Enlarged model of lambda phage ontogenesis

An enlarged threshold model of the Regulatory System of Development of λ -Phage (RSDP λ-2) is built. It includes 15 synthetic blocks of proteins and mRNAs and four blocks corresponding to the other ontogenetic processes: two-stage replication, integration and excision of phage genome, formation of oligomers of regulatory proteins, regulation of bacteriallysis. By way of computer simulation of the RSDP A-2 model the dynamics of concentrations of all main proteins, respective fractions of mRNAs and DNA are described in the lytic and lysogenic regimes of phage ontogenesis. Results obtained are in good agreement with available experimental data. The dependence of a portion (%) of lysogenic responses on the multiplicity $\text{k}$ of phage infection of bacterial culture, is built. This curve has a maximum point in accordance with the experimental data of P. Kourilsky (1973).     

Regulation of the in vitro synthesis of the alpha-peptide of beta-galactosidase directed by a restriction fragment of the lactose operon.

The regulation of the $\text{in vitro}$ synthesis of the N-terminal portion of the β-galactosidase molecule (α-peptide) has been investigated using DNA fragments of the lactose operon as template. DNA fragments of about 789 base pairs were isolated after endonuclease (Hin II) digestion of either λplac5, λh80dlacp^s or λh80dlacUV5 phage DNA or DNA from the recombinant plasmid PMC3. The regulation of the expression of these fragments is similar to that observed for the synthesis of β-galactosidase using total phage or plasmid DNA as template, indicating that the regulatory regions on the fragments are intact and functional. Thus, the synthesis of the α-peptide required an inducer due to the presence of $\text{lac}$ repressor in the $\text{E. coli}$ S-30 extract used. In addition a dependency on adenosine 3′,5′-cyclic monophosphate (cAMP)¹ for α-peptide synthesis was obtained with the fragments isolated from λplac5 and λh80dlacp^s DNAs, whereas little effect of cAMP was seen with the fragment isolated from λh80dlacUV5 phage DNA or PMC3 plasmid DNA containing a UV5 promotor region. However, a significant difference in the effect of guanosine-3′-diphosphate-5′-diphosphate (ppGpp) was observed. With the total phage DNA as template, ppGpp resulted in a 2–4 fold stimulation whereas with the fragment, or PMC3 plasmid DNA, directed synthesis of the α-peptide no significant stimulation by ppGpp was seen.     

Prophage P2 does not kill recB bacteria.

$\text{Escherichia}\text{coli}$ carrying a temperature-sensitive $\text{recB}$ mutation and lysogenic for phage P2 was able to grow normally even at 42°C, at which temperature the bacteria are phenotypically $\text{recB}{}^{\mathrm{?}}$. At this temperature, the bacteria were, however, unable to support the growth of λspi^? phages.     

Identification of an Escherichia coli inner membrane polypeptide specified by a lambda-tonB transducing.

Analysis by polyacrylamide gel electrophoresis of the proteins coded by a λ$\text{ton}$B transducing phage, after infection of UV-irradiated bacteria, revealed the presence of at least 7 new polypeptides. Three of these were identified as proteins of the $\text{trp}$ operon whilst three others were deleted by a spontaneous mutation in the $\text{ton}$B region carried by the phage. A single polypeptide, molecular weight 40,000 was absent from a phage carrying a proflavine induced mutation in $\text{ton}$B. We conclude that this protein, which was localised in the inner membrane by sarkosyl fractionation of the envelope, is the $\text{tonB}$ product.     

The separation of phage promoter from bacterial lac promoter for beta-galactosidase expression in transducing phage lambda plac5.

The replication defective transducing phage λp$\text{lac}\text{5}\text{O}\text{29}\text{P}$3 carries a portion of the $\text{E.}\text{coli}\text{lac}$ operon in the $\text{b}$2 region of the lambda phage. This $\text{lac}$ operon segment contains the $\text{lac}$ promoter, the $\text{lac}$ operator, and the β-galactosidase $\text{z}$ gene, but does not contain the $\text{lac}$ repressor $\text{i}$ gene. The $\text{z}$ gene can be expressed from both the inserted $\text{lac}$ promoter and the phage promoter. When $\text{E.}\text{coli}$ strain 594 ($\text{z}$^?, $\text{i}$⁺) or JC6256 (Δ$\text{lac}$) is infected by λp$\text{lac}\text{5}\text{O}\text{29}\text{P}$3 in the absence of additional cyclic AMP, β-galactosidase synthesis is shown to be expressed from the phage promoter. When 594 (λ⁺) or JC6256 (λ⁺) is infected by λp$\text{lac}\text{5}\text{O}\text{29}\text{P}$3 in the presence of additional cyclic AMP and IPTG, β-galactosidase synthesis is shown to be expressed from the inserted $\text{lac}$ promoter.The ability to separate the phage promoter from the inserted $\text{lac}$ promoter for β-galactosidase expression will simplify the interpretation whenever λp$\text{lac}$5 is used.     

