Induction threshold of the Q - mutant of bacteriophage λ in Escherichia coli

Temperature induction of bacteriophage in Escherichia coli depends on bacterial population density. The lowest rate of viability loss at the temperature threshold results in maximal gene expression of . -Infection causes bacterial cells to lose cell viability and thus decrease temperature induction efficiency. In addition, shifting-up in temperature increases the probability of progeny ; thus, the mortality of bacterial hosts increases and the expression of recombinant proteins by naked significantly decrease.     

Uptake of bacteriophage λ DNA in Escherichia coli by electroporation: An alternative to in vitro packaging

Summary By using a high field strength DC pulse of 15 kV/cm and a pulse duration of 5 ms for the transfection of E. coli by bacteriophage DNA, we obtained efficiencies of 1.1 × 10⁶ (pfu/g bacteriophage , DNA). This represents a 100-fold improvement over the traditional CaCl₂/heat shock method and is a viable alternative to the more costly in vitro packaging of recombinant bacteriophage DNA for the production of cDNA and genomic libraries.     

Two-stage continuous operation of recombinant Escherichia coli using the bacteriophage λ Q − vector

A two-stage continuous culture of Escherichia coli in combination with a bacteriophage λ system was performed in order to overcome the intrinsic plasmid instability that is frequently observed in recombinant fermentation. A phage λ vector with a Q ⁻ mutation was used to enhance the expression of the λ system. The optimal values of the important operational variables such as the substrate concentration, the dilution rate, and the mean residence time on the expression of the cloned gene were determined in both batch and continuous cultures. For all culturing modes, the full induction of the cloned gene was observed 4 h after the temperature shift. In the two stage continuous culture, the overproduction reached their maxima at D=0.25 h⁻¹ with 1.5 S ₀ of the medium supply. The maximum productivity of the total β-galactosidase was 16.3×10⁶ U l⁻¹ h⁻¹, which was approximately seven times higher than that in the single-copy lysogenic stage. The recombinant cells were stable in the lysogenic state for more than 260 h, while they were stable for 40 h in the lytic state. The instability that developed rapidly in the second tank is believed to be due to the accumulation of lysis proteins as a result of vector leakage during the operation.     

The Bacteriophage λ Attachment Site in Wild Strains of Escherichia coli

The attachment site (attlambda) of bacteriophage lambda was examined in wild strains of Escherichia coli. Although the att region is non-coding, the DNA sequence was invariant in the 13 strains examined. Two other non-coding regions showed nine changes, all associated with a single strain. In four of 33 strains, sequences were inserted in or near the attlambda site and in two of these the insert was related to lambda. Among strains that can be lysogenized by lambda, integration was via the attlambda site in all cases. Some resistant strains can be lysogenized, and these have been termed "lenient." Most of these fail to give normal phage yield after induction. In some cases rare lysogens have been formed in cells that belong to a mutant subpopulation.     

Enhancement of bacteriophage λ stability using a λQ − S − mutant in the continuous culture of Escherichia coli

In this study, we used a bacteriophage λQ ⁻ S ⁻ mutant that increased the stability of recombinant Escherichia coli during continuous culture. The operation was conducted in two stages: the first stage was carried out to promote cell growth, and the second stage was performed for product formation. The productivity of recombinant proteins depends on the substrate concentration of the fresh medium supplied to the second stage (S ₃) and dilution rate of the second stage (D ₂). With the optimal value of S ₃ and D ₂, the first and second stages were stably maintained for 170 and 80 h, respectively. To further improve this process, a three-stage continuous process was conducted with an additional induction stage between the growth and production stages. Compared with the two-stage operation, the stable production period was extended by 1.7 fold, and the recombinant protein production increased by 1.3 fold.     

