Computational protocol: Identification of the Genes Involved in Riemerella anatipestifer Biofilm Formation by Random Transposon Mutagenesis

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Protocol publication

[…] The genomic DNA of the mutant strains was extracted with a TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit (TaKaRa, Dalian, China). The amplification of the DNA region at the site of Tn4351 insertion was performed using either inverse PCR or genomic walking. Inverse PCR was performed as described previously . Briefly, the genomic DNA was digested with Hind III and then re-ligated, which resulted in the formation of circular molecules. Pairs of primers specific for Tn4351 (primer 340 and primer 341; primer TN-1 and primer IS4351-F) were used to amplify the sequences adjacent to the insertion site using a TaKaRa LA PCR kit (TaKaRa, Dalian, China). Genomic walking was performed with a genomic walking kit (TaKaRa) using a variety of arbitrary primers (AP1, AP2, AP3, AP4) provided in the kit and three specific primers (SP1, SP2 and SP3), according to the manufacturer’s instructions.Each mutated gene was PCR amplified. The PCR products were cloned into the pGEM®-T easy vector (Promega, Madison, WI, USA). DNA sequencing was performed on an Applied Biosystems DNA sequencer (ABI PRISM 3730) by Invitrogen Co, Ltd (Invitrogen, Shanghai, China). The sequences of the identified genes were searched for on the BLASTX server (http://www.ncbi.nlm.nih.gov/BLASTX/) to find homologous sequences and putative functions, and searched for using the online software PSORT v.3.0 (http://www.psort.org/) to predict the subcellular localization of the proteins. Functional characterization of the proteins was predicted using the online software COGnitor (http://www.ncbi.nlm.nih.gov/COG/old/xognitor.html), by comparison of the sequence to the Clusters of Orthologous Groups of proteins (COGs) database, which is based on COG functional categories. The sequences of genes that were not found in the genome of R. anatipestifer DSM15868 (accession number: CP002346), but were present in that of strain CH3, were submitted to GenBank. […]

Pipeline specifications

Software tools BLASTX, PSORT
Databases COGs
Application Protein sequence analysis
Organisms Anas platyrhynchos, Riemerella anatipestifer
Diseases Communicable Diseases