In vivo quantification of the secretion rates of the hemolysin A Type I secretion system

Abstract

Type 1 secretion systems (T1SS) of Gram-negative bacteria secrete a broad range of substrates into the extracellular space. Common to all substrates is a C-terminal secretion sequence and nonapeptide repeats in the C-terminal part that bind Ca(2+) in the extracellular space, to trigger protein folding. Like all T1SS, the hemolysin A (HlyA) T1SS of Escherichia coli consists of an ABC transporter, a membrane fusion protein and an outer membrane protein allowing the one step translocation of the substrate across both membranes. Here, we analyzed the secretion rate of the HlyA T1SS. Our results demonstrate that the rate is independent of substrate-size and operates at a speed of approximately 16 amino acids per transporter per second. We also demonstrate that the rate is independent of the extracellular Ca(2+) concentration raising the question of the driving force of substrate secretion by T1SS in general.

Figure 1. Total cell fluorescence of Cy3.

Immunofluorescent labeled E. coli cells expressing eGFP-HlyAc in the presence (left bar) and absence (right bar) of HlyB and HlyD, respectively. Error bars represent the standard deviation of the Cy3 fluorescence of at least three biological replicates.

Figure 2. HlyB and HlyD expression levels.

Western blot analysis of E. coli cells demonstrated that the expression levels of HlyB and HlyD were equal for cells expressing and/or secreting either eGFP-HlyAc, HlyA or HlyAc.

Figure 3. Detection of the surface exposed HlyA fragment of eGFP-HlyA by confocal laser scanning microscopy.

E. coli cells expressing eGFP-HlyA, HlyD, HlyB or HlyB-H662A in different combinations of the proteins. Shown is the eGFP fluorescence (left panel) of the fusion proteins, the HlyA mediated Cy3 fluorescence at the cell surface (second left panel), merged images of eGFP and Cy3 fluorescence (second right panel) and differential interference contrast (DIC) images of the cells (right panel). The different combinations of proteins employed are indicated to the left.

Figure 4. Relative amount of secreted HlyA and HlyAc in the presence of different extracellular Ca²⁺ concentrations.

The relative amount of secreted HlyA (a) and HlyAc (b) in the presence of different extracellular Ca²⁺ concentration after 2 h (dark grey bars) and 4 h (light grey bars) is summarized. Values are normalized to the highest mean value of secreted HlyA (a) or HlyAc (b) after 4 h of secretion. Error bars represent the standard deviation of at least three biological replicates.

Figure 5. The secretion rate of HlyA and HlyAc is independent of the extracellular Ca²⁺ concentration.

Determined secretion rates of HlyA (blue cubes) and HlyAc (black dots) in the presence of different Ca²⁺ concentration. Shown are the mean secretion rates of triplicate secretion experiments as amino acids (aa) T1SS⁻¹ s⁻¹. Error bars represent the standard deviation of at least three biological replicates.