Oxygen-triggered synchronisized variant formation of the S-layer carrying Bacillus stearothermophilus PV72 during continuous cultivation

Abstract

The crystalline cell surface layer (S-layer) of Bacillus stearothermophilus PV72 shows hexagonal lattice symmetry and is composed of a single protein species with a molecular weight of 130 000, termed SbsA. Stable synthesis of the SbsA on synthetic PVIII-medium was achieved during continuous cultivation of the wild-type strain at constant DO of 20%. When the DO was controlled at 50%, an apparent steady state was achieved after 2 volume exchanges after starting continuous culture. After 2–3 further volume exchanges, the respiratoric activity and culture fluorescence peaked markedly while the redox potential showed a steady increase. The increase in the optical density did not correlate with an increase in biomass concentration but rather reflected differences in the morphology of the cells and a slight sporulation. In addition to the physiological and morphological changes, a switch in S-layer protein synthesis was observed. Instead of the SbsA from the wild-type strain, an S-layer protein with a molecular weight of 97 000, termed SbsB, was produced which assembled into an oblique lattice type. The decrease in the wild-type S-layer protein content in biomass samples harvested during variant formation, freeze-etching and immunogold-labelling of whole cells confirmed that expression of SbsA was synchronously stopped in most, if not in all individual cells of the culture and was completely replaced by synthesis of the SbsB. Peptide mapping, N-terminal sequencing and Western blotting confirmed that both types of S-layer proteins were encoded by different genes. The hypothesis of oxygen-triggered synchronisized variant formation was finally supported by computer simulations.