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

Abstract

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.

Keywords: Streptococcus bovis S1; catabolite control protein A; glucose concentration; metabolism regulation; transcriptome.

FIGURE 1

Growth curve of wild-type strain (WT) and ccpA-knockout strain (KO) of S. bovis S1 in glucose-excess or -limited conditions measured as optical density at 600 nm (OD₆₀₀). Error bars indicate SD. The maximal growth rate (μ_max) was estimated and shown in the figure. Values marked with different superscript uppercase letters (AB) indicate those are statistically significant differences (P < 0.05) between wild-type strain (WT) and ccpA-knockout strain (KO); values marked with different superscript lowercase letters (ab) indicate those are statistically significant differences (P < 0.05) between strains grown in glucose-excess or -limited conditions. LGWT, the wild-type strain grown in the media with 5 mM glucose; LGKO, the ccpA-knockout strain grown in the media with 5 mM glucose; HGWT, the wild-type strain grown in the media with 50 mM glucose; HGKO, the ccpA-knockout strain grown in the media with 50 mM glucose.

FIGURE 2

Intracellular concentration of FBP in wild-type strain (WT) and ccpA-knockout strain (KO) of S. bovis S1 in glucose-excess or -limited conditions. HGWT, the wild-type strain grown in the media with 50 mM glucose; HGKO, the ccpA-knockout strain grown in the media with 50 mM glucose; LGWT, the wild-type strain grown in the media with 5 mM glucose; LGKO, the ccpA-knockout strain grown in the media with 5 mM glucose. **Means P < 0.01.

FIGURE 3

Distribution of upregulated and downregulated genes in the four pair-wise comparisons based on KEGG pathway categories. (A) The comparison between wild-type and its ccpA mutant grown in glucose-excess condition; (B) the comparison between wild-type and its ccpA mutant grown in glucose-limited condition; (C) the comparison between wild-type strain grown in glucose-excess and -limited conditions; (D) the comparison between ccpA mutant grown in glucose-excess and -limited conditions.

FIGURE 4

Overview of the key genes and related pathways changed in the transcriptomic analysis. 1, the comparison between wild-type strain grown in glucose-excess and -limited conditions; 2, the comparison between ccpA mutant grown in glucose-excess and -limited conditions. 3, the comparison between wild-type and its ccpA mutant grown in glucose-excess condition; 4, the comparison between wild-type and its ccpA mutant grown in glucose-limited condition. Gene annotation: scrA, sucrose PTS system EIIBCA or EIIBC component; scrB, beta-fructofuranosidase; scrK, fructokinase; amyA, alpha-amylase; malQ, 4-alpha-glucanotransferase; mapA, maltose phosphorylase; pgmB, beta-phosphoglucomutase; ptsG, glucose PTS system EIICB or EIICBA component; pgi, glucose-6-phosphate isomerase; manXa, mannose PTS system EIIA component; manA, mannose-6-phosphate isomerase; pfkA, 6-phosphofructokinase; fba, fructose-bisphosphate aldolase; tpi, triosephosphate isomerase; gapA, glyceraldehyde 3-phosphate dehydrogenase; gapN, glyceraldehyde-3-phosphate dehydrogenase (NADP+); pgk, phosphoglycerate kinase; gpmA, 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase; eno, enolase; pyk, pyruvate kinase; ldh, L-lactate dehydrogenase; pfl, formate C-acetyltransferase; pdh, pyruvate dehydrogenase complex (pyruvate dehydrogenase E1 component, dihydrolipoamide dehydrogenase, pyruvate dehydrogenase E2 component); adhE, acetaldehyde dehydrogenase/alcohol dehydrogenase; pta, phosphate acetyltransferase; acyP, acylphosphatase; ackA, acetate kinase; acc, acetyl-CoA carboxylase; fabD, [acyl-carrier-protein] S-malonyltransferase; fabF, 3-oxoacyl-[acyl-carrier-protein] synthase II; fabG, 3-oxoacyl-[acyl-carrier protein] reductase; fabZ, 3-hydroxyacyl-[acyl-carrier protein] dehydratase; fabK, enoyl-[acyl-carrier protein] reductase II; tkt, transketolase; rpe, ribulose-phosphate 3-epimerase; rpiA, ribose 5-phosphate isomerase A; prs, ribose-phosphate pyrophosphokinase; hisG, ATP phosphoribosyltransferase; hisZ, ATP phosphoribosyltransferase regulatory subunit; hisI, phosphoribosyl-AMP cyclohydrolase; hisA, phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase; hisF, imidazole glycerol-phosphate synthase subunit; hisB, imidazoleglycerol-phosphate dehydratase; hisC, histidinol-phosphate aminotransferase; hisD, histidinol dehydrogenase.