Effects of plasmid presence on growth and enzyme activity of Escherichia coli DH5α

Summary The effects of recombinant DNA propagation and gene expression on the physiology of the host cell was investigated using a series of copy number mutant plasmids. The plasmids at copy numbers of 30, 57, 115 and 501 per chromosome equivalent encoded constitutive production of the enzyme -lactamase. Ribose phosphate isomerase activity was relatively unaffected by plasmid presence, and glucose-6-phosphate dehydrogenase, fructose 1,6-diphosphate aldolase and fructose 1,6-diphosphatase activities were lower in plasmid-containing cells than in the plasmid-free host strain. Increasing copy number resulted in increased depression of enzyme activity levels. The results indicate that plasmid presence mediates subtle changes in the net expression of host enzymes involved in carbon metabolism. Responses of Escherichia coli DH5 in Evans medium to these plasmids differed substantially from responses of E. coli HB101 in rich medium.Offprint requests to: J. E. Bailey     

Metabolic engineering of Escherichia coli for α-farnesene production

Sesquiterpenes are important materials in pharmaceuticals and industry. Metabolic engineering has been successfully used to produce these valuable compounds in microbial hosts. However, the microbial potential of sesquiterpene production is limited by the poor heterologous expression of plant sesquiterpene synthases and the deficient FPP precursor supply. In this study, we engineered E. coli to produce α-farnesene using a codon-optimized α-farnesene synthase and an exogenous MVA pathway. Codon optimization of α-farnesene synthase improved both the synthase expression and α-farnesene production. Augmentation of the metabolic flux for FPP synthesis conferred a 1.6- to 48.0-fold increase in α-farnesene production. An additional increase in α-farnesene production was achieved by the protein fusion of FPP synthase and α-farnesene synthase. The engineered E. coli strain was able to produce 380.0 mg/L of α-farnesene, which is an approximately 317-fold increase over the initial production of 1.2 mg/L.     

How does plasmid DNA penetrate cell membranes in artificial transformation process of Escherichia coli?

Artificial transformation of Escherichia coli with plasmid DNA in presence of CaCl2 is a widely used technique in recombinant DNA technology. However, exact mechanism of DNA transfer across cell membranes is largely obscure. In this study, measurements of both steady state and time-resolved anisotropies of fluorescent dye trimethyl ammonium diphenyl hexatriene (TMA-DPH), bound to cellular outer membrane, indicated heat-pulse (0 degrees C42 degrees C) step of the standard transformation procedure had lowered considerably outer membrane fluidity of cells. The decrease in fluidity was caused by release of lipids from cell surface to extra-cellular medium. A subsequent cold-shock (42 degrees C0 degrees C) to the cells raised the fluidity further to its original value and this was caused by release of membrane proteins to extra-cellular medium. When the cycle of heat-pulse and cold-shock steps was repeated, more release of lipids and proteins respectively had taken place, which ultimately enhanced transformation efficiency gradually up to third cycle. Study of competent cell surface by atomic force microscope showed release of lipids had formed pores on cell surface. Moreover, the heat-pulse step almost depolarized cellular inner membrane. In this communication, we propose heat-pulse step had two important roles on DNA entry: (a) Release of lipids and consequent formation of pores on cell surface, which helped DNA to cross outer membrane barrier, and (b) lowering of membrane potential, which facilitated DNA to cross inner membrane of E. coli.     

Modulation of guanosine 5′-diphosphate-d-mannose metabolism in recombinant Escherichia coli for production of guanosine 5′-diphosphate-l-fucose

Guanosine 5’-diphosphate (GDP)-l-fucose, an activated form of a nucleotide sugar, plays an important role in a wide range of biological functions. In this study, the enhancement of GDP-l-fucose production was attempted by supplementation of mannose, which is a potentially better carbon source to be converted into GDP-l-fucose than glucose, and combinatorial overexpression of the genes involved in the biosynthesis of GDP-d-mannose, a precursor of GDP-l-fucose. Supply of a mannose and glucose led to a 1.3-fold-increase in GDP-l-fucose concentration (52.5 ± 0.8 mg l^?1) in a fed-batch fermentation of recombinant E. coli BL21star(DE3) overexpressing the gmd and wcaG genes, compared with the case using glucose as a sole carbon source. A maximum GDP-l-fucose concentration of 170.3 ± 2.3 mg l^?1, corresponding to a 4.4-fold enhancement compared with the control strain overexpressing gmd and wcaG genes only, was achieved in a glucose-limited fed-batch fermentation of a recombinant E. coli BL21star(DE3) strain overexpressing manB, manC, gmd and wcaG genes. Further improvement of GDP-l-fucose production was not obtained by additional overexpression of the manA gene.     

