Fig. 1.

Amidase domains and plasmid and phage inserts.

A. Signal sequences of E. coli periplasmic murein amidases AmiA, B and C (SWISSPROT accession P36548, P26365 and Q46929), as predicted by signalp version 1.1 (Nielsen et al., 1997). Potential Tat-targeting motifs (Berks et al., 2000; DeLisa et al., 2002) in AmiA and AmiC are underlined.

B–D. Domain organization of AmiA (B), AmiC (C) and AmiB (D), plasmid and phage inserts and summary of functionality and sublocalization studies. Predicted signal sequences (SS), septal targeting (T) and catalytic (C) domains, relevant residue numbers and the approximate position of a native amiC promoter are indicated in the diagrams. Plasmid and phage inserts (solid lines), the lac promoter (arrows), gfpmut2 (boxes) and the presence of a non-native ribosome binding site (*) are indicated below the diagrams. The amidase domains were drawn based on the Pfam database (Bateman et al., 2002). Columns on the right-hand side summarize the functionality and sublocalization studies. TB53 [ΔamiA ΔamiC] cells carrying the indicated construct were grown in the presence of 0, 50, 100, 250 and 500 μM IPTG, and their division phenotypes (Phen.) were examined. ccc, severe chaining phenotype, over 80% of cells in chains of 4–14 units; c, mild chaining phenotype, 10–30% of cells in chains of 4–6 units; wt, wild type, less than 5% of cells in chains. Minimum IPTG concentrations (in μM) required to attain the indicated phenotype were: 0 (pTB27, λTB27, pTB39), 50 (pTB28, pTB41), 100 (λTB28), 250 (pTB32) and 500 (pTB33). Plasmids pTB34 and pTB37 failed to suppress chaining at all concentrations. The localization patterns (Loc.) of the fusion proteins are given in the second column: P, peripheral in all cells; P/R, accumulated at the septal ring in constricting cells, peripheral otherwise; ?, cytoplasmic fluorescence but localization unreliable because of excessive degradation of the fusion protein.