Ed higher levels of extracellular nuclease. This data supports the hypothesis that there is a straindependent variation of the importance of eDNA as a component of the Pemafibrate chemical information biofilm matrix. Accumulation of extracellular DNA occurs through controlled cell death, regulated in S. aureus by the lysis-promoting cidABC operon and the lysisopposing lrgAB operon [98]. Maintaining a balance of this process is critical for biofilm development, as disruption of cidA resulted in reduced biofilm adherence, abnormal biofilm structure and reduced accumulation of extracellular DNA in the biofilm Metformin (hydrochloride)MedChemExpress Metformin (hydrochloride) matrix [61,62]. A lrgAB mutant, on the other hand, displayed enhanced adherence and greater accumulation of eDNA in the biofilm [61]. Extracellular nuclease activity also impacts accumulation of eDNA in S. aureus biofilms, as mutations of nuc1 and/or nuc2 have been shown to enhance biofilm formation in vitro, leading to thicker biofilms with alteredPLOS ONE | www.plosone.orgSwine MRSA Isolates form Robust BiofilmsFigure 8. Gene expression. Quantitative real-time PCR was used to determine mRNA expression of icaA, icaR, nuc1 and nuc2 in the indicated S. aureus strains relative to strain USA300. Each gene was normalized to the expression of the 16S rRNA and fold change is plotted as the mean of two experiments. Error bars represent the SEM.doi: 10.1371/journal.pone.0073376.gbiofilm architecture, and overexpression of nuc suppressed biofilm formation [61,71,72]. These results demonstrate that proper control of extracellular nuclease activity is important in development of normal biofilm structure. A biofilm is not a homogenous structure; localized microenvironments exist within the biofilm that result in subpopulations of bacterial cells expressing different physiological states [48,99?01]. As the biofilm grows and matures, distinct three-dimensional structural features develop, typically described as towers and channels. Formation of these structures has been linked to controlled cell death and lysis in a number of bacterial species and spatial and temporal regulation of cid and lrg expression has been demonstrated in S. aureus biofilms [55,102,103]. In S. aureus biofilms eDNA is predominately associated with the tower structures and mutations in cidA, lrgAB or nuc altered the distribution of eDNA throughout the biofilm [61,102]. The extracellular nuclease activity detected in our biofilm cultures may function alongside the cid/lrg system to modulate the accumulation of eDNA and help maintain proper biofilm structure.Different laboratories have reported conflicting results concerning the composition of the biofilm matrix and its sensitivity to various enzymatic treatments. In particular, the role of the PNAG polysaccharide has been disputed. Early investigations in S. aureus identified the presence of the ica locus and production of PNAG as crucial for biofilm formation [69]. Later work demonstrated the presence of proteins and eDNA in the S. aureus biofilm matrix [59,77,79,104]. The relative importance of these three factors, polysaccharide, protein and eDNA, has been a matter of some debate and has been shown to vary depending on the specific strains tested and the biofilm growth conditions. In particular, media composition appears to strongly influence the composition of the biofilm matrix [60,79,105]. For these experiments, we chose to focus on a single growth condition, using tryptic soy broth (TSB) supplemented with 0.5 glucose and 3 NaCl as the media and polyst.Ed higher levels of extracellular nuclease. This data supports the hypothesis that there is a straindependent variation of the importance of eDNA as a component of the biofilm matrix. Accumulation of extracellular DNA occurs through controlled cell death, regulated in S. aureus by the lysis-promoting cidABC operon and the lysisopposing lrgAB operon [98]. Maintaining a balance of this process is critical for biofilm development, as disruption of cidA resulted in reduced biofilm adherence, abnormal biofilm structure and reduced accumulation of extracellular DNA in the biofilm matrix [61,62]. A lrgAB mutant, on the other hand, displayed enhanced adherence and greater accumulation of eDNA in the biofilm [61]. Extracellular nuclease activity also impacts accumulation of eDNA in S. aureus biofilms, as mutations of nuc1 and/or nuc2 have been shown to enhance biofilm formation in vitro, leading to thicker biofilms with alteredPLOS ONE | www.plosone.orgSwine MRSA Isolates form Robust BiofilmsFigure 8. Gene expression. Quantitative real-time PCR was used to determine mRNA expression of icaA, icaR, nuc1 and nuc2 in the indicated S. aureus strains relative to strain USA300. Each gene was normalized to the expression of the 16S rRNA and fold change is plotted as the mean of two experiments. Error bars represent the SEM.doi: 10.1371/journal.pone.0073376.gbiofilm architecture, and overexpression of nuc suppressed biofilm formation [61,71,72]. These results demonstrate that proper control of extracellular nuclease activity is important in development of normal biofilm structure. A biofilm is not a homogenous structure; localized microenvironments exist within the biofilm that result in subpopulations of bacterial cells expressing different physiological states [48,99?01]. As the biofilm grows and matures, distinct three-dimensional structural features develop, typically described as towers and channels. Formation of these structures has been linked to controlled cell death and lysis in a number of bacterial species and spatial and temporal regulation of cid and lrg expression has been demonstrated in S. aureus biofilms [55,102,103]. In S. aureus biofilms eDNA is predominately associated with the tower structures and mutations in cidA, lrgAB or nuc altered the distribution of eDNA throughout the biofilm [61,102]. The extracellular nuclease activity detected in our biofilm cultures may function alongside the cid/lrg system to modulate the accumulation of eDNA and help maintain proper biofilm structure.Different laboratories have reported conflicting results concerning the composition of the biofilm matrix and its sensitivity to various enzymatic treatments. In particular, the role of the PNAG polysaccharide has been disputed. Early investigations in S. aureus identified the presence of the ica locus and production of PNAG as crucial for biofilm formation [69]. Later work demonstrated the presence of proteins and eDNA in the S. aureus biofilm matrix [59,77,79,104]. The relative importance of these three factors, polysaccharide, protein and eDNA, has been a matter of some debate and has been shown to vary depending on the specific strains tested and the biofilm growth conditions. In particular, media composition appears to strongly influence the composition of the biofilm matrix [60,79,105]. For these experiments, we chose to focus on a single growth condition, using tryptic soy broth (TSB) supplemented with 0.5 glucose and 3 NaCl as the media and polyst.