Table 3. Construction and characterization of BacA-BlaM hybrid proteins.
Truncated forms of the bacA gene were amplified by PCR by using the forward primer bacA-for and one of the reverse primers listed here whose sequences are reported in S1 Table. These fragments were inserted into the pNF150 vector and the resulting plasmids were transformed in the DH5α strain. The levels of C55-PP phosphatase and β-lactamase activities detected in membrane extracts of these transformants, as well as their susceptibility to ampicillin, were then determined.
| Plasmid | 3’end primer a | Fusion site b | Ampicillin resistance c | C55-PP phosphatase activity d | β-lactamase activity e |
|---|---|---|---|---|---|
| pNF150 vector | - | - | S | 0.32 | ND |
| pB42TEM | bacA-G42 | Gly42 | R | 0.35 | 185 |
| pB88TEM | bacA-G88 | Gly88 | S | 0.39 | 203 |
| pB110TEM | bacA-H110 | His110 | R | 0.34 | 114 |
| pB140TEM | bacA-K140 | Lys140 | S | 0.36 | 321 |
| pB143TEM | bacA-E143 | Glu143 | S | 0.38 | 253 |
| pB144TEM | bacA-P144 | Pro144 | S | 0.31 | 715 |
| pB176TEM | bacA-G176 | Gly176 | R | 0.38 | 103 |
| pB191TEM | bacA-A191 | Ala191 | R | 0.39 | 32 |
| pB194TEM | bacA-E194 | Glu194 | R | 0.33 | 68 |
| pB215TEM | bacA-S215 | Ser215 | R | 0.30 | 43 |
| pB244TEM | bacA-T244 | Thr244 | S | 0.33 | 85 |
| pB251TEM | bacA-R251 | Arg251 | S | 0.39 | 126 |
| pBacATEM | bacA-F273 | Phe273 | R | 12.44 | 1563 |
| pNF150::bacA | - | - | S | 8.95 | ND |
a The sequences of oligonucleotides are shown in S1 Table.
b The fusion site corresponds to the C-terminal amino acid residue of the truncated BacA protein that has been fused to the mature form of β-lactamase via an intermediate peptide linker AVPHAISSSPLR originating from the pNF150 vector sequence.
c DH5α strains carrying these different BacA-BlaM fusion-expressing plasmids were grown in 2YT- kanamycin liquid medium and dilutions (ca. 200 cells) were spread onto 2YT plates containing or not ampicillin at 100 μg.ml-1. Their resistance (R) or sensitivity (S) towards ampicillin was judged after 24 h of incubation at 37°C.
d The total C55-PP phosphatase activity detected in membrane extracts of DH5α cells carrying these plasmids was determined as described in Materials and Methods and is expressed in nmol.min-1.mg-1 protein.
e The β-lactamase activity detected in membrane extracts of DH5α cells carrying these plasmids was determined as described in Materials and Methods and is expressed in units.mg−1 protein. ND, not detected.