Functional characterization of sucrose phosphorylase and scrR, a regulator of sucrose metabolism in Lactobacillus reuteri

Zhao

et al. 2015

Appl Environ Microbiol

dAlthough fructooligosaccharides (FOS) can selectively stimulate the growth and activity of probiotics and beneficially modulate the balance of intestinal microbiota, knowledge of the molecular mechanism for FOS metabolism by probiotics is still limited. Here a combined transcriptomic and physiological approach was used to survey the global alterations that occurred during the logarithmic growth of Lactobacillus plantarum ST-III using FOS or glucose as the sole carbon source. A total of 363 genes were differentially transcribed; in particular, two gene clusters were induced by FOS. Gene inactivation revealed that both of the clusters participated in the metabolism of FOS, which were transported across the membrane by two phosphotransferase systems (PTSs) and were subsequently hydrolyzed by a ␤-fructofuranosidase (SacA) in the cytoplasm. Combining the measurements of the transcriptome-and membrane-related features, we discovered that the genes involved in the biosynthesis of fatty acids (FAs) were repressed in cells grown on FOS; as a result, the FA profiles were altered by shortening of the carbon chains, after which membrane fluidity increased in response to FOS transport and utilization. Furthermore, incremental production of acetate was observed in both the transcriptomic and the metabolic experiments. Our results provided new insights into gene transcription, the production of metabolites, and membrane alterations that could explain FOS metabolism in L. plantarum. Prebiotics are defined as nondigestible food ingredients that selectively stimulate the growth and activity of beneficial microbial strains residing in the host gastrointestinal tract (GIT) (1). Among the sugars that are qualified as prebiotics, fructooligosaccharides (FOS) are fructose polymers of diverse lengths that can be either derivatives of simple fructose polymers or fructose moieties attached to a sucrose molecule (2). Because of the linkage configuration, FOS are not digested in the upper GIT and have been shown in vivo to beneficially modulate the composition of the intestinal microbiota by preferentially increasing the numbers of bifidobacteria and lactobacilli (3, 4).Despite considerable commercial and research interest in the beneficial effects of FOS, the molecular basis of FOS metabolism by specific members of the intestinal microbiota has only recently been examined. In order to understand the influence of environmental conditions on genome-wide gene expression levels, wholegenome DNA microarrays have often been used to survey the gene expression patterns of strains in the presence and absence of oligosaccharides (2, 5-8). On the basis of in silico analysis of the Lactobacillus acidophilus NCFM genome sequence, Barrangou et al. (2) identified a multiple-sugar metabolism (msm) operon that was involved in the metabolism of FOS. The msm operon encodes an ATP-dependent binding cassette-type transport system and a cytoplasmic ␤-fructosidase, which mediates FOS uptake and intracellular hydrolysis. Moreover, expression of the operon was i...

Section: Resultsmentioning

confidence: 99%

Metabolism of Fructooligosaccharides in Lactobacillus plantarum ST-III via Differential Gene Transcription and Alteration of Cell Membrane Fluidity

Zhao

et al. 2015

Appl Environ Microbiol

“…In these two clusters, the local regulators SacR1 and SacR2, which are also members of LacI-GalR family, are cotranscribed with other FOS-related genes. In the presence of FOS, SacR1 and SacR2 may be induced by their substrates and maintain their own expression at a certain level to help bacteria to adjust sugar utilization to their metabolic capacities ( Robert et al, 2008 ; Teixeira et al, 2013 ). Although the effects of CcpA have been confirmed, the interactions between specific local operators and the putative binding sites in these clusters are not directly proven and the exact binding site is not yet clear.…”

Section: Discussionmentioning

confidence: 99%

CcpA-Dependent Carbon Catabolite Repression Regulates Fructooligosaccharides Metabolism in Lactobacillus plantarum

Wang

et al. 2018

Front. Microbiol.

