C. James Newbold

Rumen ciliate protozoa contain high concentrations of conjugated linoleic acids and vaccenic acid, yet do not hydrogenate linoleic acid or desaturate stearic acid

Conjugated linoleic acids (CLA) have been shown to improve human health. They are derived from the microbial conversion of dietary linoleic acid (cis-9,cis-12-18 : 2 (LA)) in the rumen. An investigation was undertaken to determine the role of ruminal ciliate protozoa v. bacteria in the formation of CLA and its precursor in animal tissues, vaccenic acid (trans-11-18 : 1 (VA)). Mixed protozoa from the sheep rumen contained at least two to three times more unsaturated fatty acids, including CLA and VA, than bacteria. Different species had different composition, with larger fibrolytic species such as Epidinium ecaudatum caudatum containing more than ten times more CLA and VA than some small species, including Entodinium nanellum. In incubations with ruminal microbial fractions (bacterial fraction (BAC), protozoal fraction (PRO)), LA metabolism was very similar in strained ruminal fluid (SRF) and in the BAC, while the PRO had LA-metabolising activity an order of magnitude lower. Using PCR-based methods, no genes homologous to fatty acid desaturase genes were found in cDNA libraries from ruminal protozoa. The absence of an alternative route of VA/CLA formation via desaturation of stearate was confirmed by incubations of SRF, BAC or PRO with [14C]stearate. Thus, although protozoa are rich in CLA and VA, they appear to lack the ability to form these two fatty acids from LA or stearate. The most likely explanation is that protozoa preferentially incorporate CLA and VA formed by bacteria. The implication of the present findings is that the flow of unsaturated fatty acids, including CLA and VA, from the rumen could depend on the flow of protozoa rather than bacteria.

Incorporation of [15N]ammonia by the cellulolytic ruminal bacteria Fibrobacter succinogenes Bl2, Ruminococcus albus SY3, and Ruminococcus flavefaciens 17

ABSTRACT The origin of cell nitrogen and amino acid nitrogen during growth of ruminal cellulolytic bacteria in different growth media was investigated by using 15NH3. At high concentrations of peptides (Trypticase, 10 g/liter) and amino acids (15.5 g/liter), significant amounts of cell nitrogen ofFibrobacter succinogenes BL2 (51%), Ruminococcus flavefaciens 17 (43%), and Ruminococcus albusSY3 (46%) were derived from non-NH3-N. With peptides at 1 g/liter, a mean of 80% of cell nitrogen was from NH3. More cell nitrogen was formed from NH3 during growth on cellobiose compared with growth on cellulose in all media. Phenylalanine was essential for F. succinogenes, and its15N enrichment declined more than that of other amino acids in all species when amino acids were added to the medium.

Terminator Operon Reporter : Combining a transcription termination switch with reporter technology for improved gene synthesis and synthetic biology applications

Synthetic biology is characterized by the development of novel and powerful DNA fabrication methods and by the application of engineering principles to biology. The current study describes Terminator Operon Reporter (TOR), a new gene assembly technology based on the conditional activation of a reporter gene in response to sequence errors occurring at the assembly stage of the synthetic element. These errors are monitored by a transcription terminator that is placed between the synthetic gene and reporter gene. Switching of this terminator between active and inactive states dictates the transcription status of the downstream reporter gene to provide a rapid and facile readout of the accuracy of synthetic assembly. Designed specifically and uniquely for the synthesis of protein coding genes in bacteria, TOR allows the rapid and cost-effective fabrication of synthetic constructs by employing oligonucleotides at the most basic purification level (desalted) and without the need for costly and time-consuming post-synthesis correction methods. Thus, TOR streamlines gene assembly approaches, which are central to the future development of synthetic biology.