Heterologous Expression of the Streptococcus mutans Mannitol PTS Genes

Streptococcus mutans translocates carbohydrates into the cell by means of the phosphoenolpyruvate-dependent phospho-transferase system (PTS), which is the major mechanism for carbohydrate uptake in this organism. The hexitol mannitol is one sugar that is transported by system. Objectives: To determine if the S. mutans PTS genes could be expressed in a heterologous system, we have cloned the mannitol PTS gene cluster into a shuttle vector and have assayed for expression of these genes in Streptococcus thermophilus. This organism does not normally utilize mannitol as a carbon source. Methods: PCR primers that flank the mannitol PTS gene cluster were designed using the genomic sequence of S. mutans UA159. The PCR product was cloned into the shuttle vector pALH292. The resulting construct was electroporated into S. thermophilus 119. Transformants were screened by PCR for the presence of the vector antibiotic resistance genes and the cloned insert. An appropriate transformant was identified and assayed for the ability to ferment mannitol. Mannitol fermentation was determined using Purple Broth Base agar plates containing mannitol as the sole carbohydrate source. In addition, growth in mannitol media was compared for the S. thermophilus wild-type parental strain and the S. thermophilus strain containing the cloned mannitol genes. Results: The S. thermophilus strain containing the mannitol PTS genes from S. mutans was able to ferment mannitol while the wild-type parental strain was not. The comparison of overnight growth determined that the strain containing the S. mutans mannitol genes could reach an optical density approximately one-half the value of that obtained by growth on lactose, the preferred carbohydrate of S. thermophilus. Conclusion: The S. mutans mannitol PTS genes are adequately expressed in the heterologous host S. thermophilus to a level that enables the host to ferment mannitol as a carbon source while reaching a significant optical density.