A decrease of the absorbance a t 578 nm of a cell wall suspension of mid log phase E . coli occurs when the suspension is incubated with trypsin. The reaction is so rapid that 55O/, of the total decrease is obtained within the first 2 min [ratio of enzyme to total cell wall protein = I : 50 (w/w), room temperature]. The rate of the reaction is specific for trypsin when compared with other proteases, different lipases, lysozyme and other glycosidases. A peptide bond especially sensitive to trypsin could be localized within the complex cell wall by the demonstration that the decrease of the absorbance is paralleled by the splitting of the protein from the murein.This protein could be shown to be a lipoprotein with a part of the lipid probably covalently bound to the protein. It is called murein-lipoprotein. The link between the lipoprotein and the murein is lysine. After trypsin digestion lysine is the only additional amino acid remaining a t the murein. The ratio of the amount of lysine to the known constituents of the murein demonsstrates that on the average one lipoprotein molecule is covalently bound to every tenth repeating unit of the murein (N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-glutamic acidmeso-diaminopimelic acid-D-alanine). After 3 min incubation with trypsin, the isolated lipoprotein molecules have a lysine to arginine ratio of 4:4 as compared with 5 : 4 in the native molecule. The lipoprotein has an unusual amino acid composition since it contains about 65O/, polar amino acids and no glycine, cysteine, proline, phenylalanine, and histidine could be found. On a weight basis the lipoprotein accounts for more than 40°/, of the rigid layer. Since the murein is held together exclusively by covalent bonds one can get a fairly accurate idea of the distribution of the lipoprotein molecules in the cell wall. About lo5 lipoprotein molecules per cell should be distributed 103 A apart along the glycosidic chains of the murein.The lipoprotein has an important function in stabilizing the total structure of the cell wall.It seems that cleavage of only one peptide bond adjacent to the lysine link between the lipoprotein and the murein causes the rapid decrease of the absorbance and as shown by electron microscopic examination of ultrathin sections of trypsin treated cell walls, two separated membrane structures appear which otherwise are closely adjacent to one another.There are several lines of evidence suggesting that cell walls are more than mere cell envelopes protecting the essential cell functions which are supposed to take place in the cytoplasm. For example cell walls of E. coli not only contain the systems of active transport but interact with colicins (bacteriocins) in such a way that suggests that the whole complex memUnwnutl Abbreviation. Dansyl-, l-dimethylaminonaphthalene-5-sulfonyl-.Enzymes. Trypsin (EC 3.4.4.4); chymotrypsin (EC 3.4.4.5) ; papain (EC 3.4.4.10) ; pronase (EC 3.4.4) ; lysozyme (EC 3

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Iron in the form of ferric siderophore complexes and vitamin B12 are transported through the outer membrane of Gram-negative bacteria by a mechanism which consumes energy. There is no known energy source in the outer membrane or in the adjacent periplasmic space so that energy is provided by the electrochemical potential across the cytoplasmic membrane. Energy flows from the cytoplasmic into the outer membrane via a complex consisting of the TonB, ExbB and ExbD proteins which are anchored in the cytoplasmic membrane. It is proposed that the TonB--ExbB--ExbD complex opens--via an energized conformation of the TonB protein--channels in the outer membrane, formed by proteins which serves as highly specific binding sites for the various ferric siderophores and vitamin B12. In addition, outer membrane receptors together with the TonB--ExbB--ExbD complex are directly involved in induction of the transcription of ferric citrate and pseudobactin transport genes of Escherichia coli and Pseudomonas putida, respectively.

Sideromycins are antibiotics covalently linked to siderophores. They are actively transported into gram-positive and gram-negative bacteria. Energy-coupled transport across the outer membrane and the cytoplasmic membrane strongly increases their antibiotic efficiency; their minimal inhibitory concentration is at least 100-fold lower than that of antibiotics that enter cells by diffusion. This is particularly relevant for gram-negative bacteria because the outer membrane, which usually forms a permeability barrier, in this case actively contributes to the uptake of sideromycins. Sideromycin-resistant mutants can be used to identify siderophore transport systems since the mutations are usually in transport genes. Two sideromycins, albomycin and salmycin, are discussed here. Albomycin, a derivative of ferrichrome with a bound thioribosyl-pyrimidine moiety, inhibts seryl-t-RNA synthetase. Salmycin, a ferrioxamine derivative with a bound aminodisaccharide, presumably inhibts protein synthesis. Crystal structures of albomycin bound to the outer membrane transporter FhuA and the periplasmic binding protein FhuD have been determined. Albomycin and salmycin have been used to characterize the transport systems of Escherichia coli and Streptococcus pneumoniae and of Staphylococcus aureus, respectively. The in vivo efficacy of albomycin and salmycin has been examined in a mouse model using Yersinia enterocolitica, S. pneumoniae, and S. aureus infections. Albomycin is effective in clearing infections, whereas salmycin is too unstable to lead to a large reduction in bacterial numbers. The recovery rate of albomycin-resistant mutants is lower than that of the wild-type, which suggests a reduced fitness of the mutants. Albomycin could be a useful antibiotic provided sufficient quantities can be isolated from streptomycetes or synthesized chemically.