| Rumen acidosis is a common nutritional metabolic disease in intensive ruminant production.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 depending on the source of carbon.The main cause of rumen acidosis is the high concentration of readily fermentable carbohydrates in diet,however,whether the regulation of CcpA on the production of organic acids and expression of target genes of S.bovis is dependent on carbon concentrations,remains unknown.In the present study,the regulation of CcpA on the fermentation pathway organic acids and its regulation mechanism were investigated,which may provide scientific basis for prevention and control of ruminant rumen acidosis in the future.Trial 1:Construction of S.bovis S1 ccpA knockout strain and its physiological characteristicsIn this study,the ccpA-knockout strain of S.bovis S1 was constructed by homologous recombination.Strains were cultured in batch with 3 g/L glucose as an energy source.Cell growth,organic acids production,the activities of lactate dehydrogenase and acetate kinase were analyzed.The results showed as follows:the absence of ccpA gene significantly prolonged the delay period and decreased the maximum growth rate of S.bovis S1(P<0.05);compared with the wild strain,the percentage of lactic acid in ccpA-knockout strain was significantly decreased(P<0.05),while the percentage of formic acid and acetic acid was significantly increased(P<0.05);the activities of lactate dehydrogenase in ccpAknockout strain significantly decreased(P<0.05),and the activities of acetate kinase significantly increased(P<0.05).Taken together,ccpA gene deletion could inhibit the proliferation of S.bovis S1 and change its organic acid fermentation mode by regulating the enzyme activities.Trial 2:Regulation of CcpA on the physiological characteristics and transcriptome of S.bovis S1 at different glucose concentrationsIn this study,9 g/L or 0.9 g/L glucose was provided as an energy source,respectively.The transcriptomic and physiological characteristics of S.bovis S1 wild strain and ccpA deletion strain were analysed,and the activities of some key enzymes screened according to the transcriptome results were measured.The results showed as follows:1)The absence of ccpA gene significantly prolonged the delay period and decreased the maximum growth rate of S.bovis S1 at both high and low glucose concentrations(P<0.05);the maximum growth rate of the two strains at high glucose concentration was significantly higher than that at low glucose concentration(P<0.05),and the lag period was longer than that at low glucose concentration(P<0.05).2)Compared with the wild strain,the percentage of lactic acid in ccpA-knockout strain was significantly decreased(P<0.05),while the percentage of formic acid and acetic acid was significantly increased(P<0.05)at both high and low glucose concentrations;as expected,the organic acid yield of the two strains was higher at high glucose concentration(P<0.05),but the percentage of formic acid and acetic acid was significantly lower than that at low glucose concentration(P<0.05).3)Transcriptomic results showed that about 46%of the total genes were differentially transcribed between the wild-type strain and ccpA mutant strain grown at high glucose concentration;while only 12%genes were differentially transcribed at low glucose concentration.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 at high glucose concentration;this effect was attenuated at low glucose concentration.4)At high glucose concentration,the activities of fructose-1,6-diphosphate aldolase(FBA),pyruvate kinase(PYK)and LDH decreased significantly(P<0.05),while the activities of pyruvate dehydrogenase(PDH),NADH oxidase(NOX)and ACKA increased significantly(P<0.05);at low glucose concentration,the activities of FBA,PYK and PDH activities decreased significantly(P<0.05),and there was no significant difference in others(P>0.05);the activities of FBA,PYK and LDH of wild strains at high glucose condition were significantly higher than those at low glucose condition(P<0.05),while the activities of PDH,NOX and ACKA were significantly lower than those at low glucose condition(P<0.05);the activities of FBA,LDH and NOX in ccpA knockout strains were significantly higher than those at low glucose conditions(P<0.05),and the activities of PDH and ACKA were significantly lower than those at low glucose conditions(P<0.05).Taken together,both CcpA and substrate glucose concentration could regulate the proliferation,organic acid production and transcription of related genes of S.bovis S1,and the regulation of CcpA on S.bovis S1 is affected by substrate concentration.Trial 3:Regulation of CcpA on the proteome of S.bovis S1 at different glucose concentrationsIn this study,the differential expression of proteome of S.bovis S1 wild strain and ccpA deletion strain at different glucose concentrations was analyzed using TMT chemical labeling quantitative proteomics technology.The results showed as follows:1)At high glucose concentration,1076 differentially expressed proteins(535 up-regulated and 541 down regulated)were screened from S.bovis S1 wild strain compared with ccpA knockout strain,while at low glucose concentration,there were only 744 differentially expressed proteins(399 up-regulated and 375 down regulated)between the two strains,indicating that the regulation of