Table 1. Topological location of selected WcaJ amino acid residues based on reporter fusions and PEGylation.
| Amino Acid position | Reporter fusion | Plate assay | Miller Units | PEGylation | Location |
|---|---|---|---|---|---|
| V39 | PhoA | + | 2112 ± 95 | OUT | PL1 |
| V39 | LacZ | − | UD | ||
| G74 | PhoA | − | 4.0 ± 2 | IN | CL1 |
| G74 | LacZ | + | 0.2 ± 0.1 | ||
| D106 | PhoA | + | 938 ± 70 | OUT | PL2 |
| D106 | LacZ | − | UD | ||
| S120 | PhoA | + | 97 ± 2 | TM | TMIV |
| S120 | LacZ | + | UD | ||
| A134 | PhoA | − | 15 ± 1 | IN | CL2 |
| A134 | LacZ | − | UD | ||
| N143 | PhoA | − | 15 ± 4 | IN | CL2 |
| N143 | LacZ | + | 1 ± 0.2 | ||
| M160 | PhoA | − | 13 ± 3 | IN | CL2 |
| M160 | LacZ | + | 1 ± 0.2 | ||
| P180 | PhoA | − | UD | IN | CL2 |
| P180 | LacZ | − | 0.3 ± 0.1 | ||
| L223 | PhoA | − | UD | IN | CL2 |
| L223 | LacZ | + | 65.8 ± 3.2 | ||
| N254 | PhoA | − | U.D. | IN | CL2 |
| N254 | LacZ | + | 15 ± 0.6 | ||
| L272 | PhoA | − | UD | BORDER | CL2 |
| L272 | LacZ | + | 22 ± 0.4 | ||
| A276 | ND | ND | ND | TM | TMV |
| A282 | ND | ND | ND | TM | TMV |
| P291 | ND | ND | ND | UD | ? |
| C294 | ND | ND | ND | TM | TMV |
| K301 | PhoA | + | 611 ± 44 | IN | CT |
| K301 | LacZ | + | 5 ± 0.5 | ||
| S303 | ND | ND | ND | BORDER IN | CT |
| G316 | PhoA | − | UD | IN | CT |
| G316 | LacZ | + | 13 ± 1 | ||
| S360 | ND | ND | ND | IN | CT |
WcaJ-LacZ/PhoA fusion proteins were expressed in E. coli DH5α or CC118 (see Methods). Blue colonies were assessed on plates containing XGal or XP. Alkaline phosphatase and β-galactosidase activities were determined (in triplicate) by measuring the hydrolysis of p-nitrophenyl phosphate and ortho-nitrophenyl-β-galactoside, respectively, and the results calculated as Miller units (mean ± standard deviation). PEGylation of WcaJCysless with ectopic cysteine residues at the indicated positions was done as described in Methods (see also Fig. 5). UD, undetectable; ND, not done; ?, location could not be established. PL, periplasmic loop; CL, cytoplasmic loop; CT, C-terminal domain.