Table_1_Transcriptome Analysis Reveals Catabolite Control Protein A Regulatory Mechanisms Underlying Glucose-Excess or -Limited Conditions in a Ruminal Bacterium, Streptococcus bovis.XLSX

Ruminants may suffer from rumen acidosis when fed with high-concentrate diets due to the higher proliferation and overproduction of lactate by Streptococcus bovis. The catabolite control protein A (CcpA) regulates the transcription of lactate dehydrogenase (ldh) and pyruvate formate-lyase (pfl) in S. bovis, but its role in response to different carbon concentrations remains unclear. To characterize the regulatory mechanisms of CcpA in S. bovis S1 at different levels of carbon, herein, we analyzed the transcriptomic and physiological characteristics of S. bovis S1 and its ccpA mutant strain grown in glucose-excess and glucose-limited conditions. A reduced growth rate and a shift in fermentation pattern from homofermentation to heterofermentation were observed under glucose-limited condition as compared to glucose-excess condition, in S. bovis S1. Additionally, the inactivation of ccpA significantly affected the growth and end metabolites in both conditions. For the glycolytic intermediate, fructose 1,6-bisphosphate (FBP), the concentration significantly reduced at lower glucose conditions; its concentration decreased significantly in the ccpA mutant strain. Transcriptomic results showed that about 46% of the total genes were differentially transcribed between the wild-type strain and ccpA mutant strain grown in glucose-excess conditions; while only 12% genes were differentially transcribed in glucose-limited conditions. Different glucose concentrations led to the differential expression of 38% genes in the wild-type strain, while only half of these were differentially expressed in the ccpA-knockout strain. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the substrate glucose concentration significantly affected the gene expression in histidine metabolism, nitrogen metabolism, and some carbohydrate metabolism pathways. The deletion of ccpA affected several genes involved in carbohydrate metabolism, such as glycolysis, pyruvate metabolism, fructose and mannose metabolism, as well as in fatty acid biosynthesis pathways in bacteria grown in glucose-excess conditions; this effect was attenuated under glucose-limited conditions. Overall, these findings provide new information on gene transcription and metabolic mechanisms associated with substrate glucose concentration and validate the important role of CcpA in the regulation of carbon metabolism in S. bovis S1 at differential glucose availability.

反刍动物饲喂高精料日粮时，可能罹患瘤胃酸中毒（rumen acidosis），这一病症由牛链球菌（Streptococcus bovis）过度增殖并过量合成乳酸所引发。分解代谢物控制蛋白A（catabolite control protein A, CcpA）可调控牛链球菌中乳酸脱氢酶（lactate dehydrogenase, ldh）与丙酮酸甲酸裂解酶（pyruvate formate-lyase, pfl）的转录过程，但其在响应不同碳源浓度中的作用仍有待阐明。为解析不同碳水平下牛链球菌S1中CcpA的调控机制，本研究对在葡萄糖过量与葡萄糖限制培养条件下培养的牛链球菌S1及其ccpA突变株的转录组与生理特征进行了分析。相较于葡萄糖过量培养条件，葡萄糖限制条件下的牛链球菌S1生长速率下降，发酵模式从同型发酵转向异型发酵。此外，ccpA基因的失活会显著影响两种培养条件下的菌体生长与末端代谢产物谱。对于糖酵解中间产物果糖-1,6-二磷酸（fructose 1,6-bisphosphate, FBP），其浓度在低葡萄糖条件下显著降低；同时在ccpA突变株中，该代谢物的浓度也显著下降。转录组分析结果显示，在葡萄糖过量培养条件下，野生型菌株与ccpA突变株间约有46%的总基因存在转录差异；而在葡萄糖限制条件下，仅12%的基因呈现转录差异。野生型菌株中，不同葡萄糖浓度会导致38%的基因差异表达，而在ccpA敲除菌株中，仅半数此类基因存在差异表达。京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析表明，底物葡萄糖浓度显著影响组氨酸代谢、氮代谢以及部分碳水化合物代谢通路的基因表达。在葡萄糖过量培养条件下，ccpA的缺失会影响若干参与碳水化合物代谢的基因，包括糖酵解、丙酮酸代谢、果糖与甘露糖代谢，以及细菌的脂肪酸生物合成通路；而在葡萄糖限制条件下，这种影响会被减弱。综上，本研究结果为与底物葡萄糖浓度相关的基因转录与代谢机制提供了新的认知，并验证了CcpA在不同葡萄糖可利用性条件下，对牛链球菌S1碳代谢调控的重要作用。