Assembly of the Type II Secretion System such as Found in Vibrio cholerae Depends on the Novel Pilotin AspS
(A) The conserved domain architecture tool (CDART) was used to map the regions of PulS from Klebsiella oxytoca 10-5250 (EHT07154.1), OutS from Dickeya dadantii 3937 (YP_003883937.1), EtpO from Shiga toxin-producing E. coli (STEC) O157:H7 (CAA70966.1) and YacC (CAS07673.1) from EPEC. Numbers refer to the amino acids of each protein sequence, and the broken blue bar denotes that the N-terminal and C-terminal residues of YacC diverge from the consensus features of the PulS-OutS family. Pairwise sequence alignment over 80 residues showing similarity of the previously characterized EtpO (CAA70966.1) and YacC (CAS07673.1). Identical residues are highlighted between the two sequences, and conserved substitutions are shown (+). (B) CLANS analysis graphically depicts homology in large datasets of proteins, utilizing all-against-all pairwise BLAST to cluster representations (colored dots) of individual protein sequences in three-dimensional space. Lines are shown between the most similar sequences, with an E-value cut-off of 1e−5. The analysis shows that proteins from diverse species cluster into two groups: the PulS/OutS group and YacC-related proteins (blue), and the AspS-related proteins (red). There are numerous relationships between the PulS/OutS proteins and YacC proteins, but no relationship links these to the AspS group of proteins. (C) Wild-type EPEC (WT), and the indicated mutants of EPEC were grown in culture and post-cell supernatants containing secreted proteins (200 µg of protein) were analyzed by SDS-PAGE and Coomassie blue staining. Mass spectrometry was used to identify SslE, FliC and EspC, consistent with a previous study .