@article{wetzel_ehrenshaft_denslow_daub_2004, title={Functional complementation between the PDX1 vitamin B-6 biosynthetic gene of Cercospora nicotianae and pdxJ of Escherichia coli}, volume={564}, ISSN={["1873-3468"]}, DOI={10.1016/S0014-5793(04)00329-1}, abstractNote={The pathway for de novo vitamin B 6 biosynthesis has been characterized in Escherichia coli , however plants, fungi, archaebacteria, and most bacteria utilize an alternative pathway. Two unique genes of the alternative pathway, PDX1 and PDX2 , have been described. PDX2 encodes a glutaminase, however the enzymatic function of the product encoded by PDX1 is not known. We conducted reciprocal transformation experiments to determine if there was functional homology between the E. coli pdxA and pdxJ genes and PDX1 of Cercospora nicotianae . Although expression of pdxJ and pdxA in C. nicotianae pdx1 mutants, either separately or together, failed to complement the pyridoxine mutation in this fungus, expression of PDX1 restored pyridoxine prototrophy to the E. coli pdxJ mutant. Expression of PDX1 in the E. coli pdxA mutant restored very limited ability to grow on medium lacking pyridoxine. We conclude that the PDX1 gene of the alternative B 6 pathway encodes a protein responsible for synthesis of the pyridoxine ring.}, number={1-2}, journal={FEBS LETTERS}, author={Wetzel, DK and Ehrenshaft, M and Denslow, SA and Daub, ME}, year={2004}, month={Apr}, pages={143–146} }
 @article{chung_ehrenshaft_wetzel_daub_2003, title={Cercosporin-deficient mutants by plasmid tagging in the asexual fungus Cercospora nicotianae}, volume={270}, ISSN={["1617-4623"]}, DOI={10.1007/s00438-003-0902-7}, abstractNote={We have successfully adapted plasmid insertion and restriction enzyme-mediated integration (REMI) to produce cercosporin toxin-deficient mutants in the asexual phytopathogenic fungus Cercospora nicotianae. The use of pre-linearized plasmid or restriction enzymes in the transformation procedure significantly decreased the transformation frequency, but promoted a complicated and undefined mode of plasmid integration that leads to mutations in the C. nicotianae genome. Vector DNA generally integrated in multiple copies, and no increase in single-copy insertion was observed when enzymes were added to the transformation mixture. Out of 1873 transformants tested, 39 putative cercosporin toxin biosynthesis ( ctb) mutants were recovered that showed altered levels of cercosporin production. Seven ctb mutants were recovered using pre-linearized plasmids without the addition of enzymes, and these were considered to be non-REMI mutants. The correlation between a specific insertion and a mutant phenotype was confirmed using rescued plasmids as gene disruption vectors in the wild-type strain. Six out of fifteen rescued plasmids tested yielded cercosporin-deficient transformants when re-introduced into the wild-type strain, suggesting a link between the insertion site and the cercosporin-deficient phenotype. Sequence analysis of a fragment flanking the insert site recovered from one insertion mutant showed it to be disrupted in sequences with high homology to the acyl transferase domain of polyketide synthases from other fungi. Disruption of this polyketide synthase gene ( CTB1) using a rescued plasmid resulted in mutants that were defective in cercosporin production. Thus, we provide the first molecular evidence that cercosporin is synthesized via a polyketide pathway as previously hypothesized.}, number={2}, journal={MOLECULAR GENETICS AND GENOMICS}, author={Chung, KR and Ehrenshaft, M and Wetzel, DK and Daub, ME}, year={2003}, month={Oct}, pages={103–113} }