Changes in the nature of the sensitivity of an E. coli M strain carrying Inc-group R plasmids, to coliphages T3 and T7

After the transfer of prototype plasmids R6K (IncX), R387 (IncK), R27 (IncH1) and T (IncN) to E. coli M nalr the appearance of histidine-dependent mutants (R27, T), histidine-leucine-dependent mutants (R6K), methionine-proline-dependent mutants (R387) was observed among the resulting transconjugates. The mutations of E. coli M nalr R+ cells induced by the introduction of the plasmids were accompanied by the transformation of the cells from the S-form into the R-form. In contrast to the prototrophs E. coli M nalr, the auxotrophs carrying plasmids R6K, R27, T acquired sensitivity to phage T7, and the methionine-proline-dependent mutant became sensitive to phages T and T7. The above-mentioned plasmids rendered E. coli M cells capable of synthetizing the donor pili. But the adsorption of phages T3 and T7 on the auxotrophic cells, both with and without plasmids, occurred due to their interaction with the cell-wall receptors.     

Reconstitution of denatured E. coli alkaline phosphatase with E. coli ribosome.

E. coli alkaline phosphatase was denatured by physical/chemical means. In vitro reconstitution of this denatured enzyme was assisted by 70S E. coli ribosome, as shown by the recovery of its catalytic competence. Almost total recovery of activity of the totally inactivated enzyme was obtained in presence of equimolar concentration of 70S ribosome at 50 degrees C.     

Enzymatic degradation of uracil-containing deoxyribonucleic acid. V. Survival of Escherichia coli and coliphages treated with sodium bisulfite.

A number of mutants of Escherichia coli defective in the ung gene (structural gene for uracil-deoxyribonucleic acid [ura-DNA] glycosylase) are shown to be abnormally sensitive to treatment with sodium bisulfite when compared with congenic ung+ strains. These results provide further evidence that sodium bisulfite causes the deamination of cytosine to uracil in DNA and that ura-DNA glycosylase is required for the repair of U-G mispairs. The effect of the chemical is apparently selective with respect to base damage; coliphages containing cytosine in their DNA are inactivated by treatment with sodium bisulfite, whereas those containing hydroxymethylcytosine are not. ura-DNA glycosylase and the major apurinic-apyrimidinic endonuclease of E. coli may function in the same repair pathway, since the extent of inactivation of a congenic set of strains which are ung xth (structural gene for the major apurinic-apyrimidinic endonuclease of E. coli) or ung xth+ is the same.     

E．coli分泌表达载体的构建和人表皮生长因子在E．coli中的分泌性表达

采用ＰＣＲ技术从Ｅ．ｃｏｌｉ基因组片段中克隆出碱性磷酸酯酶（ＰｈｏＡ）的启动子和信号肽序列．在ＰｈｏＡ启动予５＇端设计了ＥｃｏＲⅠ酶切位点,在信号肽编码序列３＇端设计了ＨｉｎｄⅢ酶切位点．将ＰＣＲ产物酶切后ＥｃｏＲⅠ－ＨｉｎｄⅢ片段克隆至ｐＢＲ３２２的ＥｃｏＲⅠ－ＨｉｎｄⅢ位点,组构出含有ＰｈｏＡ启动子和信号肽序列的分泌表达载体ｐＢＭ－Ｐｈｏ－１．之后将人表皮生长因子的成熟肽基因克隆至该载体,使之在Ｅ．ｃｏｌｉ中获得分泌表达,另采用ｐＩＮⅢ载体系统以分泌方式表达了人表皮生长因子。     

l-Asparaginase from E. coli

l-Asparaginase [EC 3.5.1.1], antitumor enzyme, was purified to a crystalline form from the cell free extract of Escherichia coli A-l-3 KY3598, by ethanol fractionation and chromatographies on DEAE cellulose and CM Sephadex. The crystalline enzyme was homogeneous by the criteria of ultracentrifugation: s_{20, w} was 7.87S.

The molecular weight was estimated to be 141,000 by the short column method. The pI of the enzyme protein was 4.75 according to isoelectric electrofocusing.

Amino acid analysis revealed the absence of cysteine or cystine residues in the molecule.

The enzyme exhibited optimal activity between pH 6 and 8. It was stable in the pH range 5.5 ~ 9.0.

The enzyme activity was cleared very slowly in the plasma of dog. Intravenous administration of the enzyme caused a complete regression of the Gardner lymphoma implanted in the C3H mice.     

Mistranslation in E. coli.