Comparative proteomic and genetic analyses reveal unidentified mutations in Escherichia coli XL1-Blue and DH5α

Escherichia coli has been used widely in laboratory and the biotech industry. However, the genetic and metabolic characteristics remain inadequately studied, particularly for those strains with extensive genetic manipulations that might have resulted in unknown mutations. Here, we demonstrate a comparative proteomics and genetics approach to identify unknown mutations in E. coli K-12 derivatives. The comparative proteomic and genetic analyses revealed an IS5 disruption of the kdgR gene in two commonly used derivative strains of E. coli K-12, XL1-Blue and DH5α, compared with K-12 wild-type strain W3110. In addition, a controversial deoR mutation was clarified as a wild type in E. coli DH5α using the same approach. This approach should be useful in characterizing the unknown mutations in various mutant strains developed. At the same time, comparative proteomic analysis also revealed the distinct metabolic characteristic of the two derivatives: higher biosynthetic flux to purine nucleotides. This is potentially beneficial for the synthesis of plasmid DNA.     

Pathway engineering of Escherichia coli for α-ketoglutaric acid production

α-Ketoglutaric acid (α-KG) is a multifunctional dicarboxylic acid in the tricarboxylic acid (TCA) cycle, but microbial engineering for α-KG production is not economically efficient, due to the intrinsic inefficiency of its biosynthetic pathway. In this study, pathway engineering was used to improve pathway efficiency for α-KG production in Escherichia coli. First, the TCA cycle was rewired for α-KG production starting from pyruvate, and the engineered strain E. coli W3110Δ4-PCAI produced 15.66 g/L α-KG. Then, the rewired TCA cycle was optimized by designing various strengths of pyruvate carboxylase and isocitrate dehydrogenase expression cassettes, resulting in a large increase in α-KG production (24.66 g/L). Furthermore, acetyl coenzyme A (acetyl-CoA) availability was improved by overexpressing acetyl-CoA synthetase, leading to α-KG production up to 28.54 g/L. Finally, the engineered strain E. coli W3110Δ4-P_(H)CAI_(H)A was able to produce 32.20 g/L α-KG in a 5-L fed-batch bioreactor. This strategy described here paves the way to the development of an efficient pathway for microbial production of α-KG.     

High-density Escherichia coli cultures for continuous l(−)-carnitine production

The use of a biological procedure for l-carnitine production as an alternative to chemical methods must be accompanied by an efficient and highly productive reaction system. Continuous l-carnitine production from crotonobetaine was studied in a cell-recycle reactor with Escherichia coli O44 K74 as biocatalyst. This bioreactor, running under the optimum medium composition (25 mM fumarate, 5 g/l peptone), was able to reach a high cell density (26 g dry weight/l) and therefore to obtain high productivity values (6.2 g l-carnitine l⁻¹ h⁻¹). This process showed its feasibility for industrial l-carnitine production. In addition, resting cells maintained in continuous operation, with crotonobetaine as the only medium component, kept their biocatalytic capacity for 4 days, but the biotransformation capacity decreased progressively when this particular method of cultivation was used. Received: 10 December 1998 / Received revision: 19 February 1999 / Accepted: 20 February 1999     

Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies

Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex in vitro or in vivo assembly reactions followed by plasmid propagation in recombination-impaired Escherichia coli strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six double-stranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids in vivo. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processes associated with most common applications of DNA assembly. We demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of E. coli may enable productivity gain across disciplines involving recombinant DNA work.     

