Distribution of SecA in <i>S.</i><i>agalactiae</i> NEM316.

Abstract

<p>Bacteria grown overnight in 10 ml of TH (OD<sub>600</sub>≈2) were diluted to get an initial OD<sub>600</sub> of 0.05 (1/40 dilution) and grown at 37°C until OD<sub>600</sub> reached 0.5 and re-diluted again in TH (1/10) until reaching an OD<sub>600</sub> of 0.5 and diluted again (1/10) before final collection at mid-exponential phase (OD<sub>600</sub> of 0.3) to get a homogenous population of exponentially growing cells. Bacteria were pretreated with lysozyme (1 mg/mL final concentration) for 15 min at 37°C and then permeabilized with PBS-Triton X-100 (0.4%) for 5 min at RT, washed twice with PBS and then fixed with PBS containing 3% paraformaldehyde for 15 min at RT. (A) Differential interference contrast (DIC) and immunofluorescence microscopy (IFM) of bacteria harvested in mid-exponential phase and visualized with rabbit anti-SecA pAb (red) or fluorescent vancomycin (green) plus rabbit anti-SecA pAb (red). White arrows in the last panel indicate potential constriction septa. Image representative of at least 800 GBS chains analyzed (B) Deconvolution images of sequential z-sections (0.3 µm) of NEM316 cells labeled with rabbit anti-SecA pAb presented as maximum intensity projections.</p

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