There was no difference in biofilm formation when the strains were cultured with THY medium (data not shown). Together the results indicate that CodY has a relatively minor, but reproducible, influence

S. pyogenes biofilm formation under specific environmental conditions, perhaps due to changes in extracellular nuclease LY294002 in vitro activity. Figure 5 Static biofilm formation is diminished in the codY mutant when cultured with CDM. Biofilm formation was compared between the wild and codY mutant strains. The strains were cultured with CMD and static biofilm formation determined. An asterisk indicates the difference between the means is statistically significant (P < 0.05). Deletion of codY affects the production of a putative zinc permease and CAMP factor A constellation of at least four variants of the uncharacterized protein AdcA (proteins spots 7608, 8612, 8611, and 8610) was more abundant in supernatants from the codY mutant strain (Figure 3, Table 1). A significant difference in adcA transcripts was not previously identified using DNA microarrays in either the exponential or stationary phases of growth [23].

The predicted 515 amino acid protein (Spy49_0549) has a putative signal peptide, a histidine rich motif, and is annotated as a zinc binding transporter [25]. It is part of the TroA superfamily, the members of which are involved in the transport of zinc into the cytoplasm. An AdcA orthologue in Streptococcus Thiamine-diphosphate kinase pneumoniae is a Zn2+ permease involved in the development of natural competence for DNA transformation

[29, 30] and the orthologue in S. pneumoniae and S. gordonii AZD1152 supplier is required for both biofilm formation and competence [29–31]. We note that while AdcA was more abundant in the mutant strain, which did not form significant biofilms when cultured with CDM, the protein was detected in samples from the wild-type strain and thus production may have been sufficient to support biofilm production. In addition, two positional variants of CAMP factor (Cfa; 7311 and 8306) were less abundant in CSPs obtained from the codY mutant strain compared to the wild-type strain (Figure 3, Table 1). The results correlated with those obtained previously by measuring cfa transcripts [24]. Cfa is encoded as a 257 amino acid protein with a type II signal peptide. In a CAMP test, Cfa acts synergistically with the β hemolysin of Staphylococcus aureus to lyse CHIR98014 erythrocytes. The CAMP test was used to compare Cfa activity between the two strains and the results showed that deletion of codY decreased Cfa activity (Figure 6). While it remains possible that potential differences in growth between the two strains on blood agar plates may contribute to the difference in CAMP factor activity the results are consistent with those obtained with proteomic analyses (Figure 3) and those obtained previously by measuring transcripts [23, 24]. Figure 6 Decreased Cfa activity in the codY mutant. S.