Uncoupling of mitochondrial oxidative phosphorylation by thallium.

The mechanism of integration of λ$\text{bio}$ll, which is deleted of all the known λ recombination genes, was studied using $\text{bio}$ deleted hosts as recipients. The presence of $\text{rec}$BC DNase and $\text{exo}$I in the recipient cells affected the fate of λ$\text{bio}$ll DNA. In nine of ten $\text{imm}\text{λ}{}^{\mathrm{+}}$ transductants, insertion of the λ$\text{bio}$ll genome took place somewhere between J and N and the remaining one had abnormally permuted prophage λ. In this lysogen (#42), the sequence of prophage genes was similar to that of vegetative phage λ. The properties of lysogen #42 were compared with those of other lysogens.     

An analysis of repressor overproduction by the lambda cIIIs mutant.

Efficient lysogenization of Escherichia coli K12 by bacteriophage λ requires the high level of synthesis of the phage repressor shortly after infection. This high level of synthesis of repressor requires the action of the λ eII and cIII proteins. Certain mutants of λ (λcIIIs) appear to have excess $\text{c}\text{II}\text{c}\text{III}$ activity and can lysogenize more efficiently than λ⁺. The basis for the enhanced lysogenization is that, while two or more infecting phage are necessary for λ⁺ to lysogenize, a single infecting λcIIIs particle is sufficient for lysogenization. Also, repressor levels in cells infected with λcIIIs are higher than in those infected with λ⁺. I report here that repressor overproduction by λcIIIs (1) is due to a much higher rate of repressor synthesis than that of λ⁺; (2) is most marked at low multiplicities of infection, possibly because λcIIIs produces repressor much more efficiently than λ⁺ as a singly infecting phage.     

Properties and structure of a gene 24-controlled T4 giant phage

Giant T4 bacteriophage were found by Doermann et al. (1973a) with point mutants in gene 23 and by Cummings et al. (1973) after l-canavanine induction followed by an arginine chase. We now find T4 giant phage with 14 out of 15 tested temperature-sensitive mutants in gene 24 grown at intermediate temperatures between 33 °C and 37 °C.For one of these mutants, T4,24(tsB86), we found that (a) the optimum temperature for giant phage production is 34.8 °C, (b) the head-length distribution peaks sharply between 10 and 12 normal T4 phage head lengths, (c) about 75% of our giant phage have two tails, (d) the buoyant density in CsCl is greater than that of normal phage, (e) they are infectious and show an increased u.v. resistance, (f) their sodium dodecyl sulphate gel electrophoresis pattern is qualitatively similar to that of normal T4 phage, although the relative intensities of some of the bands are different, showing for example, a decreased $\text{P24}{}^1\text{P23}{}^1$² ratio, (g) optical diffraction and filtering of the flattened cylindrical part of the giant heads show a p6 surface net with a lattice constant of approximately 130 Å, a unique $\text{u}\text{v}$ ratio of $\text{15}\text{5}$ and a capsomer morphology of the type 1 + 6 + 6.Mixed infections with T4 wild type and T4.24(amN65) also yield giant phage. These are produced in highest amounts with a multiplicity of infection ratio of 5:5; no giants are observed at ratios of 1:9 or 9:1, suggesting that their formation may be caused by a dosage effect of P24.     

Growth of phages lambda, phiX174, and Ml3 requires the dnaZ (previously dnaH) gene product of Escherichia coli

A functional $\text{dnaZ}$ (previously designated $\text{dnaH}$) product, known to be involved in DNA polymerization, is required for phage λ, ØX174, and M13, but not T4 or T7, growth.     

