Vivo Analyses Research Articles

In Vivo Analyses of Interactions between SecE and SecY, Core Components of the Escherichia coli Protein Translocation Machinery

We have carried out structure-function studies on the cytoplasmic membrane protein, SecE, a component of the Escherichia coli secretion machinery. SecE, along with SecY, form a complex in the cytoplasmic membrane essential for protein translocation. By directed mutagenesis, we altered highly conserved residues of the second cytoplasmic domain (CD2) and of the COOH-terminal periplasmic region (PD2) of SecE. These mutants, as well as previously constructed mutations in the third membrane-spanning segment of SecE (MSS3), were tested for their ability to complement a secE null mutation, for their effects on protein export in vivo, and for their ability to form a stable complex with SecY. Most single mutations at the conserved positions in CD2 caused secretion defects, but had little effect on growth at 37 degrees C. Double mutations in CD2, or the introduction or removal of proline residues, affected growth and protein translocation more severely. Co-immunoprecipitations of SecE and SecY revealed that all mutant proteins, except those altered in PD2, destabilized the SecE-SecY complex. These results suggest that several regions contribute to the formation of a stable SecE-SecY complex but the elimination of a single contact point does not necessarily affect the functionality of the complex.

In Vitro and in Vivo Analyses of Hays of Switchgrass Strains Selected for High and Low in Vitro Dry Matter Digestibility1

In a previous report the progress made in altering the in vitro dry matter digestibility (IVDMD) of switchgrass (Panicum virgatum L.) in one cycle of divergent selection was described. In this study, the detergent system of analyses was used in an attempt to determine what plant constituents had been altered by the selection for IVDMD. Hays from five switchgrass strains differing in IVDMD harvested over a 3‐year period were analyzed for protein content, neutral detergent fiber (NDF), acid detergent fiber (ADF), ash, and lignin. Although there were differences (p ≤ 0.05) among strains for IVDMD as determined by two different procedures (with and without additional N), there were no differences among strains for ADF, ash, lignin, and protein. The strains ranked the same with the two IVDMD procedures but the IVDMD‐N (with additional N) test averaged 6.7 percentage points higher in digestibility. There were small differences (p ≤ 0.1) among strains for NDF in 2 of the 3 years. The strain with the highest IVDMD had the lowest NDF, suggesting that, in part, the improved IVDMD was due to a decrease in the cell wall component of the harvested forage. Hays of three of these strains, high‐IVDMD PC, low‐IVDMD PC, and ‘Pathfinder’ from the 1980 harvest were ground and fed to sheep to compare in vivo and in vitro digestibilities. After grinding, the hays had IVDMD‐N values of 49.9,45.5, and 46.7%, respectively. There were no differences among strains in the sheep feeding trial for in vivo digestibility, dry matter intake, and fiber digestibility. The in vivo digestibilities for these strains were: high‐ IVDMD PC 50.4%, low‐IVDMD PC 49.0%, and Pathfinder 50.8%. In contrast to a previous report in the literature, the IVDMD‐N values were generally similar to in vivo digestibilities for switchgrass hays. Parameters of the conventional detergent system of analyses, while useful in identifying factors responsible for changes in digestibility of switchgrass hays, would probably be of little value as selection criteria in a breeding program. Because of the large number of animals required to obtain statistical significance in in vivo digestibility studies, the IVDMD procedure would be preferable in studies in which breeding material is being evaluated and small differences in digestibility are expected.