Behavior of λ Bacteriophage in a Recombination Deficient Strain of Escherichia coli

The behavior of lambda phage in the Rec(-) strain JC-1569 is compared with that in the Rec(+) strain JC-1557. No difference deemed significant was noted in the adsorption rate, latent period, burst size, frequency of lysogenization, and frequency of vegetative phage recombination. The location of the prophage and its mode of insertion in the Rec(-) lysogen of wild-type lambda (lambda(+)) were inferred to be normal from the results of conjugational crosses. Spontaneous and ultraviolet (UV) irradiation induction of lambda(+) were markedly reduced in the Rec(-) lysogen. On the other hand, thermal induction of a mutant lambda (lambdacI857) lysogen of the Rec(-) strain was not reduced and was only slightly affected by UV irradiation. Phage subject to inhibition by lambda immunity failed to multiply in UV-irradiated cells of the Rec(-) lambda(+) lysogen, whereas those not inhibited by this immunity did multiply. It was concluded that the failure of UV to induce lambda(+) in the Rec(-) lysogen was not due to damage to the prophage, but rather to the inability of the irradiated cells to respond by lifting immunity. Preliminary evidence indicates that a single mutation confers recombination deficiency and the inability to lift immunity after UV irradiation. Possible relationships between recombination and the lifting of immunity are enumerated.     

Loss of Host-controlled Restriction of λ Bacteriophage in Escherichia coli Following Methionine Deprivation

lambda Bacteriophages produced in Escherichia coli C (designated as lambda . C) are restricted in their ability to grow in E. coli K-12. The rare successful infections that arise in the K-12 population occur in "special" cells which have lost their capacity to restrict lambda . C. These infections yield modified progeny phage (designated as lambda . K) which, unlike lambda . C, plate equally well on E. coli C and E. coli K-12. When methionine, but no other amino acid, was removed from the growth medium of a mutant strain of E. coli K-12, the number of special cells rapidly increased 500- to 3,000-fold. These new special cells retain their capacity to produce modified lambda . K progeny. This conversion of restricting cells into special cells does not require the synthesis of new protein. The special cells formed when methionine was removed from the culture did not revert into restricting cells when methionine was restored. Such cells have also lost the ability to divide for at least 4 hr after methionine supplementation. When methionine was restored, the remaining restricting cells, but not the special cells, immediately resumed growth. Removing methionine from cultures of E. coli B caused a similar increase in the number of special cells able to support the growth of lambda . C and lambda . K. However, when E. coli K-12 (P1) cultures were deprived of methionine, the number of special cells increased for lambda . C but not for lambda . K. Thus, retention of the P1-restriction system, unlike the B- and the K-12-systems, does not require the presence of methionine.     

Formation and Stability of the Bacteriophage λ Replication Complexes in UV-Irradiated Escherichia coli

Bacteriophage λ replication complex, containing the phage-encoded O initiator protein protected from proteases by other elements of this complex, is a stable structure that can be inherited by one of the two daughter λ DNA copies after a replication round in Escherichia coli. In normal growth conditions in bacteria bearing a plasmid derived from bacteriophage λ, such a complex may be stable for many cell generations. However, it was found that this stable structure is disassembled under certain conditions, namely, after heat shock. Therefore, we asked whether other environmental stresses may cause disassembly of the λ replication complex. We found that UV irradiation of the host cells prevented formation of the stable λ replication complex (though not preventing phage replication), while the same UV doses did not affect the stability of the replication complex assembled prior to the irradiation. These results indicate that the stable λ replication complex, although sensitive to heat shock, is resistant to some other environmental stresses and that formation of at least two types of λ replication complexes is possible. Both stable and unstable λ replication complexes are functional because replication of λ DNA under conditions preventing formation of the stable complex proceeds efficiently. Received: 18 January 2000 / Accepted: 2 March 2000     

Extracellular β-lactamase from Escherichia coli

Summary Escherichia coli strain O 127: K63: (B8): H—was grown in nutrient broth (Difco). Penicillinase activity was found in the culture supernatant after only five hours of incubation, i.e. during the exponential phase of growth. At this phase the levels of typical intracellular markers, did not indicate cell lysis or gross cell damage. The bioautographic revelation of penicillin-splitting enzymes on electropherograms of cell-free liquids confirmed the presence of one basic broad-spectrum -lactamase. This possibly extracellular -lactamase seems to be also present in the cellular extracts where it coexists with several other cell-bound penicillinases.     

The Escherichia coli CRISPR system protects from λ lysogenization, lysogens, and prophage induction

We show that phage lysogenization, lysogens, and prophage induction are all targeted by CRISPR. The results demonstrate that genomic DNA is not immune to the CRISPR system, that the CRISPR system does not require noncytoplasmic elements, and that the system protects from phages entering and exiting the lysogenic cycle.     

Repressor and int synthesis of bacteriophage λ in the E. coli host mutant ER437

Molecular Genetics and Genomics - Analysis of λ phage infection of the host mutant ER437 by SDS polyacrylamide gel electrophoresis and autoradiography has revealed altered expression of...     

