CYCLOPHILIN AND TRIGGER FACTOR FROM BACILLUS-SUBTILIS CATALYZE IN-VITRO PROTEIN-FOLDING AND ARE NECESSARY FOR VIABILITY UNDER STARVATION CONDITIONS

Cyclophilin (the product of the ppiB gene) and the trigger factor (the product of the tig gene) are the only cytosolic peptidyl-prolyl cis-t rans isomerases that are known in Bacillus subtilis. Both enzymes cata lyze the in vitro refolding of ribonuclease T1, a reaction that is lim ited in rate by a prolyl cis/trans isomerization. The efficiency of cy clophilin as a folding catalyst is only modest with a k(cat)/K-M value of 3.8 x 10(4) M-1 s(-1), but the trigger factor shows an almost 40-f old higher specific activity with a k(cat)/K-M value of 1.4 x 10(6) M- 1 s(-1). This high catalytic activity originates from the tight bindin g to the protein substrate as reflected in both the low K-M value of 0 .5 mu M and in the strong inhibition of the trigger factor by unfolded proteins. By use of a protein-folding assay, the concentrations of cy clophilin and the trigger factor in the cytosol of B. subtilis could b e determined as 26 and 35 mu M, respectively. Together they account fo r the entire folding activity that is detectable in crude extracts of wild-type B. subtilis cells. The genes encoding cyclophilin and the tr igger factor in the B. subtilis chromosome were disrupted individually and simultaneously. Even in combination, these disruptions had no eff ect on cell viability in rich medium or under several stress condition s, such as heat, osmotic, or oxidative stress. However, in poor medium and, in particular, in the absence of amino acids, the growth of the double mutant strain was strongly decelerated, indicating that the pro lyl isomerases become essential for growth under starvation conditions . It is not yet known whether this function relates to the catalysis o f the proline-limited folding of essential proteins.