Strains used in this study.

<p>Strains used in this study.</p

Production of extracellular nuclease.

<p>Results were assessed after overnight incubation of the indicated strains overnight on DNase test agar plates. Numbers refer to UAMS strain designations (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003361#pone-0003361-t001" target="_blank">Table 1</a>).</p

Effect of <i>alsSD</i> and <i>sarA</i> mutations on biofilm formation.

<p>Biofilm formation was assessed using the microtiter plate assay. Results represent the mean and standard deviation of 24 replicates. Strain designations are the same as those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003361#pone-0003361-g001" target="_blank">Figs. 1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003361#pone-0003361-g002" target="_blank"></a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003361#pone-0003361-g003" target="_blank">3</a> with the addition of the <i>sarA/alsSD</i> double mutant (UAMS-1300).</p

Zymogram analysis of protease production in the presence of protease inhibitors.

<p>Analysis of the production of specific proteases was assessed using casein (upper panel) and gelatin gels (lower panel) in UAMS-1 (WT) and its UAMS-929 <i>sarA</i> mutant. Analysis was done after growth in biofilm medium in the absence of any inhibitor (Φ), the presence of the inhibitor cocktail (CT), or the presence of each inhibitor alone and in combination with each other. Inhibitor designations are the same as in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003361#pone-0003361-g010" target="_blank">Fig. 10</a>. The presumed identity of individual proteases is indicated to the right. In the case of SspA, this was confirmed by analysis of the corresponding <i>sarA/sspA</i> mutant (data not shown). In the case of ScpA and SspB, identification is presumptive based on their identity as cysteine proteases, inhibition of both by E-64, and the fact that ScpA has greater activity than SspB on casein while the opposite is true on gelatin (Dr. Jan Potempa, personal communication).</p

Biofilm formation in the presence of individual inhibitors alone and in combination with each other.

<p>Biofilm formation in the UAMS-929 <i>sarA</i> mutant was assessed in the absence of protease inhibitors (Φ), the presence of the protease inhibitor cocktail (CT), or in presence of individual inhibitors alone and in paired combinations with each other. Inhibitor designations are E-64 (E), DIC (D) and phenanthroline (P). Results represent the mean±standard deviation of 24 replicates.</p

Effect of <i>alsSD</i> and <i>sarA</i> mutations on stationary phase survival.

<p>Each strain was grown in the presence of 35 mM glucose. Aliquots were removed at the indicated times to assess the number of colony-forming units (CFU) per ml (panel A) and culture density (panel B). Strain designations: UAMS-1 (▪), UAMS-929 (▴), UAMS-969 (•), UAMS-1489 (X), UAMS-1551 (♦).</p

Primers used in this study.

<p>Primers used in this study.</p

Effect of protease inhibitors on biofilm formation.

<p>Panel A: The microtiter plate assay of <i>in vitro</i> biofilm formation was performed with bacteria grown in biofilm medium (gray) or biofilm medium containing the inhibitor cocktail (black). Results represent the mean±standard deviation of 24 replicates. Panel B: Results of the biofilm assay shown in panel A represented as the ratio of biofilm formation in the presence of the inhibitor cocktail/biofilm formation in the absence of inhibitors.</p

Effect of protease inhibitors on growth.

<p>Growth of the UAMS-929 <i>sarA</i> mutant in biofilm medium without protease inhibitors (♦) was compared to growth in the same medium containing 1 mM DIC (▴), 1 mM E-64 (•), 10 µM 1,10-phenanthroline (+) or a cocktail containing all three inhibitors (X). Growth was monitored at hourly intervals for 10 hours. Results indicate the OD₆₀₀±standard deviation of 3 replicates.</p

Effect of nuclease on biofilm formation.

<p>Biofilm formation was assessed using the microtiter plate assay. Results represent the mean±standard deviation of 24 replicates. Strains designations are the same as in previous figures with the addition of UAMS-1725 and UAMS-1726, which are the <i>sarA/nuc</i> mutant complemented with <i>nuc</i> and <i>sarA</i> respectively.</p