Molecular characterization of the tox operon involved in toxoflavin biosynthesis of Burkholderia glumae

Two toxoflavin biosynthesis-related proteins (TRP-1, TRP-2) from wild strains of the phytopathogen Burkholderia glumae were previously identified, and toxA was determined to encode TRP-1, which has characteristics of a methyltransferase. An 8.2-kb region in the chromosomal DNA of B. glumae that contains the tox operon (toxABCDE) and an upstream regulatory gene (toxR) involved in phytotoxin toxoflavin biosynthesis was cloned and sequenced in this study. The sequence downstream of toxA contains four open reading frames - toxB, toxC, toxD and toxE - which encode polypeptides with calculated mole-cular masses of 23.3, 61.6, 34.9, and 38.3thinspkDa, respectively, all having the same transcriptional orientation as toxA. Mutants disrupted in the tox operon lost their ability to produce toxoflavin and did not induce typical chlorosis on infected rice panicles. Based on results from reverse transcription-polymerase chain reaction experiments, the message encoded by the tox operon may be polycistronic for all five genes. Also, the toxR gene was located upstream of this operon. The toxR gene product, with a calculated molecular mass of 37.5thinspkDa, might be a member of the LysR family of regulatory molecules and an activator that allows transcription of the tox operon. Furthermore, the deduced proteins of ToxB and ToxE had significant similarity to the GTP cyclohydrolase II and the deaminase, respectively, involved in riboflavin synthesis in several organisms. These results suggest that toxoflavin is synthesized in part through a biosynthetic pathway common to the synthesis of riboflavin, starting with GTP as the precursor. [PUBLICATION ABSTRACT]