. altitudinis, whereas Cluster B consisted of three isolates attributed to B. safensis. This is the first MLSA and rep-PCR study on B. pumilus group, which provides an in-depth understanding of the diversity of these rubber-pathogenic isolates in Malaysia.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.135.2-136
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• 2003

To characterize plasmids in Xanthomonu axonopodis pv. glycines, we isolated plasmids pAG1 from the strain AG1 and pXAG81 and PXAG82 from the strain Bra, respectively, and sequenced three plasmids. The size of plasmids, pAG1, pXAG81, and pXAG82 was 15,149-base pairs (bp), 26,727-bp, and 1,496-bp, respectively Fifteen and twenty six possible open reading frames (ORFs) were present in pAG1 and pXAG81, respectively. Only one ORF homologous to a rep gene of Xylella fastidiosa was present in pXAG82. pAG1 contained genes homologous to avrBs3, tnpA, tnpR, repA, htrA, three parA genes, M.XmaI, R.XmaI, and six hypothetical proteins. pXAG81 contained genes homologous to avrBs3, tnpA, tnpR, repA, htrA, two parA genes, pemI, pemK, mobA, mobB, mobC, mobD, mobE, trwB, traF, traH, ISxac2, and eleven hypothetical proteins. Based on DNA sequence analysis, we presume that pXAG81 is a conjugal plasmid. Interestingly, we found 0.5-kb truncated avirulence gene similar to aurXacE3 on the right border of avrBs3 homolgs of pAG1 and pXAG81. Two hundred twenty five isolates were analyzed to find aurBS3 or tra gene homologs by Southern hybridization. The numbers of avrBs3 homolog varied from 3 in AG1 to 8 in AG166. Two hundred seventeen isolates appeared to can conjugative plasmids (pXAG81 type), and thirty eight isolates appeared to carry non-conjugative plamids (pAGl type). This indicated that aurBs3 gene homologs might be spread by conjugation in X. axonopodis pv. glycines.

• Korean Journal of Clinical Laboratory Science
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• v.48 no.4
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• pp.327-334
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• 2016

Recently, antimicrobial resistance of pathogenic bacteria has been increasing due to excessive use of antimicrobial agents in both humans and livestock. PCR amplification and nucleotide sequence analyses were conducted to investigate16S ribosomal RNA methyltransferase (RMTase) genes and integrons in P. mirabilis strains isolated from clinical specimens and chickens in an area of the Chungcheong providence. In addition, clonality analysis of P. mirabilis strains was performed using a repetitive extragenic palindromic sequence-based PCR (REP-PCR) method. Of the total 38 P. mirabilis isolates, 7 (18.4%) strains were isolated from clinical specimens contained in the RMTase genes and showed resistance to amikacin, tobramycin, and gentamicin. A total of 23 (60.5%) isolates carried class 1 integrons, but no isolates in our study harbored class 2 and class 3 integrons. Class 1 integrons detected in our study harbored genes encoding resistance to aminoglycosides (aadA2, aadA5, aadA7, and aacCA5), ${\beta}$-lactams ($bla_{PSE}$), erythromycin (ereA), lincosamides (linF), and trimethoprim (dfrA12, dfrA17, and dfrA32). We confirmed that the RMTase genes had spread among only the P. mirabilis isolates from clinical specimens, but class 1 integrons had widely disseminated among P. mirabilis isolates from clinical specimens and chickens. In addition, identical REP-PCR banding patterns were evidenced in only P. mirabilis isolates from chickens. Our results suggest the horizontal spreading of P. mirabilis isolates in the chicken farm. To prevent further spreading of antimicrobial resistant genes among P. mirabilis isolates, monitoring and clinical policing will be required.

• Journal of Microbiology and Biotechnology
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• v.19 no.4
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• pp.403-408
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• 2009

A number of bifidobacterial species of human origin were screened for the presence of cryptic plasmids. One strain, Bifidobacterium longum FI10564, harbored plasmids of approximately 2.2 kb, 3.6 kb, and 4.9 kb in size. The smallest plasmid, pFI2576(2,197 bp), was studied in detail and its complete nucleotide sequence was determined. Computer-assisted analysis of this novel plasmid(G+C content 62%) identified 9 putative open reading frames(orfs), 3 of which were shown to be probable genes. These putative genes are arranged in an operon-like structure, in which the overlapping orfs 1 and 2 encode putative Rep proteins and are highly homologous to the rep genes of the B. longum plasmid pMBI(1,847 bp). The mechanism of replication of pFI2576 was investigated using Southern blot analysis of whole cell lysates, with and without S1 nuclease treatment, and atomic force microscopy(AFM). The results indicate that pFI2576 is likely to use the theta mode of replication.

• Journal of Life Science
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• v.34 no.2
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• pp.86-93
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• 2024

Since replication of plasmids must be strictly controlled, plasmids that generally perform rolling circle replication generally maintain a constant copy number by strictly controlling the replication initiator Rep at the transcriptional and translational levels. Plasmid pJB01 contains three orfs (copA, repB, repC or repABC) consisting of a single operon. From analysis of amino acid sequence, pJB01 CopA was homologous to the Cops, as a copy number control protein, of other plasmids. When compared with a CopG of pMV158, CopA seems to form the RHH (ribbon-helix-helix) known as a motif of generalized repressor of plasmids. The result of gel mobility shift assay (EMSA) revealed that the purified fusion CopA protein binds to the operator region of the repABC operon. To examine the functional role of CopA on transcriptional level, 3 point mutants were constructed in coding frame of copA such as CopA R16M, K26R and E50V. The repABC mRNA levels of CopA R16M, K26R and E50V mutants increased 1.84, 1.78 and 2.86 folds more than that of CopA wt, respectively. Furthermore, copy numbers owing to mutations in three copA genes also increased 1.86, 1.68 and 2.89 folds more than that of copA wt, respectively. These results suggest that CopA is the transcriptional repressor, and lowers the copy number of pJB01 by reducing repABC mRNA and then RepB, as a replication initiator.

