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Figure 1 | BMC Microbiology

Figure 1

From: Directed evolution and targeted mutagenesis to murinize listeria monocytogenes internalin A for enhanced infectivity in the murine oral infection model

Figure 1

Nisin inducibe InlA plasmid constructs and the expression of InlAWT on the surface of L. lactis. A. Lactococcal nisin inducible plasmid pNZB with the entire (i) inlAWT gene from EGD-e cloned upstream of the nisin inducible nisA promoter (P). The labels for the InlA domains are described in the introduction text. The naturally occuring Bgl II/Bst XI restriction sites within the inlA gene encompass the entire LRR required for the interaction with E-cadherin. These two sites were used for the cloning of the (ii) the murinized version of inlA (inlAm*) with the amino acid changes as described by Wollert et al. [17] (created by site directed mutagenesis) and (iii) four randomly mutated banks of inlA generated by error prone PCR. B. A lysozyme cell wall extract was isolated from L. lactis InlAWT grown under the conditions used for invasion assay. Exponential phase cells (OD = 0.6) were cultured for 1 h in the presence (+) or absence (-) of nisin (approx 10 ng/ul). Proteins were run on 10% SDS-PAGE and either stained with coomassie blue or blotted and detected with a InlA monoclonal antibody [23].

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