A proteome reference map and proteomic analysis of Bifidobacterium longum NCC2705

A comprehensive proteomic study was carried out to identify and characterize proteins expressed by Bifidobacterium longum NCC2705. A total of 708 spots representing 369 protein entries were identified by MALDI-TOF-MS and/or ESI-MS/MS. Isoelectric point values estimated by gel electrophoresis matched closely with their predicted ones, although some discrepancies exist suggesting that post-translational protein modifications might be common in B. longum. The identified proteins represent 21.4% of the predicted 1727 ORFs in the genome and correspond to 30% of the predicted proteome. Moreover 95 hypothetical proteins were experimentally identified. This is the first compilation of a proteomic reference map for the important probiotic organism B. longum NCC2705. The study aimed to define a number of cellular pathways related to important physiological processes at the proteomic level. Proteomic comparison of glucose- and fructose-grown cells revealed that fructose and glucose are catabolized via the same degradation pathway. Interestingly the sugar-binding protein specific to fructose (BL0033) and Frk showed higher levels of expression in cells grown on fructose than on glucose as determined by semiquantitative RT-PCR. BL0033 time course and concentration experiments showed that the induction time and fructose concentration correlates to increased expression of BL0033. At the same time, an ABC (ATP-binding cassette) transporter ATP-binding protein (BL0034) was slightly up-regulated in cells grown on fructose compared with glucose. All of the above results suggest that the uptake of fructose into the cell may be conducted by a specific transport system in which BL0033 might play an important role.     

长双歧杆菌NCC2705葡萄糖与果糖代谢的比较蛋白质组学

为揭示长双歧杆菌NCC2705 (Bifidobacterium longum NCC2705)果糖代谢途径, 建立其果糖发酵模型。以本实验室前期构建的长双歧杆菌NCC2705菌株蛋白质参考图谱为基础, 进行了果糖和葡萄糖生长的菌体比较蛋白质组学研究, 利用MALDI-TOF和ESI-MS/MS鉴定差异蛋白, 进一步通过半定量RT-PCR验证二者显著差异表达蛋白。果糖生长的菌体蛋白中鉴定到了所有葡萄糖降解途径中的酶和蛋白质, 另外鉴定到3倍以上差异蛋白点9个, 其对应的5个蛋白在果糖发酵中上调。半定量RT-PCR验证显著差异蛋白, 显示在果糖发酵中具有高水平表达是ABC 转运系统的果糖特异性-结合蛋白BL0033和ATP结合蛋白BL0034。果糖的发酵时间和浓度梯度试验显示诱导时间越长、浓度越高, BL0033的表达量越高。第一, 比较蛋白谱证明果糖和葡萄糖以相同途径降解。第二, BL0033的表达是受果糖诱导的, 果糖的吸收可能是通过一个特殊的转运系统, 即ABC转运系统将果糖从胞外转运到胞内, 其中BL0033和BL0034共同作为系统元件扮演了重要角色。     

长双歧杆菌NCC2705葡萄糖与乳糖代谢的比较蛋白质组学

[目的]以本实验室前期构建的长双歧杆菌NCC2705菌株蛋白质参考图谱为基础,研究长双歧杆菌发酵乳糖和葡萄糖的比较蛋白质组学.[方法]采用ImageMaster 2D Elite Platnum Version 5.0比较分析3倍以上蛋白差异点;利用MALDI-TOF进行差异蛋白鉴定,每个蛋白质点的肽指纹图谱在长双歧杆菌NCC2705菌株的蛋白质数据库用Mascot进行检索;采用Pro-Q磷酸化试剂进行磷酸化蛋白的染色.[结果]鉴定到31个蛋白表达发生显著变化,在乳糖发酵中14个蛋白上调17个蛋白下调.这些蛋白为亲水性酸性蛋白,它们基因的CAI值均在0.5以上,主要包括糖代谢相关蛋白、应激蛋白、转录和翻译相关蛋白,还有一些未知功能的蛋白.此外,有两个蛋白:转醛缩酶(BL0715,transaldolase,tal)L3蛋白点和丙酮酸激酶(BL0988,pyruvate kinase,pyk)G9蛋白点发生了磷酸化作用.[结论]长双歧杆菌NCC2705在乳糖中生长快于葡萄糖,它们的降解途径是相同的;转醛缩酶和丙酮酸激酶发生了翻译后修饰作用,推测转醛缩酶在43T和47S发生了磷酸化,而丙酮酸激酶在65S发生了磷酸化.     

