FIGURE 4. (A) CLSM images of S. aureus wildtype and S. aureus Δvwbp biofilms producing Coa:msfGFP. The composite image (left) is displayed along with the channel containing only signal from Coa:msfGFP (middle) and a zoomed in image of that channel (right). Coa:msfGFP localised to the surface of bacteria within the fibrin pseudocapsule. (B) The parental strains of S. aureus that produce unmodified Coa when imaged with the same imaging settings as modified bacteria producing Coa:msfGFP. No fluorescence was detected, which confirms that the fluorescence in Figure 4A originates from msfGFP and not from autofluorescence. (C) A double mutant lacking both Coa and vWbp (ΔcoaΔvwbp) forms no fibrin at all. Biofilms were grown in BHI containing 50% human plasma for 2 h. Bacteria (wildtype and Δvwbp) were visualised by staining with SYTO 41 (blue), fibrin was visualised by amending Alexa 647-conjugated fibrinogen to media (red), and Coa was visualised by fluorescence emitted from msfGFP (green). The double mutant expressed gfp from a plasmid pCM29 [54] (blue), and fibrin was also visualised by addition of Alexa-647-conjugated fibrinogen to the media (red). Brightness for each colour channel for each image were increased equally using Fiji ImageJ to visualise the data for both (A) and (B).

54. Qazi SNA, Rees CED, Mellits KH, and Hill PJ (2001). Development of gfp vectors for expression in Listeria monocytogenes and other low G+C gram positive bacteria. Microb Ecol 41(4): 301–309. 10.1007/s002480000091