TRI12, a trichothecene efflux pump from Fusarium sporotrichioides:: gene isolation and expression in yeast
TRI12, a trichothecene efflux pump from Fusarium sporotrichioides:: gene isolation and expression in yeast
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DOI：
10.1007/s004380051046
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发表时间：
1999-07-01
期刊：
影响因子：
--
通讯作者：
Hohn, TM
中科院分区：
文献类型：
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作者：
Alexander, NJ;McCormick, SP;Hohn, TM
孢子毛状镰刀菌中参与毛霉毒素生物合成的许多基因存在于一个基因簇中。在这里，我们报道了tr12基因的完整序列，该基因编码一个毛孢子菌外排泵，位于孢子毛孢子菌的毛孢子菌基因簇中。据推测，TRI12编码一个含有598个残基的多肽，其序列与主要促进物超家族（MFS）成员相似，预计包含14个跨膜片段。破坏tr12会导致在复杂培养基上的生长减少和毛藻烯产量降低。在含有毛霉烯的培养基上，tri12突变体的生长受到抑制，提示tri12可能在孢子毛霉对毛霉烯的自我保护中起作用。通过与编码毛霉烯15- o-乙酰转移酶的基因（TRI3）共转化的酵母菌株中表达TRI12，进行了功能分析。在TRI3底物（15-decalonectrin）存在的情况下，携带TRI12和TRI3的酵母菌培养物积累的乙酰化产物（calonectrin）水平远高于单独携带TRI3的酵母菌培养物。PDR5是ABC超家族的一种转运体，已知可介导酵母对毛霉烯的抗性，在TRI12/TRI3酵母菌株中增加了calonectrin的积累，而在TRI3菌株中没有。这些结果证实了TRI12参与与毒素生物合成相关的毛霉烯外排，并证明了酵母作为mfs型转运体研究的宿主系统的有效性。
Many of the genes involved in trichothecene toxin biosynthesis in Fusarium sporotrichioides are present within a gene cluster. Here we report the complete sequence for TRI12, a gene encoding a trichothecene efflux pump that is located within the trichothecene gene cluster of F. sporotrichioides. TRI12 encodes a putative polypeptide of 598 residues with sequence similarities to members of the major facilitator superfamily (MFS) and is predicted to contain 14 transmembrane-spanning segments. Disruption of TRI12 results in both reduced growth on complex media and reduced levels of trichothecene production. Growth of tri12 mutants on trichothecene-containing media is inhibited, suggesting that TRI12 may play a role in F. sporotrichioides self-protection against trichothecenes. Functional analysis of TRI12 was performed by expressing it in yeast strains that were co-transformed with a gene (TRI3) encoding a trichothecene 15-O-acetyl-transferase. In the presence of the TRI3 substrate, 15-decalonectrin, cultures of yeast strains carrying TRI12 and TRI3 accumulated much higher levels of the acetylated product, calonectrin, than was observed for strains carrying TRI3 alone. PDR5, a transporter of the ABC superfamily, which is known to mediate trichothecene resistance in yeast, increased calonectrin accumulation in TRI12/TRI3 yeast strains but not in TRI3 strains. These results confirm the involvement of TRI12 in the trichothecene efflux associated with toxin biosynthesis, and demonstrate the usefulness of yeast as a host system for studies of MFS-type transporters.