Cyanobacteria are potentially useful photosynthetic microorganisms for bioremediation under oligotrophic environments. Here, the biphenyl degradation pathway genes of b-proteobacterium Acidovorax sp. strain KKS102 were co-expressed in cyanobacterium Synechocystis sp. PCC6803 cells under control of the photo-inducible psbE promoter. In the KKS102 cells, biphenyl is dioxygenated by bphA1 and bphA2 gene products complex using electrons supplied from NADH via phA4 and bphA3 gene products (BphA4 and BphA3，respectively), and converted to benzoic acid by bphB , BphC and bphD gene products. Unexpectedly, biphenyl was effectively hydroxylated in oligotrophic BG11 medium by co-expressing the bphA3 , bphA1 and bphA2 genes without the bphA4 gene, suggesting that endogenous cyanobacteria-derived protein(s) can supply electrons to reduce BphA3 at the start of the biphenyl degradation pathway. Furthermore, biphenyl was converted to benzoic acid by cyanobacterial cells co-expressing bphA3 , bphA1 , bphA2 , bphB , bphC and bphD . Structural gene-screening using recombinant Escherichia coli cells co-expressing bphA3 , bphA1 , bphA2 , bphB and bphC suggested that petH , which encodes long- and short-type NADP-ferredoxin oxidoreductase isomers (FNR L and FNR S , respectively), and slr0600 , which is annotated as an NADPH-thioredoxin reductase gene in CyanoBase, were BphA3-reducible proteins. Purified FNR L and FNR S , and the slr0600 gene product showed BphA3 reductase activity dependent on NADPH and the reduced form of glutathione, respectively, potentially shedding light on the physiological roles of the slr0600 gene product in cyanobacterial cells. Collectively, our results demonstrate the utility of Synechocystis sp. PCC6803 cells as a host for bioremediation of biphenyl compounds under oligotrophic environments without an organic carbon source.
We have found that tissue-type transglutaminase (tTG), also called TGc, TGase2 and Gα h , has the activity of protein disulphide isomerase (PDI). We have shown that tTG converts completely reduced/denatured inactive RNase A molecule to the native active enzyme. The PDI activity of tTG was strongly inhibited by bacitracin, which is a frequently used inhibitor of conventional PDI activity. It was substantially inhibited by the simultaneous presence of other potential substrate proteins such as completely reduced BSA, but not by native BSA. This activity was especially high in the presence of GSSG, but not GSH. The addition of GSH to the reaction mixture in the presence of GSSG at a fixed concentration up to at least 200-fold excess did not very substantially inhibit the PDI activity. It is possible that tTG can exert PDI activity in a fairly reducing environment like cytosol, where most of tTG is found. It is quite obvious from the following observations that PDI activity of tTG is catalysed by a domain different from that used for the transglutaminase reaction. Although the alkylation of Cys residues in tTG completely abolished the transglutaminase activity, as was expected, it did not affect the PDI activity at all. This PDI activity did not require the presence of Ca 2+ . It was not inhibited by nucleotides including GTP at all, unlike the other activity of tTG.