Search

Serratia marcescens hemTUV genes encoding a potential heme permease were cloned in Escherichia coli recombinant mutant FB827 dppF::Km(pAM 238-hasR). This strain, which expresses HasR, a foreign heme outer membrane receptor, is potentially capable of using heme as an iron source. However, this process is invalidated due to a dppF::Km mutation which inactivates the Dpp heme/peptide permease responsible for heme, dipeptide, and [delta]-aminolevulinic (ALA) transport through the E. coli inner membrane. We show here that hemTUV genes complement the Dpp permease for heme utilization as an iron source and thus are functional in E. coli. However, hemTUV genes do not complement the Dpp permease for ALA uptake, indicating that the HemTUV permease does not transport ALA. Peptides do not inhibit heme uptake in vivo, indicating that, unlike Dpp permease, HemTUV permease does not transport peptides. HemT, the periplasmic binding protein, binds heme. Heme binding is saturable and not inhibited by peptides that inhibit heme uptake by the Dpp system. Thus, the S. marcescens HemTUV permease and, most likely, HemTUV orthologs present in many gram-negative pathogens form a class of heme-specific permeases different from the Dpp peptide/heme permease characterized in E. coli.

On the basis of the three-dimensional model of the heme/hemophore TonB-dependent outer membrane receptor HasR, mutants with six-residue deletions in the 11 putative extracellular loops were generated. Although all mutants continued to be active TonB-dependent heme transporters, mutations in three loops abolished hemophore HasA binding both in vivo and in vitro.

The Serratia marcescens hemophore-specific outer membrane receptor HasR is a member of the TonB-dependent family of autoregulated receptors. It can transport either heme itself or heine bound to the hemophore HasA. On the basis of sequence and functional similarities with other TonB-dependent outer membrane receptors whose three-dimensional structures have been determined, a HasR structure model was proposed. The mature HasR protein comprises a 99-residue amino-terminal extension necessary for hasR transcription, followed by a plug domain of 139 amino acids and a [beta]-barrel domain inserted in the outer membrane, the lumen of which is closed by the plug. This model was used to generate hasR deletions encoding HasR proteins with the native signal peptides but lacking either the N-terminal regulatory extension or encoding the plug or the [beta]-barrel alone. The protein lacking the N-terminal extension, HasR [DELTA]11-91, was incorporated in the outer membrane and was fully functional for active uptake of free and hemophore-bound heme. The HasR [beta]-barrel, [DELTA]11-192, was also incorporated in the outer membrane and bound the hemophore but expressed no active heme transport properties. The HasR plug remained in the periplasm. Coexpression of the plug and the [beta]-barrel allowed partial plug insertion in the outer membrane, demonstrating that these two HasR domains interact in vivo. The [beta]-barrel and the plug also interact in vitro. Nevertheless, the two domains did not complement each other to reconstitute an active TonB-dependent receptor for free or hemophore-bound heme uptake. Production of the [beta]-barrel alone selectively increased passive diffusion of heme but not of other exogenous compounds. A mutation at histidine 603, which is required for heme uptake through the wild-type receptor, abolished heme diffusion, showing that HasR [DELTA]11-192 forms a specific heme channel.

Many gram-negative bacteria have specific outer membrane receptors for free heme, hemoproteins, and hemophores. Heme is a major iron source and is taken up intact, whereas hemoproteins and hemophores are not transported: the iron-containing molecule has to be stripped off at the cell surface, with only the heme moiety being taken up. The Serratia marcescens hemophore-specific outer membrane receptor HasR can transport either heme itself or heme bound to the hemophore HasA. This second mechanism is much more efficient and requires a higher TonB-ExbB-ExbD (TonB complex) concentration than does free or hemoglobin-bound heme uptake. This requirement for more of the TonB complex is associated with a higher energy requirement. Indeed, the sensitivity of heme-hemophore uptake to the protonophore carbonyl cyanide m-chlorophenyl hydrazone is higher than that of heme uptake from hemoglobin. We show that a higher TonB complex concentration is required for hemophore dissociation from the receptor. This dissociation is concomitant with heme uptake. We propose that increasing the TonB complex concentration drives more energy to the outer membrane receptor and speeds up the release of empty hemophores, which, if they remained on receptors, would inhibit heme transport.

The genetic, biochemical and crystallographic studies are conducted to find out how most of the organisms including bacteria fulfill their iron needs. The results allowed substantial progress in describing molecular mechanisms of siderophore and hemophore interactions with the outer membrane receptors, transport through the inner membrane, iron storage, and regulation of genes encoding biosynthesis and uptake proteins.

Circular dichroism and NMR spectroscopy help investigate C-terminal secretion signal encoding in the signal peptide-independent pathway of Erwinia chrysanthemi protease. The 26-29 and 50-62 peptide residues in the protease form a stable helix and the 13-63 residue region contains a random structure equilibrium with a helix structure. The C-terminal secretion signal encodes an alpha-helix near the tetrapeptide of the C-terminal.

Research has been conducted on Serratia marcescens heme aquisition system's HasA secreted hemophore. The genuine chaperone involved in HasA secretion and the role of this chaperone have been investigated, and the results are reported.