CcpA on the protein expression of S.bovis S1 may be weakened with the decrease of substrate glucose concentration.Even so,at both high and low glucose concentrations,the differentially expressed proteins caused by ccpA deletion were enriched in KEGG pathways related to carbohydrate metabolism,such as starch and sucrose metabolism,glycolysis/gluconeogenesis,galactose metabolism and propionate metabolism.The deletion of ccpA gene also leaded to the differential protein expression of key enzymes downstream of pyruvate metabolism,in which LDH was down-regulated and PFL and ACKA were up-regulated,with the result that the organic acids fermentation of S.bovis S1 changed.2)Comparing the differentially expressed proteins at different glucose concentrations,total of 1055 differentially expressed proteins(519 up-regulated and 536 down regulated)were screened by S.bovis S1 wild strain,while 977 differentially expressed proteins(438 up-regulated and 539 down regulated)were screened by ccpA deletion strain.KEGG enrichment analysis showed that these differential proteins were mainly involved in glycolysis/gluconeogenesis,pyruvate metabolism,TCA cycle and some amino acid metabolism.Taken together,both CcpA and substrate glucose concentration regulate the carbohydrate metabolism of S.bovis S1 at the protein expression level.Trial 4:Regulation of CcpA on the energy metabolism of S.bovis S1 at different glucose concentrationsThe results of transcriptome and proteome analysis showed that both CcpA and substrate glucose concentration played an important role on the central carbon metabolism(also known as energy metabolism)of S.bovis S1.In this study,therefore,the intracellular energy metabolites of S.bovis S1 wild strain and ccpA deficient strain were analyzed at different glucose concentrations by using targeted energy metabolism quantitative detection technology.The results showed as follows:1)At high glucose concentration,a total of 23 differential metabolites(20 down-regulated and 3 up-regulated)were screened from S.bovis S1 wild strain compared with ccpA deficient strain,while at low glucose concentration,there were 17 differential metabolites(15 down-regulated and 2 up-regulated)between the two strains,indicating that CcpA mainly plays a positive regulatory role in the central carbon metabolism of S.bovis S1.At both high and low glucose concentration,the down regulated differential metabolites caused by ccpA gene deletion included fructose-1,6-diphosphate(FBP),dihydroxyacetone phosphate,phosphoenolpyruvate,pyruvate,lactic acid and some amino acid and nucleotide derivatives,which mainly involved in KEGG metabolic pathways such as glycolysis/gluconeogenesis pathway,alanine,aspartate and glutamate metabolism,pentose phosphate pathway.After ccpA gene deletion,the intracellular concentration of Sedum heptanose 7-phosphate was up-regulated under both high and low glucose conditions,which was mainly related to pentose phosphate pathway.2)There were 13 different metabolites(10 down-regulated and 3 up-regulated)between S.bovis S1 wild strain at different glucose concentrations.The down regulated differential metabolites were mainly involved in KEGG metabolic pathways such as nicotinic acid and nicotinamide metabolism,propionate metabolism,fructose and mannose metabolism,TCA cycle and so on;the up regulated differential metabolites were mainly involved in arginine biosynthesis,arginine and proline metabolism,nitrogen metabolism.A total of 21 differential metabolites(10 down-regulated and 11 up-regulated)were screened from ccpA deletion strains at different glucose concentrations.The down regulated differential metabolites were mainly enriched in KEGG metabolic pathways such as amino acid biosynthesis,pentose phosphate pathway,carbon metabolism,glycolysis/gluconeogenesis pathway;the up-regulated differential metabolites were mainly involved in amino acid biosynthesis,aminoacyl tRNA biosynthesis,protein digestion and absorption.Taken together,at the metabolites level,CcpA could promote the glycolysis of S.bovis S1 and the synthesis of some amino acids and nucleotides,regulate carbon overflow metabolism,and inhibit some pentose phosphate pathway;low glucose concentration would inhibit the glycolysis and TCA activity of S.bovis S1,promote the synthesis of some amino acids,and regulate nucleotide metabolism.In conclusion,ccpA gene deletion could inhibit the proliferation of S.bovis S1 and switch the fermentation pattern from lactic acid fermentation to formic acid and acetic acid fermentation;after ccpA gene deletion,the transcription and protein expression of key enzymes in glycolysis pathway were down-regulated,and the gene and protein expression of key enzymes in pyruvate metabolism were changed,in which LDH(ldh)was down-regulated,PFL(pfl)and ACKA(acka)were up-regulated;additionally,the concentration of intracellular glycolytic intermediate metabolites and lactic acid decreased after the ccpA gene deletion.Comprehensively,CcpA regulated the transcription and protein expression of key enzymes of carbohydrate metabolism of S.bovis S1,so as to change the proportion of organic acids in fermentation products.The regulation of CcpA on organic acids production from glucose metabolism by S.bovis S1 is more effective at high glucose concentration. |
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