The bacterial serine-threonine protein kinase HipA promotes multidrug tolerance by phosphorylating the glutamate-tRNA ligase (GltX), leading to a halt in translation, inhibition of growth, and induction of a physiologically dormant state (persistence). The HipA variant HipA7 substantially increases persistence despite being less efficient at inhibiting cell growth. We postulated that this phenotypic difference was caused by differences in the substrates targeted by both kinases. We overproduced HipA and HipA7 in Escherichia coli and identified their endogenous substrates by SILAC-based quantitative phosphoproteomics. We confirmed that GltX was the main substrate of both kinase variants and likely the primary determinant of persistence. When HipA and HipA7 were moderately overproduced from plasmids, HipA7 targeted only GltX, but HipA phosphorylated several additional substrates involved in translation, transcription, and replication, such as ribosomal protein L11 (RplK) and the negative modulator of replication initiation, SeqA. HipA7 showed reduced kinase activity compared to HipA and targeted a substrate pool similar to that of HipA only when produced from a high–copy number plasmid. The kinase variants also differed in autophosphorylation, which was substantially reduced for HipA7. When produced endogenously from the chromosome, HipA showed no activity because of inhibition by the antitoxin HipB, whereas HipA7 phosphorylated GltX and phage shock protein PspA. Initial testing did not reveal a connection between HipA-induced phosphorylation of RplK and persistence or growth inhibition, suggesting that other HipA-specific substrates were likely responsible for growth inhibition. Our results contribute to the understanding of HipA7 action and present a resource for elucidating HipA-related persistence.

细菌丝氨酸-苏氨酸蛋白激酶HipA通过将谷氨酸-tRNA连接酶（GltX）磷酸化来提高多药耐受性，从而导致翻译停止，生长抑制和诱导生理休眠状态（持久性）。HipA变体HipA7尽管抑制细胞生长的效率较低，但仍显着增加了持久性。我们推测该表型差异是由两种激酶靶向的底物差异引起的。我们在大肠杆菌中过量生产了HipA和HipA7并通过基于SILAC的定量磷酸化蛋白质组学鉴定了它们的内源底物。我们证实，GltX是这两种激酶变体的主要底物，并且可能是持久性的主要决定因素。当从质粒中适度过量生产HipA和HipA7时，HipA7仅靶向GltX，但HipA磷酸化了参与翻译，转录和复制的其他几种底物，例如核糖体蛋白L11（RplK）和复制起始的负调控子SeqA。与HipA相比，HipA7显示出降低的激酶活性，并且仅在由高拷贝数质粒生产时才能靶向类似于HipA的底物库。激酶变体在自身磷酸化方面也有所不同，对于HipA7而言，磷酸化显着降低。当从染色体内生时，由于抗毒素HipB的抑制，HipA没有活性，而HipA7磷酸化了GltX和噬菌体休克蛋白PspA。最初的测试并未揭示HipA诱导的RplK磷酸化与持久性或生长抑制之间的联系，这表明其他HipA特异性底物可能是生长抑制的原因。我们的研究结果有助于人们了解HipA7的作用，并为阐明与HipA相关的持久性提供了资源。