• Korean Journal of Clinical Laboratory Science
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• v.48 no.2
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• pp.94-101
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• 2016

Acinetobacter species isolates are important opportunistic pathogens and commonly implicated in nosocomial infections. The therapeutic options for treatment of the bacterial infections are limited because the bacteria isolates are usually multidrug resistant (MDR). In the current study, we investigated various carbapenemase genes in 68 Acinetobacter species isolates. Antimicrobial susceptibilities were tested using the disk diffusion method. Screening of carbapenemase genes was performed via multiplex PCR. In addition, PCR and DNA sequencing were used to identify the carbapenemase genes. Repetitive extragenic palindromic-PCR (REP-PCR) was also performed to assess the clonality of isolates. In our study, A. baumannii isolates were highly resistant to all agents tested while all non-A. baumannii isolates were susceptible to all agents tested, with the exception of aztreonam and cefotaxime. All 51 A. baumannii isolates contained the $bla_{OXA-51}$ gene and 37 (72.5%) isolates also harbored the $bla_{OXA-23}$ gene. In addition, 39 MDR A. baumannii isolates were identified in our study and 37 isolates contained the $bla_{OXA-23}$ gene. The 37 MDR strains harboring $bla_{OXA-23}$ showed type I (n=22) or type II (n=15) banding patterns on their REP-PCR profiles. Our results suggest clonal relation and horizontal spreading of MDR A. baumannii isolates containing the $bla_{OXA-23}$ gene at the hospital located in Daejeon. Continuous investigation of antimicrobial resistant determinants and monitoring emergence and dissemination of MDR isolates is required to prevent and control infection and colonization of MDR A. baumannii isolates.

• Journal of Digital Convergence
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• v.14 no.10
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• pp.349-354
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• 2016

The emergence and dissemination of extended-spectrum ${\beta}$-lactamse (ESBL) producing Klebsiella pneumoniae isolates make it more difficult to treatment of bacterial infections. In our study, we detected ESBL genes and investigated antimicrobial susceptibility of K. pneumoniae isolates in Chungcheong province. In addition, clonality among the isolates was analyzed by repetitive element sequence (REP)-PCR. Twenty-one of 102 K. pneumoniae isolates produced CTX-M-14 and/or CTX-M-15 and showed high level (over 70%) resistance to third cephalosporins. CTX-M type ESBL producing K. pneumoniae strains isolated in our study showed diverse clonality and some of the isolates have been disseminated in the community. Enhancing infection control will be needed to prevent dissemination of the K. pneumoniae isolates. In addition, for more effective control of resistant bacteria it is considered necessary to monitor the database constructed through convergence of biological investigation and statistical analysis of antimicrobial resistance genes.

• Korean Journal of Clinical Laboratory Science
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• v.45 no.2
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• pp.43-47
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• 2013

This study was designed to evaluate the prevalence of ESBL genes and monitor antimicrobial resistance pattern in Escherichia coli, isolated from a hospital and a community. We tested 200 E. coli strains isolated in the hospitals and community in Chungcheong area from January to March 2012. Antimicrobial susceptibilities were tested by using the disk diffusion method. A search for ESBL genes was conducted by PCR amplification, and the genotypes were determined by direct nucleotide sequence analysis of the amplified products. An Epidemiologic study was performed by repetitive extragenic palindromic sequence-based PCR (REP-PCR). The percentage of ESBL-producing isolates was 17% for hospital associated E. coli and 11% for community associated E. coli. The ESBL gene sequencing results showed that the most common ESBL in E. coli was CTX-M-14 (19/28), followed by CTX-M-15 (9/28). The REP-PCR study also showed the genetic diversity, but there was no difference between the hospital and community associated E. coli. In this study, the most common types of class A ESBLs identified were CTX-M in the hospital and the community in Chungcheong area. ESBL-producing E. coli isolates showed diverse clonality.

• Parasites, Hosts and Diseases
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• v.43 no.4 s.136
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• pp.149-156
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• 2005

Four hundred and sixty five randomly selected clones from a cDNA library of Blattella germanica were partially sequenced and searched using BLAST as a means of analyzing the transcribed sequences of its genome. A total of 363 expressed sequence tags (ESTs) were generated from 465 clones after editing and trimming the vector and ambiguous sequences. About $42\%$ (154/363) of these clones showed significant homology with other data base registered genes. These new B. germanica genes constituted a broad range of transcripts distributed among ribosomal proteins, energy metabolism, allergens, proteases, protease inhibitors, enzymes, translation, cell signaling path-ways, and proteins of unknown function. Eighty clones were not well-matched by database searches, and these rep-resent new B. germanica-specific ESTs. Some genes which drew our attention are discussed. The information obtained increases our understanding of the B. germanica genome.