Over-expression of rexA nullifies T4rII exclusion in Escherichia coli K(lambda) lysogens

Dosage and relative cellular levels of RexA and RexB proteins encoded by the rexA-rexB genes of a lambda prophage are important for the Rex+ phenotype, which was nullified when greater RexA or RexB was provided than was necessary for the complementation of a rexA- or a rexB- prophage.     

Characterization of a novel beta-galactosidase from Bifidobacterium adolescentis DSM 20083 active towards transgalactooligosaccharides

This paper reports on the effects of both reducing and nonreducing transgalactooligosaccharides (TOS) comprising 2 to 8 residues on the growth of Bifidobacterium adolescentis DSM 20083 and on the production of a novel beta-galactosidase (beta-Gal II). In cells grown on TOS, in addition to the lactose-degrading beta-Gal (beta-Gal I), another beta-Gal (beta-Gal II) was detected and it showed activity towards TOS but not towards lactose. beta-Gal II activity was at least 20-fold higher when cells were grown on TOS than when cells were grown on galactose, glucose, and lactose. Subsequently, the enzyme was purified from the cell extract of TOS-grown B. adolescentis by anion-exchange chromatography, adsorption chromatography, and size-exclusion chromatography. Beta-Gal II has apparent molecular masses of 350 and 89 kDa as judged by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating that the enzyme is active in vivo as a tetramer. Beta-Gal II had an optimal activity at pH 6 and was not active below pH 5. Its optimum temperature was 35 degrees C. The enzyme showed highest V(max) values towards galactooligosaccharides with a low degree of polymerization. This result is in agreement with the observation that during fermentation of TOS, the di- and trisaccharides were fermented first. Beta-Gal II was active towards beta-galactosyl residues that were 1-->4, 1-->6, 1-->3, and 1 <--> 1 linked, signifying its role in the metabolism of galactooligosaccharides by B. adolescentis.     

Influence of alternansucrase-derived oligosaccharides and other carbohydrates on alpha-galactosidase and alpha-glucosidase activity in Bifidobacterium adolescentis.

AIMS: To determine the influence of alternansucrase-derived oligosaccharides (AOS) and other carbohydrates on alpha-galactosidase and alpha-glucosidase activity in Bifidobacterium adolescentis. METHODS AND RESULTS: Activities for alpha-galactosidase and alpha-glucosidase were determined from cell extracts of B. adolescentis grown on 18 test carbohydrates including AOS. alpha-galactosidase activity was enhanced on a variety of alpha-linked or beta-linked carbohydrates regardless of a galactoside or glucoside. alpha-glucosidase, however, was enhanced only on alpha-linked carbohydrates. AOS significantly enhanced enzyme activity compared with most of the carbohydrates tested. Most of the AOS showed significant increases in activity for both enzymes over that displayed by their corresponding acceptor carbohydrates. CONCLUSIONS: alpha-galactosidase may serve as a biomarker for microbial metabolic activity within the large intestine for potential prebiotics composed of alpha-linked or beta-linked oligosaccharides whereas alpha-glucosidase activity may be restricted to assessing the influence of only alpha-linked carbohydrates. The AOS synthesis process provided a value-added component to carbohydrates by increasing metabolic activity (via alpha-galactosidase and alpha-glucosidase) over certain acceptor carbohydrates. SIGNIFICANCE AND IMPACT OF THE STUDY: Fundamental knowledge of enzyme activity in Bifidobacterium may aid in the design of more effective prebiotics and may also help identify enzyme indicators of metabolic activity when assessing influence within the intestine.     

Synthesis of α-galacto-oligosaccharides by a cloned α-galactosidase from Bifidobacterium adolescentis

A genomic library of Bifidobacterium adolescentis was constructed in Escherichia coli and a gene encoding an -galactosidase was isolated. The identified open reading frame showed high similarity and identity with bacterial -galactosidases, which belong to Family 36 of the glycosyl hydrolases. For the purification of the enzyme from the medium a single chromatography step was sufficient. The yield of the recombinant enzyme was 100 times higher than from B. adolescentis itself. In addition to hydrolytic activity the -galactosidase showed transglycosylation activity and can be used for the production of -galacto-oligosaccharides.     

