pC-pep was constructed to model the sequence in the native genome but deleting most of the structural adhesin gene. Amplified by PCR, the construct contained, in order, the predicted promoter region of sspA, the signal sequence, the coding region for the first eight amino acids of the mature SspA, the four amino acids preceding the LPXTG motif, the LPXTG motif, the sequence encoding C-pep, and the stop codon. The oligonucleotides JKR1-B and JKR1-Sup introduced restriction enzyme cleavage sites for Bam HI and Sal I into the amplified promoter-signal peptide fragment. Similarly, the PCR primers JKR1-Sdown and JKR1-H introduced restriction enzyme cleavage sites for Hind III and Sal I into the amplified fragment encoding LPXTG–C-pep. Each fragment was further cloned into pGEM-T (Promega, Madison, WI) and sequenced. For genes without a homologous partner, sgo_0707 and pavB (sgo_1182), we deleted the nucleotide region between the signal peptide sequence and the LPXTG motif using the protocol for whole gene deletion as above, creating strains SGO_0707SLC and PavBSLC.

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