High-Level Production of Plasmid DNA by Escherichia coli DH5α ΩsacB by Introducing inc Mutations

For small-copy-number pUC-type plasmids, the inc1 and inc2 mutations, which deregulate replication, were previously found to increase the plasmid copy number 6- to 7-fold. Because plasmids can exert a growth burden, it was not clear if further amplification of copy number would occur due to inc mutations when the starting point for plasmid copy number was orders of magnitude higher. To investigate further the effects of the inc mutations and the possible limits of plasmid synthesis, the parent plasmid pNTC8485 was used as a starting point. It lacks an antibiotic resistance gene and has a copy number of ∼1,200 per chromosome. During early stationary-phase growth in LB broth at 37°C, inc2 mutants of pNTC8485 exhibited a copy number of ∼7,000 per chromosome. In minimal medium at late log growth, the copy number was found to be significantly increased, to approximately 15,000. In an attempt to further increase the plasmid titer (plasmid mass/culture volume), enzymatic hydrolysis of the selection agent, sucrose, at late log growth extended growth and tripled the total plasmid amount such that an approximately 80-fold gain in total plasmid was obtained compared to the value for typical pUC-type vectors. Finally, when grown in minimal medium, no detectable impact on the exponential growth rate or the fidelity of genomic or plasmid DNA replication was found in cells with deregulated plasmid replication. The use of inc mutations and the sucrose degradation method presents a simplified way for attaining high titers of plasmid DNA for various applications.     

Engineering central metabolic modules of Escherichia coli for improving β-carotene production

ATP and NADPH are two important cofactors for production of terpenoids compounds. Here we have constructed and optimized β-carotene synthetic pathway in Escherichia coli, followed by engineering central metabolic modules to increase ATP and NADPH supplies for improving β-carotene production. The whole β-carotene synthetic pathway was divided into five modules. Engineering MEP module resulted in 3.5-fold increase of β-carotene yield, while engineering β-carotene synthesis module resulted in another 3.4-fold increase. The best β-carotene yield increased 21%, 17% and 39% after modulating single gene of ATP synthesis, pentose phosphate and TCA modules, respectively. Combined engineering of TCA and PPP modules had a synergistic effect on improving β-carotene yield, leading to 64% increase of β-carotene yield over a high producing parental strain. Fed-batch fermentation of the best strain CAR005 was performed, which produced 2.1 g/L β-carotene with a yield of 60 mg/g DCW.     

Combinatorial expression of different β-carotene hydroxylases and ketolases in Escherichia coli for increased astaxanthin production

Journal of Industrial Microbiology & Biotechnology - In natural produced bacteria, β-carotene hydroxylase (CrtZ) and β-carotene ketolase (CrtW) convert β-carotene into...     

Recombinant protein production in cultures of an Escherichia coli trp − strain

Fermentation conditions were developed in order to achieve simultaneously a high biomass concentration and high-level expression of a hybrid cI-human insulin B peptide gene. In our system, this hybrid gene is under control of the Escherichia coli trp promoter, in a trp ^– derivative strain of E. coli W3110. The dual role of tryptophan concentration on cellular growth and hybrid gene regulation was studied in 10-l batch fermentations. In the best batch conditions, a biomass concentration of 12 g dry weight/l can be obtained, and 0.53 g/l of cI-insulin B hybrid protein is produced. Tryptophan in the culture medium is consumed by the growing culture, until a level is reached that causes induction of the hybrid gene. Plasmid loss was detected, as only 62% of the cells retained the recombinant plasmid. In order to increase the hybrid protein production level, a fed-batch culture strategy was developed whereby the specific growth rate of the cells was restrained. Using the same amount of nutrients as in the batch fermentations, it was possible to increase the final biomass concentration to 20 g/l, plasmid-bearing cells in the population to 90% and recombinant hybrid protein to 1.21 g/l. Correspondence to: F. Bolivar     

Engineering Escherichia coli for propionic acid production through the Wood–Werkman cycle

Native to propionibacteria, the Wood–Werkman cycle enables propionate production via succinate decarboxylation. Current limitations in engineering propionibacteria strains have redirected attention toward the heterologous production in model organisms. Here, we report the functional expression of the Wood–Werkman cycle in Escherichia coli to enable propionate and 1-propanol production. The initial proof-of-concept attempt showed that the cycle can be used for production. However, production levels were low (0.17 mM). In silico optimization of the expression system by operon rearrangement and ribosomal-binding site tuning improved performance by fivefold. Adaptive laboratory evolution further improved performance redirecting almost 30% of total carbon through the Wood–Werkman cycle, achieving propionate and propanol titers of 9 and 5 mM, respectively. Rational engineering to reduce the generation of byproducts showed that lactate (∆ldhA) and formate (∆pflB) knockout strains exhibit an improved propionate and 1-propanol production, while the ethanol (∆adhE) knockout strain only showed improved propionate production.     

