Dipicolinic acid-less mutants of Bacillus cereus.

When J. Powell (Biochem. J. 54:210, 1953) established dipicolinic acid (DPA) as an important constituent of bacterial spores, microbiologists became concerned about the function this compound serves and about its biosynthetic pathway. It is now generally considered that DPA is involved in the heat resistance of spores, but the mechanism is unknown. A number of schemes have been proposed for its biosynthesis. J. Powell and R. E. Strange (Nature 184:878, 1959) proposed a, E-diketopimelic acid as a precursor with dihydrodipicolinic acid (DHDPA) as an intermediate that could be oxidized to DPA. However, when they found that DHDPA could be oxidized to DPA nonenzymatically in air, they had some doubts as to the validity of this pathway. H. H. Martin and J. W. Foster (J. Bacteriol. 76:167, 1958) suggested, and Y. Yugari and C. Gilvarg (J. Biol. Chem. 240:4710, 1965) published supporting evidence for, the involvement of the lysine pathway in the synthesis of DPA. In this pathway, DHDPA is an intermediate that could yield DPA through oxidation. Srinivasan (unpublished data), working in Halvorson's laboratory in Urbana, Ill., found that Bacillus cereus growing in a medium containing ethyloxamate produced spores devoid of DPA, and from such cultures he isolated a compound that he thought might be an intermediate in the synthesis of DPA. He believed this compound was formed through condensation of acetoacetyl coenzyme A with alanine and that methylpicolinic acid was then formed by cyclization. This is another possible pathway. Since we still lack convincing evidence as to the actual pathway, we felt it would be helpful to find mutants of spore-formers that lack the ability to synthesize DPA. The fact that DPAdeficient spores can be produced by use of inhibitors, encouraged us to try to find such mutants. We first subjected cultures of B. cereus to a variety of mutagenic agents and then allowed the cultures to grow until they formed spores. We treated these cultures with octyl alcohol to kill the vegetative cells and made spread plates of the survivors. When an examination of several colonies indicated that spores had been formed, we made replicate plates with velvet pads; first to one plate, and then after exposing the pad to a heat lamp to kill heat-sensitive colonies, another replicate plate was made. After growth, we examined the paired plates and looked for colonies that appeared only on plates made from the unheated pad. We found a number of such colonies, but we were apparently selecting for late spore-formers rather than mutants that formed heat-sensitive spores. After spending considerable effort over a period of several months with negative results, we abandoned this procedure. H. Tamir and C. Gilvarg (J. Biol. Chem. 241: 1085, 1966) reported a method for isopycnic gradient centrifugation of spores and showed that B. megaterium spores produced in calciumdeficient sporulation media were lighter in density than normal spores. We assumed that mutants which could not synthesize DPA would produce spores which also lack calcium, and therefore, would be less dense than the majority of the spore population. For these density gradients, Renografin-76 (N,N' diacetyl 3,5 diamino 2,4,6 triiodobenzoate, an X-ray contrasting agent purchased from Squibb) was obtained in 20-ml sterile bottles as a 76% solution. Linear, 5-ml gradients were formed at room temperature from 50% (1:1 dilution of Renografin with water) to 100% (highest obtainable concentration of Renografin) and were placed at 4C for 15 to 20 min. Washed spores from cultures irradiated with ultraviolet were suspended in 0.2% Tween 80, and, after adding an equal volume of 100% Renografin, the suspension was kept at 4 C for 15 to 20 min before layering on top of the gradient. Centrifugation was carried out in a Spinco model L ultracentrifuge with a SW-39 swinging bucket rotor at 4 C for 45 min at 15,000 rev/min. After centrifugation, a visible band of spores appeared about two-thirds down the tube. In such gradients, vegetative cells were found to form a layer on the surface, while germinated spores banded several millimeters from the top. When 109 spores were layered on a gradient, the majority of the spores were present in a