Behavior of coliphage lambda in hybrids between Escherichia coli and Salmonella

Salmonella typhosa hybrids able to adsorb lambda were obtained by mating S. typhosa recipients with Escherichia coli K-12 donors. After adsorption of wild-type lambda to these S. typhosa hybrids, no plaques or infective centers could be detected. E. coli K-12 gal(+) genes carried by the defective phage lambdadg were transduced to S. typhosa hybrids with HFT lysates derived from E. coli heterogenotes. The lysogenic state which resulted in the S. typhosa hybrids after gal(+) transduction differed from that of E. coli. Ability to produce lambda, initially present, was permanently segregated by transductants of the S. typhosa hybrid. S. typhosa lysogens did not lyse upon treatment for phage induction with mitomycin C, ultraviolet light, or heat in the case of thermoinducible lambda. A further difference in the behavior of lambda in Salmonella hybrids was the absence of zygotic induction of the prophage when transferred from E. coli K-12 donors to S. typhosa. A new lambda mutant class, capable of forming plaques on S. typhosa hybrids refractory to wild-type lambda, was isolated at low frequency by plating lambda on S. typhosa hybrid WR4254. Such mutants have been designated as lambdasx, and a mutant allele of lambdasx was located between the P and Q genes of the lambda chromosome. Plaques were formed also on the S. typhosa hybrid host with a series of lambda(i21) hybrid phages which contain the N gene of phage 21. The significance of these results in terms of Salmonella species as hosts for lambda is discussed.     

Calorimetric analysis of Escherichia coli in continuous culture

The heat evolution rate was measured in continuous culture of Escherichia coli k-12 on glucose-limiting medium. The specific heat production rate based on the dry cell mass increased in proportion to increase of dilution rate. The specific heat production rate based on a viable cell increased nonlinearly because the cell size varied in each dilution rate. Several thermochemical data were calculated for each dilution rate from the stoichiometric equation which was derived from elementary analysis of the cells. Growth yields based on energy measured directly was found to be less influenced by dilution rate than the growth yield based on consumed glucose. This suggested that the energy efficiency of utilization of catabolic energy for anabolism was almost constant.     

Direct transfer of coliphage lambda DNA from Escherichia coli to cellular slime mold Dictyostelium discoideum

Dictyostelium discoideum myxamoebae were cultured with Escherichia coli cells infected with lambda phage in the presence of chloramphenicol. After eliminating the uningested bacteria by repeated centrifugation in a Percoll gradient, we examined the myxamoeba cytoplasm (not the food vacuole) for the presence of phage DNA. A significant amount of DNA extracted from the myxamoebae was hybridizable with purified phage lambda DNA, and capable of forming phage particles when packaged in vitro with phage lambda proteins. The EcoRI restriction maps of the phages recovered from the plaques were identical to that of the infecting phage. These results strongly suggest that phage DNA molecules were taken up by the cellular slime mold cells and that at least some fraction existed in intact form.     

Escherichia coli dnaA initiation function is required for replication of plasmids derived from coliphage lambda

The dnaA gene function, indispensable for the initiation of Escherichia coli replication from oriC is not essential for the growth of phage lambda. The in-vitro replication of plasmids derived from phage lambda does not seem to require DnaA protein either. However, we present evidence that in vivo the normal replication of lambda plasmids is dnaA-dependent. After inactivating the dnaA gene function, half of the plasmid molecules may enter a single round of replication. Rifampicin sensitivity of this abortive, as well as normal, replication indicates involvement of RNA polymerase. The rifampicin resistance of the normal replication of lambda plasmids in E. coli carrying the dnaAts46 or dnaAts5, but not the dnaAts204 allele at 30 degrees C implies the interaction of DnaA protein and RNA polymerase in this process. We propose that DnaA protein co-operates with RNA polymerase in the initiation of replication at ori lambda. The dispensability of DnaA in the growth of phage lambda and in lambda plasmid replication in vitro is discussed.     

Oxygen-limited continuous culture and respiratory energy conservation in Escherichia coli.