Enhanced production of human FcγRIIa receptor by high cell density cultivation of Escherichia coli

Human Fc receptors (FcγR) are membrane glycoproteins that are expressed on all immunologically active cells and have a well-defined role in regulating innate and adaptive immune responses by binding to the immunoglobulin G (IgG) antibody. Among the several classes of Fc receptors, FcγRIIa is the most widely expressed, and it serves as an important reagent in antibody engineering. Here, we report on high cell density cultivations (HCDC) of Escherichia coli for preparative scale production of FcγRIIa in a 6.6L bioreactor. Briefly, a pH-stat feeding strategy was employed, and two different cell densities (OD(600) of 46 and 100) were examined for the induction of FcγRIIa gene expression. When cells were induced at a high cell density (OD(600) of 100), the cell density increased to an OD(600) of 234 within 9h after induction, and a 2-fold higher production yield was obtained compared with that of induction at low cell density (OD(600) of 46). After simple purification steps including denaturation and refolding, 87.7 mg of soluble FcγRIIa that was more than 95% pure was obtained from a 20-mL culture with high recovery yield (≈54%). The biological activity of purified FcγRIIa was also confirmed by evaluating its interaction with all subclasses of IgG antibodies using an ELISA bioassay.     

The structure of Escherichia coli ExoIX—implications for DNA binding and catalysis in flap endonucleases

Escherichia coli Exonuclease IX (ExoIX), encoded by the xni gene, was the first identified member of a novel subfamily of ubiquitous flap endonucleases (FENs), which possess only one of the two catalytic metal-binding sites characteristic of other FENs. We have solved the first structure of one of these enzymes, that of ExoIX itself, at high resolution in DNA-bound and DNA-free forms. In the enzyme–DNA cocrystal, the single catalytic site binds two magnesium ions. The structures also reveal a binding site in the C-terminal domain where a potassium ion is directly coordinated by five main chain carbonyl groups, and we show this site is essential for DNA binding. This site resembles structurally and functionally the potassium sites in the human FEN1 and exonuclease 1 enzymes. Fluorescence anisotropy measurements and the crystal structures of the ExoIX:DNA complexes show that this potassium ion interacts directly with a phosphate diester in the substrate DNA.     

Engineering Escherichia coli for production of C12–C14 polyhydroxyalkanoate from glucose

Demand for sustainable materials motivates the development of microorganisms capable of synthesizing products from renewable substrates. A challenge to commercial production of polyhydroxyalkanoates (PHA), microbially derived polyesters, is engineering metabolic pathways to produce a polymer with the desired monomer composition from an unrelated and renewable source. Here, we demonstrate a metabolic pathway for converting glucose into medium-chain-length (mcl)-PHA composed primarily of 3-hydroxydodecanoate monomers. This pathway combines fatty acid biosynthesis, an acyl-ACP thioesterase to generate desired C₁₂ and C₁₄ fatty acids, β-oxidation for conversion of fatty acids to (R)-3-hydroxyacyl-CoAs, and a PHA polymerase. A key finding is that Escherichia coli expresses multiple copies of enzymes involved in β-oxidation under aerobic conditions. To produce polyhydroxydodecanoate, an acyl-ACP thioesterase (BTE), an enoyl-CoA hydratase (phaJ3), and mcl-PHA polymerase (phaC2) were overexpressed in E. coli ΔfadRABIJ. Yields were improved through expression of an acyl-CoA synthetase resulting in production over 15% CDW – the highest reported production of mcl-PHA of a defined composition from an unrelated carbon source.     

Restoration of Growth Phenotypes of Escherichia coli DH5α in Minimal Media through Reversal of a Point Mutation in purB