A partially purified β-glucosidase from Bifidobacterium adolescentis converts cycasin to a mutagenic compound

Y.J. CHOI, C.J. KIM AND G.E. JI. 1996. β-Glucosidase was extracted from sonicated Bifidobacterium adolescentis Int-57 and partially purified by Sepharose CL-6B gel-filtration and DEAE-cellulose ion-exchange chromatography. The partially purified enzyme was confirmed to convert cycasin to a mutagen in the Ames and SOS chromotests. β-Glucosidase negative strains were unable to activate cycasin mutagenically.     

Two routes of metabolic cross-feeding between Bifidobacterium adolescentis and butyrate-producing anaerobes from the human gut

Dietary carbohydrates have the potential to influence diverse functional groups of bacteria within the human large intestine. Of 12 Bifidobacterium strains of human gut origin from seven species tested, four grew in pure culture on starch and nine on fructo-oligosaccharides. The potential for metabolic cross-feeding between Bifidobacterium adolescentis and lactate-utilizing, butyrate-producing Firmicute bacteria related to Eubacterium hallii and Anaerostipes caccae was investigated in vitro. E. hallii L2-7 and A. caccae L1-92 failed to grow on starch in pure culture, but in coculture with B. adolescentis L2-32 butyrate was formed, indicating cross-feeding of metabolites to the lactate utilizers. Studies with [(13)C]lactate confirmed carbon flow from lactate, via acetyl coenzyme A, to butyrate both in pure cultures of E. hallii and in cocultures with B. adolescentis. Similar results were obtained in cocultures involving B. adolescentis DSM 20083 with fructo-oligosaccharides as the substrate. Butyrate formation was also stimulated, however, in cocultures of B. adolescentis L2-32 grown on starch or fructo-oligosaccharides with Roseburia sp. strain A2-183, which produces butyrate but does not utilize lactate. This is probably a consequence of the release by B. adolescentis of oligosaccharides that are available to Roseburia sp. strain A2-183. We conclude that two distinct mechanisms of metabolic cross-feeding between B. adolescentis and butyrate-forming bacteria may operate in gut ecosystems, one due to consumption of fermentation end products (lactate and acetate) and the other due to cross-feeding of partial breakdown products from complex substrates.     

The detection of Bifidobacterium adolescentis by colony hybridization as an indicator of human faecal pollution

AIMS: To develop an improved method for the detection of Bifidobacterium adolescentis as an indicator of human faecal pollution. METHODS AND RESULTS: Bifidobacterium medium (BFM) was identified as the optimal medium for the recovery of bifidobacteria from human effluent. Dilutions of faeces and effluent from both humans and animals were filtered, grown on BFM and human specific B. adolescentis identified via colony hybridization with a digoxigenin (DIG)-labelled oligonucleotide probe. CONCLUSIONS: The combination of BFM with colony probing allows the detection of B. adolescentis, a specific indicator of human faecal pollution. SIGNIFICANCE AND IMPACT OF THE STUDY: It is now technically feasible to use B. adolescentis as indicators of human faecal pollution, and studies to examine the survival and appropriateness of bifidobacteria in this role can be initiated.     

Rapid detection of human fecal contamination in estuarine environments by PCR targeting of Bifidobacterium adolescentis

Detection of Bifidobacterium adolescentis was used as an effective genetic marker of human fecal contamination in Georgia estuaries. Enterococci enumerations on mEI media indicated that a tributary to the Little Satilla River with 516 CFU/100 ml was the most polluted of all the rivers tested. Extracted DNA from eight river water samples was subjected to a two-step nested PCR protocol using genus and species specific primers for Bifidobacterium spp. and B. adolescentis. B. adolescentis was detected from extracted DNA in Dunbar River, Black Bank Creek, and in a Little Satilla River tributary which demonstrates the presence of human fecal contamination in these three rivers. In the five other estuaries tested including West Point-Federica River and the Altamaha River, which both had less than 16 CFU/100 ml of enterococci, B. adolescentis was not detected.     