Genome comparison of Bifidobacterium longum strains NCC2705 and CRC-002 using suppression subtractive hybridization

Because probiotic effects are strain dependent, genomic explanations of these differences will contribute to understanding their mechanisms of action. The genomic sequence of the Bifidobacterium longum probiotic strain NCC2705 was determined, but little is known about the genetic diversity between strains of this species. Suppression subtractive hybridization (SSH) is a powerful method for generating a set of DNA fragments differing between two closely related bacterial strains. The purpose of this study was to identify genetic differences between genomes of B. longum strains NCC2705 and CRC-002 using PCR-based SSH. Strain CRC-002 produces exopolysaccharides whereas NCC2705 is not known for reliable exopolysaccharide production. Thirty-five and 30 different sequences were obtained from the SSH libraries of strains CRC-002 and NCC2705, respectively. Specific CRC-002 genes found were predicted to be involved in the biosynthesis of exopolysaccharides and metabolism of other carbohydrates, and these genes were not present in the genome of strain NCC2705. The identification of an endo-1,4-beta-xylanase gene in the CRC-002 SSH library is an important difference because xylanase genes have previously been proposed as a defining characteristic of the NCC2705 strain. The results demonstrate that the SSH technique was useful to highlight potential genes involved in complex sugar metabolism that differ between the two probiotic strains.     

长双歧杆菌NCC2705株果糖结合蛋白BL0033基因的克隆及其在大肠杆菌中的表达

目的：克隆长双歧杆菌NCC2705株果糖结合蛋白BL0033的基因,利用大肠杆菌表达GST-BL0033融合蛋白并纯化。方法：以长双歧杆菌NCC2705株基因组为模板,PCR扩增BL0033基因,并将其插入pGEX-4T-1表达载体,转化至大肠杆菌DH5α;提取质粒,经PCR、质粒双酶切及测序鉴定后,转入大肠杆菌BL21,并对表达条件进行摸索;用谷胱甘肽-Sepharose4B树脂对可溶性GST-BL0033融合蛋白进行纯化。结果：PCR扩增的BL0033基因长度接近1000bp,与预期值一致;重组菌在IPTG浓度为0.05mmoL/L的条件下,于16℃诱导过夜后,SDS-PAGE分析可见可溶性表达条带,相对分子质量约60×103,与预期值一致;亲和纯化后,SDS-PAGE结果显示单一的表达条带。结论：克隆了BL0033蛋白的基因,并表达纯化了融合蛋白GST-BL0033,为进一步研究长双歧杆菌NCC2705株BL0033蛋白功能奠定了基础。     

Kinetics and metabolism of Bifidobacterium adolescentis MB 239 growing on glucose, galactose, lactose, and galactooligosaccharides

The kinetics and the metabolism of Bifidobacterium adolescentis MB 239 growing on galactooligosaccharides (GOS), lactose, galactose, and glucose were investigated. An unstructured unsegregated model for growth in batch cultures was developed, and kinetic parameters were calculated with a recursive algorithm. The growth rate and cellular yield were highest on galactose, followed by lactose and GOS, and were lowest on glucose. Lactate, acetate, and ethanol yields allowed the calculation of carbon fluxes toward fermentation products. Distributions between two- and three-carbon products were similar on all the carbohydrates (55 and 45%, respectively), but ethanol yields were different on glucose, GOS, lactose, and galactose, in decreasing order of production. Based on the stoichiometry of the fructose-6-phosphate shunt and on the carbon distribution among the products, the ATP yield was calculated. The highest yield was obtained on galactose, while the yields were 5, 8, and 25% lower on lactose, GOS, and glucose, respectively. Therefore, a correspondence among ethanol production, low ATP yields, and low biomass production was established, demonstrating that carbohydrate preferences may result from different distributions of carbon fluxes through the fermentative pathway. During the fermentation of a GOS mixture, substrate selectivity based on the degree of polymerization was exhibited, since lactose and the trisaccharide were the first to be consumed, while a delay was observed until longer oligosaccharides were utilized. Throughout the growth on both lactose and GOS, galactose accumulated in the cultural broth, suggesting that beta(1-4) galactosides can be hydrolyzed before they are taken up.     

