The succinate:menaquinone reductase of Bacillus cereus: characterization of the membrane-bound and purified enzyme.

Utilization of external succinate by Bacillus cereus and the properties of the purified succinate:menaquinone-7 reductase (SQR) were studied. Bacillus cereus cells showed a poor ability for the uptake of and respiratory utilization of exogenous succinate, thus suggesting that B. cereus lacks a specific succinate uptake system. Indeed, the genes coding for a succinate-fumarate transport system were missing from the genome database of B. cereus. Kinetic studies of membranes indicated that the reduction of menaquinone-7 is the rate-limiting step in succinate respiration. In accordance with its molecular characteristics, the purified SQR of B. cereus belongs to the type-B group of SQR enzymes, consisting of a 65-kDa flavoprotein (SdhA), a 29-kDa iron-sulphur protein (SdhB), and a 19-kDa subunit containing 2 b-type cytochromes (SdhC). In agreement with this, we could identify the 4 conserved histidines in the SdhC subunit predicted by the B. cereus genome database. Succinate reduced half of the cytochrome b content. Redox titrations of SQR-cytochrome b-557 detected 2 components with apparent midpoint potential values at pH 7.6 of 79 and -68 mV, respectively; the components were not spectrally distinguishable by their maximal absorption bands as those of Bacillus subtilis. The physiological properties and genome database analyses of B. cereus are consistent with the cereus group ancestor being an opportunistic pathogen.

Haem O and a putative cytochrome bo in a mutant of Bacillus cereus impaired in the synthesis of haem A.

In a spontaneous mutant (PYM1) of Bacillus cereus impaired in the synthesis of haem A, no haem-A-containing cytochromes were detected spectroscopically. The haem A deficiency was compensated by high levels of haem O and a CO-reactive cytochrome o in membranes; no other oxidases were detected. In contrast, the wild-type strain had considerable amounts of haem A and negligible levels of haem O. The mutant PYM1 exhibited normal colony morphology, growth, and sporulation in nonfermentable media, whereas on fermentable media, the mutant overproduced acid, which led to poor growth and inhibition of sporulation. External control of the pH of the medium in fermentable media allowed close-to-normal growth and massive sporulation of the mutant. The presence of membrane-bound cytochrome caa3-OII and aa3-II subunits in strain PYM1 was confirmed by Western blots and haem C staining (COII subunit). Western blotting also revealed that in contrast to the wild-type - strain PYM1 contained the membrane-bound subunits caa3-COI and aa3-I, but in low amounts. The effect of several respiratory inhibitors on the respiratory system of strain PYM1 suggested that the terminal oxidase is highly resistant to KCN and CO and that a c-type cytochrome might be involved in the electron transfer sequence to the putative cytochrome bo.