Selective plating underestimates abundance and shows differential recovery of bifidobacterial species from human feces

The aim of the present work was to compare the efficacies and levels of selectivity of different culture-dependent and -independent methods for analyzing bifidobacteria in human stool samples. The three different culture media used here significantly differed from each other, particularly with regard to the recovery of Bifidobacterium adolescentis. Bifidobacterium medium failed to recover B. adolescentis; Beerens medium recovered some B. adolescentis organisms (17% of total bifidobacteria), whereas tomato-Eugon medium recovered mainly B. adolescentis organisms (58% of total bifidobacteria). A culture-independent method that combines GC fractionation of bacterial community DNA and 16S rRNA sequencing indicated that B. adolescentis organisms accounted for 85% of all bifidobacteria. Methodological biases, such as those described in this paper, should be taken into account in interpreting earlier studies and designing future experiments.     

Antioxidative properties and inhibitory effect of Bifidobacterium adolescentis on melanogenesis

Melanin is a dark pigment produced by melanocytes. Tyrosinase is a key enzyme which catalyzes the rate-limiting step of melanogenesis. However, accumulation of melanin leads to various skin hyperpigmentation disorders. To find a novel skin-whitening agent, the antioxidant capacity of Bifidobacterium adolescentis culture filtrate and inhibitory effect on melanogenesis were investigated. The antioxidant effects of B. adolescentis culture filtrate include 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging capacity, 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid)(ABTS) radical cation scavenging activity and reducing power were measured spectrophotometrically. The reducing power is a useful index for the evaluation of potential antioxidants which carry out reduction of ferricyanide to ferrocyanide. Furthermore, the inhibitory effects of the bacterial culture filtrate on mushroom tyrosinase, B16F10 intracellular tyrosinase activity and melanin content were also determined. The results revealed that B. adolescentis culture filtrate (2.5, 5.0 and 7.5?%; v/v) effectively scavenged DPPH and ABTS radicals, and lower concentrations of the bacterial culture filtrates (0.5, 1.0 and 1.5?%; v/v) showed potent reducing power in a dose-dependent pattern. Additionally, the bacterial culture filtrate suppressed murine tyrosinase activity and decreased the amount of melanin in a dose-dependent manner. Our results demonstrated that B. adolescentis culture filtrate decreases the melanogenesis process of melanoma cells by inhibiting tyrosinase activity, which we suggest may be mediated through its antioxidant activity.     

Interactions between salivary Bifidobacterium adolescentis and other oral bacteria: in vitro coaggregation and coadhesion assays

Coaggregation assays were performed to investigate interactions between oral Bifidobacterium adolescentis and other oral bacterial species. Bifidobacterium adolescentis OLB6410 isolated from the saliva of healthy humans did not coaggregate with Actinomyces naeslundii JCM8350, Streptococcus mitis OLS3293, Streptococcus sanguinis JCM5708, Veillonella parvula ATCC17745 or Porphyromonas gingivalis OB7124, but it did coaggregate with Fusobacterium nucleatum JCM8532. Subsequent examination of biofilm formation on saliva-coated hydroxyapatite discs using FISH revealed that B. adolescentis OLB6410 could not directly adhere to the coated discs. It did, however, adhere to biofilms of A. naeslundii, V. parvula, and F. nucleatum, although it did not coaggregate with A. naeslundii nor with V. parvula. These results suggest that the adhesion of B. adolescentis to tooth surfaces is mediated by other oral bacteria. Heat- or proteinase K-treated F. nucleatum could not coaggregate with B. adolescentis. Similarly, the coaggregation and coadhesion of proteinase K-treated B. adolescentis were strongly inhibited. It is therefore probable that proteinaceous factors on the cellular surface of B. adolescentis and F. nucleatum are involved in their interaction. The data presented in this study add to our understanding of bifidobacterial colonization in the human oral cavity.     

