Bile salt hydrolase (BSH) activity was shown to be constitutive and substrate-specific: the BSH isogenic Lactobacillus plantarum wild type (LP80 WT) and BSH overproducing LP80 (pCBH1) strains preferentially hydrolysed glycodeoxycholic acid (GDCA), whereas the hamster Lact. animalis isolates H362 and H364 showed a higher affinity for taurodeoxycholic acid (TDCA). In viability studies in the presence of nutrients, it was demonstrated that GDCA exerted a higher toxicity than TDCA in a pH-dependent manner. This toxicity was inversely proportionate to the BSH activity level of the strains tested, indicating that BSH activity contributed towards bile salt resistance when appropriate nutrients were available. The high toxicity of GDCA relative to TDCA was suggested to be caused by their weak and strong acid properties respectively. It was therefore hypothesized that the protonated form of bile salts exhibited toxicity as it imported protons in the cell. This puts an energy-burden on BSH- lactobacilli which undergo intracellular acidification. BSH+ cells primarily protect themselves through the formation of the weaker DCA compound, which can help negate the pH-drop by recapturing and exporting the co-transported proton. However, since DCA is more toxic than its conjugated counterparts, an additional energy-dependent detoxification of DCA is suggested.

The recently developed SHIME reactor (Simulator of the Human Intestinal Microbial Ecosystem) was validated by analysing a number of microorganism-associated activities. Data from the reactor were compared with values from the literature and results obtained from the analysis of faecal material of eight healthy persons. The fermentation patterns of four polysaccharides were studied. Arabinogalactan, xylan and pectin gave fermentation patterns in vitro indistinguishable from in vivo. Five different enzymatic activities were compared with in vivo experiments. All had activities which were of the order of those observed in vitro. The pro-drug sulphasalazine, whose active compound is released through microbial modification in the large intestine, was used as a reference compound. A fair correlation between in vivo and in vitro was found for the latter transformation. In addition the effect of Lactobacillus plantarum on the autochthonous microbial populations was investigated. Administration of L plantarum resulted in an in virro decrease in gram-negative anaerobic bacteria and total anaerobes. The same effect has been reported in vivo. The results obtained by the various validation experiments indicate that the reactor can be used to study the microbial communities of the gastrointestinal tract.

The effect of feeding live Lactobacillus reuteri cells containing active bile salt hydrolase (BSH) on plasma cholesterol levels was studied in pigs. During an experiment lasting 13 weeks, twenty pigs were fed on a high-fat, high-cholesterol, low-fibre diet for the first 10 weeks, and a regular pig diet for the last 3 weeks. One group of animals received, twice daily, 11.25 (SD 0.16) loglo colony forming units of the potential probiotic bacteria for 4 weeks (from week 3 until week 7). From week 8 onwards, the treated group was again fed on the same diet as the control group without additions. The total faecal Lactobacillus counts were only significantly higher in the treated pigs during the fist 2 weeks of L. reuteri feeding. Based on limited data, it was suggested that the administered LactobacilZus species had caused a temporary shift within the indigenous Lactobacillus population rather than permanently colonizing the intestinal tract. The probiotic feeding brought about significant lowering (P 5 0.05) of total and LDL-cholesterol concentrations in the treated pigs compared with the control pigs, while no change in HDLcholesterol concentration was observed. The data for faecal output of neutral sterols and bile salts were highly variable between the animals of each group, yet they indicated an increased output in the treated pigs. Although the blood cholesterol levels went up in both groups during the 3 weeks following the Lactobacillus administration period, significantly lower serum total and LDL-cholesterol levels were observed in the treated pigs. During the final 3 weeks of normalization to the regular diet, cholesterol concentrations significantly decreased in both animal groups and the differences in total and LDL-cholesterol concentrations between the groups largely disappeared.

