Isolation and characterization of a cDNA from soybean and its homolog from Escherichia coli, which both complement the light sensitivity of Escherichia coli hemH mutant strain VS101.

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  • Kanjo Naoko
    Department of Biophysics, Graduate School of Science, Kyoto University
  • Nakahigashi Kenji
    Department of Biophysics, Graduate School of Science, Kyoto University
  • Oeda Kenji
    Biotechnology Laboratory, Sumitomo Chemical Co. Ltd,
  • Inokuchi Hachiro
    Department of Biophysics, Graduate School of Science, Kyoto University

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Using Escherichia coli strain VS101, whose hemH gene encoding the ferrochelatase is partially defective, we isolated and analyzed a clone (designated λ WH-1) from a λ phage library of soybean (Glycine max) cDNA, which exhibited weak complementation activity against the light sensitivity of VS101. In VS101 bacteria lysogenized with λWH-1, a significant decrease in accumulation of protoporphyrin IX (PROTO IX) was detected as compared with that in non-lysogenic bacteria. On the other hand, in the wild-type E. coli strains lysogenized with λ WH-1, significant accumulation of δ-aminolevulinic acid (ALA) was observed, although accumulation of other intermediates such as uroporphyrinogen III (UROGEN III) and coproporphyrinogen III (COPROGEN III), was not observed. The growth of the wild-type bacteria in which the insert cDNA from λWH-1 had been introduced via a plasmid vector was markedly inhibited. By constructing, testing and sequencing a series of deletion clones of the insert, it was found that the insert encodes two proteins, a trancated LepA and a hypothetical protein ORF296, and that only ORF296 possesses the ability to block the heme biosynthetic pathway. ORF296 showed about 30% identity with the E. coli hypothetical protein YicL. By cloning and examining the gene for YicL in E. coli, we found that YicL shows the same effect as that of the soybean cDNA. From these findings, we concluded that the clone from soybean and yicL from E. coli block a step in an early stage of the heme biosynthetic pathway (probably the step catalyzed by HemB). Consequently, we postulate that the VS101 bacteria harboring these genes became light resistant as a result of a decrease in accumulated PROTO IX, and that the growth of the bacteria harboring these genes was inhibited because of the inhibition of heme biosynthesis at the step catalyzed by HemB.

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