Yang Tsuey-Ching, Wu Shao-Chi, Yeh Ting-Yu, Lu Hsu-Feng, Lin Yi-Tsung, Li Li-Hua
Department of Biotechnology and Laboratory Science in Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan, Republic of China.
Department of Medical Laboratory Science and Biotechnology, Asia University, Taichung, Taiwan, Republic of China.
BMC Microbiol. 2025 Mar 7;25(1):122. doi: 10.1186/s12866-025-03840-9.
Outer membrane protein OmpA is composed of two domains, an N-terminal β-barrel structure embedded in the outer membrane and a C-terminal globular domain noncovalently associated with the peptidoglycan layer in periplasm. Stenotrophomonas maltophilia KJ is a clinical isolate. In our recent study, we disclosed that KJ∆OmpA an OmpA C-terminal deletion mutant, compromised menadione tolerance. Furthermore, the involvement of σ, σ, and ompO in the ∆ompA-mediated phenotype was proposed. In that study, we hypothesized that there was an unidentified σ-regulated candidate responsible for ∆ompA-mediated menadione tolerance decrease, and the candidate was disclosed in this study.
Transcriptome analysis of wild-type KJ and KJ∆OmpA revealed that a five-gene cluster, smlt4023-smlt4019 (annotated as nagPIBAF), was upregulated in KJ∆OmpA. Reverse transcription-PCR (RT-PCR) confirmed the presence of the nagPIBAF operon. The expression of the nagPIBAF operon was negatively regulated by NagI and σ, and triggered by N-acetylglucosamine. In-frame deletion mutant construction and menadione tolerance assay demonstrated that nagP, nagB, and nagA upregulation in KJ∆OmpA connected with ∆ompA-mediated menadione tolerance decrease. The intracellular reactive oxygen species (ROS) level assay further verified that in the presence of external oxidative stress such as menadione treatment, nagPIBAF operon upregulation and ompO inactivation synergistically increased intracellular ROS levels, which exceeded the capacity of bacterial oxidative stress alleviation systems and resulted in a decrease of menadione tolerance.
Loss of interaction between OmpA C-terminus and peptidoglycan causes envelope stress and activates σ regulon. ompO and rpoN are downregulated in response to σ activation. rpoN downregulation further derepresses nagPIBAF operon, which can favor the metabolism route of glycolysis, TCA cycle, and electron transport chain. nagPIBAF upregulation and OmpO downregulation synergistically increase intracellular ROS levels and result in menadione tolerance decrease.
Not applicable.
外膜蛋白OmpA由两个结构域组成，一个N端β桶结构嵌入外膜，一个C端球状结构域与周质中的肽聚糖层非共价结合。嗜麦芽窄食单胞菌KJ是一株临床分离株。在我们最近的研究中，我们发现KJ∆OmpA（一种OmpA C端缺失突变体）的甲萘醌耐受性受损。此外，还提出了σ、σ和ompO参与∆ompA介导的表型。在该研究中，我们假设存在一个未鉴定的受σ调控的候选基因，它负责∆ompA介导的甲萘醌耐受性降低，并且该候选基因在本研究中被揭示。
对野生型KJ和KJ∆OmpA的转录组分析表明，一个五基因簇smlt4023-smlt4019（注释为nagPIBAF）在KJ∆OmpA中上调。逆转录PCR（RT-PCR）证实了nagPIBAF操纵子的存在。nagPIBAF操纵子的表达受NagI和σ负调控，并由N-乙酰葡糖胺触发。框内缺失突变体构建和甲萘醌耐受性测定表明，KJ∆OmpA中nagP、nagB和nagA的上调与∆ompA介导的甲萘醌耐受性降低有关。细胞内活性氧（ROS）水平测定进一步证实，在甲萘醌处理等外部氧化应激存在的情况下，nagPIBAF操纵子上调和ompO失活协同增加细胞内ROS水平，超过了细菌氧化应激缓解系统的能力，导致甲萘醌耐受性降低。
OmpA C端与肽聚糖之间相互作用的丧失导致包膜应激并激活σ调控子。ompO和rpoN因σ激活而下调。rpoN下调进一步解除对nagPIBAF操纵子的抑制，这有利于糖酵解、三羧酸循环和电子传递链的代谢途径。nagPIBAF上调和OmpO下调协同增加细胞内ROS水平，导致甲萘醌耐受性降低。
不适用。
