• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.529.2-529
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• 1986

pSp-Si (1.6kbp) was originally found in pediococcus halophilus to be a cryptic multicopy-plasmid. Hoping that the plasmid can also replicate in Bacillus subtilis, protoplast transformation of strain 207-25 (recE) was performed using pSP-Sl onto which was added the marker of tmrB8 (on 4.9 kbp EcoRI fragment ) or tmrB+ (on 0.9 kbp xbaI fragment) gene. Though the tmrB8 gene can expres tunicamycin-resistance at the single copy state, and the tmrB+ gene exerts the resistance only at the multicopy state, we could not confirm the replication of pSP-Sl (tmrB8) or pSP-Sl(tmrB+) in B. subtilis. During the experiment, however, we unexpectedly found that the circularized 0.9 kbp xgaI fragment (tmrB+) itself, which had no replication origin, could transform strain 207-25 to tunicamycin-resistant by protoplast transformation. Southern hybridization analyses with tmrB+ and other probes revealed the integration of the fragment at a single copy state into a position other than the homologous tmrB gene. This recE independent integration of another tmrB+ gene into the chromosome may contribute to the tunicamycinresistance in the transformants.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2004.06a
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• pp.323-327
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• 2004

Leuconostoc citreum CBUE isolated from kimchi proved to harbor a small cryptic plasmid, pNS75. The complete nucleotide sequence of pNS75 was 1,821 bp and had a low G+C content of 39.2%. Computer analysis using DNASIS revealed one open reading frame (ORF), having ATG as putatitive start condon and potentially encoding proteins with molecular mass of 38 kDa. The chimeric plasmid pLeuCM was first constructed wih pNS75, pUC19 and chroamphenicol acetyltransferase (CAT) from Staphylococcus sp.. pLeuCM replicated and expressed chroamphenicol acetyltransferase in Leuconostoc citerum CBNF after transformation. To test the availability of shuttle vector as cloning vehicle of foreign gene, $\alpha$-amylase gene of Streptococcus bovis was cloned and all transformants secreated the $\alpha$-amylase successfully. The result indicates that pLeuCM is a potential shuttle vector for Leuconostoc spp. and lactic acid bacteria.

• Journal of Microbiology and Biotechnology
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• v.16 no.1
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• pp.149-152
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• 2006

Leuconostoc mesenteroides SY1, a strain isolated from cabbage Kimchi, was transformed with pCW4, a shuttle vector based on a cryptic plasmid from Lactobacillus paraplantarum C7. $\alpha-Amylase$ gene, amyL, from Bacillus licheniformis was cloned into pCW4, resulting in $pCW4T{\alpha},\;and\;pCW4T{\alpha}$ was introduced into SY1 by electroporation. Transformation efficiency was $10^2cells/{\mu}g$ plasmid DNA. L. mesenteroides cells harboring $pCW4T{\alpha}$ did not show amylase activity, although amyL transcript was synthesized as determined by slot blot experiment. $pCW4T{\alpha}$ was stably maintained in SY1 in the presence of erythromycin (Em, $5\;{\mu}g/ml$) but rapidly lost when Em was omitted. Less than $1\%$ of the cells maintained $pCW4T{\alpha}$ after 5 days at $30^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• v.17 no.11
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• pp.1841-1847
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• 2007

We previously identified the origin of replication of p703/5, a small cryptic plasmid from the KBL703 strain of Enterococcus faecalis. The origin of replication contains putative regulatory cis-elements required for replication and a replication initiator (RepA) gene. The replicon of p703/5 is similar in its structural organization to theta-type plasmids, and RepA is homologous to a family of Rep proteins identified in several plasmids from Gram-positive bacteria. Here, we report molecular interactions between RepA and the replication origin of p703/5. DNase I footprinting using recombinant RepA together with electrophoretic mobility shift assays confirmed the binding of RepA to the replication origin of p703/5 via iterons and an inverted repeat. We also demonstrated the formation of RepA dimers and the different binding of RepA to the iteron and the inverted repeat using gel filtration chromatographic analysis, a chemical crosslinking assay, and electrophoretic mobility shift assays in the presence of guanidine hydrochloride. Our results suggest that RepA plays a regulatory role in the replication of the enterococcal plasmid p703/5 via mechanisms similar to those of typical iteroncarrying theta-type plasmids.

• Microbiology and Biotechnology Letters
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• v.23 no.4
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• pp.390-396
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• 1995

Bacillus sphaericus 1593 is a larvicidal toxin-producing mosquitocidal bacterium. The toxin contains a parasporal crystalline inclusion which is composed of a protein that is activated under alkaline condition. To enhance alkaline environment around toxin protein, cryptic plasmid cured, B. sphaericus 1593 was transformed by the Bacillus pasteurii urease gene which generate ammonia from urea. Transformant produced urease at about 80% more than wild type strain. B. sphaericus 1593, and the urease gene was stably maintained. It also produced crystalline toxin protein at the same level as the wild type strain B. sphaericus 1593.

