DairySafe Group, Department of Technology and Biotechnology of Dairy Products, IPLA-CSIC, Carretera de Infiesto s/n, Apdo. 85, 33300 Villaviciosa, Asturias, Spain.
Appl Environ Microbiol. 2011 Nov;77(21):7576-85. doi: 10.1128/AEM.06107-11. Epub 2011 Sep 2.
pBL1 is a Lactococcus lactis theta-replicating 10.9-kbp plasmid that encodes the synthetic machinery of the bacteriocin Lcn972. In this work, the transcriptomes of exponentially growing L. lactis strains with and without pBL1 were compared. A discrete response was observed, with a total of 10 genes showing significantly changed expression. Upregulation of the lactococcal oligopeptide uptake (opp) system was observed, which was likely linked to a higher nitrogen demand required for Lcn972 biosynthesis. Strikingly, celB, coding for the membrane porter IIC of the cellobiose phosphoenolpyruvate-dependent phosphotransferase system (PTS), and the upstream gene llmg0186 were downregulated. Growth profiles for L. lactis strains MG1363, MG1363/pBL1, and MG1363 ΔcelB grown in chemically defined medium (CDM) containing cellobiose confirmed slower growth of MG1363/pBL1 and MG1363 ΔcelB, while no differences were observed with growth on glucose. The presence of pBL1 shifted the fermentation products toward a mixed acid profile and promoted substantial changes in intracellular pool sizes for glycolytic intermediates in cells growing on cellobiose as determined by high-pressure liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). Overall, these data support the genetic evidence of a constriction in cellobiose uptake. Notably, several cell wall precursors accumulated, while other UDP-activated sugar pools were lower, which could reflect rerouting of precursors toward the production of structural or storage polysaccharides. Moreover, cells growing slowly on cellobiose and those lacking celB were more tolerant to Lcn972 than cellobiose-adapted cells. Thus, downregulation of celB could help to build up a response against the antimicrobial activity of Lcn972, enhancing self-immunity of the producer cells.
pBL1 是一种乳球菌 theta 复制的 10.9-kbp 质粒，它编码细菌素 Lcn972 的合成机制。在这项工作中，比较了含有和不含有 pBL1 的乳球菌指数生长期的转录组。观察到一个离散的反应，总共有 10 个基因表现出显著变化的表达。乳球菌寡肽摄取（opp）系统的上调被观察到，这可能与 Lcn972 生物合成所需的更高氮需求有关。引人注目的是，编码细胞二糖磷酸烯醇丙酮酸依赖磷酸转移酶系统（PTS）膜载体 IIC 的 celB 和上游基因 llmg0186 下调。在含有细胞二糖的化学定义培养基（CDM）中生长的乳球菌 MG1363、MG1363/pBL1 和 MG1363ΔcelB 的生长曲线证实了 MG1363/pBL1 和 MG1363ΔcelB 的生长速度较慢，而在葡萄糖上生长则没有差异。pBL1 的存在将发酵产物转向混合酸谱，并通过高压液相色谱（HPLC）和核磁共振（NMR）确定，在细胞二糖上生长时，细胞内糖酵解中间产物的池大小发生了实质性变化。总的来说，这些数据支持细胞二糖摄取受到限制的遗传证据。值得注意的是，几种细胞壁前体积累，而其他 UDP 激活的糖池较低，这可能反映出前体向结构或储存多糖的生产重新布线。此外，在细胞二糖上生长缓慢的细胞和缺乏 celB 的细胞比适应细胞二糖的细胞对 Lcn972 的耐受性更强。因此，celB 的下调可以帮助建立对 Lcn972 抗菌活性的反应，增强生产细胞的自我免疫能力。
