Allison G, Gough K, Rogers L, Smith A
Institute of Biological Sciences, University of Wales Aberystwyth, Wales.
Mol Gen Genet. 1997 Jul;255(4):392-9. doi: 10.1007/s004380050511.
Gabaculine (2,3-dihydro 3-amino benzoic acid) is a potent inhibitor of tetrapyrrole biosynthesis in organisms that use the C5 pathway for the synthesis of delta-aminolaevulinic acid. Glutamate semialdehyde aminotransferase (GSA-AT), the enzyme catalysing the formation of this key precursor of tetrapyrroles, is normally inhibited by concentrations of gabaculine in the order of 5 microM. However, in Synechococcus 6301 strain GR6, a cyanobacterium that is resistant to 100 microM gabaculine, this enzyme has undergone two changes in structure: a deletion of three amino acids from positions 5 to 7 and the substitution of isoleucine for methionine at position 248. To establish the effect in vivo of these specific changes in the gene for GSA-AT (hemL), a suicide vector (pHS7) containing an antibiotic cassette was constructed to achieve the replacement, by homologous recombination, of the wild-type hemL gene in the chromosome by a modified form of the gene. Recombinant strains of Synechococcus 7942 obtained using pHS7-hemLGR6 were indistinguishable from Synechococcus 6301 GR6 in terms of the resistance of growth and of chlorophyll accumulation to high concentrations of gabaculine, while a wild-type recombinant produced using pHS7-hemLWT had retained its sensitivity. Southern hybridisation using gene probes for hemL, ampr and cmr confirmed that chromosomal integration of the plasmids had occurred in both WT and GR6 recombinants. Growth and chlorophyll accumulation in equivalent strains with the hemL gene containing either the deletion or the transition characteristic of Synechococcus 6301 GR6 were inhibited by 10 microM gabaculine. Consequently, resistance in vivo to high concentrations of this compound is dependent on both the changes in gene/enzyme structure. This investigation has established the effectiveness of the suicide vector pHS7 for studying the effect in vivo of specific changes in the hemL gene. It has also demonstrated that replacement of the wild-type gene by that from Synechococcus 6301 GR6 is sufficient to confer resistance in vivo to high concentrations of gabaculine.
加巴库林（2,3 - 二氢 - 3 - 氨基苯甲酸）是一种在利用C5途径合成δ-氨基乙酰丙酸的生物体中，对四吡咯生物合成具有强效抑制作用的物质。谷氨酸半醛氨基转移酶（GSA - AT）是催化形成四吡咯这种关键前体的酶，通常会被约5微摩尔浓度的加巴库林所抑制。然而，在对100微摩尔加巴库林具有抗性的蓝细菌聚球藻6301菌株GR6中，这种酶发生了两个结构变化：从第5位到第7位缺失了三个氨基酸，并且在第248位异亮氨酸取代了甲硫氨酸。为了确定GSA - AT基因（hemL）中这些特定变化在体内的影响，构建了一个含有抗生素盒的自杀载体（pHS7），通过同源重组，用该基因的修饰形式替换染色体中的野生型hemL基因。使用pHS7 - hemLGR6获得的聚球藻7942重组菌株，在生长抗性以及叶绿素积累对高浓度加巴库林的抗性方面，与聚球藻6301 GR6没有区别，而使用pHS7 - hemLWT产生的野生型重组菌株则保留了其敏感性。使用针对hemL、ampr和cmr的基因探针进行的Southern杂交证实，质粒已在野生型和GR6重组体中发生了染色体整合。含有聚球藻6301 GR6特征性缺失或转变的hemL基因的等效菌株，其生长和叶绿素积累受到10微摩尔加巴库林的抑制。因此，体内对高浓度该化合物的抗性取决于基因/酶结构的这两个变化。这项研究确立了自杀载体pHS7在研究hemL基因特定变化在体内影响方面的有效性。它还证明了用聚球藻6301 GR6的基因替换野生型基因足以使体内对高浓度加巴库林产生抗性。
