Preparation of PduA-Specific Antiserum

      To determine the specificity of the polyclonal antiserum generated against the His6PduA protein, western blots were performed on boiled cell lysates. Anti-PduA polyclonal antiserum recognized a major protein band at 9.5 kDa in the wild type strain but not in strain BE182 (which contains a nonpolarpduA deletion mutation) (Figure 33A, lanes 1 and 2). This indicated that the band at 9.5 kDa corresponded to the native PduA protein. Anti-PduA antiserum also recognized a protein band at 9.5 kDa in strain BE233 (Figure 3-3A, lane 4) which carries a plasmid with thepduA gene under control of an IPTG inducible promoter but lacks the entirepdu operon due to a deletion mutation. No band at 9.5 kDa was observed in an isogenic strain carrying the same plasmid without an insert (BE232) (Figure 3-3A, lane 3). These results confirmed that the band at 9.5 kDa was the native PduA protein.

      The anti-PduA antiserum preparation also reacted with an 8 kDa protein

expressed by the wild type strain and BE182 (Figure 3-3A, lanes 1 and 2), and a 7.5 kDa protein band expressed by BE233 and BE232 (Figure 3-3A, lanes 3 and 4). Additional western blots of strain BE6 apdu operon deletion mutant and strain BE23 1, which overexpresses a PduJ-His8 fusion protein, indicated that this 8 kDa protein was the PduJ protein (Figure 3-3C, lane 3). The observed 7.5 kDa protein band was apparently plasmid encoded as this band was detected in western blots of cells carrying plasmid pTA749, but not in blots of an isogenic strain (BE6) that lacked this plasmid (Figure 33C, lanes 1 and 2). In order to improve specificity of the polyclonal antiserum preparation an adsorption procedure was performed.

      An acetone powder was made from whole cells of strain BE23 1, a derivative of BL21 (DE3) RIL that expressed PduJ at high levels, and then used to absorb antibodies