Biphenyl degradation by recombinant photosynthetic cyanobacterium Synechocystis sp. PCC6803 in an oligotrophic environment using unphysiological electron transfer.

Cyanobacteria are potentially useful photosynthetic microorganisms for bioremediation under oligotrophic environments. Here, the biphenyl degradation pathway genes of ß-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 bphA4 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 (FNRL and FNRS, respectively), and slr0600, which is annotated as an NADPH-thioredoxin reductase gene in CyanoBase, were BphA3-reducible proteins. Purified FNRL and FNRS, 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.

Distinct roles for U-type proteins in iron-sulfur cluster biosynthesis revealed by genetic analysis of the Bacillus subtilis sufCDSUB operon.

The biosynthesis of iron-sulfur (Fe-S) clusters in Bacillus subtilis is mediated by the SUF-like system composed of the sufCDSUB gene products. This system is unique in that it is a chimeric machinery comprising homologues of E. coli SUF components (SufS, SufB, SufC and SufD) and an ISC component (IscU). B. subtilis SufS cysteine desulfurase transfers persulfide sulfur to SufU (the IscU homologue); however, it has remained controversial whether SufU serves as a scaffold for Fe-S cluster assembly, like IscU, or acts as a sulfur shuttle protein, like E. coli SufE. Here we report that reengineering of the isoprenoid biosynthetic pathway in B. subtilis can offset the indispensability of the sufCDSUB operon, allowing the resultant Δsuf mutants to grow without detectable Fe-S proteins. Heterologous bidirectional complementation studies using B. subtilis and E. coli mutants showed that B. subtilis SufSU is interchangeable with E. coli SufSE but not with IscSU. In addition, functional similarity in SufB, SufC and SufD was observed between B. subtilis and E. coli. Our findings thus indicate that B. subtilis SufU is the protein that transfers sulfur from SufS to SufB, and that the SufBCD complex is the site of Fe-S cluster assembly.