Computational protocol: Massive Expansion of Ubiquitination-Related Gene Families within the Chlamydiae

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Protocol publication

[…] The proteome of Protochlamydia was used as query (tBLASTn) against the intergenic regions. The hits were conservatively filtered based on size (>50 aa), bitscore (>50), and E value (<10−10). For each intergenic region when two query proteins overlap, only the best hit was considered. In addition, if different parts of the same protein had hits in one intergenic space only, the top scoring (bitscore) was considered. Putative domains in the intergenic regions were detected using InterProScan 5 (). [...] For each domain of interest, we downloaded the Pfam HMM model () and scanned all chlamydial proteomes using the hmmscan program from the HMMER3 suite (). In the case of the F-box and F-box-like domains, the results were combined into a nonredundant list. The data for the other taxa were obtained through the Pfam website under the species distribution tab for each domain or clan as in the case for the F-box. The networks were created using the “arcdiagram” package in R. [...] Fusion proteins containing the 5′-part of the genes of interest (including the first 20 codons) and the adenylate cyclase Cya were expressed in Sh. Flexneri SF401 and SF620, derivatives of the wild-type strain M90T, in which the mxiD and ipaB genes have been inactivated (). The 5′-part of the target genes were amplified by polymerase chain reaction and cloned in the puc19cya vector as described (). Secretion assays were performed on 30 ml of exponentially grown cultures as described previously (). Antibodies against CRP, a cytosolic marker, were used to estimate the contamination of supernatant fractions with bacterial proteins as a result of bacterial lysis. Antibodies against IpaD, a type-III-secreted protein of Shigella, were used to verify that type III secretion occurred normally in the transformed strains. A monoclonal antibody against Cya and polyclonal antibodies against CRP and IpaD were generously provided by Drs N. Guiso, A. Ullmann, and C. Parsot, respectively (Institut Pasteur, Paris). Prediction of type III secretion was performed via the Effective database () and the PSORTb webserver (). […]

Pipeline specifications

Software tools TBLASTN, InterProScan, HMMER hmmscan, HMMER, PSORTb
Databases Pfam
Applications Protein sequence analysis, Amino acid sequence alignment
Organisms Bacteria, Escherichia coli