Flagellin, the protomeric subunit of bacterial flagella, contains no cysteine. We have detected the incorporation of trace quantities of 35S-cysteine into flagellin, highly purified and then resolved by SDS polyacrylamide gel electrophoresis, to measure mistranslation in vivo. Under normal conditions, this value is about 6 X 10(-4) pmoles cysteine per pmole flagellin. This value is greatly increased during growth in low concentrations of streptomycin and neomycin, antibiotics which are known to stimulate misreading in vitro. Of the specific types of misreading which streptomycin stimulates in vitro, only misreading of the CGU and CGC arginine codons could give rise to illegitimate incorporation of cysteine. In agreement, partial arginine starvation increases the incorporation of 35S-cysteine into flagellin in a relA- mutant, with or without streptomycin, but has no such effect in its isogenic relA+ partner- Assuming from these results that 35S-cysteine incorporation into flagellin reflects misreading of CGU/C coda, we deduce a misreading probability per codon in the range of 10(-4).     

Sequences of the E. coli uvrB gene and protein.

The UvrB protein is one of the three subunits of the E. coli ABC excinuclease. We have reported the sequences of the other two subunits, the UvrA and UvrC proteins. In this paper the sequence of the UvrB protein is presented. The protein sequence was determined from the DNA sequence of the uvrB gene and was confirmed by sequencing the NH2-terminus of the UvrB protein and analyzing its overall amino acid composition. The coding region of uvrB is 2019 basepairs, specifying a protein of 672 amino acids and Mr of 76,118. The sequence of the UvrB protein shows a moderate level of homology to that of the UvrC protein and to the ATP binding site of the UvrA protein. During purification of UvrB protein a proteolytic product, UvrB, is produced in high quantities. We find that UvrB results from removal of about 40 amino acids from the COOH-terminus of the UvrB protein. The uvrB gene has complex regulatory features. On the 5' side, the coding region is preceded by 3 promoters, a DnaA box and an SOS box. On the 3' side the gene is followed by an REP (Repetitive Extragenic Palindrome) sequence which has been implicated in gene regulation by an unknown mechanism.     

Isolation and selection of coliphages as potential biocontrol agents of enterohemorrhagic and Shiga toxin‐producing E. coli(EHEC and STEC) in cattle

Aims: To isolate, characterize and select phages as potential biocontrol agents of enterohemorrhagic and Shiga toxin‐producing Escherichia coli (EHEC and STEC) in cattle. Methods and Results: Sixteen STEC and EHEC coliphages were isolated from bovine minced meat and stool samples and characterized with respect to their host range against STEC, EHEC and other Gram‐negative pathogens; their morphology by electron microscopy; the presence of the stx1, stx2 and cI genes by means of PCR; RAPD and rep‐PCR profiles; plaque formation; and acid resistance. Six isolates belonged to the Myoviridae and 10 to the Podoviridae families. The phages negative for stx and cI that formed large, well‐defined plaques were all isolated using EHEC O157:H7 as host. Among them, only CA911 was a myophage and, together with CA933P, had the broadest host range for STEC and EHEC; the latter phage also infected Shigella and Pseudomonas. Isolates CA911, MFA933P and MFA45D differed in particle morphology and amplification patterns by RAPD and rep‐PCR and showed the highest acidity tolerance. Conclusions: Myophage CA911 and podophages CA933P, MFA933P and MFA45D were chosen as the best candidates for biocontrol of STEC and EHEC in cattle. Significance and Impact of the Study: This work employs steps for a rational selection and characterization of bacteriophages as therapeutic agents. This report constitutes the first documentation of STEC and EHEC phages isolated in Argentina and proposes for the first time the use of rep‐PCR as a complement of RAPD on DNA fingerprinting of phages.     

alpha-Hemolysin of E. coli]

The activity of alpha-hemolysin increased at the log growth phase in the culture of E. coli P678 Hly+ hemolytic strain; this activity diminished with the change into the stationary phase, and then fell sharply. Replacement of the culture medium in the stationary growth phase by fresh one led to restoration of the hemolytic activity of the culture. The culture fluid separated from the cells at the stationary growth phase produced an inhibitory action on the alpha-hemolysin Ca ions activated and stabilized the alpha-hemolysin. Sodium citrate and sucrose served as hemolysis inhibitors. The action of alpha-hemolysin was maximal against human erythrocytes at pH 6.5. Hemolytic activity was characterized in time by a distinct lag-phase and the phase of the greatest rate of reaction. The duration of the lag-phase and also the rate of hemolysis depended on the concentration of alpha-hemolysin (with the increase of the hemolysin concentration lag-phase was shortened and the reaction was accelerated). There proved to be a linear relationship between the amount of erythrocytes taken into the reaction and the rate of hemoglobin release, and also there was noted a temperature activation of the hemolytic reaction.