TABLE 2.
Plasmids and hybrid proteins
| Plasmida | Terminus encodedb
|
Primersc | Junction sequenced | Complementatione | |
|---|---|---|---|---|---|
| Amino | Carboxyl | ||||
| pAG6 | Wild-type PBP 6 | Wild-type PBP 6 | |||
| pPJ5C | Wild-type PBP 5 | Wild-type PBP 5 | |||
| pAGP-7 | 1-47 PBP 5 | 48-378 PBP 6 | A, H, E, D | DPASLTK | − |
| pAGR-10 | 1-199 PBP 5 | 200-378 PBP 6 | A, I, F, D | NRNRLLW | − |
| pAGS-29 | 1-228 PBP 5 | 229-378 PBP 6 | A, J, G, D | AGYNLVASA | + |
| pAGE-1 | 1-47 PBP 6 | 48-374 PBP 5 | C, N, K, B | DPASLTK | + |
| pAGG-1 | 1-199 PBP 6 | 200-374 PBP 5 | C, O, L, B | NRNGLLW | + |
| pAGH-1 | 1-228 PBP 6 | 229-374 PBP 5 | C, P, M, B | AGYNLVASA | − |
Plasmids were constructed by cloning DNA fragments into the NheI and HindIII sites of pBAD18-CAM.
Amino acids and source of the protein encoded by each cloned DNA fragment. Each plasmid expressed a single hybrid PBP consisting of the segments indicated (those derived from PBP 5 are underlined). Numbers refer to the amino acid residues present in the mature protein and correspond to the numbering system used in the crystal structure of PBP 5 (9). Not included in the numbering scheme is the 29-amino-acid signal peptide that is removed from the mature PBP 5 protein. Likewise, the 27-residue signal peptide of PBP 6 is omitted in numbering the mature protein that corresponds to the crystal structure of PBP 5.
Oligonucleotide primers used to construct the two segments of each of the cloned DNA fragments are presented in the order P1, P2, P3, and P4, as described in Materials and Methods. The sequence of each oligonucleotide is given in Table 1.
Partial amino acid sequences (one-letter code) at the junction sites of the hybrid PBPs. Because the fusions were constructed at sequences that were identical or nearly identical between PBPs 5 and 6, no additional or foreign residues are present in the expressed proteins. The pAGR-10 junction is the PBP 6 sequence at that point, and the pAGG-1 junction is the PBP 5 sequence at that point. These two sequences differ only at residue 200 (R or G) (underlined).
Expression of the hybrid PBP did (+) or did not (−) return the aberrant morphology of CS703-1 to normal or near normal.