Supplementary Material for: Manganese Affects <i>Streptococcus mutans</i> Virulence Gene Expression

<i>Background/Aims:</i> Studies of trace metals in drinking water and tooth enamel have suggested a caries-promoting potential for manganese (Mn). Additionally, Mn has been shown to be essential for the expression of mutans streptococci virulence factors such as the glucan-binding lectin (GBL) of <i>Streptococcus sobrinus</i>. The <i>Streptococcus mutans</i> glucan-binding protein (Gbp) GbpC is the functional analogue of the <i>S. sobrinus</i> GBL. <i>S. mutans</i> Gbps have been shown to contribute to biofilm architecture and virulence. This study was undertaken to examine the effects of Mn on the transcription of genes encoding <i>S. mutans</i> Gbps, including <i>gbpC</i>, along with other critical <i>S. mutans</i> virulence genes. <i>Methods:</i> Microarray analyses suggested the potential for an Mn effect on Gbp genes. Further investigation of the Mn effects on selected genes was undertaken by performing Northern blots, Western blots, and RT-PCR under conditions of planktonic and biofilm growth in Mn-depleted media or in media containing 50 µ<i>M</i> Mn. <i>Results:</i> Mn resulted in increased expression of <i>gbpC</i> and <i>gtfB</i>, and decreased expression of <i>wapA</i>, in both planktonic and biofilm cultures. The expression levels of <i>gbpA</i> and <i>gbpD</i> were also decreased in the presence of Mn, but only in biofilms. The expression of <i>gtfC</i> was increased in the presence of Mn only in planktonic cultures. The <i>spaP</i> gene was expressed more highly in Mn-supplemented planktonic cultures but less in Mn-supplemented biofilms. <i>Conclusion:</i> Mn availability affects the expression of multiple <i>S. mutans</i> genes involved in adhesion and biofilm formation. Furthermore, these effects depend on the growth state of the organism.