Identification ofEscherichia coli spheroplasts by the fluorescent-antibody staining technique

Spheroplasts ofEscherichia coli were produced by penicillin or lysozyme-ethylenediaminetetraacetic acid and examined by the direct fluorescent-antibody staining technique. Most spheroplasts stained with somatic-O fluorescent antibody exhibited brilliant peripheral fluorescence with localized areas of irregular staining. Electron micrographs showed that these spherical structures had considerable amounts of cell wall fragments associated with them. Two strains ofE. coli employed in the present study required different concentrations of penicillin for the conversion of all cells in an exponential culture to spheroplasts. Slight differences in lysozyme sensitivity were also encountered with these strains. The direct fluorescent-antibody staining technique was effective for the rapid identification ofE. coli spheroplasts in mixed cultures.     

Mutant ofEscherichia coli sensitive to proteinase

A mutant ofEscherichia coli sensitive to proteinase (MP2) has been isolated and characterized briefly. MP2 cells were impaired by Pronase, showing a decrease of the cell turbidity both in growing cell and in cell suspension, while the parental cells remained unimpaired. Pronase conceivably digested cellular protein, but DNA remained with cells. This Pronase action was prevented by the inhibitor of protein synthesis, while it was stimulated by bacitracin. The mutant was susceptible to an externally added repressor. A transient repression of arginine enzyme synthesis was observed only when the cell extract of a repressible strain was added, not by the extract of a nonrepressible strain. It was concluded that the proteinase-sensitive mutant has a lower permeability barrier to macromolecules.     

Production and purification ofEscherichia coli hemolysin

Eight strains of Escherichia coli, isolated from patients with a urinary tract infection were investigated for production of hemolysin. Six of these produced hemolysin and one revealed maximum hemolytic activity. Three urinary and two faecal isolates were positive for mannose-resistant hemagglutination. One isolate positive for hemagglutination and giving maximum hemolytic activity was then used. Hemolysin was present in the supernatant broth and the medium of choice to obtain the optimum yield was the alkaline meat extract broth followed by brain heart infusion broth. The highest yield appeared in the exponential phase of growth. Hemolysin is a heat-labile protein, being produced optimally at pH 8. A three-stage procedure was the best method for its purification.     

Adaptation ofEscherichia coli to chlortetracycline during continuous cultivation

При трехступенчатой проточной культивации штаммаЕscherichia coli SZÚ В питательной среде с повышениением концентраци хлортетрациклина быстро наступает адаптаця к этому антибитику. Процесс адаптации протекает интенсивнее у ?изиологически более молодых клеток в первых двух ступенях культивации, но достигает максимума в конечной стадии развития клеточой поиуляции. Полученные результаты говораят микроорганизмов к антибиотикам, а, быть может, и к другим ингибиторам.     

Overproduction and purification ofEscherichia coli tRNALeu

Chemically synthesized genes encodingEscherichia coli tRNA ₁ ^Leu and tRNA ₂ ^Leu were ligated into the plasmid pTrc99B. then transformed intoEscherichia coli MT102, respectively. The positive transformants, named MT-Leu1 and MT-Leu2, were confirmed by DNA sequencing, and the conditions of cultivation for the two transformants were optimized. As a result, leucinc accepting activity of their total tRNA reached 810 and 560 pmol/A₂₆₀, respectively: the content of tRNA ₁ ^Leu was 50% of total tRNA from MT-Leu1, while that of tRNA ₂ ^Leu was 30% of total tRNA from MT-Leu2. Both tRNA^Leus from their rotal tRNs were fractionated to 1 600 pmol/A₂₆₀ after DEAE-Sepharose and BD-cellulose column chromatography. The accurate kinetic constants of aminoacylation of the two isoacceptors of tRNA^Leu catalyzed by leucyl-tRNA synthetase were determined.     

