Functional analysis of the role of CcpA in Lactobacillus plantarum grown on fructooligosaccharides or glucose: a transcriptomic perspective

Abstract

Background: The catabolite control protein A (CcpA) is a master regulator of many important cellular processes in Gram-positive bacteria. In Lactobacillus plantarum, CcpA directly or indirectly controls the transcription of a large number of genes that are involved in carbohydrate metabolism, aerobic and anaerobic growth, stress response and metabolite production, but its role in response to different carbon sources remains unclear.

Results: Here a combined transcriptomic and physiological approach was used to survey the global alterations that occurred during the logarithmic growth phase of wild-type and ccpA mutant strains of L. plantarum ST-III using fructooligosaccharides (FOS) or glucose as the sole carbon source. The inactivation of ccpA significantly affected the growth and production of metabolites under both carbon sources. About 15% of the total genes were significantly altered between wild-type and ccpA strains grown on glucose and the value is deceased to 12% when these two strains were compared on FOS, while only 7% were obviously changed due to the loss of CcpA when comparing strains grown on glucose and FOS. Although most of the differentially expressed genes mediated by CcpA are glucose dependent, FOS can also induce carbon catabolite repression (CCR) through the CcpA pathway. Moreover, the inactivation of ccpA led to a transformation from homolactic fermentation to mixed fermentation under aerobic conditions. CcpA can control genes directly by binding in the regulatory region of the target genes (mixed fermentation), indirectly through local regulators (fatty acid biosynthesis), or have a double effect via direct and indirect regulation (FOS metabolism).

Conclusion: Overall, our results show that CcpA plays a central role in response to carbon source and availability of L. plantarum and provide new insights into the complex and extended regulatory network of lactic acid bacteria.

Keywords: Carbohydrate metabolism; Catabolite control protein A; Fatty acid biosynthesis; Lactobacillus plantarum; Mixed fermentation; Transcriptome.

Fig. 1

Growth curves of the wild-type and ccpA mutant in CDM containing glucose or FOS. Sampling point was chosen for the transcriptomic analysis, the metabolites measurement and RT-qPCR analysis. The μ_max for each condition was also calculated and shown in the figure. Data presented are mean values based on two replicate fermentations. Error bars indicate standard deviations

Fig. 2

Distribution of upregulated and downregulated genes by KEGG pathway categories of the four pair-wise comparisons. a The ccpA mutant strain grown on glucose versus the wild-type strain grown on glucose. b The ccpA mutant strain grown on FOS versus the wild-type strain grown on FOS. c The wild-type strain grown on FOS versus the wild-type strain grown on glucose. d The ccpA mutant strain grown on FOS versus the ccpA mutant strain grown on glucose. KEGG pathway categories: 1, carbohydrate metabolism; 2, energy metabolism; 3, lipid metabolism; 4, nucleotide metabolism; 5, amino acid metabolism; 6, metabolism of other amino acids; 7, glycan biosynthesis and metabolism; 8, metabolism of cofactors and vitamins; 9, metabolism of terpenoids and polyketides; 10, biosynthesis of other secondary metabolites; 11, xenobiotic biodegradation and metabolism; 12, enzyme families; 13, transcription; 14, translation; 15, folding, sorting and degradation; 16, replication and repair; 17, RNA family; 18, membrane transport; 19, signal transduction; 20, signaling molecules and interaction; 21, transport and catabolism; 22, cell motility; 23, cell growth and death; 24, Cellular community—prokaryotes

Fig. 3

Venn diagrams of the number of genes differentially altered in the four pair-wise comparisons. a Number of differentially altered genes in ccpA mutant compared to the wild-type, grown on glucose (dotted line) and FOS (full line). b Number of differentially altered genes in response to FOS compared with glucose of the wild-type (dotted line) and ccpA mutant strains (full line)

Fig. 4

Overview of the key genes and related pathways changed in the transcriptomic analysis. 1, the ccpA mutant strain grown on glucose versus the wild-type strain grown on glucose; 2, the ccpA mutant strain grown on FOS versus the wild-type strain grown on FOS; 3, the ccpA mutant strain grown on FOS verus the ccpA mutant strain grown on glucose. Gene annotation downloaded from NCBI: pts, phosphotransferase system; sacK, fructokinase; pgi, glucose-6-phosphate isomerase; fbp, fructose-1,6-bisphosphatase; pfkA, 6-phosphofructokinase; fbpA, fructose-bisphosphate aldolase; tpi, triose-3-phosphate isomerase; gapB, glyceraldehyde-3-phosphate dehydrogenase; pgk, phosphoglycerate kinase; pgm, phosphoglycerate mutase; eno, enolase; pyk, pyruvate kinase; ldh, l-lactate dehydrogenase; pfl, pyruvate formate-lyase; pdh, pyruvate dehydrogenase; pox, pyruvate oxidase; pta, phosphotransacetylase; ack, acetate kinase; acc, acetyl-CoA carboxylase; fabD, ACP-S-malonyltransferase; fabF, 3-oxoacyl-ACP synthase II; fabH, 3-oxoacyl-ACP synthase III; fabG, 3-oxoacyl-ACP reductase; fabZ, (3R)-hydroxymyristoyl-ACP dehydratase; fabI, enoyl-ACP reductase