双歧杆菌对裸鼠腹腔巨噬细胞NO形成的调节作用

给裸小鼠腹腔注射青春型双歧杆菌,每天一次,连续５天,以Ｇｒｉｅｓ试剂测定了裸鼠腹腔巨噬细胞分泌ＮＯ的含量。结果表明：双歧杆菌注射组其腹腔巨噬细胞产生ＮＯ的量显著高于对照组,具有显著的统计学意义（Ｐ＜００１）。提示青春型双歧杆菌可激活巨噬细胞,使之产生一定量的ＮＯ,ＮＯ在介导双歧杆菌的多种生理功能方面起重要作用     

青春双歧杆菌DM8504菌株对荷瘤小鼠体内NO诱生作用的研究

应用处死的青春双歧杆菌ＤＭ８５０４菌株皮下免疫荷瘤小鼠,按Ｇｒｉｅｓ反应原理测定一氧化氮（ＮＯ）的含量。结果表明,双歧杆菌能提高荷瘤小鼠体内ＮＯ的含量,较对照组显著升高,肿瘤组织的坏死程度在处理组与对照组之间也具有显著性差异。提示：在双歧杆菌抗肿瘤作用中,除ＴＮＦ—α外,ＮＯ也发挥重要作用。     

Use of specific primers based on the 16S-23S internal transcribed spacer (ITS) region for the screening Bifidobacterium adolescentis in yogurt products and human stool samples

Effective methods for the identification and enumeration of lactic acid producing bacteria (LAB) cells are important for the quality control and assurance of probiotic products. In this study, we designed a polymerase chain reaction (PCR) primer set from the sequence in 16S-23S internal transcribed spacer (ITS) region and used it for the specific detection of Bifidobacterium adolescentis, one of the Bifidobacterium species used in probiotics. Specificity of the PCR primers, i.e., bits-1/bits-2, was assured by assay strains of B. adolescentis, other Bifidobacterium species, and strains of non-Bifidobacterium spp. Coupled with the use of a known primer set specific for Bifidobacterium species, Bifidobacterium strains and B. adolescentis could be identified from LAB strains in fermented dairy products and human fecal samples.     

Stimulation of the secretion of pro-inflammatory cytokines by Bifidobacterium strains

To characterize the ability of bifidobacteria to affect the production of macrophage-derived cytokines, a murine macrophage-like cell line, J774.1, was cultured in the presence of 27 strains of heat-inactivated bifidobacteria. Bifidobacterium adolescentis and B. longum, known as adult-type bifidobacteria, induced significantly more pro-inflammatory cytokine secretion, IL-12 and TNF-alpha, by J774.1 cells, than did the infant-type bifidobacteria, B. bifidum, B. breve, and B. infantis (P<0.01). In contrast, B. adolescentis did not stimulate the production of anti-inflammatory IL-10 from J774.1 cells as the other tested bacteria did. The results suggest that the adult-type bifidobacteria, especially B. adolescentis, may be more potent to amplify but less able to down-regulate the inflammatory response.     

Bifidobacterial diversity and the development of new microbial source tracking indicators

Many studies suggest a close relationship between species of Bifidobacterium and their hosts. Thus, species such as B. adolescentis and B. thermacidophilum subsp. porcinum have been proposed as potential indicators of human and porcine fecal pollution. The diversity of bifidobacteria in wastewaters (human and animal) and slurries is analyzed using nested PCR followed by denaturant gradient gel electrophoresis (DGGE). The sewage samples showed similar DGGE patterns. The predominant bands were recognized as B. adolescentis, B. longum, and two unidentified species related to B. adolescentis. A single band detected in poultry samples was identified as B. saeculare. Bifidobacterial diversity was higher within porcine and bovine samples. The main bands in porcine samples were identified as B. minimum, an unknown species, and B. thermophilum/B. thermacidophilum subsp. porcinum. The latter species was also identified among the main bands in bovine samples together with B. pseudolongum and B. ruminantium. We then attempted to isolate the host-specific strains. DGGE bands were examined to develop specific probes to screen environmental samples by colony hybridization and further isolation of strains from positively hybridized colonies. Bifidobacterial strains that are host associated by DGGE bands to human and pig were successfully isolated from the environment: B. adolescentis from human sewage samples and the unidentified species related to pig from slurries and slaughterhouse wastewater. Neither the poultry-associated B. saeculare nor the ruminant-associated B. pseudolongum could be isolated with the current methodology, suggesting either a low prevalence in the samples or failure of the culture to grow in the media used.     