Kinetic analysis for the isomerization of glucose,fructose, and mannose in subcritical aqueous ethanol

Fructose, glucose, and mannose were treated with subcritical aqueous ethanol for ethanol concentrations ranging from 0 to 80% (v/v) at 180–200 °C. The aldose–ketose isomerization was more favorable than ketose–aldose isomerization and glucose–mannose epimerization. The isomerization of the monosaccharides was promoted by the addition of ethanol. In particular, mannose was isomerized most easily to fructose in subcritical aqueous ethanol. The apparent equilibrium constants for the isomerizations of mannose to fructose, K_eq,M→F, and glucose to fructose, K_eq,G→F, were independent of ethanol concentration and increased with increasing temperature. Moreover, the K_eq,M→F value was much larger than the K_eq,G→F value. The enthalpies for the isomerization of mannose to fructose, ΔH_M→F, and glucose to fructose, ΔH_G→F, were estimated to be 18 and 24 kJ/mol, respectively, according to van’t Hoff equation. Subcritical aqueous ethanol can be used to produce fructose from glucose and mannose efficiently.     

Diauxic growth of Clostridium thermosaccharolyticum on glucose and xylose

Abstract Clostridium thermosaccharolyticum growing on medium containing glucose and xylose exhibits classical diauxic growth in which glucose is utilized during the first phase. The lag period between growth phases is associated with induction of synthesis of a xylose transport system together with the enzymes xylose isomerase and xylukokinase. Xylose metabolism by this organism is therefore shown to be inducible and subject to repression by glucose. Xylose utilization by cells adapted to this carbon source is also prevented immediately upon addition of glucose to the culture, suggesting a direct inhibitory effect of glucose on xylose uptake or metabolism.     

Composition and toxicity of lipids from Rhodococcus rhodochrous grown on medium containing galactose, glucose or mannose

Rhodococcus rhodochrous, a producer of mycolic acid of approx. C40, exhibited a higher cellular mass yield when grown on glucose than when grown on galactose or mannose. The cellular content of the diethyl ether-soluble lipids in microorganisms cultivated on glucose or mannose varied with the incubation time, while that of microorganisms grown on galactose remained constant. The lipids extracts from cells cultivated on different hexoses and collected at the exponential phase of growth were more toxigenic; this property was related in general to the content of glycolipid. On the other hand, cells cultivated on galactose or mannose had a higher quantity of glycolipid in the exponential phase, while the glycolipid content of those grown on glucose remained approximately constant. Amongst the components of the lipid extract, the glycolipid fraction was the sole fraction bearing toxic property. Neutral plus fatty acids and phospholipids displayed no similar characteristic.     

Studies of the effect of the sugars ribose, xylose and fructose on the retrogradation of wheat starch gels by x-ray diffraction

The effects of the sugars ribose, xylose and fructose on the retrogradation of wheat starch gels were investigated by measuring the area under the strong 0·516 nm diffraction peak (characteristic of B-type crystalline retrograded starch) as a function of storage time for a series of gels containing different amounts of added sugars. Retrogradation was monitored as the increase in peak area with storage time. The results obtained suggested that all three sugars altered crystallisation and hence retrogradation of the gels. For the concentration regimes studied, xylose and ribose acted by progressively reducing crystallisation with increasing sugar concentration. In the case of fructose two effects were noted. The fructose led to an increase in both thermally reversible and thermally irreversible crystallisation upon storage. For xylose and ribose the increase in crystallisation upon storage was almost totally thermoreversible suggesting that the retrogradation upon storage was dominated by amylopectin crystallisation.     

In vitro effects of phosphatidylcholine and transgalactooligosaccharides on the production of 1,2-sn-diacylglycerol by Bifidobacterium longum biovar infantis

Aims: To investigate the production of the tumour promoter 1,2-sn-diacylglycerol (DAG) by a human gut isolate of Bifidobacterium longum biovar infantis. Methods and Results: Bifidobacterium longum biovar infantis was grown in vitro using anaerobic static batch cultures in the presence of phosphatidylcholine (PC) and trans-galactooligosaccharides (TOS). Production of DAG was found to be dependent upon the presence of PC, while TOS had a reducing effect. Considerable differences in morphology, growth and metabolic end products from the micro-organism were observed under the different culture conditions. Conclusions: Our results have provided evidence that B. longum biovar infantis can produce DAG in vitro and that a prebiotic exerted a reducing effect upon this production. Significance and Impact of the Study: The results presented in this study demonstrate an ability of ostensibly beneficial member of the colonic environment to produce unwanted compounds under certain conditions. Therefore, it may be important that a combination of substrates and other factors are assessed when studying the behaviour of any bacterial group or species, especially when designing the dietary interventions.     

