Stability and storage ofEscherichia coli mutants hyperproducing β-galactosidase

Summary Enzyme activity of the hyperproducing mutants isolated from a chemostat decreases by passaging under nonselective conditions to about one half of the original value, and then remains stable. High activity can be quickly restored by transfer to chemostat selective conditions. The elaborated storage method prevents the decrease of enzyme activity after 2–3 years.     

Polyamines and protein synthesis: Studies in various polyamine-requiring mutants ofEscherichia coli

Summary DifferentEscherichia coli mutants auxotrophic for polyamines were studied in order to investigate the relationships among polypeptide synthesis in cell-free systems, ribosomal distribution profiles and endogenous polyamine pools. Thein vitro protein synthetic activity and the polyribosomal content were reduced in extracts from putrescine-starved cells of the double mutants MA 255 and MA 261, but not in the arginine-conditional auxotroph DK 6. Putrescine addition to the cultures of all these strains previously starved for polyamines, provoked a shift towards monomers in the equilibrium involving ribosomal particles. Concomitant changes in the intracellular levels of polyamines were observed: putrescine and spermidine increased markedly, and cadaverine disappeared.Dedicated to ProfessorLuis F. Leloir on the occasion of ths 70^th birthday.     

Physicochemical characterization ofEscherichia coli

EightEscherichia coli strains were characterized by determining their adhesion to xylene, surface free energy, zeta potential, relative surface charge, and their chemical composition. The latter was done by applying X-ray photoelectron spectroscopy (XPS) and infrared spectroscopy (IR). No relationship between the adhesion to xylene and the water contact angles of these strains was found. Three strains had significantly lower surface free energies than the other strains. Surface free energies were either obtained from polar and dispersion parts or from Lifshitz-van der Waals and acid/base parts of the surface free energy. A correlation (r=0.97) between the polar parts and the electron-donor contributions to the acid/base part of the surface free energy was found. The zeta potentials of all strains, measured as a function of pH (2–11), were negative. Depending on the zeta potential as a function of pH, three groups were recognized among the strains tested. A relationship (r=0.84) was found between the acid/base component of the surface free energy and the zeta potential measured at pH=7.4. There was no correlation between results of XPS and IR studies. Data from the literature of XPS and IR studies of the gram-positive staphylococci and streptococci were compared with data from the gram-negativeE. coli used in this study. It appeared that in these three groups of bacteria, the polysaccharide content detected by IR corresponded well with the oxygen-to-carbon ratio detected by XPS.     

Survival of Campylobacter jejuni in chicken meat at frozen storage temperatures

The aim of this study was to determine the survival of Campylobacter jejuni in chicken meat samples at frozen temperatures and given length of incubation and to determine the impact of aerobic bacteria on the survival of C. jejuni. The chicken meat samples were inoculated with C. jejuni NCTC 11351 suspensions and stored in bags at temperatures of -20°C and -70°C. The mean value of C. jejuni from meat samples decreased from 7.52 log10 CFU/g after 30 minutes of incubation at ambient temperature, to 3.87 log10 CFU/g on the eighth week of incubation at -20°C, and to 3.78 log10 CFU/g at incubation at -70°C after the same incubation period. Both freezing temperatures, -20°C and -70°C, decreased the number of campylobacters. The presence of aerobic mesophilic bacteria did not influence the survival of C. jejuni in chicken meet samples. Keeping poultry meat at freezing temperatures is important for the reduction of C. jejuni, which has a strong influence on the prevention of occurrence of campylobacteriosis in humans.     

