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筛选和功能表征异柠檬酸裂解酶 AceA 在生物膜形成中的作用。

Screening and functional characterization of isocitrate lyase AceA in the biofilm formation of .

机构信息

School of Marine Sciences, Ningbo University, Ningbo, P. R. China.

出版信息

Appl Environ Microbiol. 2024 Nov 20;90(11):e0069724. doi: 10.1128/aem.00697-24. Epub 2024 Oct 8.

Abstract

Biofilm is a well-known sessile lifestyle for bacterial pathogens, but a little is known about the mechanism on biofilm formation in . In this study, we screened strains with strong biofilm formation ability from coastal seawater. The antibiotic resistance of the biofilm cells (BFs) was higher than that of the planktonic cells (PTs). To study the genes and pathways involved in biofilm formation, we performed transcriptome analysis of the BFs and PTs of R9. A total of 685 differentially expressed genes (DEGs) were upregulated, and 517 DEGs were downregulated in the BFs. The upregulated DEGs were significantly enriched in several pathways including glyoxylate and dicarboxylate metabolism, while the downregulated genes were significantly enriched in the flagellar assembly pathways. The key gene involved in glyoxylate shunt, , was cloned, and Δ mutant was constructed to determine the function of AceA in carbon source utilization, biofilm formation, and virulence. Real-time reverse transcription PCR showed that the expression of was higher at the mature stage but lower at the disperse stage of biofilm formation, and the expression of the flagellar related genes was upregulated in Δ. This is the first study to illustrate the global gene expression profile during the biofilm formation of , and isocitrate lyase AceA, the key enzyme involved in glyoxylate shunt, was shown to maintain biofilms accompanied by downregulation of flagellation but promoted dispersal of BFs at the late stage.IMPORTANCEBiofilms pose serious public problems, not only protecting the cells in it from environmental hazard but also affecting the composition and abundance of bacteria, algae, fungi, and protozoa. The important opportunistic pathogen is extremely ubiquitously present in seawater, and it also exhibited a strong ability to form biofilm; thus, investigation on the biofilm formation of at molecular level is fundamental for the deeper exploration of the environmental concerns arose by biofilm. In this study, transcriptome analysis of biofilm cells (BFs) and planktonic cells (PTs) from was performed and AceA was screened to play an important role in biofilm formation. AceA was shown to maintain biofilms accompanied by downregulation of flagellation but promoted dispersal of BFs at the disperse stage. This method was helpful to further understand the ability and mechanism of biofilm formation and provide clues for prevention of infection.

摘要

生物膜是细菌病原体的一种众所周知的固着生活方式,但对 中生物膜形成的机制知之甚少。在这项研究中,我们从沿海海水中筛选出具有强生物膜形成能力的 菌株。生物膜细胞 (BFs) 的抗生素抗性高于浮游细胞 (PTs)。为了研究生物膜形成涉及的基因和途径,我们对 R9 的 BFs 和 PTs 进行了转录组分析。结果共鉴定到 685 个差异表达基因 (DEGs),其中 517 个基因上调,168 个基因下调。上调的 DEGs 在几个途径中显著富集,包括乙醛酸和二羧酸代谢途径,而下调的基因在鞭毛组装途径中显著富集。克隆了参与乙醛酸分流的关键基因 ,并构建了 Δ 突变体,以确定 AceA 在碳源利用、生物膜形成和毒力中的作用。实时逆转录 PCR 显示,在生物膜形成的成熟阶段, 的表达水平较高,而在分散阶段较低,并且 Δ 中的鞭毛相关基因表达上调。这是首次阐明 生物膜形成过程中的全基因表达谱,并且发现乙醛酸分流关键酶异柠檬酸裂解酶 AceA 维持生物膜的形成,同时下调鞭毛的表达,但在后期促进 BFs 的分散。

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