SEQUENCE ANALYSIS AND MOLECULAR CLONING OF A STREPTOCOCCUS MUTANS ORTHOPHOSPHATE PERMEASE.

Daniela Eleuterio da Luz (ICBII-USP); Roberto Nepomuceno de Souza Lima (ICBII-USP); Beny Spira (ICBII-USP); Luis Carlos de Souza Ferreira (ICBII-USP); Rita de Cássia Café Ferreira (ICBII-USP)

Inorganic phosphate (Pi) is an ubiquitous molecule, present in all living beings and with important roles in different biological molecules, such as nucleics acids , phospholipids and ATP. In Escherichia coli  and other microorganisms phosphate  is taken up through two main specific carriers, a low affinity system, represented by Pit and a high-affinity Pst system which belongs to the family of ABC-transportes and is induced under phosphate starvation. The aim of the present study was the in silico characterization of the Streptococcus mutans pst operon and cloning of the Phosphate-biding protein PstS. Analysis of the genome sequence of the S. mutans UA159 revealed an operon with six genes encompassing pstS (phosphate-binding protein), pstC and pstC1 (transmembrane proteins),  pstB and SM1134 (ATP-binding proteins) and phoU (function unknown). The PstS amino acid sequence shared the highest similarity with Streptococcus sanguinis (80% identity) ortholog, although there was no evidence of horizontal gene transfer between the two species. Streptococcus mutans pstS gene was amplified and cloned in Escherichia coli expression vectors (pQE-30 and pET28a),  but the recombinant protein could not expressed in this bacterium. Successful expression of the Streptococcus mutans PstS was achieved in Bacillus subtilis, using vector pHT08, which allowed the intracellular accumulation of the recombinant protein. BALB/c mice inoculated with the recombinant protein raised specific anti-PstS antibodies that recongnized the native protein expressed in S. mutans strains. Identification and cloning of the Pst proteins will contribute to the understanding of the physiological role of the Pst system in the nutrition strategies and patogenicity of Streptococcus mutans.