Co-utilization of glucose and xylose by evolved Thermus thermophilus LC113 strain elucidated by 13C metabolic flux analysis and whole genome sequencing

We evolved Thermus thermophilus to efficiently co-utilize glucose and xylose, the two most abundant sugars in lignocellulosic biomass, at high temperatures without carbon catabolite repression. To generate the strain, T. thermophilus HB8 was first evolved on glucose to improve its growth characteristics, followed by evolution on xylose. The resulting strain, T. thermophilus LC113, was characterized in growth studies, by whole genome sequencing, and ¹³C-metabolic flux analysis (¹³C-MFA) with [1,6-¹³C]glucose, [5-¹³C]xylose, and [1,6-¹³C]glucose+[5-¹³C]xylose as isotopic tracers. Compared to the starting strain, the evolved strain had an increased growth rate (~2-fold), increased biomass yield, increased tolerance to high temperatures up to 90 °C, and gained the ability to grow on xylose in minimal medium. At the optimal growth temperature of 81 °C, the maximum growth rate on glucose and xylose was 0.44 and 0.46 h⁻¹, respectively. In medium containing glucose and xylose the strain efficiently co-utilized the two sugars. ¹³C-MFA results provided insights into the metabolism of T. thermophilus LC113 that allows efficient co-utilization of glucose and xylose. Specifically, ¹³C-MFA revealed that metabolic fluxes in the upper part of metabolism adjust flexibly to sugar availability, while fluxes in the lower part of metabolism remain relatively constant. Whole genome sequence analysis revealed two large structural changes that can help explain the physiology of the evolved strain: a duplication of a chromosome region that contains many sugar transporters, and a 5x multiplication of a region on the pVV8 plasmid that contains xylose isomerase and xylulokinase genes, the first two enzymes of xylose catabolism. Taken together, ¹³C-MFA and genome sequence analysis provided complementary insights into the physiology of the evolved strain.     

Heterofermentative metabolism of glucose and ribose and utilisation of citrate by the smooth biotype of Lactobacillus amylovorus NCFB 2745

Lactobacillus amylovorus NCFB 2745 exhibits a rough colony morphology, ferments glucose homofermentatively and cannot utilise ribose. After five transfers in de Man Rogosa and Sharpe media (containing glucose and citrate) Lb. amylovorus 2745 appears smooth on agar plates; smooth cultures reverted to rough by culturing in aerobic conditions. The smooth type shows patterns of fermentation that are typical of a heterofermentative lactobacillus. Thus, the smooth morphotype produces CO₂ and ethanol in addition to lactate and is able to ferment ribose. The switch in metabolism to the smooth form is accompanied by an increase in phosphoketolase and a reduction in aldolase enzyme activities. Citrate also has effects on growth rates and end-metabolites.     

Characterization and compartmentation,in green leaves,of hexokinases with different specificities for glucose,fructose, and mannose and for nucleoside triphosphates

