Abstract
Type 1 secretion systems (T1SS) of Gram-negative bacteria are responsible for the secretion of various proteases, lipases, S-layer proteins or toxins into the extracellular space. The paradigm of these systems is the hemolysin A (HlyA) T1SS of Escherichia coli. This multiple membrane protein complex is able to secrete the toxin HlyA in one step across both E. coli membranes. Common to all secreted T1SS substrates is a C-terminal secretion sequence being necessary as well as sufficient for secretion. However, it is not known whether transport occurs directionally, i.e. the N- or the C-terminus of T1SS substrates is secreted first. We have addressed this question by constructing HlyA fusions with the rapidly folding eGFP resulting in a stalled T1SS. Differential labeling and subsequent fluorescence microscopic detection of C- and N-terminal parts of the fusions allowed us to demonstrate vectorial transport of HlyA through the T1SS with the C-terminus appearing first outside the bacterial cells.
Highlights
Gram-negative bacteria transport a broad range of compounds ranging from small molecules to intact proteins into the extracellular space
Similar behavior has been observed for the Has secretion system where only unfolded HasA could be exported by its cognate ATP-binding cassette (ABC) transporter, whereas the presence of folded cytosolic HasA resulted in an inhibition of secretion of its unfolded isoform[29]
This assumption is in line with the fact that calcium ions are required for folding of hemolysin A (HlyA), which is prevented in the cytoplasm due to the low concentration of calcium ions[16,30,31]
Summary
Gram-negative bacteria transport a broad range of compounds ranging from small molecules to intact proteins into the extracellular space. Transport occurs via dedicated cellular nanomachineries either directly from the cytoplasm to the extracellular space or via periplasmic intermediates Among these nanomachineries, type 1 secretion systems (T1SS) adopt the most simple architecture consisting of an ATP-binding cassette (ABC) transporter and a membrane fusion protein (MFP) located in the inner membrane and an outer membrane protein (OMP). T1SS substrates include adenylate cyclases, lipases, proteases, surface layer proteins and toxins They vary in size from relatively small proteins such as the hemophore HasA (19 kDa, 188 amino acids) from S. marescens to large proteins of approximately 900 kDa (8682 amino acids) such as the adhesion factor LapA from P. fluorescence[3,4,5]. High levels of secretion were obtained with a fragment consisting of the 218 C-terminal amino acids of HlyA (HlyAc) containing three GG repeats and the secretion signal[17]
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