The argECBH-bearing EcoRI segment of the Escherichia coli chromosome

The transducing phage λd$\text{arg}\text{14}$, carrying a portion of the $\text{E. coli}$ chromosome including $\text{argECBH}$, is derived from the heat-inducible, lysis-defective strain λy199, which has the $\text{b}\text{519}$ and $\text{b}\text{515}$ deletions. Cleavage of λy199 DNA by $\text{Eco}$RI endonuclease, followed by agarose slab gel electrophoresis, results in bands corresponding to the known C, D, E, and F segments of λ, and a segment A′ (A plus B minus $\text{b}\text{519}$ minus $\text{b}\text{515}$, the cleavage site between A and B being eliminated). Cleavage of λd$\text{arg}\text{14}$ DNA by $\text{Eco}$RI yields the expected D, E, and F segments of λ and four other segments, termed 14-1 through 14-4, whose length is 17.5, 6.2, 3.0, and 2.0 kilobases, respectively, as determined by electron microscopy and corroborated by electrophoretic mobility. Heteroduplex analysis shows that the $\text{E. coli argECBH}$ cluster is on the 14-1 segment.     

Yet another way that phage λ manipulates its Escherichia coli host: λrexB is involved in the lysogenic–lytic switch

The life cycle of phage λ has been studied extensively. Of particular interest has been the process leading to the decision of the phage to switch from lysogenic to lytic cycle. The principal participant in this process is the λcI repressor, which is cleaved under conditions of DNA damage. Cleaved λcI no longer acts as a repressor, allowing phage λ to switch from its lysogenic to lytic cycle. The well‐known mechanism responsible for λcI cleavage is the SOS response. We have recently reported that the Escherichia coli toxin‐antitoxin mazEF pathway inhibits the SOS response; in fact, the SOS response is permitted only in E. coli strains deficient in the expression of the mazEF pathway. Moreover, in strains lysogenic for prophage λ, the SOS response is enabled by the presence of λrexB. λRexB had previously been found to inhibit the degradation of the antitoxin MazE, thereby preventing the toxic action of MazF. Thus, phage λ rexB gene not only safeguards the prophage state by preventing death of its E. coli host but is also indirectly involved in the lysogenic–lytic switch.     

Why the Lysogenic State of Phage λ Is So Stable: A Mathematical Modeling Approach

We develop a mathematical model of the phage λ lysis/lysogeny switch, taking into account recent experimental evidence demonstrating enhanced cooperativity between the left and right operator regions. Model parameters are estimated from available experimental data. The model is shown to have a single stable steady state for these estimated parameter values, and this steady state corresponds to the lysogenic state. When the CI degradation rate (γ_cI) is slightly increased from its normal value (γ_cI 0.0 min⁻¹), two additional steady states appear (through a saddle-node bifurcation) in addition to the lysogenic state. One of these new steady states is stable and corresponds to the lytic state. The other steady state is an (unstable) saddle node. The coexistence these two globally stable steady states (the lytic and lysogenic states) is maintained with further increases of γ_cI until γ_cI 0.35 min⁻¹, when the lysogenic steady state and the saddle node collide and vanish (through a reverse saddle node bifurcation) leaving only the lytic state surviving. These results allow us to understand the high degree of stability of the lysogenic state because, normally, it is the only steady state. Further implications of these results for the stability of the phage λ switch are discussed, as well as possible experimental tests of the model.     

Endopeptidase Activity of Phage λ-Endolysin

JACOB and Fuerst^1,2 demonstrated the presence of a bacteriolytic enzyme (λ-endolysin) in the induced cultures of lysogenic Escherichia coli K12 (λ). The enzyme was later identified as the product of gene R; of phage λ³ which is involved in bacterial lysis at the end of a latent period. The enzyme is apt to form spheroplast-like structures in E. coli² and one would therefore expect its substrate to be murein.     

Outer membrane of gram-negative bacteria. XVII. Secificity of transport process catalyzed by the lambda-receptor protein in Escherichia coli.

The outer membrane of Gram-negative bacteria contains (a) “porin” proteins that form transmembrane channels and allow diffusion of various hydrophilic, small molecules (Nakae, J. Biol. Chem., 251, 2176–2178, 1976), and (b) proteins which catalyze the specific transport of unique classes of compounds, e.g. the λ-receptor protein facilitates the diffusion of maltose and maltotriose (Szmelcman et al., Eur. J. Biochem., 65, 13–19, 1976). When strains of $\text{Escherichia coli}$, $\text{B}\text{r}$ and K-12 containing the λ-receptor but not porin were constructed and compared with those containing neither of them, it was found that in the former strains the transmembrane diffusion of glucose and lactose, but not of histidine and 6-aminopenicillanic acid, was significantly accelerated. These results suggest that λ-receptor may facilitate the diffusion of sugars other than maltose.