Fructooligosaccharides (FOSs) metabolism in Lactobacillus plantarum is controlled by two gene clusters, and the global regulator catabolite control protein A (CcpA) may be involved in the regulation. To understand the mechanism, this study focused on the regulation relationships of CcpA toward target genes and the binding effects on the catabolite responsive element (cre). First, reverse transcription-PCR analysis of the transcriptional organization of the FOS-related gene clusters showed that they were organized in three independent polycistronic units. Diauxic growth, hierarchical utilization of carbohydrates and repression of FOS-related genes were observed in cultures containing FOS and glucose, suggesting carbon catabolite repression (CCR) control in FOS utilization. Knockout of ccpA gene eliminated these phenomena, indicating the principal role of this gene in CCR of FOS metabolism. Furthermore, six potential cre sites for CcpA binding were predicted in the regions of putative promoters of the two clusters. Direct binding was confirmed by electrophoretic mobility shift assays in vitro and chromatin immunoprecipitation in vivo. The results of the above studies suggest that CcpA is a vital regulator of FOS metabolism in L. plantarum and that CcpA-dependent CCR regulates FOS metabolism through the direct binding of CcpA toward the cre sites in the promoter regions of FOS-related clusters.

“…The sacC gene mutant strain showed three-fold higher levansucrase (SacB) activity than the wild-type strain and the levan titer increased from 15.5 g/L to 21.2 g/L. Similar genetic modification was a performed in Lactobacillus reuteri , demonstrating that disruption of the sucrose phosphorylase gene scrP , (encoding a sucrose hydrolysis enzyme), also increased levan production 14 . Shida et al 15 studied the effects of disrupting the levanase gene sacC on levan production in Bacillus subtilis 327UH strain.…”

mentioning

confidence: 90%

Recruiting a new strategy to improve levan production in Bacillus amyloliquefaciens

Feng

Quan

et al. 2015

Sci Rep

Microbial levan is an important biopolymer with considerable potential in food and medical applications. Bacillus amyloliquefaciens NK-ΔLP strain can produce high-purity, low-molecularweight levan, but production is relatively low. To enhance the production of levan, six extracellular protease genes (bpr, epr, mpr, vpr, nprE and aprE), together with the tasA gene (encoding the major biofilm matrix protein TasA) and the pgsBCA cluster (responsible for poly-γ-glutamic acid (γ-PGA) synthesis), were intentionally knocked out in the Bacillus amyloliquefaciens NK-1 strain. The highest levan production (31.1 g/L) was obtained from the NK-Q-7 strain (ΔtasA, Δbpr, Δepr, Δmpr, Δvpr, ΔnprE, ΔaprE and ΔpgsBCA), which was 103% higher than that of the NK-ΔLP strain (ΔpgsBCA) (15.3 g/L). Furthermore, the NK-Q-7 strain also showed a 94.1% increase in α-amylase production compared with NK-ΔLP strain, suggesting a positive effect of extracellular protease genes deficient on the production of endogenously secreted proteins. This is the first report of the improvement of levan production in microbes deficient in extracellular proteases and TasA, and the NK-Q-7 strain exhibits outstanding characteristics for extracellular protein production or extracellular protein related product synthesis.Microbial levan, one of the two main types of fructan biopolymers, is mainly polymerized via β -(2 → 6) bonds 1 and has been isolated from Gram-negative bacteria, Gram-positive bacteria and some fungi 2 . Levan has many favourable properties, and is used in variety of industrial applications including in foods, cosmetics and pharmaceuticals [3][4][5] .Previous work has revealed that microbial levan is synthesized in the medium by the secreted levansucrase (EC: 2.4.1.10) from the sucrose substrate 6 . Microorganisms synthesize higher-molecular-weight levan at the beginning of fermentation, after which the molecule is hydrolyzed to lower-molecular-weight levan products in the presence of the β -2,6-fructofuranoside linkage-hydrolyzing enzyme, levanase 5 .Bacillus amyloliquefaciens NK-1 has the ability to co-produce γ -PGA and levan during fermentation. The pgsBCA genes (responsible for γ -PGA synthesis) deletion strain B. amyloliquefaciens NK-Δ LP can produce high-purity levan, with the highest titer observed being 14 g/L 7 . The molecular weight of levan obtained from the NK-Δ LP strain is around 5 kDa, which is much lower than other reported levan products 8 . The Bacillus amyloliquefaciens strain was isolated from fermented food 9 , thus its levan product was supposed to have the potential to be the dietary supplements 10 . However, the low levan production by this strain is unable to meet industrial demands. Therefore, further strain improvement through metabolic engineering is required.