Co-culture of Bifidobacterium adolescentis and Bacteroides thetaiotaomicron in arabinogalactan-limited chemostats: effects of dilution rate and pH

The effects of dilution rate (D = 0.04-0.38/h) and pH (5.0-6.5) on co-cultures of Bifidobacterium adolescentis and Bacteroides thetaiotaomicron were studied in arabinogalactan-limited chemostats. B. thetaiotaomicron outcompeted B. adolescentis at all dilution rates at culture pH values between 5.0 and 6.0, although the bifidobacterium was always detected in the fermenters. At pH 6.5, however, B. adolescentis predominated in co-cultures at dilution rates above 0.24/h. Arabinogalactan degrading enzymes (beta-galactosidase, alpha-arabinofuranosidase) were strongly catabolite repressed in bacteroides at high dilution rates, but were constitutive and growth rate-associated in B. adolescentis. The increased competitiveness of B. adolescentis at high specific growth rates was not related to its ability to synthesise increased levels of depolymerising enzymes. Measurements of residual carbohydrate in pure and mixed culture chemostats showed that the bacteroides extensively digested the galactose backbone of the polymer, and to a lesser degree, the arabinose sidechains. Nevertheless, arabinose monomers and oligosaccharides (d.p. < 10) accumulated in these cultures under all growth conditions. In contrast, the bifidobacterium utilized considerably less arabinogalactan than the bacteroides, and this was reflected in the mixed culture studies. These experiments demonstrate that B. thetaiotaomicron was able to compete most successfully for this plant cell wall polysaccharide under nutritional, physiological and environmental conditions broadly similar to those encountered in the human colon, and indicate the existence of synergistic interactions between the two organisms that were growth rate dependent.     

青春双歧杆菌对2型糖尿病模型大鼠免疫功能和肾脏的影响

目的通过观察青春双歧杆菌对2型糖尿病模型大鼠血清中细胞因子IL-2、IL-6和IFN-γ活性的影响,以及血清及尿中的NO与ET-1的变化,探讨青春双歧杆菌对2型糖尿病模型免疫功能和肾脏的影响。方法采用青春双歧杆菌灌胃2型糖尿病模型大鼠,取血液和尿液,ELISA法检测细胞因子IL-2、IL-4、IL-6、IFN-γ和ET-1活性,硝酸酶还原法测定NO水平。结果青春双歧杆菌提高IL-2、IL-4水平,降低IL-6、IFN-γ和ET-1活性,NO水平在病程中动态变化。结论青春双歧杆菌具有平衡2型糖尿病模型大鼠免疫功能,抑制ET-1,调节NO水平的作用,从而预防肾小球硬化的发生。     

Bifidobacterial diversity in human feces detected by genus-specific PCR and denaturing gradient gel electrophoresis

We describe the development and validation of a method for the qualitative analysis of complex bifidobacterial communities based on PCR and denaturing gradient gel electrophoresis (DGGE). Bifidobacterium genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same length from different bifidobacterial species showed good separation upon DGGE. DGGE of fecal 16S rDNA amplicons from five adult individuals showed host-specific populations of bifidobacteria that were stable over a period of 4 weeks. Sequencing of fecal amplicons resulted in Bifidobacterium-like sequences, confirming that the profiles indeed represent the bifidobacterial population of feces. Bifidobacterium adolescentis was found to be the most common species in feces of the human adult subjects in this study. The methodological approach revealed intragenomic 16S rDNA heterogeneity in the type strain of B. adolescentis, E-981074. The strain was found to harbor five copies of 16S rDNA, two of which were sequenced. The two 16S rDNA sequences of B. adolescentis E-981074(T) exhibited microheterogeneity differing in eight positions over almost the total length of the gene.