Fruit constitutes a major component of human diets, providing fiber, vitamins, and phytonutrients. Carotenoids are a major class of compounds found in many fruits, providing nutritional benefits as precursors to essential vitamins and as antioxidants. Although recent gene isolation efforts and metabolic engineering have primarily targeted genes involved in carotenoid biosynthesis, factors that regulate flux through the carotenoid pathway remain largely unknown. Characterization of the tomato high-pigment mutations (hp1 and hp2) suggests the manipulation of light signal transduction machinery may be an effective approach toward practical manipulation of plant carotenoids. We demonstrate here that hp1 alleles represent mutations in a tomato UV-DAMAGED DNA-BINDING PROTEIN 1 (DDB1) homolog. We further demonstrate that two tomato light signal transduction genes, LeHY5 and LeCOP1LIKE, are positive and negative regulators of fruit pigmentation, respectively. Down-regulated LeHY5 plants exhibit defects in light responses, including inhibited seedling photomorphogenesis, loss of thylakoid organization, and reduced carotenoid accumulation. In contrast, repression of LeCOP1LIKE expression results in plants with exaggerated photomorphogenesis, dark green leaves, and elevated fruit carotenoid levels. These results suggest genes encoding components of light signal transduction machinery also influence fruit pigmentation and represent genetic tools for manipulation of fruit quality and nutritional value.

Escherichia coli TonB protein is an energy transducer, coupling cytoplasmic membrane energy to active transport of vitamin B12 and iron-siderophores across the outer membrane. TonB is anchored in the cytoplasmic membrane by its hydrophobic amino terminus, with the remainder occupying the periplasmic space. In this report we establish several functions for the hydrophobic amino terminus of TonB TonB protein is anchored in the cytoplasmic membrane by its uncleaved amino terminus, with the bulk of the protein occupying the periplasmic space, the aqueous compartment bounded by the outer and cytoplasmic membranes (14,39,41

Alkaline phosphatase (PhoA) fusions to TonB amino acids 32, 60, 125, 207, and 239 (the carboxy terminus) all showed high PhoA activity; a PhoA fusion to TonB amino acid 12 was inactive. The full-length TonB-PhoA fusion protein was associated with the cytoplasmic membrane and retained partial TonB function. These results support a model in which TonB is anchored in the cytoplasmic membrane by its hydrophobic amino terminus, with the remainder of the protein, including its hydrophobic carboxy terminus, extending into the periplasm.The tonB gene product is required for energy-dependent transport of large (>600 Da) nutrients across the outer membrane of Escherichia coli. TonB couples the electrochemical potential of the cytoplasmic membrane to the active transport of vitamin B12 and ferric siderophores across the outer membrane, to the energy-dependent steps of bacteriophage +80 and Ti infection, and to the entry of B-group colicins into bacteria. Thus, TonB functions to transduce energy between the cytoplasmic and outer membranes (for a review, see reference 16).To understand the mechanism of TonB-dependent energy transduction, it is important to know the disposition of TonB within the cell envelope. Hydropathy plots of the TonB amino acid sequence indicate that it is markedly hydrophilic, with the notable exceptions of short hydrophobic regions at both the amino and carboxy termini (17). One or both of these regions could serve as membrane anchors. We recently showed that TonB is membrane associated and that it extends into the periplasm sufficiently to be accessible to proteinase K (18). In addition, we showed that the amino terminus of TonB functions as an export signal but is not proteolytically cleaved (18,21). Previous studies had shown that TonB expressed during infection of UV-irradiated bacteria with XtonB transducing phage is associated with the cytoplasmic membrane based on Sarkosyl fractionation (15). These results suggested that the amino terminus of TonB serves to anchor TonB in the cytoplasmic membrane, with the bulk of the polypeptide extending into the periplasm. However, the location of the carboxy terminus remained uncertain. The 11-residue hydrophobic region near the carboxy terminus is shorter than the 21 residues thought to be required to span the lipid bilayer in an a-helix. Nevertheless, the hydropathy profile of this region is similar to that of penicillin-binding protein 5, which is known to be anchored in the cytoplasmic membrane by its carboxy terminus (19). Thus, it seemed possible that the short hydrophobic region at the carboxy terminus of TonB could be a membrane anchor.To further investigate the membrane topology of TonB, we constructed tonB-phoA gene fusions by oligonucleotidedirected deletion mutagenesis (1,2). To avoid problems with potentially toxic TonB-PhoA fusion proteins (1,3,21) (4) and CAGE/ GEM (5) software packages. Starting with pKP287, a precise deletion of the 5' tetA coding region, the tonB promoter, and sequences 5' to the tonB coding region was made by oligonucleot...

We have developed a selection for mutations in a trpC-tonB gene fusion that takes advantage of the properties of the plasmid-encoded TrpC-TonB hybrid protein. The TrpC-TonB hybrid protein consists of amino acids 1 through 25 of the normally cytoplasmic protein, TrpC, fused to amino acids 12 through 239 of TonB. It is expressed from the trp promoter and is regulated by the trpR gene and the presence or absence of tryptophan. Under repressing conditions in the presence of tryptophan, the trpC-tonB gene can restore phi 80 sensitivity to a tonB deletion mutant, which indicates that TrpC-TonB can be exported and is functional. High-level expression of TrpC-TonB protein in the absence of tryptophan results in virtually immediate cessation of growth for strains carrying the trpC-tonB plasmid. By selecting for survivors of the induced growth inhibition (overproduction lethality), we have isolated a variety of mutations. Many of the mutations decrease expression of the TrpC-TonB protein, as expected. In addition, three independently isolated mutants expressing normal levels of TrpC-TonB protein result in a Gly----Asp substitution within the hydrophobic amino terminus of TonB. The mutant proteins are designated TrpC-TonBG26D. The mutations are suppressed by prlA alleles, known to suppress export (signal sequence) mutations. TrpC-TonB proteins carrying the Gly----Asp substitution accumulate in the cytoplasm. We conclude that the Gly----Asp substitution is an export mutation. TrpC-TonBG26D protein has been purified and used to raise polyclonal antibodies that specifically recognize both TrpC-TonB protein and wild-type TonB protein.

Magnesium is unique among biological cations. Its volume change from hydrated cation to atomic ion is over an order of magnitude larger than that of any other biological cation. This volume change presents particular problems for a magnesium transport system and suggests that these systems may be significantly different from other classes of ion transport systems. Detailed study of Mg2+ transport in complex organisms is limited by severe technical problems. However, molecular genetic techniques have enabled the isolation of three Mg2+ transport systems from the Gram-negative bacterium Salmonella typhimurium. The MgtA and MgtB transport systems are members of the P-type ATPase superfamily of transporters but possess unique characteristics among members of this family. The CorA transport protein is the first member of an entirely new class of transport proteins. In addition, another completely new family of Mg2+ transport proteins have been identified that is present in both Gram-negative and Gram-positive bacteria. Characterization of these transporters should provide substantial insight into Mg2+ transport and cellular Mg2+ homeostasis.