A method is described to determine simultaneously the effect of any changes in the ribosome-binding site (RBS) of mRNA on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. The approach was used to analyse systematically the influence of spacing between the Shine-Dalgarno sequence and the initiation codon, the three different initiation codons, and RBS secondary structure on translational yields in the two organisms. Both B. subtilis and E. coli exhibited similar spacing optima of 7-9 nucleotides. However, B. subtilis translated messages with spacings shorter than optimal much less efficiently than E. coli. In both organisms, AUG was the preferred initiation codon by two- to threefold. In E. coli GUG was slightly better than UUG while in B. subtilis UUG was better than GUG. The degree of emphasis placed on initiation codon type, as measured by translational yield, was dependent on the strength of the Shine-Dalgarno interaction in both organisms. B. subtilis was also much less able to tolerate secondary structure in the RBS than E. coli. While significant differences were found between the two organisms in the effect of specific RBS elements on translation, other mRNA components in addition to those elements tested appear to be responsible, in part, for translational species specificity. The approach described provides a rapid and systematic means of elucidating such additional determinants.

No abstract

No abstract

An average of 11 (range, 2 to 47) μmoles of formate per g per hr was produced and used in whole bovine rumen contents incubated in vitro, as calculated from the product of the specific turnover rate constant, k , times the concentration of intercellular formate. The latter varied between 5 and 26 (average, 12) nmoles/g. The concentration of formate in the total rumen contents was as much as 1,000 times greater, presumably owing to formate within the microbial cells. The concentration of formate in rumen contents minus most of the plant solids was varied, and from the rates of methanogenesis the Michaelis constant, K m , for formate conversion to CH 4 was estimated at 30 nmoles/g. Also, the dissolved H 2 was measured in relation to methane production, and a K m of 1 nmole/g was obtained. A pure culture of Methanobacterium ruminantium showed a K m of 1 nmole of H 2 /g, but the K m for formate was much higher than the 30 nmoles for the rumen contents. It is concluded that nonmethanogenic microbes metabolize intercellular formate in the rumen. CO 2 and H 2 are the principal substrates for rumen methanogenesis. Eighteen per cent of the rumen methane is derived from formate, as calculated from the intercellular concentration of hydrogen and formate in the rumen, the Michaelis constants for conversion of these substrates by rumen liquid, and the relative capacities of whole rumen contents to ferment these substrates.