Survival ofEscherichia coli andYersinia enterocolitica in stream water: Comparison of field and laboratory exposure

Experiments were done to compare the influence of three aquatic exposure methods on the behavior of pathogenic and nonpathogenic enteric bacteria (Yersinia enterocolitica andEscherichia coli). Bacterial suspensions were exposed to stream water in membrane diffusion chambers in situ as well as in the laboratory using a large vessel of stream water and in enclosed bottles. The persistence of culturability of the bacterial suspensions was dependent upon the method of aquatic exposure. This difference was most apparent during the initial six days of each experiment. A steady decline in colony forming units was seen after a short stationary period in chambers in situ, while there was an abrupt increase in bacteria within chambers exposed in the laboratory. A rapid initial decrease was observed in the experimental variation using bottles, accompanied by higher levels of injury inE. coli and reduced expression of plasmid-borne virulence phenotypes inY. enterocolitica. However, there were no changes in the plasmid profiles of either organism throughout the 21-day duration of the experiments. In addition, the survival and injury of pathogenic and nonpathogenic strains of both test bacteria was very similar with aquatic exposure. These results suggest that the response of enteric bacteria in aquatic environments is influenced by experimental design as well as other factors and that the comparison of survival data should only be attempted when similar methods are used.     

Voltage-sensitive ion channel ofEscherichia coli

Summary A voltage-sensitive, cation-selective ion channel ofEscherichia coli has been reconstituted into liposomes and studied with the patch-clamp method. The single channel conductance was 91 pS in symmetric solutions of 150mm KCl. Many channels were open most of the time, with frequent brief transitions to closed levels. Multiple conducting units could close and reopen simultaneously, and this apparent cooperativity in gating was increases with depolarizing voltages. Above a voltage threshold, the channels closed irreversibly, often in groups.     

Signal peptide mutants ofEscherichia coli

Numerous secretory proteins of the Gram-negative bacteriaE. coli are synthesized as precursor proteins which require an amino terminal extension known as the signal peptide for translocation across the cytoplasmic membrane. Following translocation, the signal peptide is proteolytically cleaved from the precursor to produce the mature exported protein. Signal peptides do not exhibit sequence homology, but invariably share common structural features: (1) The basic amino acid residues positioned at the amino terminus of the signal peptide are probably involved in precursor protein binding to the cytoplasmic membrane surface. (2) A stretch of 10 to 15 nonpolar amino acid residues form a hydrophobic core in the signal peptide which can insert into the lipid bilayer. (3) Small residues capable of -turn formation are located at the cleavage site in the carboxyl terminus of the signal peptide. (4) Charge characteristics of the amino terminal region of the mature protein can also influence precursor protein export. A variety of mutations in each of the structurally distinct regions of the signal peptide have been constructedvia site-directed mutagenesis or isolated through genetic selection. These mutants have shed considerable light on the structure and function of the signal peptide and are reviewed here.     

Thesec andprl genes ofEscherichia coli

Two general approaches have been used to define genetically the genes that encode components of the cellular protein export machinery. One of these strategies identifies mutations that confer a conditional-lethal, pleiotropic export defect (sec,secretion). The other identifies dominant suppressors of signal sequence mutations (prl,proteinlocalization). Subsequent characterization reveals that in at least three cases,prlA/secY,prlD/secA, andprlG/secE, both types of mutations are found within the same structural gene. This convergence is satisfying and provides compelling evidence for direct involvement of these gene products in the export process.     

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.     

Adenyl cyclase in cell-free extracts ofEscherichia coli

The adenyl cyclase enzyme system was detected in the cells ofEscherichia coli disrupted by sonic treatment. This enzyme activity is located mainly in the cell fraction sedimenting at 2,000×g, i.e. in the cytoplasmic membrane fraction. A prolonged sonication treatment of the cell suspension was followed by the disappearance of activity in the membrane preparation. The pH optimum of the adenyl cyclase inEscherichia coli was on the alkaline side, around pH 9.     

