Operon structure and expression of the genes for benzylsuccinate synthase in Thauera aromatica strain K172.

The first step in anaerobic toluene degradation is the addition of a fumarate cosubstrate to the methyl group of toluene, as catalyzed by the glycyl radical enzyme benzylsuccinate synthase. The bssDCAB genes code for the subunits of benzylsuccinate synthase (BssA, B and C) and an additional enzyme implicated in activating the enzyme by introducing the glycyl radical (BssD). Quantitation of the amounts of benzylsuccinate synthase and activating enzyme showed that both proteins are only synthesized in toluene-grown cells, and that the activating enzyme is present in about 14-fold lower amounts. Two mRNA species of the bss gene cluster were identified, one beginning in front of bssD, and a second in front of bssC. Only the first mRNA 5'-end correlates with a toluene-induced promoter, which is similar to that preceding the bbs operon coding for the further enzymes of toluene catabolism of the same strain. The second mapped 5'-end appears to be generated by endonucleolytic processing. The mRNA segment containing the bssD gene is very short-lived, while that containing the bssCAB genes is more stable. The RNA stability data are consistent with the observed amounts of encoded gene products. Furthermore, the previously known bssDCAB genes are apparently cotranscribed with a fifth gene ( bssE) whose product may function as a putative ATP-dependent chaperone for assembly and/or activation of benzylsuccinate synthase.