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Properties and construction of plasmid pFW213, a shuttle vector with the oral Streptococcus origin of replication.
Appl Environ Microbiol 2011; 77(12):3967-74AE

Abstract

Streptococcus parasanguinis is among the most successful colonizers of the human body. Strain FW213 harbors a 7.0-kb cryptic plasmid, pFW213, with a copy number at 5 to 10 per chromosome. Sequence and functional analyses of pFW213 revealed that the open reading frame (ORF) encoding the replication protein (Rep) is essential for the replication of pFW213, and the putative plasmid addiction system (RelB and RelE) and an ORF (ORF6) with no known function are required for its stability. The minimal replicon of pFW213 contains the rep gene and its 5'-flanking 390-bp region. Within the minimal replicon, an A/T-rich region followed by 5 contiguous 22-bp repeats was located 5' of the ATG of rep. No single-stranded replication intermediates were detected in the derivatives of pFW213, suggesting that pFW213 replicates via the theta replication mechanism. The minimal replicon was unstable in streptococcal hosts without selection, but the stability was greatly enhanced in derivatives containing the intact relBE genes. A Streptococcus-Escherichia coli shuttle vector, pCG1, was constructed with the pFW213 replicon. Plasmid pCG1 features a multiple cloning region and a spectinomycin resistance determinant that is expressed in both Streptococcus spp. and E. coli. Various streptococcal DNA fragments were cloned in pCG1, and the recombinant constructs were stably maintained in the streptococcal hosts. Since pCG1 is compatible with the most widely used streptococcal replicon, pVA380-1, pCG1 will provide a much needed tool allowing the cloning of two genes that work in concert in the same host.

Authors+Show Affiliations

Department of Microbiology and Immunology, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan, Taiwan. mchen@mail.cgu.edu.twNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

21531841

Citation

Chen, Yi-Ywan M., et al. "Properties and Construction of Plasmid pFW213, a Shuttle Vector With the Oral Streptococcus Origin of Replication." Applied and Environmental Microbiology, vol. 77, no. 12, 2011, pp. 3967-74.
Chen YY, Shieh HR, Lin CT, et al. Properties and construction of plasmid pFW213, a shuttle vector with the oral Streptococcus origin of replication. Appl Environ Microbiol. 2011;77(12):3967-74.
Chen, Y. Y., Shieh, H. R., Lin, C. T., & Liang, S. Y. (2011). Properties and construction of plasmid pFW213, a shuttle vector with the oral Streptococcus origin of replication. Applied and Environmental Microbiology, 77(12), pp. 3967-74. doi:10.1128/AEM.02828-10.
Chen YY, et al. Properties and Construction of Plasmid pFW213, a Shuttle Vector With the Oral Streptococcus Origin of Replication. Appl Environ Microbiol. 2011;77(12):3967-74. PubMed PMID: 21531841.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Properties and construction of plasmid pFW213, a shuttle vector with the oral Streptococcus origin of replication. AU - Chen,Yi-Ywan M, AU - Shieh,Hui-Ru, AU - Lin,Cheng-Tzer, AU - Liang,Shin-Yi, Y1 - 2011/04/29/ PY - 2011/5/3/entrez PY - 2011/5/3/pubmed PY - 2011/9/13/medline SP - 3967 EP - 74 JF - Applied and environmental microbiology JO - Appl. Environ. Microbiol. VL - 77 IS - 12 N2 - Streptococcus parasanguinis is among the most successful colonizers of the human body. Strain FW213 harbors a 7.0-kb cryptic plasmid, pFW213, with a copy number at 5 to 10 per chromosome. Sequence and functional analyses of pFW213 revealed that the open reading frame (ORF) encoding the replication protein (Rep) is essential for the replication of pFW213, and the putative plasmid addiction system (RelB and RelE) and an ORF (ORF6) with no known function are required for its stability. The minimal replicon of pFW213 contains the rep gene and its 5'-flanking 390-bp region. Within the minimal replicon, an A/T-rich region followed by 5 contiguous 22-bp repeats was located 5' of the ATG of rep. No single-stranded replication intermediates were detected in the derivatives of pFW213, suggesting that pFW213 replicates via the theta replication mechanism. The minimal replicon was unstable in streptococcal hosts without selection, but the stability was greatly enhanced in derivatives containing the intact relBE genes. A Streptococcus-Escherichia coli shuttle vector, pCG1, was constructed with the pFW213 replicon. Plasmid pCG1 features a multiple cloning region and a spectinomycin resistance determinant that is expressed in both Streptococcus spp. and E. coli. Various streptococcal DNA fragments were cloned in pCG1, and the recombinant constructs were stably maintained in the streptococcal hosts. Since pCG1 is compatible with the most widely used streptococcal replicon, pVA380-1, pCG1 will provide a much needed tool allowing the cloning of two genes that work in concert in the same host. SN - 1098-5336 UR - https://www.unboundmedicine.com/medline/citation/21531841/Properties_and_construction_of_plasmid_pFW213_a_shuttle_vector_with_the_oral_Streptococcus_origin_of_replication_ L2 - http://aem.asm.org/cgi/pmidlookup?view=long&pmid=21531841 DB - PRIME DP - Unbound Medicine ER -