Milner Centre for Evolution, Department of Life Sciences, University of Bath, Claverton Down, Bath BA2 7AY, UK.
Division of Evolution and Genomic Sciences, School of Biological Sciences, University of Manchester, Manchester, UK.
Microbiology (Reading). 2023 Aug;169(8). doi: 10.1099/mic.0.001378.
Evolutionary innovation of transcription factors frequently drives phenotypic diversification and adaptation to environmental change. Transcription factors can gain or lose connections to target genes, resulting in novel regulatory responses and phenotypes. However the frequency of functional adaptation varies between different regulators, even when they are closely related. To identify factors influencing propensity for innovation, we utilise a SBW25 strain rendered incapable of flagellar mediated motility in soft-agar plates via deletion of the flagellar master regulator (). This bacterium can evolve to rescue flagellar motility via gene regulatory network rewiring of an alternative transcription factor to rescue activity of FleQ. Previously, we have identified two members (out of 22) of the RpoN-dependent enhancer binding protein (RpoN-EBP) family of transcription factors (NtrC and PFLU1132) that are capable of innovating in this way. These two transcription factors rescue motility repeatably and reliably in a strict hierarchy – with NtrC the only route in a ∆ background, and PFLU1132 the only route in a ∆∆ background. However, why other members in the same transcription factor family have not been observed to rescue flagellar activity is unclear. Previous work shows that protein homology cannot explain this pattern within the protein family (RpoN-EBPs), and mutations in strains that rescued motility suggested high levels of transcription factor expression and activation drive innovation. We predict that mutations that increase expression of the transcription factor are vital to unlock evolutionary potential for innovation. Here, we construct titratable expression mutant lines for 11 of the RpoN-EBPs in . We show that in five additional RpoN-EBPs (FleR, HbcR, GcsR, DctD, AauR and PFLU2209), high expression levels result in different mutations conferring motility rescue, suggesting alternative rewiring pathways. Our results indicate that expression levels (and not protein homology) of RpoN-EBPs are a key constraining factor in determining evolutionary potential for innovation. This suggests that transcription factors that can achieve high expression through few mutational changes, or transcription factors that are active in the selective environment, are more likely to innovate and contribute to adaptive gene regulatory network evolution.
转录因子的进化创新经常推动表型多样化和适应环境变化。转录因子可以获得或失去与靶基因的连接，从而产生新的调控反应和表型。然而，即使它们是密切相关的，不同调节因子的功能适应频率也会有所不同。为了确定影响创新倾向的因素，我们利用 SBW25 菌株通过缺失鞭毛主调控因子 () 来破坏软琼脂平板中的鞭毛介导的运动。这种细菌可以通过基因调控网络重布线来拯救替代转录因子的鞭毛运动，从而拯救 FleQ 的活性。以前，我们已经鉴定出 22 个依赖 RpoN 的增强子结合蛋白 (RpoN-EBP) 家族转录因子中的两个成员 (NtrC 和 PFLU1132) 能够以这种方式进行创新。这两个转录因子在严格的层次结构中重复且可靠地拯救运动——在 ∆ 背景下只有 NtrC 是唯一途径，在 ∆∆ 背景下只有 PFLU1132 是唯一途径。然而，为什么在同一个转录因子家族中没有观察到其他成员拯救鞭毛活性还不清楚。以前的工作表明，蛋白质同源性不能解释该蛋白家族 (RpoN-EBPs) 内的这种模式，并且拯救运动的突变株中的突变表明高水平的转录因子表达和激活驱动创新。我们预测，增加转录因子表达的突变对于释放创新的进化潜力至关重要。在这里，我们在 中构建了 11 个 RpoN-EBP 的可滴定表达突变株。我们表明，在另外 5 个 RpoN-EBP（FleR、HbcR、GcsR、DctD、AauR 和 PFLU2209）中，高水平表达导致不同的突变赋予运动拯救，表明存在替代的重布线途径。我们的结果表明，RpoN-EBP 的表达水平（而不是蛋白质同源性）是决定创新进化潜力的关键约束因素。这表明，能够通过少数突变获得高表达的转录因子，或在选择环境中活跃的转录因子，更有可能创新并有助于适应性基因调控网络的进化。