A point mutation (E115K) resulting in slower growth of Escherichia coli DH5α and XL1-Blue in minimal media was identified in the purB gene, coding for adenylosuccinate lyase (ASL), through complementation with an E. coli K-12 genomic library and serial subcultures. Chromosomal modification reversing the mutation to the wild type restored growth phenotypes in minimal media.The Escherichia coli DH5α strain possesses many beneficial genotypes (recA, deoR, gyrA, and endA1) and has been widely used for many purposes, such as gene cloning and protein production (5). However, E. coli DH5α also exhibits inferior growth phenotypes, especially in minimal media, compared to other E. coli strains. As such, the utilization of this bacterium has been limited to the laboratory despite its numerous advantages. We can assume that these inferior growth phenotypes have resulted from unknown accumulated mutations during the strain development process (5). Some of those mutations, which might impact growth in minimal media, have been characterized, including the phenotypes for thiamine requirement and relaxed amino acid synthesis (5). Still, there may be other uncharacterized mutations whose interactions hamper the growth of E. coli DH5α in minimal media.Based on successful identifications (6, 7) of gene targets for metabolic engineering (3), we performed serial subcultures of E. coli DH5α transformants with an E. coli K-12 genomic library based on a multicopy plasmid (9) to isolate genes that improve growth phenotypes in minimal media. The M9 minimal medium and R medium (11) were chosen for enrichment experiments because of their popular use in metabolic engineering (1, 2, 7) and in high-cell-density fermentation (8, 10, 11). After 11 serial transfers of the transformants in the M9 medium, and 27 transfers in the R medium, cultured cells were diluted and plated onto LB agar for single-colony isolation. Although more than 10 colonies were picked, only three distinctive plasmids, containing different inserts, were isolated from the transformants enriched in M9 medium. In the case of R medium enrichment, all isolated plasmids were identical. Sequencing of the isolated plasmids revealed the exact genome coordinates of each insert. A diagram of the inserts in the context of the E. coli genome sequence is shown in Fig. ​Fig.1.1. Interestingly, all of the isolated plasmids contained similar regions of genomic DNA. mnmA (tRNA 5-methylaminomethyl-2-thiouridylate-methyltransferase), purB (adenylosuccinate lyase), and hflD (lysogenization regulator) were the annotated genes in the overlapping region among distinctive isolated fragments. However, since the N-terminal portions of mnmA and hflD were truncated in some of the inserts, we selected only the M3 and R1 plasmids for further experimentation. These two plasmids were retransformed into E. coli DH5α for confirmation of their beneficial effects on growth of E. coli in minimal media. The newly transformed strains showed growth phenotypes almost identical to those of the previously isolated transformants. When cultured in flasks, the specific growth rate of E. coli DH5α with the R1 plasmid was 1.5-fold higher (0.53 versus 0.36 h⁻¹) than the rate of cells transformed with a control plasmid (pZE). The R1 transformant reached the stationary phase much earlier, arriving at an optical density at 600 nm (OD₆₀₀) of 10 within 16 h, whereas the control transformant reached this cell density after 24 h. However, the final cell densities were almost equivalent. Acetate accumulation, as well as glucose consumption, by the R1 transformant was much higher than that of the control transformant (2.2 versus 0.3 g acetate/liter). The increased accumulation of acetate could be the result of increased cell density. These findings confirm that the enhanced growth phenotypes of the isolated transformants were conferred not by accumulated spontaneous mutations in the genome during enrichment but by the introduced plasmids.Open in a separate windowFIG. 1.Diagram of open reading frames in the identified genomic DNA fragments. M1, M2, and M3 were isolated from the serial subculture using M9 medium. R1 was isolated from the serial subculture using R medium.The open reading frame (ORF) of purB was amplified and cloned into a multicopy plasmid under the control of a strong promoter (rrnB). Transformation of the resulting plasmid (pZE-purB) into E. coli DH5α resulted in a growth phenotype almost identical to that of the R1 transformant. This result suggested that overexpression of purB is a specific genetic perturbation improving growth phenotypes of E. coli DH5α in minimal media. We also performed 1-liter batch fermentation experiments with three DH5α transformants: one containing the control plasmid (pZE), one with the isolated plasmid (R1), and a third with the purB overexpression plasmid (pZE-purB). Growth phenotypes of these strains were very similar to results obtained from shaker flask experiments (Fig. ​(Fig.2).2). Next, we tested whether the overexpression of purB is beneficial to the growth of other E. coli strains by introducing the R1 and pZE-purB plasmids into various other strains (K-12, BL21, and XL1-Blue) that are commonly used in biotechnological research. Among the four strains tested in our various experiments, the positive effects of purB overexpression on growth phenotypes were observed only in DH5α and XL1-Blue, both of which have been favored in molecular cloning. These results suggest that an uncharacterized mutation might have been introduced into both strains during strain development. This unknown mutation might cause growth inhibition, which can be suppressed by the overexpression of purB. Therefore, we concluded that expression of an exogenous, K-12-derived copy of the purB gene under a constitutive promoter can enhance growth phenotypes of E. coli DH5α and XL1-Blue strains in minimal media.Open in a separate windowFIG. 2.Comparison of levels of cell growth (♦) (OD₆₀₀), glucose consumption (▪) (g/liter), and acetate production (▴) (g/liter) by E. coli DH5α transformants with a control plasmid (A), the isolated R1 plasmid (B), and the pZE-purB plasmid (C) in R medium with glucose in a bioreactor.However, it is plausible that a mutation is located in the purB locus of DH5α and XL1-Blue that decreases the activity of the encoded enzyme. In order to identify a putative mutation in purB, we sequenced the chromosomal purB gene of DH5α and XL1-Blue. A point mutation resulting in the transition of nucleotide 343 of purB from guanine (G) to adenine (A) was identified in the genomes of both strains. This mutation causes a change of the 115th residue of adenylosuccinate lyase from glutamate to lysine (E115K). This finding explains why the expression of exogenous, K-12-derived purB in DH5α and XL1-Blue strains enhances growth phenotypes in minimal media. The E115K mutation of purB was named purB20 for simple notation.Chromosomal modification of the mutant allele in E. coli DH5α or XL1-Blue might be desirable for practical applications. To this end, the purB20 mutant allele was replaced by purB amplified from E. coli K-12 through recombination based on phage lambda Red recombinase (4). The resulting strain (SC1) showed growth phenotypes similar to those of E. coli DH5α strains harboring the pZE-purB or R1 plasmid. The specific growth rate of SC1 in M9 medium was 40% higher than that of DH5α (0.50 versus 0.36 h⁻¹). These results show that what we had originally interpreted as overexpression of the purB gene was actually complementation of the mutant purB20 allele with wild-type purB. We also tested whether the modification from purB20 to wild-type purB elicits a change in the transformation efficiency. Chemically induced competent SC1 cells exhibited approximately 2.5-fold lower transformation efficiency than E. coli DH5α cells did when induced under identical conditions (1.8 ± 0.1 × 10⁶ versus 4.6 ± 0.3 × 10⁶ CFU/μg pUC19 DNA). Still, the transformation efficiency of the SC1 strain was of the same order of magnitude as that of E. coli DH5α, suggesting that the SC1 strain would be useful for many biotechnological applications, such as the mass production of DNA vectors and recombinant proteins.     