Shewanella oneidensis couples anaerobic oxidation of lactate, formate, and pyruvate to the reduction of vanadium pentoxide (V V ). The bacterium reduces V V (vanadate ion) to V IV (vanadyl ion) in an anaerobic atmosphere. The resulting vanadyl ion precipitates as a V IV -containing solid.Vanadium is a transition metal which, at neutral pH, can exist in two oxidation states, V IV (vanadyl ion, cationic species VO 2ϩ ), and V V (vanadate ion, anionic species, H 2 VO 4 Ϫ ) (10, 11).The environmental chemistry of vanadium is complex. Vanadium is an abundant element that has proven to be a valuable resource for different industrial applications such as vanadium alloys, oxidation catalysis in sulfuric acid manufacturing and automobile catalytic converters, photographic development, textile dyeing, and ceramic coloring. A number of bacteria are able to reduce metal compounds, most commonly iron and manganese, through anaerobic reduction. Some organisms are known to be able to reduce other metals such as arsenic, mercury, selenium, uranium, technetium, chromium, molybdenum, gold, silver, and copper (4,8,13). The microbial reduction of vanadate has also been reported (1,14). To date, two Pseudomonas strains have been described to be capable of reducing vanadium (5,15). In this study we demonstrate that the gram-negative facultative anaerobic nonfermenting bacterium Shewanella oneidensis (6) can also reduce vanadium V V . Vanadium uptake on solid media. Colonies of S. oneidensis were grown on Luria-Bertani 1% agar plates containing 5 mM vanadate at 28°C for 48 h. Plates were then exposed to hydrogen sulfide in a sealed container. Upon formation of metal sulfide, plates were examined for changes in the regions surrounding or inside bacterial colonies. A halo could be seen indicating possible sequestration of the metal. Darkening of the cells indicated accumulation or possible reduction of the metal (data not shown).Vanadate detection. To facilitate the detection and quantification of the reduction of vanadate to V IV , a vanadate detection assay was designed, based on a detection assay for chromium described by Sandel (12). Vanadate was thereby detected on the basis of its reaction with diphenylcarbazide (DPC) in acid. The assay solution was made by addition of 1% (wt/vol) DPC in acetone to an equal volume of 2 M H 2 SO 4 . A volume of 500 l of diluted sample was added to the same volume of assay solution. Absorbance was measured at 320 nm after 15 min. A standard curve was made with vanadium pentoxide dilutions. Vanadyl ions do not react in this assay.Anaerobic vanadium reduction. S. oneidensis was grown aerobically on a rotary shaker (150 rpm) at 28°C in LB medium containing 2 or 10 mM V 2 O 5 and anaerobically with a Coy anaerobic chamber (Coy Laboratories, Grass Lake, Mich.) in phosphate-buffered defined medium (7) containing 10 mM lactate as the electron donor and 2 or 10 mM V 2 O 5 as the electron acceptor. An increase in lag phase could be detected in cultures grown with 10 mM but not with 2 mM V 2 O 5 compared to c...

No abstract

Exogenous plasmid isolation was used to assess the presence of mobilizing plasmids in several soils and activated sludges. Triparental matings were performed with Escherichia coli (a member of the y subgroup of the Proteobacteria) as the donor of an IncQ plasmid (pMOL155, containing the heavy metal resistance genes CZC: Cor, Znr, and Cdr), Alkaligenes eutrophus (a member of the P subgroup of the Proteobacteria) as the recipient, and indigenous microorganisms from soil and sludge samples as helper strains. We developed an assay to assess the plasmid mobilization potential of a soil ecosystem on the basis of the number of transconjugants obtained after exogenous isolations. After inoculation into soil of several concentrations of a helper strain (E. coli CM120 harboring IncP [IncPl] mobilizing plasmid RP4), the log numbers of transconjugants obtained from exogenous isolations with different soil samples were a linear function of the log numbers of helper strain CM120(RP4) present in the soils. Four soils were analyzed for the presence of mobilizing elements, and mobilizing plasmids were isolated from two of these soils. Several sludge samples from different wastewater treatment plants yielded much higher numbers of transconjugants than the soil samples, indicating that higher numbers of mobilizing strains were present. The mobilizing plasmids isolated from Gent-O sludge and one plasmid isolated from Eislingen soil hybridized to the repP probe, whereas the plasmids isolated from Essen soil did not hybridize to a large number of rep probes (repFIC, repHIl, repHI2, repL/M, repN, repP, repT, repU, repW, repX). This indicates that in Essen soil, broad-host-range mobilizing plasmids belonging to other incompatibility groups may be present.

No abstract

Growth and bile salt hydrolase (BSH) activity of the isogenic LactobacilZus plantarum 80 (LP80) strains were studied in vitro. In pure culture experiments viability and growth performance of the BSH-LP80 strain was negatively affected by the presence of conjugated bile salts. The LP80 wild type (WT) and BSH overproducing LP80 (pCBH1) strains did not show a die-off upon supplementation of bile salts. The latter strains hydrolysed glyco-conjugated deoxycholate (GDCA) more readily than tauro-conjugated deoxycholate (TDCA), indicating substrate specificity of the enzyme. BSH activities towards TDCA of LP80 WT and LP80 @CBHl) stationary phase cells were 0.17 and 1.02 FmoUmg CDW.h respectively; activities towards GDCA of the respective strains were 3.52 and 54%0 FmoUmg CDW.h respectively. The study of BSH activity as a function of growth revealed a marked difference in behaviour between LP80 WT and LP80 (pCBH1) with LP80 WT hydrolysing GDCA when reaching the exponential phase, whereas LP80 (pCBH 1) immediately started to hydrolyse GDCA. TDCA hydrolysis increased after GDCA hydrolysis was completed. BSH activity of LP80 (pCBH1) in a mixed microbial association, resembling that of the small intestine, was comparable to that determined under pure culture conditions, indicating that BSH activity will probably not be influenced by the presence of the normal intestinal microbiota. Based on the BSH activity of LP80 (pCBH 1) and on physiological data on the bile salt-cholesterol metabolism interrelationship, it was calculated that a daily intake of a realistic amount of highly BSH active Lactobacillus cells, e.g. in the form of yoghurt, might lead to a significant reduction of cholesterol. Hence, this in vitro study indicates that altering BSH activity can be a valid (micro) biological alternative treatment for patients with severe hypercholesterolaemia.