Pseudomonas aeruginosa is a ubiquitous gram-negative bacterium in the environment and a leading cause of nosocomial infections worldwide. Therefore, it is listed by the WHO as a human pathogen that urgently needs the development of new antibacterial drugs. Recent findings have demonstrated that eukaryote-type Ser/Thr protein kinases play a vital role in regulating various bacterial physiological processes by catalyzing protein phosphorylation. Stk1 has proven to be a Ser/Thr protein kinase in P. aeruginosa. However, the regulatory roles of Stk1 have not yet been revealed. Thus, we constructed a stk1 knockout mutant (∆stk1) from the P. aeruginosa PAO1 strain and employed a Tandem Mass Tag (TMT) labeling-based quantitative proteomic strategy to characterize proteome-wide changes in response to the stk1 knockout. In total, 620 differentially expressed proteins, among which 288 proteins were upregulated and 332 proteins were downregulated, were identified in ∆stk1 compared with P. aeruginosa PAO1. A detailed bioinformatics analysis of these differentially expressed proteins was performed, including GO annotation, protein domain profile, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, subcellular localization and enrichment analysis. Notably, the downregulation of type IV pilus-related proteins and upregulation of T6SS-H1-related proteins were found in the ∆stk1 strain, and the results were corroborated by quantitative PCR at the mRNA level. Further experiments confirmed that the loss of stk1 weakens bacterial twitching motility and promotes a growth competition advantage, which are, respectively, mediated by type IV pilus-related proteins and T6SS-H1-related proteins. These findings contribute to a better understanding of the physiological role of Stk1, and proteomic data will help further investigations of the roles and mechanisms of Stk1 in P. aeruginosa, although the detailed regulation and mechanism of Stk1 still need to be revealed.

铜绿假单胞菌是环境中普遍存在的革兰氏阴性细菌，并且是全世界医院感染的主要原因。因此，它被世界卫生组织列为急需开发新型抗菌药物的人类病原体。最近的研究结果表明，真核生物型 Ser/Thr 蛋白激酶通过催化蛋白质磷酸化在调节各种细菌生理过程中发挥着至关重要的作用。Stk1 已被证明是一种丝氨酸/苏氨酸蛋白激酶铜绿假单胞菌. 然而，Stk1 的调节作用尚未揭示。于是，我们构建了一个stk1 敲除突变体（Δstk1） 来自 铜绿假单胞菌 PAO1 菌株并采用基于串联质量标签 (TMT) 标记的定量蛋白质组学策略来表征蛋白质组范围内的变化，以响应 stk1昏死。共鉴定出 620 个差异表达蛋白，其中 288 个蛋白上调，332 个蛋白下调。stk1 和....相比 铜绿假单胞菌PAO1。对这些差异表达的蛋白质进行了详细的生物信息学分析，包括 GO 注释、蛋白质结构域概况、京都基因和基因组百科全书 (KEGG) 通路分析、亚细胞定位和富集分析。值得注意的是，在 ∆ 中发现了 IV 型菌毛相关蛋白的下调和 T6SS-H1 相关蛋白的上调。stk1菌株，结果通过 mRNA 水平的定量 PCR 得到证实。进一步的实验证实，损失stk1削弱细菌抽搐运动并促进生长竞争优势，这分别由 IV 型菌毛相关蛋白和 T6SS-H1 相关蛋白介导。这些发现有助于更好地理解 Stk1 的生理作用，蛋白质组学数据将有助于进一步研究 Stk1 在铜绿假单胞菌，虽然 Stk1 的详细调控和机制仍有待揭示。