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Applied and Environmental Microbiology, May 2008, p. 3216-3228, Vol. 74, No. 10
0099-2240/08/$08.00+0     doi:10.1128/AEM.02631-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118{triangledown} ,{dagger}

Fang Fang,1,2 Sarah Flynn,1 Yin Li,1,2,{ddagger} Marcus J. Claesson,1,2 Jan-Peter van Pijkeren,1,2 J. Kevin Collins,1,2 Douwe van Sinderen,1,2 and Paul W. O'Toole1,2*

Department of Microbiology,1 Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland2

Received 20 November 2007/ Accepted 24 March 2008

The genome of Lactobacillus salivarius UCC118 comprises a 1.83-Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids of 20 kb (pSF118-20) and 44 kb (pSF118-44). Annotation and bioinformatic analyses suggest that both of the smaller plasmids replicate by a theta replication mechanism. Furthermore, it appears that they are transmissible, although neither possesses a complete set of conjugation genes. Plasmid pSF118-20 encodes a toxin-antitoxin system composed of pemI and pemK homologs, and this plasmid could be cured when PemI was produced in trans. The minimal replicon of pSF118-20 was determined by deletion analysis. Shuttle vector derivatives of pSF118-20 were generated that included the replication region (pLS203) and the replication region plus mobilization genes (pLS208). The plasmid pLS203 was stably maintained without selection in Lactobacillus plantarum, Lactobacillus fermentum, and the pSF118-20-cured derivative strain of L. salivarius UCC118 (strain LS201). Cloning in pLS203 of genes encoding luciferase and green fluorescent protein, and expression from a constitutive L. salivarius promoter, demonstrated the utility of this vector for the expression of heterologous genes in Lactobacillus. This study thus expands the knowledge base and vector repertoire of probiotic lactobacilli.

* Corresponding author. Mailing address: Department of Microbiology, University College Cork, Cork, Ireland. Phone: 353 21 490 3997. Fax: 353 21 4903101. E-mail: pwotoole{at}ucc.ie

{triangledown} Published ahead of print on 4 April 2008.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} Present address: Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China.

Applied and Environmental Microbiology, May 2008, p. 3216-3228, Vol. 74, No. 10
0099-2240/08/$08.00+0     doi:10.1128/AEM.02631-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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