K88 fimbriae are ordered polymeric protein structures at the surface of enterotoxigenic Escherichia coli cells. Their production and assembly requires a molecular chaperone located in the periplasm (FaeE) and a molecular usher located in the outer membrane (FaeD). FaeC is the tip component of the K88 fimbriae. We studied the expression of the subcloned faeC gene, the subcellular localization of FaeC and its interaction with the chaperone and the outer membrane usher. In the absence of the chaperone or the usher, FaeC could not be detected in E. coli cells harbouring the faeC gene and its ribosome binding site under contol of the IPTG inducible lpp/lac promoter/operator. The expression of FaeC was detectable in the presence of chaperone FaeE, but a direct interaction between the chaperone and FaeC was not found. The expression of FaeC was also detectable in cells co-expressing the outer membrane usher FaeD. Overexpression of FaeC after changing the faeC ribosome binding site appeared to induce lethality. Expression of subcloned FaeC in the absence of FaeE or FaeD could be detected when faeC was cloned under the tight control of the ara promoter/operator and when lethality induction was avoided. The direct interaction of FaeC with outer membranes containing the usher FaeD was studied by cell fractionation, isopycnic sucrose density gradient centrifugation, SDS-PAGE and immunoblotting. FaeC was found to bind to outer membranes containing FaeD or a FaeD-PhoA hybrid construct containing 215 amino-terminal residues of FaeD. This binding was not observed when control outer membranes without FaeD were used. No other K88 specific proteins were required for this interaction. The direct interaction between FaeC and FaeD in the outer membranes was shown by affinity blotting experiments. FaeE was not required for this interaction. Together these data indicate that the minor fimbrial subunit FaeC, unlike FaeG, H and F, does not have a strong interaction with the chaperone FaeE in the E. coli periplasm, but directly binds to the outer membrane molecular usher FaeD.
NAD- and glutathione-dependent formaldehyde dehydrogenase (GD-FALDH) of Paracoccus denitrificans has been purified as a tetramer with a relative molecular mass of 150 kDa. The gene encoding GD-FALDH (flhA) has been isolated, sequenced, and mutated by insertion of a kanamycin resistance gene. The mutant strain is not able to grow on methanol, methylamine, or choline, while heterotrophic growth is not influenced by the mutation. This finding indicates that GD-FALDH of P. denitrificans is essential for the oxidation of formaldehyde produced during methylotrophic growth.
Bovine viral diarrhea virus (BVDV) is a significant problem in the cattle industry. The disease affects both beef and dairy herds. Infection in unprotected animals leads to gastrointestinal (GI) lymphoid tissue depletion, GI mucosal lining destruction, and animal death. Persistently infected (PI) animals result when a cow is infected during gestation; its calf never reaches its production potential and is a lifelong carrier of the virus. Commercial vaccines are available but yield only moderate results. Understanding the mechanisms of the acquired immune response is crucial for further advancements to be made. In the immune response, professional antigen presenting cells (APC’s) engulf, process, and present antigen on their cell surface to white blood cells, namely the T lymphocytes and B lymphocytes. In doing so, they initiate the cascade of events necessary for developing acquired immunity. Given that APC’s serve as a cornerstone to the immune response, much research focuses on their role in immunity. Isolating and preparing APC’s for experiments is rather laborious. Some immortalized, bovine macrophage (BOMAC and BoMac) cell lines had become available that had potential to be used in place of the monocyte derived macrophages (MDM’s) which have traditionally been used with success. Analysis of the BOMAC’s and BoMac’s was done by comparing them with MDM’s. Microfilament organization, phagocytic characteristics, and cell surface receptor presence was investigated. Our results indicated that these immortalized cell lines would be poor in vitro models for MDM’s. Characteristic differences between the MDM’s and both the BOMAC and BoMac cell lines leads one to believe the cell lines cannot be considered to be representative models of APC’s in vivo.
Research Article| April 01 1979 High School Biology Education in Transition Jàne Butler Kahle, Jàne Butler Kahle Search for other works by this author on: This Site PubMed Google Scholar Paul DeHart Hurd, Paul DeHart Hurd Search for other works by this author on: This Site PubMed Google Scholar Norris Harms Norris Harms Search for other works by this author on: This Site PubMed Google Scholar The American Biology Teacher (1979) 41 (4): 210–232. https://doi.org/10.2307/4446545 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Tools Icon Tools Get Permissions Cite Icon Cite Search Site Citation Jàne Butler Kahle, Paul DeHart Hurd, Norris Harms; High School Biology Education in Transition. The American Biology Teacher 1 April 1979; 41 (4): 210–232. doi: https://doi.org/10.2307/4446545 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentThe American Biology Teacher Search This content is only available via PDF. Copyright 1979 National Association of Biology Teachers Article PDF first page preview Close Modal You do not currently have access to this content.
An enzyme-linked immunosorbent assay and a whole-cell activity assay were developed which allowed detection of methanol dehydrogenase (MDH) of Paracoccus denitrificans with increased sensitivity. By these methods, it was shown that MDH was not induced by its natural substrate, methanol. Relief from a catabolite repression-like mechanism seemed responsible for low-level MDH synthesis, while product induction was the hypothesized mechanism for synthesis of high amounts of MDH. In the latter process, formaldehyde may play an important role as effector. For a variety of culture conditions, inconsistencies were observed in the relation between amounts of MDH protein synthesized and enzyme activities measured in vitro. Regulation of pyrrolo-quinoline-quinone biosynthesis or a modulation of its incorporation and stability in MDH may constitute an overriding mechanism to ensure a correct tuning between metabolic rates of methanol consumption and the required methanol oxidation rates.
A chromosomal region encoding a two-component regulatory system, FlhRS, has been isolated from Paracoccus denitrificans. FlhRS-deficient mutants were unable to grow on methanol, methylamine, or choline as the carbon and energy source. Expression of the gene encoding glutathione-dependent formaldehyde dehydrogenase (fhlA) was undetectable in the mutant, and expression of the S-formylglutathione hydrolase gene (fghA) was reduced in the mutant background. In addition, methanol dehydrogenase was immunologically undetectable in cell extracts of FhlRS mutants. These results indicate that the FlhRS sensor-regulator pair is involved in the regulation of formaldehyde, methanol, and methylamine oxidation. The effect that the FlhRS proteins exert on the regulation of C(1) metabolism might be essential to maintain the internal concentration of formaldehyde below toxic levels.
