Interestingly, it was recently reported that in MRSA S. aureus strains, biofilm development is promoted under mildly acidic growth conditions triggered by the addition of glucose to the growth medium. In this instance, it is tempting to speculate that local pH lowering resulting from sugar consumption on the cell surface may drive the Chlorhexidine hydrochloride formation of firmly adhering biofilm-like microbial communities. Third, many Gram positive pathogens modulate gene expression in response to pH changes. For instance, S. mutans survives and proliferates at low pH by up-regulating a number of genes that protect against acid stress. Moreover, microarray analysis revealed a differential expression of approximately 10 to 15% of the total number of genes in Staphylococcus aureus, S. pyogenes and S. agalactiae under mildly acidic conditions compared to neutral pH. Of note, no differences in pilus expression were detected in the S. pyogenes microarray, probably because the growth conditions investigated in that study did not lead to attain pH values under 5.4, as in our experiments. Interestingly, gene expression regulation at low versus neutral pH has been show to correlate with differential expression during exponential-phase planktonic cultures versus mature biofilm in S. aureus and, in the case of S. pyogenes, with differential gene expression during murine subcutaneous infection versus in vitro growth at neutral pH. The finding that biofilm formation was pH-dependent in some but not all GAS FCT-types, led us to further investigate whether this phenotype variation was associated with a difference in the expression of FCT-encoded genes. Indeed, Western Blot 
and RTPCR experiments revealed higher amounts of pilus proteins and fibronectin-binding adhesins in FCT-2, FCT-3 and FCT-4 M12 isolates when bacteria were grown at a starting pH of 6.4 as compared to growth at pH 7.5. In contrast, pH-independent pilus expression was observed in the case of an FCT-1 strain. Concerning pilus expression in non biofilm former strains, we observed pH-dependent expression in FCT-4 M28 as well as in M89 strains. The data suggest that M28 strains may lack a still undefined factor required, in addition to the pili, for biofilm formation. The same could be true for M89 strains, although in this case the incapacity to form biofilm could also be associated to low pilus expression. Therefore, pili appear to be necessary but not sufficient for GAS biofilm formation, confirming previous literature data indicating that this mode of growth relies on multiple bacterial components. Finally, constitutive expression of GAS pili in a FCT-3 recombinant strain resulted in pH-independent biofilm formation, in contrast to the results obtained with a recombinant strain constitutively expressing the F2 protein. This confirmed GAS pili and not F2 proteins to be directly involved in pH-dependent autoaggregation and biofilm formation on biotic and abiotic surfaces. Besides genes encoding fibronectin-binding adhesins and the pilus machinery, all FCT genomic regions contain genes coding for stand alone transcription regulators. These regulators, the RofA/Nra homologues and MsmR, coordinately control the expression of the FCT open reading frames and of important virulence factors outside this region, during different bacterial growth phases. Further on, they are involved in the response to changes of environmental parameters like oxygen pressure and temperature. Our results indicate that the expression of the FCT transcription regulators is affected by pH. In fact, in the analyzed FCT-3 M3 strain, msmR transcript amounts increased at lower pH, in parallel to the enhanced expression of pili and the F2 protein. MsmR and Nra participate in the fine tuning of FCT-3 pilus expression by exerting their function in Benzethonium Chloride opposite directions and have been shown to act either as positive or negative regulators, depending on the strain genetic background. In particular, in a FCT-3 M49 strain, expression of pili and the F2 protein were shown to be repressed by Nra and activated by MsmR.