Guerra-Lopez Denise, Daniels Lacy, Rawat Mamta
Department of Biology, California State University-Fresno, Fresno, CA 937401, USA.
Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, TX 78363, USA.
Microbiology (Reading). 2007 Aug;153(Pt 8):2724-2732. doi: 10.1099/mic.0.2006/009241-0.
Mycobacteria can tolerate relatively high concentrations of triphenylmethane dyes such as malachite green and methyl violet. To identify mycobacterial genes involved in the decolorization of malachite green, a transposon mutant library of Mycobacterium smegmatis mc2 155 was screened for mutants unable to decolorize this dye. One of the genes identified was MSMEG_5126, an orthologue of Mycobacterium bovis fbiC encoding a 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO) synthase, which is essential for the biosynthesis of the electron carrier coenzyme F420. The other gene identified was MSMEG_2392, encoding an alanine-rich protein with a DUF121 domain. The minimum inhibitory concentrations (MICs) for malachite green and methyl violet of the six fbiC mutants and two MSMEG_2392 mutants were one-third and one-fifth, respectively, of the MIC of the parent strain M. smegmatis mc2 155. Representative fbiC and MSMEG_2392 mutant strains were also sensitive to oxidative stress caused by the redox-cycling agents plumbagin and menadione, and the sensitivity was reversed in the complemented strains. HPLC analysis of representative fbiC and MSMEG_2392 strains revealed that, while the fbiC mutant lacked both coenzyme F420 and FO, the MSMEG_2392 mutant contained FO but not coenzyme F420. These results indicate that MSMEG_2392 is involved in the biosynthesis of coenzyme F420.
分枝杆菌能够耐受相对高浓度的三苯甲烷染料，如孔雀绿和甲基紫。为了鉴定参与孔雀绿脱色的分枝杆菌基因，对耻垢分枝杆菌mc2 155的转座子突变体文库进行筛选，以寻找不能使这种染料脱色的突变体。鉴定出的其中一个基因是MSMEG_5126，它是牛分枝杆菌fbiC的直系同源基因，编码一种7,8-二去甲基-8-羟基-5-脱氮核黄素（FO）合酶，该酶对电子载体辅酶F420的生物合成至关重要。鉴定出的另一个基因是MSMEG_2392，它编码一种富含丙氨酸且具有DUF121结构域的蛋白质。六个fbiC突变体和两个MSMEG_2392突变体对孔雀绿和甲基紫的最低抑菌浓度（MIC）分别是亲本菌株耻垢分枝杆菌mc2 155 MIC的三分之一和五分之一。代表性的fbiC和MSMEG_2392突变体菌株对由氧化还原循环剂白花丹素和甲萘醌引起的氧化应激也敏感，并且在互补菌株中这种敏感性得到逆转。对代表性的fbiC和MSMEG_2392菌株进行的HPLC分析表明，虽然fbiC突变体既缺乏辅酶F420也缺乏FO，但MSMEG_2392突变体含有FO但不含有辅酶F420。这些结果表明MSMEG_2392参与辅酶F420的生物合成。
