SYMBIOTIC ROLE OF THE VIABLE BUT NONCULTURABLE STATE OF VIBRIO-FISCHERI IN HAWAIIAN COASTAL SEAWATER

To achieve functional bioluminescence, the developing light organ of n ewly hatched juveniles of the Hawaiian squid Euprymna scolopes must be come colonized by luminous, symbiosis-competent Vibrio fischeri presen t in the ambient seawater. This benign infection occurs rapidly in ani mals placed in seawater from the host's natural habitat. Therefore, it was surprising that colony hybridization studies with a V. fischeri-s pecific luxA gene probe indicated the presence of only about 2 CFU of V. fischeri per ml of this infective seawater. To examine this paradox , we estimated the total concentration of V. fischeri cells present in seawater from the host's habitat in two additional ways. In the first approach, the total bacterial assemblage in samples of seawater was c ollected on polycarbonate membrane filters and used as a source of bot h a crude cell lysate and purified DNA. These preparations were then a ssayed by quantitative DNA-DNA hybridization with the luxA gene probe. The results suggested the presence of between 200 and 400 cells of V. fischeri per ml of natural seawater, a concentration more than 100 ti mes that revealed by colony hybridization. In the second approach, we amplified V. fischeri-specific luxA sequences from microliter volumes of natural seawater by PCR. Most-probable-number analyses of the frequ ency of positive PCR results from cell lysates in these small volumes gave an estimate of the concentration of V. fischeri luxA gene targets of between 130 and 1,680 copies per ml. From these measurements, we c onclude that in their natural seawater environment, the majority of V. fischeri cells become nonculturable while remaining viable and symbio tically infective. Experimental studies indicated that V. fischeri cel ls suspended in natural Hawaiian seawater enter such a state within a few days.