TABLE 2.
ClyA subcellular protein localization and cytolytic expression
| Strain/plasmid | Relevant characteristics or alteration in ClyA amino acid sequencesa | Subcellular localizationb
|
Hemolysisc | Hemolysis when coexpressed with wild-type ClyAd | ||
|---|---|---|---|---|---|---|
| P | C | M | ||||
| MWK11 | Consensus CRP binding site in the clyA promoter regions ClyA+ | ++ | + | (+) | ++ | NAe |
| YMZ19 | clyA::kan ClyA− | − | − | − | − | NA |
| BEU616 | hns::cat | ++ | (+) | + | + | NA |
| MC1061/pYMZ80 | (1-266)-DDLMLSLLKEAAKKMINTCNEYQKRHGKKTLFFVPEV (wild type) | +++ | + | (+) | +++ | NA |
| MC1061/pJON63 | (1-266)-DDLMLSLLKEAAKKMINTCNEYQKRD (Δ293-303) | − | +++ | + | − | − |
| MC1061/pSNW168 | (1-266)-DDLMLSLLKEAAKK (Δ281-303) | − | − | +++ | − | − |
| MC1061/pJON66 | (1-266)-DDLMLSLLKEAPQK (Δ281-303) | − | + | +++ | − | − |
| MC1061/pJON70 | (1-182)-(A183D G184D)-(185-303) | ++ | + | + | − | (+) |
| MC1061/pMWK16 | (1-182)-Δ(AGVV)-(187-303) | + | +++ | ++ | − | − |
The wild-type and mutant variants of ClyA were expressed from plasmid-carried clyA alleles. Amino acid alterations in comparison with the wild-type ClyA protein are indicated by boldface.
Subcellular localization was monitored by immunoblot analysis of fractions (P, periplasm; C, cytoplasm; M, membrane) prepared as described in Materials and Methods (see also Fig. 1). Western blot signals, which reflect the relative amount of ClyA present in subcellular fractions, are reported as +++ (very strong), ++ (strong), + (moderately strong), (+) (weak), or − (none).
Hemolytic activity was scored on blood agar plates. Relative activity is reported as +++ (very strong), ++ (strong), + (moderately strong), (+) (weak), or − (none).
The dominant negative effect of different plasmid-encoded ClyA variants on expression of the hemolytic phenotype of wild-type ClyA was tested by use of the ClyA+ strains BEU616 and MWK11.
NA, not applicable.