Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, P. R. China; Zhejiang Shaoxing Research Institute of Tianjin University, Shaoxing 312300, P. R. China.
Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, P. R. China.
J Dairy Sci. 2022 Oct;105(10):7940-7950. doi: 10.3168/jds.2022-21898. Epub 2022 Aug 24.
Lactococcus lactis, a lactic acid bacterium, has been widely used in the fermented dairy products. The acid tolerance of L. lactis is of great importance to food fermentation and probiotic applications. As the first barrier of bacteria, the cell wall has a protective effect on strains under many stress conditions, whereas the regulatory mechanism has rarely been reported. Here, based on the transcription analysis of 9 cell wall or membrane-related genes of L. lactis F44 under acid stress, the transcription levels of DACB, DLTD, YLBA, HRTA, WP_080613266.1 (1610), and ERFK genes were significantly increased. We constructed 9 overexpressing strains with the cell wall or membrane-related genes, respectively. It was demonstrated that the survival rates under acid stress of DACB, DLTD, and ERFK were significantly higher than that of wild-type F44. To investigate the regulatory mechanism, a DNA pull-down assay was used to identify the transcriptional regulators of these 3 genes. It was discovered that the 2-component system (TCS) transcriptional regulator TCSR7 bound to the upstream region of DLTD involved in the teichoic acid (TA) alanylation. The combination was confirmed through an electrophoretic mobility shift assay in vitro. Reverse-transcription quantitative PCR results indicated that TCSR7 upregulated the expression of DLTD gene. In addition, the transcription level of TCSR7 increased approximately 1.8-fold (log2 fold change) under acidic conditions. In summary, this study found that TCSR7 was induced by acid stress to upregulate the transcription level of the DLT operon genes, which might increase the positive charge on the cell membrane surface to increase the acid tolerance of the strain. This study lays the foundation for the regulatory mechanism of TA alanylation under acid stress.
乳球菌（Lactococcus lactis）是一种乳酸细菌，已广泛应用于发酵乳制品。乳球菌的耐酸能力对食品发酵和益生菌应用非常重要。作为细菌的第一道屏障，细胞壁在许多应激条件下对菌株具有保护作用，但其调控机制鲜有报道。本研究基于酸胁迫下乳球菌 F44 的 9 个细胞壁或膜相关基因的转录分析，发现 DACB、DLTD、YLBA、HRTA、WP_080613266.1（1610）和 ERFK 基因的转录水平显著上调。我们分别构建了 9 个与细胞壁或膜相关的基因过表达菌株，结果表明 DACB、DLTD 和 ERFK 的耐酸存活率明显高于野生型 F44。为了研究调控机制，我们采用 DNA 下拉实验鉴定了这 3 个基因的转录调控因子。发现参与磷壁酸（TA）丙氨酸化的双组分系统（TCS）转录调控因子 TCSR7 结合到 DLTD 上游区域。通过体外电泳迁移率变动分析实验证实了这种结合。反转录定量 PCR 结果表明 TCSR7 上调了 DLTD 基因的表达。此外，TCSR7 的转录水平在酸性条件下增加了约 1.8 倍（log2 倍变化）。综上所述，本研究发现 TCSR7 被酸胁迫诱导而上调 DLT 操纵子基因的转录水平，这可能增加了细胞膜表面的正电荷，从而提高了菌株的耐酸性。本研究为 TA 丙氨酸化在酸胁迫下的调控机制奠定了基础。
