Belagal Praveen
Department of Molecular Biology, School of Biological Sciences, Madurai Kamaraj University, Madurai, 625 021, India.
Mol Biol Rep. 2025 Jun 4;52(1):543. doi: 10.1007/s11033-025-10539-7.
This laboratory has been working on fit genes (factor involved in transcription) for many years. The fitA76 is temperature-sensitive (Ts) double mutant, carrying two lesions, pheS5 (pheS) and fit95 (pheT) with a combined defect at transcription. The fitB and fitC mutations were isolated as extragenic-suppressor(s) of fitA76. While fitA76 mapped at 38.7 min, fitC4 mapped at 39.01 min. The fitC4 and fitA76*, were two accompanied mutations found in temperature-insensitive derivative (JV4), isolated from fitA76 mutant. The fitC4 suppresses fitA76*(but not fitA76) and both are transcription defective. This work focuses on elucidating gene-identity of fitC4 locus, and its role in allele-specific genetic-suppression and genome-integrity.
Mutations were mobilized by P1-transductions. Marker-rescue analyses were done with Kohara-phages to narrow down the fitC4 location. Further mapping was done by complementation analyses employing various plasmids spanning that region. Complementation assays were quantitated by relative viability.
Among the Kohara phages/clones tested (321-330), the same clones 322&323 which earlier marker rescued fitA76/fit95 mutations, also marker rescued fitC4 mutation. But, fitC4 maps totally outside the overlapping region of two Kohara phages, tested positive. Complementation analyses by various plasmids spanning that region indicate that, fitC4 is same as pheS. Further, one of the clones bearing only pheT, did not complement fitC4 mutation. The implications of these results and pheST operon dynamics in gene duplication towards bacterial adaptation in context of transcription regulation and genome expansion are discussed.
The fitC4 is same as pheS allele. Only pheS but not full pheST-operon is duplicated as fitC4. The pheS gene duplication could help in allele-specific genetic suppression and adaptation of its ability as transcription factor, thus contributing to genome-stability and genome-evolution.
本实验室多年来一直在研究fit基因（参与转录的因子）。fitA76是温度敏感（Ts）双突变体，携带两个损伤，即pheS5（pheS）和fit95（pheT），在转录方面存在联合缺陷。fitB和fitC突变是作为fitA76的基因外抑制子分离出来的。虽然fitA76定位于38.7分钟处，但fitC4定位于39.01分钟处。fitC4和fitA76是在从fitA76突变体分离出的温度不敏感衍生物（JV4）中发现的两个伴随突变。fitC4抑制fitA76（但不抑制fitA76），且二者在转录方面均有缺陷。这项工作的重点是阐明fitC4基因座的基因身份及其在等位基因特异性遗传抑制和基因组完整性中的作用。
通过P1转导移动突变。用小原噬菌体进行标记拯救分析以缩小fitC4的定位范围。通过使用跨越该区域的各种质粒进行互补分析来进一步定位。通过相对活力对互补试验进行定量。
在所测试的小原噬菌体/克隆（321 - 330）中，之前对fitA76/fit95突变进行标记拯救的相同克隆322和323，也对fitC4突变进行了标记拯救。但是，fitC4完全定位于两个经测试呈阳性的小原噬菌体的重叠区域之外。通过跨越该区域的各种质粒进行的互补分析表明，fitC4与pheS相同。此外，仅携带pheT的一个克隆不能互补fitC4突变。讨论了这些结果以及pheST操纵子动力学在转录调控和基因组扩展背景下对细菌适应性基因复制的影响。
fitC4与pheS等位基因相同。作为fitC4，仅pheS而非完整的pheST操纵子被复制。pheS基因复制有助于等位基因特异性遗传抑制及其作为转录因子能力的适应性，从而有助于基因组稳定性和基因组进化。
