Figure 1.

Identifying the Newly Synthesized Proteins in the Course of Spore Revival

(A) Schematic description of the BONCAT strategy (Dieterich et al., 2007) for labeling, detection, and identification of the newly synthesized proteins tagged with AHA during spore revival. The general flow of the procedure (left) and detailed illustration of specific steps (right) are shown. Spores of LS5 (ΔmetE) strain are incubated in revival medium containing AHA (red hexagons), and the newly synthesized proteins (red curved lines) are separated from the existing spore proteins (blue curved lines). Spore proteins are extracted and tagged with TAP tag (green circles) using click chemistry (1, right). Next, newly produced proteins are isolated using avidin beads and identified by mass spectrometry (2, right). Scale bar represents 1 μm.

(B) Spores of LS5 (ΔmetE) strain were incubated in revival medium in which methionine was replaced by AHA. The sequence of events during revival is shown as captured by phase contrast images: germination is visualized as the loss of spore brightness (red line), the ripening period in which no morphological change is evident (orange line), and outgrowth is characterized by increasing cell length (green line). The average length of the reviving spores is presented below. Scale bar represents 1 μm.

(C) Spores of LS5 (ΔmetE) strain were incubated in revival medium and optical density (OD₆₀₀) was measured at the indicated time points. Data are presented as a fraction of the initial OD₆₀₀ of the phase-bright spores. Decreasing OD₆₀₀ signifies spore germination, and increasing OD₆₀₀ indicates spore outgrowth (Moir and Smith, 1990).

(D) Dot blot analysis of protein samples (in duplicates) collected throughout revival of LS5 (ΔmetE) spores at the indicated time points. Samples were diluted (1:100 and 1:200) and spotted on a membrane that was subsequently probed with anti-biotin antibodies. The obtained signal was compared with known amounts of biotinylated BSA.