Properties ofEscherichia coli grown in deuterated media

The effects on a number of parameters of transferringEscherichia coli between protonated and deuterated media were studied; these included growth, oxygen consumption and the synthesis of DNA, RNA, total protein, and β-galactosidase. Similar measurements were made on cells fully adapted to growth on deuterated media. The amino acid compositions of deuterated and protonated cellular protein were similar, but in deuterated cells the ratio protein: DNA was doubled. Deutero- and protio-β-galactosidase had similarK _M values and turnover numbers in D₂O and H₂O. The kinetics of β-galactosidase synthesis were not changed by deuteration, but it was found that lower concentrations of inducer were required to achieve particular levels of induction. Brief exposure to inducer in one medium, followed by removal of inducer and expression of enzyme-forming-potential in either D₂O or H₂O, showed that mRNA synthesized by deuterated cells was translated equally well in both media. mRNA synthesized by protonated cells was translated about twice as efficiently in H₂O. Inducible strains (but not a regulator constitutive) lost the capacity to synthesize enzymically active β-galactosidase after more than 100 generations in D₂O-acetate. The defect persisted when such cells were grown in H₂O-acetate, but enzyme activity was restored by growth in H₂O-glycerol. The failure to produce active enzyme was not due to a failure of the induction mechanism; gel electrophoresis revealed the presence of an inactive protein species. The nature of adaptation to deuteration is discussed.     

Survival of mouse embryos after being frozen in glycerol-sucrose mixture

Random bred female albino mice (6-8 weeks old) were used as a source of embryos. 8- to 16 cell embryos were dehydrated in glycerol-sucrose mixture in 0.25 ml straws at room temperature. Straws were cooled at the rate of 5 degrees C/min to -7 degrees C. Seeding was induced by touching the out side of the straw at -7 degrees C. Straws were further cooled at 0.5 degree C/min down to -35 degrees C and then plunged into liquid N2. Thawing of straws was done by direct transfer into water at 35 degrees C. Frozen-thawed embryos were cultured in a CO2 incubator maintained at 39 degrees C. Out 190 embryos (8-16 cell) initially frozen, 169 (88.94%) were recovered on thawing. 158 (93.5%) out of 169 were apparently normal and used for culture. 75 (47.46%) developed to morulae/early blastocysts and 72 (45.56%) to expanded blastocysts on 24 and 48 hr culture respectively. In conclusion, the incorporation of sucrose in the freezing medium at a concentration of 0.25 M has led us to propose a freezing, thawing and transfer method without dilution of glycerol. The technique being quite simple is worth trying in farm animals where importance of this technique in non-surgical transfer of frozen-thawed embryos will be a boon.     

Thermotolerant nalidixic acid-resistant mutants ofEscherichia coli

Nalidixic acid-resistant mutants ofEscherichia coli CGSC #6353 capable of growth at 48°C were obtained by mutagenesis withN-methyl-N-nitro-N-nitrosoguanidine. Cotransductional analyses employing phage P1 indicated that the mutation resulting in the phenotype of growth at 48°C is an allele of thegyrA structural gene. Similar thermal inactivation kinetics were observed for ribosomes isolated from a thermotolerant (T/r) mutant grown at both 37°C and 48°C and from the parental strain grown at 37°C. Cell-free extracts prepared from the T/r mutant grown at 48°C exhibited a sharp increase in protein synthesis at 55°C, whereas this effect was not displayed by extracts from the mutant or parental strains grown at 37°C. In addition, preincubation at 55°C enhanced protein synthesis at 37°C up to 15-fold in an extract prepared from the T/r mutant grown at 48°C, whereas comparable values were 2.6- to 3.0-fold for extracts from the mutant and parental strains grown at 37°C.     

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.     

Survival of Campylobacter jejuni on beef trimmings during freezing and frozen storage

AIMS: To investigate the survival of two animal isolates of Campylobacter jejuni on beef trimmings during freezing and frozen storage. METHODS AND RESULTS: Meat packs inoculated with 10(3) or 10(6) cfu g(-1) of either strain of C. jejuni were frozen to -18 degrees C, and sampled at regular intervals over 112 d storage to determine Campylobacter numbers and sublethal injury. For both strains and inoculation levels the numbers of Campylobacter decreased in the first 7 d of storage by ca. 0.6-2.2 log cfu g(-1) and then remaining constant over the remainder of the storage trial, with neither isolate exhibiting sublethal injury. CONCLUSIONS: Despite an initially significant decrease in number, these pathogens were able to survive standard freezing conditions in meat, but did not exhibit sublethal injury. SIGNIFICANCE AND IMPACT OF THE STUDY: Strict hygiene and/or the implementation of decontamination technologies are recommended as a means to assure the safety of meat with respect to this pathogen.     

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.