Glucose-induced activation of H+-ATPase in Dunaliella salina and its role in hygromycin resistance

Dunaliella salina, a eukaryotic microalga, is
known for its highly halophilic nature. The high level of
salts in growth medium for this alga has made its genetic
transformation a comparatively difficult procedure, particularly
during the selection stage. The high salt content
decreases the efficiency of most antibiotics which are being
used as selection markers. Studies pertaining to the interrelationship
between salt concentration and antibiotic sensitivity
are scarce in Dunaliella. During our previous experiment
at genetic transformation of Dunaliella, an inverse relationship
between the amount of antibiotic hygromycin and sodium
chloride in the medium was revealed. A possible link
between plasma membrane activity and the hygromycin sensitivity
was investigated in the present study by modulating
plasma membrane H+-ATPase activity using glucose.
Glucose-induced activation of H+-ATPase, reduced the tolerance
of D. salina to the antibiotic hygromycin. Hygromycin
concentration required for selection during genetic transformation
of Dunaliella was lowered from 100 to 25 mg L−1 in
the presence of 10 mM glucose. Conversely, the inhibitors of
the plasma membrane H+-ATPase, orthovanadate and diethylstilbestrol
were found to inhibit the glucose activation at
concentrations of 10 and 15 μM, respectively. The activation
of H+-ATPase by glucose was further confirmed through H+-
ATPase assay and medium acidification experiments. The
results indicated that the sensitivity of Dunaliella to antibiotic
is related to H+-ATPase and the possible involvement of pH
gradient, created through H+-ATPase activation during drug
transport.