• Journal of Microbiology and Biotechnology
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• v.23 no.4
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• pp.545-554
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• 2013

Plasmid isolation of kimchi-derived Weissella cibaria KLC140 revealed six different plasmids. The smallest plasmid, pKW2124, was DNA sequenced and characterized, showing 2,126 bp with a GC content of 36.39% and five putative open reading frames (ORFs). In silico analysis of these ORFs showed ORF1 encodes a putative replication protein similar to rolling circular replication proteins from other lactic acid bacteria. However, a single-stranded intermediate was not detected when S1 nuclease was treated, suggesting it may follow theta replication. Interestingly, the replication initiation site of this plasmid is 100% identical to other plasmids from lactic acid bacteria, suggesting it may function for replication initiation. To construct a surface layer expression vector, pTSLGFP, slpA encoding the surface layer protein from Lactobacillus acidophilus was PCR amplified and fused with the gfp gene, forming a SLGFP fused gene. The plasmid pKW2124 was cloned into the XbaI site of pUC19, forming an Weissella-E. coli shuttle vector pKUW22. NheI-linearized pTSLGFP was ligated into pKUWCAT containing pKUW22 and the chloramphenicol acetyltransferase gene from pEK104, resulting in an 8.6 kb pKWCSLGFP surface layer expression vector. After transformation of this vector into W. cibaria KLC140, a GFP fluorescence signal was detected on the surface of the transformant, substantiating production of SLGFP fused protein and its secretion. This is the first report for construction of a Weissella surface layer expression vector, which may be useful for surface layer production of beneficial proteins in Weissella.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.639-647
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• 2008

The heat-inducible expression vectors for Corynebacterium glutamicum and C. ammoniagenes were constructed by using the ${\lambda}O_L1$ and the cryptic promoters, CJ1 and CJ4 that express genes constitutively in C. ammoniagenes. Although the promoters were isolated from C. ammoniagenes, CJ1 and CJ4 were also active in C. glutamicum. To construct vectors, the $O_L1$ from the ${\lambda}P_L$ promoter was isolated and fused to the CJ1 and CJ4 promoters by recombinant PCR. The resulting artificial promoters, CJ1O and CJ4O, which have one ${\lambda}O_L1$, and CJ1OX2, which has two successive ${\lambda}O_L1$, were fused to the green fluorescent protein (GFP) gene followed by subcloning into pCES208. The expression of GFP in the corynebacteria harboring the vectors was regulated successfully by the temperature-sensitive cI857 repressor. Among them, C. ammoniagenes harboring plasmid pCJ1OX2G containing GFP fused to CJ1OX2 showed more GFP than the other ones and the expression was tightly regulated by the repressor. To construct the generally applicable expression vector using the plasmid pCJ1OX2G, the His-tag, enterokinase (EK) moiety, and the MCS were inserted in front of the GFP gene. Using the vector, the expression of pyrR from C. glutamicum was tried by temperature shift-up. The results indicated that the constructed vectors (pCeHEMG) can be successfully used in the expression and regulation of foreign genes in corynebacteria.

• Journal of Microbiology and Biotechnology
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• v.25 no.2
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• pp.196-205
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• 2015

A Sulfolobus-E. coli shuttle vector for an efficient expression of the target gene in S. acidocaldarius strain was constructed. The plasmid-based vector pSM21 and its derivative pSM21N were generated based on the pUC18 and Sulfolobus cryptic plasmid pRN1. They carried the S. solfataricus P2 pyrEF gene for the selection marker, a multiple cloning site (MCS) with C-terminal histidine tag, and a constitutive promoter of the S. acidocaldarius gdhA gene for strong expression of the target gene, as well as the pBR322 origin and ampicillin-resistant gene for E. coli propagation. The advantage of pSM21 over other Sulfolobus shuttle vectors is that it contains a MCS and a histidine tag for the simple and easy cloning of a target gene as well as one-step purification by histidine affinity chromatography. For successful expression of the foreign genes, two genes from archaeal origins (PH0193 and Ta0298) were cloned into pSM21N and the functional expression was examined by enzyme activity assay. The recombinant PH0193 was successfully expressed under the control of the gdhA promoter and purified from the cultures by His-tag affinity chromatography. The yield was approximately 1 mg of protein per liter of cultures. The enzyme activity measurements of PH0913 and Ta0298 revealed that both proteins were expressed as an active form in S. acidocaldarius. These results indicate that the pSM21N shuttle vector can be used for the functional expression of foreign archaeal genes that form insoluble aggregates in the E. coli system.

• Applied Biological Chemistry
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• v.44 no.4
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• pp.251-256
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• 2001

To develop biofertilizer solubilizing inorganic phosphate, a bacterium having high abilities to solubilize inorganic phosphate were isolated from cultivated soils. The strain was identified to Aeromonas hydrophila DA57, based on the physiological and biochemical properties. The optimum temperature and initial pH to solubilize insoluvle phosphate in sucrose minimal medium were $30^{\circ}C$ and pH 7.0, respectively. In these conditions phosphate solubilizing activities of the strain against three types of insoluble phosphate were quantitatively determined. It was possivle to distinguish between solubilization through release of gluconic acid and still unknown mechanism. Aemmonas hydrophila DA57 harbored a 4.5 kb cryptic plasmid.