Ongoing phenotypic and genomic changes in experimental coevolution of RNA bacteriophage Qβ and Escherichia coli

According to the Red Queen hypothesis or arms race dynamics, coevolution drives continuous adaptation and counter-adaptation. Experimental models under simplified environments consisting of bacteria and bacteriophages have been used to analyze the ongoing process of coevolution, but the analysis of both parasites and their hosts in ongoing adaptation and counter-adaptation remained to be performed at the levels of population dynamics and molecular evolution to understand how the phenotypes and genotypes of coevolving parasite-host pairs change through the arms race. Copropagation experiments with Escherichia coli and the lytic RNA bacteriophage Qβ in a spatially unstructured environment revealed coexistence for 54 days (equivalent to 163-165 replication generations of Qβ) and fitness analysis indicated that they were in an arms race. E. coli first adapted by developing partial resistance to infection and later increasing specific growth rate. The phage counter-adapted by improving release efficiency with a change in host specificity and decrease in virulence. Whole-genome analysis indicated that the phage accumulated 7.5 mutations, mainly in the A2 gene, 3.4-fold faster than in Qβ propagated alone. E. coli showed fixation of two mutations (in traQ and csdA) faster than in sole E. coli experimental evolution. These observations suggest that the virus and its host can coexist in an evolutionary arms race, despite a difference in genome mutability (i.e., mutations per genome per replication) of approximately one to three orders of magnitude.     

Influence of the Escherichia coli oxyR gene function on λ prophage maintenance

In Escherichia coli hosts, hydrogen peroxide is one of the factors that may cause induction of λ prophage. Here, we demonstrate that H₂O₂-mediated λ prophage induction is significantly enhanced in the oxyR mutant host. The mRNA levels for cI gene expression were increased in a λ lysogen in the presence of H₂O₂. On the other hand, stimulation of the p _M promoter by cI857 overproduced from a multicopy plasmid was decreased in the ΔoxyR mutant in the presence of H₂O₂ but not under normal growth conditions. The purified OxyR protein did bind specifically to the p _M promoter region. This binding impaired efficiency of interaction of the cI protein with the OR3 site, while stimulating such a binding to OR2 and OR1 sites, in the regulatory region of the p _M promoter. We propose that changes in cI gene expression, perhaps in combination with moderately induced SOS response, may be responsible for enhanced λ prophage induction by hydrogen peroxide in the oxyR mutant. Therefore, OxyR seems to be a factor stimulating λ prophage maintenance under conditions of oxidative stress. This proposal is discussed in the light of efficiency of induction of lambdoid prophages bearing genes coding for Shiga toxins.     

Expression of a β-galactosidase gene from Lactobacillus sake in Escherichia coli

Summary A -galactosidase gene from Lactobacillus sake coding for lactose hydrolysis was cloned and expressed in Escherichia coli. Chromosomal DNA from L. sake was partially digested with the restriction enzyme Sau3AI, and the 3–6 Kb fragment was ligated to the cloning vector pSP72 digested with BamHI. One E. coli transformant expressing -galactosidase was isolated on X-gal plates. It contained a plasmid with an insertion of approx. 4 Kb. The restriction map of the recombinant plasmid was constructed. The characteristics of the recombinant -galactosidase were compared with those of the wild type. The optima pH and temperature for both enzymes was 6.5 and 50°C, respectively. Stability of the enzymes at different temperatures and activity on lactose were determined.     

Over-production of β-carotene from metabolically engineered Escherichia coli

To produce recombinant β-carotene in vitro, synthetic operons encoding genes governing its biosynthesis were engineered into Escherichia coli. Constructs harboring these operons were introduced into either a high-copy or low-copy cloning vector. β-Carotene production from these recombinant E. coli cells was either constitutive or inducible depending upon plasmid copy number. The most efficient β-carotene production was with the low-copy based vector. The process was increased incrementally from a 5 l to a 50 l fermentor and finally into a 300 l fermentor. The maximal β-carotene yields achieved using the 50 l and 300 l fermentor were 390 mg l⁻¹ and 240 mg l⁻¹, respectively, with overall productivities of 7.8 mg l⁻¹ h⁻¹ and 4.8 mg l⁻¹ h⁻¹.