Escherichia coli B was cultured continuously in succinate-minimal medium under conditions of oxygen limitation in the phauxostat. With decreasing oxygenation and consequent decreasing growth rates, the complement of terminal cytochrome oxidases changed as follows: high growth rates, cytochrome o; intermediate growth rates, cytochromes o and d; lowest growth rates, cytochromes o, d, and a1. Respiratory kinetics exhibited by nongrowing cell suspensions obtained from continuous cultures indicated that terminal oxidase activity was exhibited by cytochrome o (Km for O2 = 0.2 micron; Vmax = 1.1 to 1.5 mumol of O2 per nmol of cytochrome o per min) and cytochrome d (Km for O2 = 0.024 micron; Vmax = 0.7 mumol of O2 per nmol of cytochrome d per min). During oxygen-limited growth, the molar growth yield referred to respiration, and corrected for maintenance respiration [Yo(max)], was 12.6 g (dry weight) per g-atom of oxygen, not significantly different from the succinate-limited value of 12.0 g (dry weight) per g-atom of oxygen. The rate of maintenance respiration of the oxygen-limited culture was only 3.4 mg-atoms of O per g (dry weight) per h, some threefold less than that of the succinate-limited culture. Respiration-driven proton extrusion did not vary with the growth rate or with the complement of terminal oxidases (H+/O = 3.7; standard deviation, 0.07). We conclude that the content of terminal oxidases is without effect on the efficiency of respiratory energy conservation.     

Glucose transport of Escherichia coli growing in glucose-limited continuous culture.

Dilute cultures of wild-type Escherichia coli K12 and of derivatives impaired in one or other Enzyme-II component of the glucose phosphotransferase system were grown in continuous culture under glucose limitation. Cells harvested from the chemostat took up [U-14C]glucose from 0.1 mM solutions at rates directly related to the rates at which those cells had grown; the activity of the phosphotransferase system in those cells, rendered permeable with optimal accounts of toluene, parallels the ability of the cells to take up glucose. The capacity of these systems was rate-limiting for growth under the negligibly low glucose concentration in the chemostat, but was adequate to account for the stimulation of respiration observed when the cells were presented suddenly with excess glucose.     

A continuous culture study of an ATPase-negative mutant of Escherichia coli

For anaerobic glucose-limited chemostat cultures of Escherichia coli a value of 8.5 was found for Y _ATP ^max . For anaerobic glucose- or ammoniumlimited chemostat cultures of the ATPase-negative mutant M2-6 of E. coli Y _ATP ^max values of 17.6 and 20.0 were found, respectively. From these data it can be concluded that in the wild type during anaerobic growth 51–58% of the total ATP production is used for energetization of the membrane. Using the Y _ATP values obtained in the anaerobic experiments a P/O ratio of 1.46 could be calculated for aerobic experiments with the wild type. It is concluded that from the energy obtained by respiration in wild type E. coli about 60% is used for membrane energetization and only about 40% for the actual formation of ATP. No dramatic difference in the maintenance requirement for ATP or glucose has been observed between glucose- and ammonium-limited chemostat cultures of the mutant. The large difference in maintenance requirement observed for such cultures of the wild type is therefore supposed to be made possible by ATP hydrolysis by the ATPase.     

Plasmid formation from bacteriophage lambda in Escherichia coli

Summary Among the survivors of Escherichia coli derivatives infected with phage c1 or vir that are unable to establish ordinal lysogeny, clones arise which perpetuate the nondefective phage genome. When the bacteria bears a mutation(s) that makes the cell tolerant to the phage multiplication, such clones appear readily.The bacteria- complex was studied genetically and chemically, and it was concluded that the intact phage genomes, about two to four circular copies per bacterial chromosome, are perpetuated in bacterial cytoplasm as plasmids or in lysogenic state in cytoplasm.Several lines of evidence suggests that the phage genome in the lysogenic state in cytoplasm is under a different regulatory system from that in the normal prophage state on chromosome.     

Antisense RNA does not significantly affect expression of the galK gene of Escherichia coli or the N gene of coliphage lambda

The effect of antisense RNA on the expression of genes galK and N was studied in vivo. These two genes were either present in the Escherichia coli chromosome, as single copies, or were cloned on plasmid vectors. Antisense RNA was supplied from multicopy vectors where the entire galK or N gene, or only their N-proximal portions, were cloned in the antisense orientation downstream from the strong PL, PR or lacZp promoters. In all of the experiments there was no significant inhibition of the galK or N expression by up to a 50-fold excess of the specific antisense RNAs, for both the in cis and in trans experimental designs. The excess of the antisense RNA was calculated as based on respective copy numbers, but was not experimentally measured. The apparent five-fold regulatory effect observed in one of the experiments was found to be artifactually caused by unexpected creation of a terminator in one of our constructs. To avoid such artifacts, all our constructs were equipped with the nut-N antitermination system. We conclude that the reported antimessenger-mediated inhibition of gene expression is not a general phenomenon, but must require some special features which are not present in the galK and N systems.     

