Involvement of an extracellular protease in algicidal activity of the marine bacterium Pseudoalteromonas sp strain A28

The marine bacterium Pseudoalteromonas sp, strain A28 was able to kill the diatom Skeletonema costatum strain NIES-324, The culture supernatant of str ain A28 showed potent algicidal activity when it was applied to a paper dis k placed on a lawn of S. costatum NIES324. The condensed supernatant, which was prepared by subjecting the A28 culture supernatant to ultrafiltration with a 10,000-M-w-cutoff membrane, showed algicidal activity, suggesting th at strain A28 produced extracellular substances capable of killing S. costa tum cells. The condensed supernatant was then found to have protease and DN ase activities. Two Pseudoalteromonas mutants lacking algicidal activity, d esignated NH1 and NH2, were selected after N-methyl-N'-nitrosoguanidine mut agenesis, The culture supernatants of NH1 and NH2 showed less than 15% of t he protease activity detected with the parental strain, A28, The protease w as purified to homogeneity from A28 culture supernatants by using ion-excha nge chromatography followed by preparative gel electrophoresis, Paper-disk assays revealed that the purified protease had potent algicidal activity. T he purified protease had a molecular mass for 50 kDa, and the N-terminal am ino acid sequence was determined to be Ala-Thr-Pro-Asn-Asp-Pro. The optimum pH and temperature of the protease were found to be 8.8 and 30 degrees C, respectively, by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substra te. The protease activity was strongly inhibited by phenylmethylsulfonyl fl uoride, diisopropyl fluorophosphate, antipain, chymostatin, and leupeptin, No significant inhibition was detected with EDTA, EGTA, phenanthroline or t etraethylenepentamine. These results suggest that Pseudoalteromonas sp, str ain A28 produced an extracellular serine protease which was responsible for the algicidal activity of this marine bacterium.