Enterobacterial sensitivity to beta-lactam antibiotics]

The susceptibility to betalactams of 868 enteric bacteria isolated from the patients at the hospital was studied. The isolated pathogens included: E. coli (549), Klebsiella sp. (195), Serratia sp. (124). Ampicillin and cefazoline demonstrated the lowest activity. Cefotaxime, ceftazidime and imipenem were active against 90 per cent of isolates. Among E. coli isolates the susceptibility to the above mentioned drugs was the following: 95.1, 96.9, 99.3 per cent, among Klebsiella sp.--89.7, 88.7, 97.9 per cent, among Proteus sp.--89.5, 90.3, 91.9 per cent respectively. Thus cefotaxime may be used in antibacterial empiric therapy if Pseudomonas aeruginosa infection is excluded.     

Production of and sensitivity to colicins among serologically classified strains of Escherichia coli

A significant proportion of 242 serologically classified strains of Escherichia coli of human origin produced colicins (33%) or were inhibited by one or more of six standard colicins (57%). The most common colicins identified were E1, I, and B; colicins B and V had greatest range of activity. Generally, neither the production of, nor sensitivity to, individual colicins was restricted to strains of a single serogroup. The coexistence of strains of one serogroup that were sensitive to the action of a colicin produced by strains of another serogroup was encountered among 2 of 21 fecal specimens containing strains of multiple serogroups. The production of colicins was not a major determinant in the acquistion of, or subsequent changes in, strains of E. coli in the feces of 10 newborn infants.     

Characterization of ompF domains involved in Escherichia coli K-12 sensitivity to colicins A and N.

Various ompF-ompC, ompC-ompF, and ompF-ompC-ompF chimeric genes were used to locate the domains of the OmpF protein involved in cellular sensitivity to colicins. Various parts of the porin participate in the entry of colicins. Colicin N receptor activity was found to require three regions: RN1, located between residues 1 and 63; RN2, located between residues 115 and 262; and RN3, located between residues 279 and 297. The central domain from residues 143 to 262 is involved during the translocation step after the binding step. A large region, including residues 1 to 262, is necessary during colicin A entry. The locations and interactions between these domains specifically required for the uptake of colicins to occur are described and discussed with regard to the homology and topology of the OmpC, OmpF, and PhoE porins.     

Studies on the enzymatic synthesis of enterochelin in Escherichia coli K-12. Four polypeptides involved in the conversion of 2,3-dihydroxybenzoate to enterochelin.

Four gene products involved in the enzymatic synthesis of enterochelin from 2,3-dihydroxybenzoate, L-serine and ATP (Luke, R.K.L. and Gibson, F. (1971) J. Bacteriol. 107,557-562; Woodrow, G.C., Young, I.G. and Gibson, F. (1975) J. Bacteriol. 124, 1-6) have been partially purified using a previously reported fractionation procedure (Bryce, G.F. and Brot, N. (1972) Biochemistry 11, 1708-1715). The products of genes E, F and G have been separated from each other and correspond to the E1, E2 and E3 activities described by Bryce and Brot. These three gene products were not completely separated from the product of gene D. We refer to these gene products as components E, F, G and D of the enzymic apparatus for biosynthesis of enterochelin. Certain properties and functions of the four semi-purified components have been investigated. The E component is involved in the activation of 2,3-dihydroxybenzoate and the F component in the activation of L-serine. The D component physically associates with the F and G components during gel filtration and chromatography on DEAE Sephadex. It is proposed that the synthesis of enterochelin from L-serine and 2,3-dihydroxybenzoic acid is catalysed in vivo by a multienzyme complex, enterochelin synthetase.     

Novel approaches to the mode of action of colicins

According to the theory of Fredericq (1949) and Nomura (1964), colicins are attached by specific receptor sites in the cell walls of sensitive bacteria, which mediate their inhibitive effects. During last years, a great variety of experimental data have been accumulated, some of which cannot be easily interpreted in terms of this theory. There exist considerable discrepancies concerning the chemical nature and molecular weight of isolated receptors. The attachment of a colicin onto its receptor need not be irreversible. The inhibition of numerous membrane-associated functions in colicin-tolerant mutants suggests their pleiotropic deletion nature. The difference between colicin resistance and colicin tolerance does not seem to be clear-cut. Cells of stable L-forms of protoplast type, completely devoid of their walls, retain in most cases the same patterns of sensitivity to colioins as rods of the same strains. Experimental changes in the relationship between the cell wall and the cytoplasmic membrane decrease colicin sensitivity of the cells. Colicin E3 has been found to be a specific endoribonuclease, able to cleave a terminal fragment from the 16 S rRNA also in isolated ribosomesin vitro: not only in ribosomes from sensitivive bacteria, but also in those from resistant ones and from eukaryotic cells. A destabilization of the DNA helix was induced by colicin E2in vitro asin vivo. It seems that there exist two distinct types of colicin receptors with different functions: those in the cell wall, and those in the cytoplasmic membrane. Only the contact of colicins with the latter ones is biologically effective and starts both stages of their inhibitive effect: the reversible and the irreversible ones.     

