Inoue Shori, Yoshimoto Shogo, Hori Katsutoshi
Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Nagoya, Aichi, Japan.
J Bacteriol. 2025 Jan 31;207(1):e0032524. doi: 10.1128/jb.00325-24. Epub 2024 Dec 11.
The methylation of ε-amino groups in protein lysine residues is an important posttranslational modification in eukaryotes. This modification plays a pivotal role in the regulation of diverse biological processes, including epigenetics, transcriptional control, and cellular signaling. Recent research has begun to reveal the potential role of methylation in modulating bacterial immune evasion and adherence to host cells. In this study, we analyzed the cell surface proteins of the toluene-degrading bacterium sp. Tol 5 by label-free liquid chromatography‒mass spectrometry and found multiple lysine methylation in its trimeric autotransporter adhesin (TAA), AtaA. Over 130 lysine residues of AtaA, consisting of 3,630 amino acids and containing 234 lysine residues, were methylated. We identified that the outer membrane protein lysine methyltransferase (OM PKMT) of Tol 5, KmtA, specifically methylates the lysine residues of AtaA. In the KmtA-deficient mutant, most lysine methylations on AtaA were absent, indicating that KmtA is responsible for the methylation of multiple lysine residues throughout AtaA. Bioinformatic analysis revealed that the OM PKMT genes were widely distributed among Gram-negative bacteria, including pathogens with TAAs that promote infectivity, such as and . Although KmtA has sequence similarities to the OM PKMTs of involved in infectivity, KmtA-like PKMTs formed a distinct cluster from those of the type according to the clustering analysis, suggesting that they are new types of OM PKMTs. Furthermore, the deletion of Tol 5 KmtA led to an increase in AtaA on the cell surface and enhanced bacterial adhesion, resulting in slower growth.
Lysine methylation has been underexplored in prokaryotes, and information on it is limited to some pathogens. Our finding is the second case of multiple lysine methylation of bacterial outer membrane (OM) proteins, following that of OmpB of . The newly found target of methylation, AtaA, a trimeric autotransporter adhesin family protein of sp. Tol 5 isolated from activated sludge, extends our understanding of OM protein methylation to non-pathogenic environmental strains. The newly identified enzyme KmtA shows higher specificity than rickettsial lysin methylases, protein lysine methyltransferases, and methylates more lysine residues of the target, which raises interest in the mechanism underlying its biological specificity. The widespread presence of KmtA-like PKMTs throughout Gram-negative bacteria suggests that lysine methylation functions more extensively in bacterial physiology than previously recognized.
蛋白质赖氨酸残基上ε-氨基的甲基化是真核生物中一种重要的翻译后修饰。这种修饰在多种生物过程的调控中起关键作用，包括表观遗传学、转录控制和细胞信号传导。最近的研究开始揭示甲基化在调节细菌免疫逃逸和黏附宿主细胞方面的潜在作用。在本研究中，我们通过无标记液相色谱 - 质谱分析法分析了甲苯降解菌Tol 5的细胞表面蛋白，发现在其三聚体自转运黏附素（TAA）AtaA中存在多个赖氨酸甲基化。AtaA由3630个氨基酸组成，含有234个赖氨酸残基，其中超过130个赖氨酸残基发生了甲基化。我们确定Tol 5的外膜蛋白赖氨酸甲基转移酶（OM PKMT）KmtA特异性地使AtaA的赖氨酸残基甲基化。在KmtA缺陷型突变体中，AtaA上的大多数赖氨酸甲基化缺失，表明KmtA负责AtaA中多个赖氨酸残基的甲基化。生物信息学分析表明，OM PKMT基因广泛分布于革兰氏阴性菌中，包括具有促进感染性的TAA的病原体，如[具体病原体1]和[具体病原体2]。尽管KmtA与参与感染性的[某种细菌]的OM PKMTs具有序列相似性，但根据聚类分析，KmtA样PKMTs与[该种细菌]类型的形成了一个不同的簇，表明它们是新型的OM PKMTs。此外，Tol 5 KmtA的缺失导致细胞表面AtaA增加并增强细菌黏附，从而导致生长缓慢。
赖氨酸甲基化在原核生物中研究较少，相关信息仅限于一些病原体。我们的发现是继[某种细菌]的OmpB之后细菌外膜（OM）蛋白多个赖氨酸甲基化的第二例。新发现的甲基化靶点AtaA是从活性污泥中分离出的Tol 5菌的三聚体自转运黏附素家族蛋白，将我们对OM蛋白甲基化的理解扩展到非致病性环境菌株。新鉴定的酶KmtA比立克次氏体赖氨酸甲基转移酶、蛋白质赖氨酸甲基转移酶具有更高的特异性，并且使靶点的更多赖氨酸残基甲基化，这引发了对其生物学特异性潜在机制的兴趣。KmtA样PKMTs在革兰氏阴性菌中的广泛存在表明赖氨酸甲基化在细菌生理学中的功能比以前认识的更广泛。
