One of the most powerful methods available to assign function to a gene is to inactivate or knockout the gene. Recently,we described the first target-selected knockout in zebrafish. Here,we report on the further improvements of this procedure,resulting in a highly efficient and easy method to do target-selected mutagenesis in zebrafish. A library of 4608 ENU-mutagenized F1 animals was generated and kept as a living stock. The DNA of these animals was screened for mutations in 16 genes by use of CEL-I-mediated heteroduplex cleavage (TILLING) and subsequent resequencing. In total,255 mutations were identified,of which 14 resulted in a premature stop codon,7 in a splice donor/acceptor site mutation,and 119 in an amino acid change. By this method,we potentially knocked out 13 different genes in a few months time. Furthermore,we show that TILLING can be used to detect the full spectrum of ENU-induced mutations in a vertebrate genome with the presence of many naturally occurring polymorphisms

Faecal microbial changes associated with ageing include reduced bifidobacteria numbers. These changes coincide with an increased risk of disease development. Prebiotics have been observed to increase bifidobacteria numbers within humans. The present study aimed to determine if prebiotic galacto-oligosaccharides (GOS) could benefit a population of men and women of 50 years and above, through modulation of faecal microbiota, fermentation characteristics and faecal water genotoxicity. A total of thirty-seven volunteers completed this randomised, double-blind, placebo-controlled crossover trial. The treatments -juice containing 4 g GOS and placebo -were consumed twice daily for 3 weeks, preceded by 3-week washout periods. To study the effect of GOS on different large bowel regions, three-stage continuous culture systems were conducted in parallel using faecal inocula from three volunteers. Faecal samples were microbially enumerated by quantitative PCR. In vivo, following GOS intervention, bifidobacteria were significantly more compared to post-placebo (P¼0·02). Accordingly, GOS supplementation had a bifidogenic effect in all in vitro system vessels. Furthermore, in vessel 1 (similar to the proximal colon), GOS fermentation led to more lactobacilli and increased butyrate. No changes in faecal water genotoxicity were observed. To conclude, GOS supplementation significantly increased bifidobacteria numbers in vivo and in vitro. Increased butyrate production and elevated bifidobacteria numbers may constitute beneficial modulation of the gut microbiota in a maturing population.

dTDP-rhamnose is an important precursor of cell wall polysaccharides and rhamnose-containing exopolysaccharides (EPS) in Lactococcus lactis. We cloned the rfbACBD operon from L. lactis MG1363, which comprises four genes involved in dTDP-rhamnose biosynthesis. When expressed in Escherichia coli, the lactococcal rfbACBD genes could sustain heterologous production of the Shigella flexneri O antigen, providing evidence of their functionality. Overproduction of the RfbAC proteins in L. lactis resulted in doubled dTDPrhamnose levels, indicating that the endogenous RfbAC activities control the intracellular dTDP-rhamnose biosynthesis rate. However, RfbAC overproduction did not affect rhamnose-containing B40-EPS production levels. A nisin-controlled conditional RfbBD mutant was unable to grow in media lacking the inducer nisin, indicating that the rfb genes have an essential role in L. lactis. Limitation of RfbBD activities resulted in the production of altered EPS. The monomeric sugar of the altered EPS consisted of glucose, galactose, and rhamnose at a molar ratio of 1:0.3:0.2, which is clearly different from the ratio in the native sugar. Biophysical analysis revealed a fourfold-greater molecular mass and a twofold-smaller radius of gyration for the altered EPS, indicating that these EPS are more flexible polymers with changed viscosifying properties. This is the first indication that enzyme activity at the level of central carbohydrate metabolism affects EPS composition.Bacterial polysaccharides can be present in the cell wall as components of the cell envelope. Information about the structure and biosynthesis pathway of these compounds is fragmented. Glucose, galactose, mannose, N-acetylglucosamine, N-acetylmannose, and rhamnose are often found to be constituents of cell wall polysaccharides (8,41).Rhamnose is a 6-deoxyhexose sugar which is widely distributed in O antigens of gram-negative bacteria as part of the lipopolysaccharide (LPS) (43). Furthermore, this compound is often found in capsular polysaccharides (CPS), which are covalently bound to the cell wall, and in exopolysaccharides (EPS), which are loosely associated with the cell wall. L-dTDPrhamnose is the sugar-nucleotide precursor of these rhamnose moieties and is formed in a four-step reaction from glucose 1-phosphate. The reaction involves the enzyme activities of glucose-1-phosphate thymidylyl transferase, dTDP-glucose-4,6-dehydratase, dTDP-4-keto-L-rhamnose-3,5-epimerase, and dTDP-L-rhamnose synthase encoded by the genes that are commonly designated rfbABCD, respectively. These genes have been found in several gram-negative bacteria, including Escherichia coli (23), Salmonella enterica (30), Xanthomonas campestris (25), and Shigella flexneri (35). Various rfb mutant strains have been described, and the mutations have various effects on the rhamnose contents of the cell wall polysaccharides produced, including a loss of O antigen production (31), a reduced level of LPS production (23), or production of LPS with a reduced amount (25) or complete lack (...