Discontinuous or semi‐discontinuous DNA replication in Escherichia coli?

The postulate that a stalled/collapsed replication fork will be generated when the replication complex encounters a UV-induced lesion in the template for leading-strand DNA synthesis is based on the model of semi-discontinuous DNA replication. A review of existing data indicates that the semi-discontinuous DNA replication model is supported by data from in vitro studies, while the discontinuous DNA replication model is supported by in vivo studies in Escherichia coli. Until the question of whether DNA replicates discontinuously in one or both strands is clearly resolved, any model building based on either one of the two DNA replication models should be treated with caution.     

Synthesis of α3 phage DNA in replication mutants of Escherichia coli

Host functions for DNA replication of bacteriophage α3, a representative of group A microvirid phages, were studied using dna and rep mutants of Escherichia coli. In dna⁺ cells, conversion of phage α3 single-stranded DNA (SS) into the double-stranded replicative form (RF) was insensitive to 30–150 μg/ml of chloramphenicol, 200 μg/ml of rifampicin, 50 μg/ml of nalidixic acid, or 200 μg/ml of novobiocin. At 43°C, synthesis of the parental RF was inhibited in dnaG and dnaZ mutants, but not in dnaE and rep strains. Replication of phage α3 progeny RF was prevented by 50 μg/ml of mitomycin C (in hcr⁺ bacteria), 50 μg/ml of nalidixic acid or 200 μg/ml of novoviocin, but neither by 30 μg/ml of chloramphenicol nor by 200 μg/ml of rifampicin. Besides dnaG and dnaZ gene products, dnaE and rep functions were essential for the progeny RF synthesis. Host factor dependence of α3 was relatively simple and, in contrast with phages øX174 and G4, α3 did not require dnaB and dnaC(D) activities.