Regulation of enterochelin synthetase in Escherichia coli K-12

Enterochelin synthetase activity is controlled by both repression and feed-back inhibition mechanisms. Inclusion of iron in growth media results in synthesis of all four (D, E, F and G) components of enterochelin synthetase being repressed. The specific inhibition of L-serine activation (partial reaction catalyzed by the F component) by the end products, ferric-enterochelin and 2,3-dihydroxybenzoylserine, is shown to inhibit overall enterochelin synthetase activity.     

Enzymatic hydrolysis of enterochelin and its iron complex in Escherichia Coli K-12. Properties of enterochelin esterase.

Properties of the enzyme which hydrolyses enterochelin (a cyclic trimer of 2,3-dihydroxy-N-benzoyl-L-serine) to 2,3-dihydroxybenzoylserine have been investigated with a view to resolving discrepancies between earlier reports. Enterochelin esterase, previously reported to consists of two components (O'Brien, I.G., Cox, G.B. and Gibson, F. (1971) Biochim. Biophys. Acta 237, 537-549), has been shown to be fully active in the absence of the so-called A component. The hydrolase described previously (Bryce, G.F. and Brot, N. (1972) Biochemistry 11, 1708-1715) as being able to break down enterochelin but not its iron complex, ferric-enterochelin, appears to be identical with the B component of enterochelin esterase. The single component enterochelin esterase corresponding to what was previously described as component B, hydrolyses both enterochelin and ferric-enterochelin. Under the assay conditions used, enterochelin is hydrolysed 2.5 times faster than the complex. Enzymatic activity is inhibited by N-ethylmaleimide and is lost rapidly at 37 degrees C. Activity is stabilized in the presence of ferric-enterochelin, enterochelin, dithiothreitol or certain protein fractions.     

Iron supply to Escherichia coli by synthetic analogs of enterochelin.

Synthetic analogs of enterochelin (enterobactin) were tested for their ability to support the growth of Escherichia coli K-12 under iron-limiting conditions. The cyclic compound MECAM [1,3,5-N.N'; N"-tris-(2,3-dihydroxybenzoyl)-triamino-methylbenzene] and its N-methyl derivative Me3MECAM promoted growth, whereas the 2,3-dihydroxy-5-sulfonyl derivatives MECAMS and Me3MECAMS were inactive. The same results were obtained with TRIMCAM [1,3,5-tris(2,3-dihydroxybenzoylcarbamido)-benzene] and TRIMCAMS (the 2,3-dihydroxy-5-sulfonyl derivative of TRIMCAM). However, the sulfonic acid-containing linear compound LICAMS [1,5,10-N,N', N"-tris(5-sulfo-2,3-dihydroxybenzoyl)-triaza-decane] supported growth. In contrast, LIMCAMC, in which the sulfonyl groups at the five position of LICAMS are replaced by carboxyl groups at the four position, was inactive. The uptake of the active analogs required the functions specified by the fepB, fesB, and tonB genes. Surprisingly, growth promotion of mutants lacking the enterochelin receptor protein in the outer membrane was observed. Only MECAM protected cells against colicin B (which kills cells after entering at the enterochelin uptake sites) and transported Fe3+ at about half the enterochelin rate.     

Change in the sensitivity to methotrexate of neoplastic cells cultivated in the presence of folic acid]

Cultivation of tumour L-cells in the presence of increasing folic acid concentrations led to the rise in the resistance of these cells population to metotrexate. With the subsequent cultivation, when the folic acid concentration was not increased the population of such cells became more sensitive to metotrexate even in comparison with the initial L-cells.     

Immunity proteins to pore-forming colicins: structure-function relationships

Colicin A and B immunity proteins (Cai and Cbi, respectively) are homologous integral membrane proteins that interact within the core of the lipid bilayer with hydrophobic transmembrane helices of the corresponding colicin channel. By using various approaches (exchange of hydrophilic loops between Cai and Cbi, construction of Cbi/Cai hybrids, production of Cai as two fragments), we studied the structure-function relationships of Cai and Cbi. The results revealed unexpectedly high structural constraints for the function of these proteins. The periplasmic loops of Cai and Cbi did not carry the determinants for colicin recognition although most of these loops were required for Cai function; the cytoplasmic loop of Cai was found to be Involved in topology and function of Cai. The immunity function did not seem to be confined to a particular region of the immunity proteins.