| Fructooligosaccharides (FOS) are polymers of fructosyl residues connected by β(2-1) orβ(2-1) glycosidic bonds with degree of polymerization ranging from2-10. As one of thepopular prebiotics, FOS can selectively stimulate the growth and activity of lactobacilli andbifidobacteria and beneficially modulate the balance of the intestinal microbiota. Although theprebiotic effects of FOS have been demonstrated both in vivo and in vitro, the molecularmechanism for FOS metabolism by lactic acid bacteria is still unclear. In this study, a systemsbiology strategy was used to describe the physiological process and metabolic regulations forFOS metabolism by Lactobacillus plantarum ST-III, including analysis the pathways for FOSmetabolism by genomics and transcriptomics approaches, validation the key genes andunderstanding the alterations both for metabolites and cells. The main results are described asfollows:(1) Whole-genome sequencing of Lb. plantarum ST-III was performed with a combinedstrategy of Roche454and Solexa paired-end sequencing technology. The complete genomesequence of ST-III is composed of a circular3,254,376bp chromosome and a53,560bpplasmid, with3,014and42coding sequences, respectively. Comparative genomics approachwas used to analysis the characteristics of the genomes of Lb. plantarum ST-III. On one hand,the genomes of ST-III carries most of the core genes and pathways of Lb.plantarum, with noknown pathogenic genes identified. On the other hand, the genome of Lb. plantarum ST-IIIdisplays several features different from others, for example, it contains genes with related tocholesterol removal ability. In addition, a remarkable degree of redundancy was observed inthe genome of Lb. plantarum ST-III for genes involved in oligosaccharides metabolism,especially several potential functional genes for FOS metabolism were found.(2) A systematic analysis was carried out for the endogenous plasmid pST-III from Lb.plantarum ST-III based on bioinformatics methods and the related features were characterized.Homology analysis for the replication protein and the typical features of the origin ofreplication suggested that pST-III replicates via the θ-type mechanism. Among the42genes,33of them have been assigned to biological functions. Especially, a kdp gene clusterencoding a high-affinity K+transport system was discovered for the first time in theLactobacillus genus. In addition, a system for osmolyte transport was also encoded in theplasmid. Analysis of the plasmid-encoded functions and the plasmid-cured experimentshowed that genes of pST-III play a significant role in hyperosmotic resistance for ST-III.(3) The differential global transcriptome of Lb. plantarum ST-III using FOS or glucoseas a sole source of carbohydrate was studied by RNA-seq in the mid-exponential phase of thestrains. A total of363genes were found to be differently expressed for Lb.plantarum ST-III inthe presence of FOS compared with glucose;324genes were up-regulated and39genes weredown-regulated. Especially, two clusters, which were7.5and4.5kb respectively, werepredicted to be involved in the FOS metabolism. To test this hypothesis, experimentalvalidation of genes encoding the β-fructosidase(SacA) and PTS systems(SacPTS1andSacPTS2) was performed by gene deletion. The results showed that FOS was transported intact across the membrane by SacPTS1and SacPTS2and was hydrolyzed intomonosaccharides by SacA in the cytoplasm. The combined results of transciptome, membranefatty acids(FAs) composition and fluidity suggested that genes involved in FAs biosynthesiswere repressed for Lb.plantarum ST-III grown on FOS; as a result, the FAs profile altered andcarbon chain shortened, and then membrane fluidity increased in response to FOS transportand utilization. In addition, according to transcriptome analysis, genes related to the proposedpathways for the production acetate were up-regulated for Lb.plantarum ST-III in thepresence of FOS and the metabolites analysis also indicated that the acetate contentsignificantly increased for cells grown on FOS compared to glucose.(4) Sequence analysis was performed for the β-fructosidase(SacA) which is necessary forFOS metabolism in Lb.plantarum ST-III. The sacA gene encodes a peptide of501amino acidswith a predicted molecular weight of56.7kDa. Sequence alignment revealed the presence ofthree highly conserved motifs, NDPNG, RDP and EC, indicating that the enzyme belongs toglycoside hydrolase family32. The three-dimensional structure of SacA protein was predictedto contain a five-blade β-propeller module and a β-sandwich domain with one specialN-terminal α-helix. Then the sacA was cloned into Escherichia coli, and the properties of therecombinant protein were examined. The optimal reaction temperature and pH of the enzymewere37℃and6.0, respectively. It was stable at low temperatures but the activity wasstrongly inhibited by Ag+, Cu2+and Hg2+. According to the analysis to the hydrolysis site,SacA enzyme hydrolyzed the β(2-1) linkages in an exo-type fashion, releasing terminalfructosyl residues from the non-reducing ends of the substrates. Kinetic parametersdemonstrated that1-kestone was the most preferred substrate for the enzyme. Additionally,the heterologous expression of the sacA gene in a non-FOS-fermenting strain, Lactobacillusrhamnosus GG, enabled the recombinant strain to metabolize FOS. |
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