African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12196

Full Length Research Paper

Cloning and heterologous expression of the plasmid-encoded shsp gene of Streptococcus thermophilus isolated from Chinese dairy

Hong-Tao Tian1,2, Le-Hui Ma2, Li-Fang Zhang2, Xin-XiGu2, Xing-Hua Guo3and Yun-Bo Luo1*
  1College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China. 2College of Food Science and Technology, Hebei Agricultural University,  Hebei Baoding 071001, China. 3Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China.
Email: [email protected]

  •  Accepted: 08 December 2010
  •  Published: 14 February 2011

Abstract

 

We first tested 12 strains of Streptococcus thermophilus isolated from China yogurt or its starter culture for their plasmid content. Two strains were found to harbor two plasmids, identified as pQC1 and pQC2, from the S. thermophilus ST-QC, as well as pHS1 and pHS2 from the S. thermophilus ST-HS. Agarose gel electrophoresisprofiles indicated that the molecular size of about 4.5 kb for the two bigger plasmids (pQC1 and pHS1) was nearly identical, same with the molecular size ofapproximately 3.5 kb for the two smaller plasmids (pQC2 and pHS2). A 765 bp DNA fragment, including a 429 bp open reading frame of the shsp gene of the smaller plasmid pQC2 from S. thermophilus St-QC, was successfully cloned and sequenced. Multiple sequence alignment revealed that the nucleotide sequence of the coding region of the shsp gene or the deduced amino acid sequence of the sHSP protein shared a high degree of identity (> 86.67 or 81.33% identity) with theshsp genes or the sHSP proteins described from other S. thermophilus plasmids. In addition, 222 bp nucleotide sequences upstream and 114 bp nucleotide sequences downstream belonging to the shsp gene coding region were alsoanalyzed. The separation of SDS-PAGE and the analysis of Western blotting for the soluble cell proteins showed that the shsp gene of plasmid pQC2 of S. thermophilus St-QC was successfully expressed in mesophilic Escherichia coli. In addition to strong heat and acid tolerance, recombinant E. coli cells overexpressing the S. thermophilus St-QC shsp gene had significantly higher resistance to ethanol stress, which is the first physiological function found to be linked to the S. thermophilus plasmid-borne shsp gene. This study will provide a basis for the cloning and expression of the shsp genes from a thermophilic microorganism in the mesophilic LAB or yeast and for further development of stress-resistant microorganism strains used in dairy fermentation and brewing wine.

 

Key words: Streptococcus thermophilus, plasmid, small heat shock protein gene (shsp gene), cloning, expression, abiotic stresses, Escherichia coli.

Abbreviation

 HSPs, Heat shock proteins; LAB, lactic acid bacteria; SDS-PAGE,sodium dodecyl sulfate; PCR, polymerase chain reaction.

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