When green leaves of spinach (Spinacia oleracea L.) were surveyed for the presence of hexokinases which utilize glucose, fructose and-or mannose as a substrate, four kinases could be distinguished by their order of elution during chromatography on diethylaminoethyl (DEAE)-cellulose: (i) a hexokinase I with a specificity for fructose, glucose, and mannose, (ii) a fructokinase I with a specificity for fructose, (iii) a hexokinase II with a specificity for glucose, fructose and mannose, and (iv) a fructokinase II with a specificity for fructose. Hexokinases I and II had high apparent K_m values for fructose (8 and 15 mM, respectively) and medium or low apparent K_m values for glucose (150 and 18 μM, respectively) and mannose (18 and 15 μM, respectively). Maximal velocities were highest with fructose, medium with glucose and lowest with mannose. That hexokinases I and II used several sugars as substrate was concluded (i) from their identical elution profiles during enzyme separation and (ii) because their activities with two or three sugars at a time was always lower than the sum of activities with one substrate, indicating competition of the sugars for the reaction with the enzymes. Fructokinases I and II were very specific for fructose (85 and 140 μM, respectively) and had only little, if any, activity with glucose or mannose. All kinases showed varying degrees of activity with nucleoside triphosphates other than ATP. In the presence of all three sugars, hexokinases I and II were considerably more active with ATP than with uridine-, cytidine-, and guanosine 5'-triphosphate (UTP, CTP, GTP) except that, in the presence of glucose, hexokinase I was almost as active with UTP as with ATP. In the presence of fructose, fructokinase I exhibited highest activity with GTP and a gradually decreasing level of activity with CTP, UTP, and ATP. The activities in the presence of the other two sugars were highest with ATP. Fructokinase II was most active with ATP and fructose and progressively less active with GTP, UTP, and CTP. Cell fractionation by isopycnic density-gradient centrifugation or differential centrifugation indicated that fructokinase II was associated with chloroplasts, hexokinase II with mitochondria, and the other two kinases with the non-particulate cell fraction. In green leaves of pea (Pisum sativum L.), only a hexokinase (II) and fructokinase (II) were present. Corn (Zea mays L.) leaves exhibited only very low hexokinase activity. Dedicated to Prof. Dr. Hans Mohr on the occasion of his 60th birthday     

Quantitative analysis of intermediary metabolism in hepatocytes incubated in the presence and absence of glucagon with a substrate mixture containing glucose, ribose, fructose, alanine and acetate.

Hepatocytes were isolated from the livers of fed rats and incubated, in the presence and absence of 100 nM-glucagon, with a substrate mixture containing glucose (10 mM), fructose (4 mM), alanine (3.5 mM), acetate (1.25 mM), and ribose (1 mM). In any given incubation one substrate was labelled with 14C. Incorporation of 14C into glucose, glycogen, CO2, lactate, alanine, glutamate, lipid glycerol and fatty acids was measured after 20 and 40 min of incubation under quasi-steady-state conditions [Borowitz, Stein & Blum (1977) J. Biol. Chem. 252, 1589-1605]. These data and the measured O2 consumption were analysed with the aid of a structural metabolic model incorporating all reactions of the glycolytic, gluconeogenic, and pentose phosphate pathways, and associated mitochondrial and cytosolic reactions. A considerable excess of experimental measurements over independent flux parameters and a number of independent measurements of changes in metabolite concentrations allowed for a stringent test of the model. A satisfactory fit to the data was obtained for each condition. Significant findings included: control cells were glycogenic and glucagon-treated cells glycogenolytic during the second interval; an ordered (last in, first out) model of glycogen degradation [Devos & Hers (1979) Eur. J. Biochem. 99, 161-167] was required in order to fit the experimental data; the pentose shunt contributed approx. 15% of the carbon for gluconeogenesis in both control and glucagon-treated cells; net flux through the lower Embden-Meyerhof pathway was in the glycolytic direction except during the 20-40 min interval in glucagon-treated cells; the increased gluconeogenesis in response to glucagon was correlated with a decreased pyruvate kinase flux and lactate output; fluxes through pyruvate kinase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase were not coordinately controlled; Krebs cycle activity did not change with glucagon treatment; flux through the malic enzyme was towards pyruvate formation except for control cells during interval II; and 'futile' cycling at each of the five substrate cycles examined (including a previously undescribed cycle at acetate/acetyl-CoA) consumed about 26% of cellular ATP production in control hepatocytes and 21% in glucagon-treated cells.     

Energetic metabolism of glucose, mannose and galactose in glucose-starved rat insulinoma cells anchored on microcarrier beads. A phosphorus-31 NMR study.

Insulin-secreting cells (RINm5F) have successfully been grown on a large scale on poly-L-lysine coated-polystyrene microcarriers, providing a high cell number in a restricted volume under conditions that respect the metabolic integrity of these anchorage-dependent cells. The energetic metabolism of the perfused cells has been followed non-invasively by phosphorus-31 nuclear magnetic resonance spectroscopy. Glucose starvation induced a rapid decrease in nucleoside triphosphates (mainly ATP) pools, correlated with an increase in Pi level. The initial ATP level was rapidly recovered when the cells were refed with glucose or with mannose, but not with galactose, even after 2 h of perfusion. These differential effects of hexoses on energetic metabolism might be related to their various insulin-release actions on tumor islet cells.