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.

Potentiation by metal ions of the efficacy of the ionophores, monensin and tetronasin, towards four species of ruminal bacteria.

Concentrations of Na(+), K(+) and Ca(2+) in the growth medium were varied within limits normally found in vivo to determine how cation concentrations affect the sensitivity of ruminal bacteria to the ionophores, monensin (a Na(+)/H(+) and K(+)/H(+) exchanger) and tetronasin (Ca(2+)/H(+)). High [Na(+)] (172 mM cf. 137 mM in control medium) enhanced the efficacy of monensin towards Eubacterium ruminantium 2388, Streptococcus bovis C277, Lactobacillus casei LB17 and Prevotella albensis M384. High [K(+)] (35 mM cf. 19 mM) alone caused a decreased potency of both ionophores, except with L. casei. Added Ca(2+) (7.4 cf. 2.8 mM) increased the potency of tetronasin when [Na(+)] was low. High [Na(+)] alone also potentiated the efficacy of tetronasin. Monensin caused intracellular [Na(+)] and [K(+)] to be decreased in the most sensitive of these organisms, E. ruminantium, whereas only intracellular [Ca(2+)] fell with tetronasin. The changes were small; however, Δp fell by only 20 mV after 2 h when ionophores caused immediate cessation of growth. ATP concentrations fell by 77% and 75% with monensin and tetronasin, respectively. Thus, altering cation concentrations might be used to potentiate the efficacy of ionophores, by increasing the rate of energy expenditure to maintain ionic homoeostasis in sensitive bacteria.

Effect of antibiotics on the bacterial population of the rabbit caecum.

The effect feeding antibiotics has on the bacterial population of the rabbit caecum was investigated. No changes in total volatile fatty acid production or total bacterial counts were observed compared with nonantibiotic treated controls. However, treatment with chlortetracycline resulted in an increase of propionate at the apparent cost of butyrate (P<0.05). Denaturing gradient gel electrophoresis analysis indicated that the two antibiotics that inhibit protein synthesis (chlortetracycline and tiamulin) exerted the most similar changes on the bacterial population structure, decreasing the diversity of the profiles. Sequence analysis of DNA from excised denaturing gradient gel electrophoresis bands was carried out. The majority of the sequences observed were most similar to bacterial sequences previously described in other gut environments, with 11% being most similar to those previously reported from the rabbit, and 95% of the sequences having 95% or greater identity to sequences already in GenBank.

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.

An NAD(+)-dependent glutamate dehydrogenase cloned from the ruminal ciliate protozoan, Entodinium caudatum.

An NAD(+)-dependent glutamate dehydrogenase (GDH; EC 1.4.1.24) was cloned from the ruminal ciliate protozoan, Entodinium caudatum. The gene had high sequence similarity to GDH genes from the Bacteroides (class)--a class of bacteria which is highly represented in the rumen. When expressed in Escherichia coli the enzyme had a high affinity for ammonia and alpha-ketoglutarate (apparent K(m) of 2.33 and 0.71 mM, respectively) and a low affinity for glutamate (apparent K(m) of 98 mM). GDH activity and GDH mRNA concentration were increased by incubating washed E. caudatum cells with ammonia and antibiotics. These results suggest that the GDH is an anabolic enzyme catalysing the assimilation of ammonia by E. caudatum in the rumen and that the gene was probably acquired by lateral gene transfer from a ruminal bacterium.

Molecular profiling of bacterial species in the rabbit caecum.

The diversity of bacteria present in the caecum of the rabbit was investigated. Partial bacterial 16S rRNA genes from a digested sample collected from the caecum of an adult rabbit were amplified by PCR. Sequence analysis of the amplified fragments indicated highest similarity was to bacterial sequences previously described from other gut environments. However, only one sequence showed significant identity (97% threshold) to any previously described bacterial 16S rRNA genes. Furthermore, most of the sequences clustered together in groups lacking representatives from sequences already described, suggesting that the rabbit caecal flora contains organisms not previously described.

