An unknown substance found in bacteria (Escherichia coli) is especially effective in attracting the vegetative amoebae of the cellular slime mold, Dictyostelium discoideum. However, the aggregating amoebae are not attracted to it at all. On the other hand, the vegetative amoebae show very little chemotactic response to cyclic adenosine monophosphate (cyclic AMP), whereas the aggregating amoebae are exceptionally responsive to it. It is suggested that the new factor may be used in food seeking, whereas cyclic AMP, the chemotactic substance responsible for aggregation, is the acrasin of this species. The important point is that the amoebae are differentially stage-specific in their responses to these two chemotactic agents.In some recent studies, we have shown that in one species of cellular slime mold, Dictyostelium discoideum, the chemotactic agent or acrasin responsible for bringing the amoebae together in aggregation is cyclic-3',5'-adenosine monophosphate (cyclic AMP; 7-9). Furthermore, this acrasin is apparently produced in large quantities only during the aggregation stage (2), and the cells become especially sensitive to it at that stage (2,5,6).It has also been known for some time that bacteria and bacterial extracts will attract amoebae (3, 6, 9, 13; T. M. Konijn, Ph.D. Thesis, University of Wisconsin, 1961). Since bacteria produce cyclic AMP (10) and cyclic AMP has some ability to attract vegetative amoebae (and also has a strong effect on aggregating amoebae), it was assumed that the amoebae used this substance, first for food location during the vegetative stage and then for aggregation during its developmental stage (2, 7-9).The new results to be reported here show that there is an additional substance present in bacteria which is very much more effective in attracting vegetative amoebae than cyclic AMP. Furthermore, the amoebae completely lose their ability to respond to this second substance as they enter the aggregation stage, the very moment at which their sensitivity to cyclic AMP increases rapidly. Therefore we shall present evidence not only for a new chemotatic agent present in bacteria, but also for stage-specific responses to the two chemotactic agents.
MATERIALS AND METHODSThese experiments were done with a haploid strain of stock number NC-4 of D. discoideum, kindly supplied by K. B. Raper. The bacterium used for the growth of the slime mold was Escherichia coli strain 281, and the extracts were obtained from E. coli strain B/r. Cellophane square test. This test for chemotaxis was devised by Bonner, Kelso, and Gillmor (3). Amoebae were grown on buffered 1% peptone-dextrose agar with E. coli at 21 C in the dark. After approximately 40 hr of incubation, the vegetative amoebae were washed free of the surface and gently centrifuged three times in 1% salt solution (1). Washed cellophane squares (5 by 5 mm) were placed in the bottom of a small dish covered with 1% salt solution. The amoebae were allowed to settle on the cellophane squares to form a dense population (20 to 30 min). The squares we...