A histidine binding protein ofEscherichia coli: a component of cystine binding protein ofEscherichia coli

Summary Commercially obtained cystine binding protein (CBP), an osmotic shock protein ofEscherichia coli, was studied in an effort to determine its binding characteristics. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) analysis of commercially obtained CBP showed three protein bands. N-terminal amino acid microsequencing and subsequent computer search revealed that the sequence of one of these proteins (25-kDa) was nearly identical to histidine binding protein (HisJ) ofSalmonella typhimurium. Purification of CBP by HPLC yielded four protein peaks, of which one bound histidine exclusively. Binding was maximal at pH 5.0 to 6.0, at 4°C, did not require calcium or magnesium ions and was not inhibited by reduction of CBP disulfide bonds. Amino acids other than histidine or cystine did not bind to CBP. These data show that commercially available CBP is not a homogenous protein; it contains a histidine as well as a cystine binding component.     

The hydrogenases and formate dehydrogenases ofEscherichia coli

Escherichia coli has the capacity to synthesise three distinct formate dehydrogenase isoenzymes and three hydrogenase isoenzymes. All six are multisubunit, membrane-associated proteins that are functional in the anaerobic metabolism of the organism. One of the formate dehydrogenase isoenzymes is also synthesised in aerobic cells. Two of the formate dehydrogenase enzymes and two hydrogenases have a respiratory function while the formate dehydrogenase and hydrogenase associated with the formate hydrogenlyase pathway are not involved in energy conservation. The three formate dehydrogenases are molybdo-selenoproteins while the three hydrogenases are nickel enzymes; all six enzymes have an abundance of iron-sulfur clusters. These metal requirements alone invoke the necessity for a profusion of ancillary enzymes which are involved in the preparation and incorporation of these cofactors. The characterisation of a large number of pleiotropic mutants unable to synthesise either functionally active formate dehydrogenases or hydrogenases has led to the identification of a number of these enzymes. However, it is apparent that there are many more accessory proteins involved in the biosynthesis of these isoenzymes than originally anticipated. The biochemical function of the vast majority of these enzymes is not understood. Nevertheless, through the construction and study of defined mutants, together with sequence comparisons with homologous proteins from other organisms, it has been possible at least to categorise them with regard to a general requirement for the biosynthesis of all three isoenzymes or whether they have a specific function in the assembly of a particular enzyme. The identification of the structural genes encoding the formate dehydrogenase and hydrogenase isoenzymes has enabled a detailed dissection of how their expression is coordinated to the metabolic requirement for their products. Slowly, a picture is emerging of the extremely complex and involved path of events leading to the regulated synthesis, processing and assembly of catalytically active formate dehydrogenase and hydrogenase isoenzymes. This article aims to review the current state of knowledge regarding the biochemistry, genetics, molecular biology and physiology of these enzymes.     

Molecular cloning of theEnvC gene ofEscherichia coli

DNA fragments complementing theenvC mutation could be isolated by cloning chromosomal DNA in the vector pUH84. When the frequencies of transformation and the frequencies of restoring theenvC ⁺ phenotype were compared, the high copy number hybrid plasmids complemented with a frequency of 10^–5. After subcloning theenvC-complementing DNA fragment into the low copy number plasmid pLG339, efficient complementation was achieved by spontaneous integration of the IS2 element ofEscherichia coli. By nucleotide sequence analysis, a potential promoter, a ribosome-binding site, and an unidentified reading frame were detected in the respective DNA fragment.     

Cell division of penicillin-induced filaments ofEscherichia coli

Reversion of cell division of penicillin-induced filamentous forms ofEscherichia coli was studied from the aspect of the function of proteosynthesis in reversion and the renewal of intact mucopeptide, and from the aspect of the function of mucopeptide in reversion. Mucopeptide renewal is an, essential condition for the initiation of reversion. If proteosynthesis is inhibited immediately after removing the penicillin, the mucopeptide is not renewed. If inhibition is delayed, it is renewed in amounts proportional to the interval of proteosynthesis. Mucopeptide renewal is probably not sufficient for completing reversion under conditions of inhibition of proteosynthesis, however. The possible existence of a protein involved in cytokinesis, whose presence or activity is determined by an intact mucopeptide structure, is discussed. From direct observation of the formation of microcolonies from filaments, we found that septa which are formed later during normal division in the absence of penicillin were formed earlier during reversion.