Characterization of XYN10B, a modular xylanase from the ruminal protozoan Polyplastron multivesiculatum, with a family 22 carbohydrate-binding module that binds to cellulose.

A new xylanase gene, xyn10B, was isolated from the ruminal protozoan Polyplastron multivesiculatum and the gene product was characterized. XYN10B is the first protozoan family 10 glycoside hydrolase characterized so far and is a modular enzyme comprising a family 22 carbohydrate-binding module (CBM) preceding the catalytic domain. The CBM22 was shown to be a true CBM. It showed high affinity for soluble arabinoxylan and is the first example of a CBM22 that binds strongly to celluloses of various crystallinities. The enzymic properties of XYN10B were also analysed. Its optimal temperature and pH for activity were 39 degrees C and 7.0 respectively; these values being close to those of the ruminal ecosystem. The phylogenetic relationships between the XYN10B CBM22 or catalytic domain and related sequences from ruminal and non-ruminal bacteria and eukaryotes are reported. The xyn10B gene is shown to lack introns.

Influence of the pattern of peptide supply on microbial activity in the rumen simulating fermenter (RUSITEC).

The source and pattern of N supply was varied in the rumen simulation technique (RUSITEC) in order to determine if continuous, rather than transient, availability of peptides was required for optimum ruminal fermentation. The energy source was fibre prepared from sugar-beet pulp. N was added as NH3 continuously infused (AC) or peptides (Bacto(R) Casitone, a pancreatic hydrolysate of casein; Difco Laboratories, Detroit, MI, USA) continuously infused (PC) or added as a single dose at the time of feeding (PS). Free peptides were detected in the fermenter liquid for 4 h after feeding in the AC treatment, for 10 h in the PS treatment, and at all times with the PC treatment. Treatments had no effect on DM degradation. Approximately 40 % of the degradation occurred during the time no peptides were detected in the PS treatment. Microbial N flow tended to be higher with the peptide additions (P<0.061), with no significant difference between the two peptides treatments. The production of liquid-associated micro-organisms (LAM) was higher in the PC treatment (P<0.05) and the proportion of LAM derived from NH3 lower (P<0.05). However, LAM only accounted for 20-30 % total microbial population. Our main conclusion was that peptides had a small stimulatory effect on the fermentation, but there was no indication that synchrony of supply of energy and amino acid-N in the fermenter promoted a more efficient fermentation than non-synchronous supply. This conclusion must be qualified, however, because some N remained in the fibre and may have become available progressively as the fibre was digested by the micro-organisms.

Effects of high-molecular-mass substrates on protein migration during sodium dodecyl sulfatepolyacrylamide gel electrophoresis.

Substrate-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has become a popular procedure for the separation and identification of active fractions present in enzyme mixtures due to its relative simplicity. Procedures including high-molecular-mass substrates within the gel, such as starch for identification of amylase activity, and protein substrates, including gelatin, casein, and collagen, for revealing protease activity, have been described. SDS-PAGE separation under denaturing conditions is dependent on the molecular mass of the proteins and on the effective pore size of the gels, the last factor being affected by the inclusion of high-molecular-mass substrates into the polyacrylamide matrix. In order to quantify the effect of the addition of increasing concentrations of such substrates on protein migration, starch, gelatin, and casein were included in gels in which polyacrylamide concentration was kept constant. High-molecular-mass substrates decreased migration of proteins ranging from 6.5 to 205 kDa, although the migration pattern, and thereby the accuracy of the assignation of relative molecular masses to proteins separated on those gels, was practically unaffected. The substitution of glycine, as the carrying ion, by Tricine in denaturing electrophoresis buffer systems resulted in an improvement of the migration of proteins in substrate-containing gels. Results suggested that zymograms including substrates remain a valuable procedure for the separation and the relative molecular mass assignation of active enzyme fractions.