Transducing lambda phages with Escherichia coli genes

The paper presents data on transducing lambdoid phages containing Escherichia coli genes. The major genetic techniques for isolating transducing phages (in vivo) are outlined. A combined table of best-studied transducing phages obtained by the methods of molecular genetics and genetic engineering lists phages genotype & basic literature references for the phages and their derivatives. The chromosome fragments of E. coli inserted in phage DNA are separately specified. Another table presents information about phages carrying E. coli fused operons and genes. The paper also provides detailed physical maps of three regions of the E. coli chromosome. The bibliography contains 300 items.     

Shiga toxin-producing Escherichia coli, faecal coliforms and coliphage in animal feeds

AIMS: Animal feeds (n = 226), collected from pastures or feeding troughs on UK farms and from feed manufacturers' bulk stores, were analysed for Escherichia coli harbouring shiga-toxin genes (stx), faecal coliforms, coliphages and stx-harbouring bacteriophages. METHODS AND RESULTS: Samples comprised of 79 fresh grasses, 26 silages and 121 dried or heat-processed feeds (DPF). Five of the 79 (6.3%) fresh grass samples contained stx(2)-E. coli. stx-E. coli were not detected in the silages or DPF that were examined. Faecal coliforms were detected in 75/79 (94.9%) of fresh grasses, 19/26 (73.1%) of silages and 36/121 (29.8%) of processed feeds. Coliphages were detected in 63/79 (79.7%) and 18/26 (69.2%) of fresh grasses and silages, respectively. Coliphages were isolated at a significantly lower prevalence of 5% (6/121) from processed feeds. Although stx(2)-phage was isolated from the enrichment of a single grass sample, stx-phages were not detected in any of the silage or processed feeds. We did not detect stx(1)-phage in any of the samples collected. CONCLUSIONS: Pastures have the potential to act as transmission vectors for stx-harbouring E. coli for grazed livestock. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to report on the prevalence of E. coli harbouring stx genes, faecal coliforms, coliphages and stx-harbouring bacteriophages in a range of feedstuffs destined for consumption by UK livestock. This study provides information on the risk of feeds to the spread of stx-phages between livestock and/or the environment.     

Temperature-dependent growth kinetics of Escherichia coli ML 30 in glucose-limited continuous culture.

Detailed comparison of growth kinetics at temperatures below and above the optimal temperature was carried out with Escherichia coli ML 30 (DSM 1329) in continuous culture. The culture was grown with glucose as the sole limiting source of carbon and energy (100 mg liter(-1) in feed medium), and the resulting steady-state concentrations of glucose were measured as a function of the dilution rate at 17.4, 28.4, 37, and 40 degrees C. The experimental data could not be described by the conventional Monod equation over the entire temperature range, but an extended form of the Monod model [mu = mu(max) x (s - s(min))/(Ks + s - s(min))], which predicts a finite substrate concentration at 0 growth rate (s(min)), provided a good fit. The two parameters mu(max) and s(min) were temperature dependent, whereas, surprisingly, fitting the model to the experimental data yielded virtually identical Ks values (approximately 33 microg liter(-1)) at all temperatures. A model that describes steady-state glucose concentrations as a function of temperature at constant growth rates is presented. In similar experiments with mixtures of glucose and galactose (1:1 mixture), the two sugars were utilized simultaneously at all temperatures examined, and their steady-state concentrations were reduced compared with to growth with either glucose or galactose alone. The results of laboratory-scale kinetic experiments are discussed with respect to the concentrations observed in natural environments.     

Restriction of bacteriophage lambda by Escherichia coli K

Derivatives of phage lambda, for which the numbers and positions of the recognition sites for endonuclease R. Eco_k are known, were used as substrates for the Escherichia coli K restriction system in vivo and in vitro. A single unmodified recognition site was sufficient for a DNA molecule to be bound and broken by the K restriction enzyme. Although discrete fragments of DNA were not produced, the breaks were made preferentially in the proximity of the recognition site. Breakage of a DNA molecule with only one recognition site required a 10 to 40-fold higher concentration of restriction enzyme than breakage of a DNA molecule with two or more recognition sites, but these substrates were all equally effective in a binding assay for the enzyme.The polynucleotide kinase reaction provided no evidence for new 5′-terminal sequences generated by restriction in vitro; the 5′ termini were either refractory to the polynucleotide kinase reaction or had no sequence specificity.     

Requirement for maturation of Escherichia coli bacteriophage lambda

During infection a λ phage that is incapable of DNA replication requires recombination for maturation. If two prophages are situated in tandem, this requirement for DNA replication and recombination is bypassed. In physical experiments using the DNA cutting assay of Freifelder et al. (1973), the DNA of a sex factor containing one or two prophages defective in both excision and DNA replication is cut efficiently only when two prophages are in tandem. We interpret this to mean that λ can only be matured from a structure of greater than unit length, and hypothesize that the structure must contain two joined ends (AR-joints).