Infection of Chlorella N1a or NC64A by the algal viruses resulted in rapid lysis of the algal cells. Dr. Meints’ laboratory developed methods to isolate the lytic enzymes and to use the lysin preparation to produce algal protoplasts (cells without cell walls). The protoplasts could be used in studies of somatic cell fusion (genetic improvement by fusion of two individuals with useful traits such as pH tolerance and high lipid production). Alternatively, the protoplasts could be used as targets in a genetic transformation system in which DNA plasmids are taken up directly into cells without walls.

A crude lysin preparation was produced by infecting Chlorella N1a with PBCV -1. After several lytic cycles (approximately 24 hours), the sample was centrifuged to remove cell debris and virus. Initially, the supernatant from this preparation was used directly to produce protoplasts from Chlorella N1a cells. Alternatively, lysin activity was precipitated from the supernatant with 65% ammonium sulfate. Cells were exposed to lysin in the presence of 1 M sorbitol as an osmoticum. (One interesting result from the protoplast studies is that algal strains exhibited significant differences in their sensitivities to osmotica commonly used for higher plant cells; i. e. mannitol, but not sorbitol, was toxic to Chlorella N1a. The sensitivity of individual algal strains to different osmotica will need to be determined empirically.) The cell wall of algal cells exposed to lysin was rapidly degraded over the entire cell surface, as judged by electron microscopy and staining of the cells with calcofluor white, which stains plant cell walls. The lysin preparation could be purified further by exposing the crude sample to an affinity matrix composed of algal cell wall fragments. Lysin activity was eluted by salt washes. Exposure of Chlorella N1a cells to this lysin sample resulted in degradation of the algal wall at specific sites; when the osmoticum was reduced to half strength, the alga protoplast was released through discrete holes in the wall. This result suggested the presence of more than one enzymatic activity in the crude lysin preparation.

Somewhat surprisingly, the protoplasts did not lyse when transferred to water. However, exposure of lysin-treated cells, but not untreated cells, to low concentrations of detergent caused the release of chlorophyll. The amounts of chlorophyll released from lysin-treated cells was used as a measure of the extent of protoplast formation in a cell sample. The protoplasts were viable, as judged by staining with fluorescein diacetate. Unfortunately, although some regeneration of the cell wall occurred, the lysin-treated cells never formed new colonies. Attempts to use the viral-lysin to produce protoplasts from other microalgal strains met with little success. A manuscript was prepared and submitted to SERI that described the progress made on the use of lysin to produce algal protoplasts, but the article was never published in a technical journal.