The Role of the Exo-Xis Region in Oxidative Stress-Mediated Induction of Shiga Toxin-Converting Prophages.

Previous studies indicated that these genetic elements could be involved in the regulation of lysogenization and prophage induction processes. The effects were dramatic in Shiga toxin-converting phage Φ24(B) after treatment with oxidative stress-inducing agent, hydrogen peroxide, while they were less pronounced in bacteriophage λ and in both phages irradiated with UV. The hydrogen peroxide-caused prophage induction was found to be RecA-dependent. Importantly, in hydrogen peroxide-treated E. coli cells lysogenic for either λ or Φ24(B), deletion of the exo-xis region resulted in a significant decrease in the levels of expression of the S.O.S. regulon genes. Moreover, under these conditions, a dramatic decrease in the levels of expression of phage genes crucial for lytic development (particularly xis, exo, N, cro, O, Q, and R) could be observed in Φ24(B)-, but not in λ-bearing cells. We conclude that genes located in the exo-xis region are necessary for efficient expression of both host S.O.S regulon in lysogenic bacteria and regulatory genes of Shiga toxin-converting bacteriophage Φ24(B).

Deciphering the DNA Damage Response.

This year's Albert Lasker Basic Medical Research Award honors Evelyn Witkin and Stephen J. Elledge, two pioneers in elucidating the DNA damage response, whose contributions span more than 40 years.

[SOS repair of 8-methoxypsoralene monoadducts in DNA of lambda bacteriophage and plasmids is mediated by MucA'2B, but not UmuD'2c (PolV) polymerase].

The light-induced action of 8-methoxypsoralen (8-MOP) on λ phage and plasmids yields monoadducts and interstrand crosslinks. The survival and clear plaque mutation frequency in the phage photosensitized with 8-MOP and irradiated with UV at wavelength > 320 nm are increased whenthe wild-type host (Escherichia coli uvr+) is subjected to UV irradiation (wavelength = 254 nm) prior to phage inoculation. These phenomena are known as "W reactivation" and "W mutagenesis." It is shown that 8-MOP monoadducts in λ DNA in- duce clear mutations in the phage inoculated to UV-irradiated excision repair mutants of E. coli only when the error-prone repair is performed by MucA B, but not PolV (UmuD'2C) polymerase. The efficiency of the SOS repair (W reactivation) of 8-MOP monoadducts in plasmid and λ phage DNA also only increases with the presence of pKM101 plasmid muc+ in E. coli uvr-.

Inactivation of recombinant bacteriophage lambda by use of chemical agents and UV radiation.

Several approaches for the inactivation of bacteriophage lambda, including UV germicidal irradiation (UVGI) and the chemical agents Virkon-S, Chloros, Decon-90, and sodium hydroxide (NaOH), were compared. Virkon, NaOH, and UVGI caused a ≥7-log(10) reduction in phage titers. This study successfully describes several methods with potential for bacteriophage inactivation in industrial settings.

Probability landscape of heritable and robust epigenetic state of lysogeny in phage lambda.

Computational studies of biological networks can help to identify components and wirings responsible for observed phenotypes. However, studying stochastic networks controlling many biological processes is challenging. Similar to Schrödinger's equation in quantum mechanics, the chemical master equation (CME) provides a basic framework for understanding stochastic networks. However, except for simple problems, the CME cannot be solved analytically. Here we use a method called discrete chemical master equation (dCME) to compute directly the full steady-state probability landscape of the lysogeny maintenance network in phage lambda from its CME. Results show that wild-type phage lambda can maintain a constant level of repressor over a wide range of repressor degradation rate and is stable against UV irradiation, ensuring heritability of the lysogenic state. Furthermore, it can switch efficiently to the lytic state once repressor degradation increases past a high threshold by a small amount. We find that beyond bistability and nonlinear dimerization, cooperativity between repressors bound to O(R)1 and O(R)2 is required for stable and heritable epigenetic state of lysogeny that can switch efficiently. Mutants of phage lambda lack stability and do not possess a high threshold. Instead, they are leaky and respond to gradual changes in degradation rate. Our computation faithfully reproduces the hair triggers for UV-induced lysis observed in mutants and the limitation in robustness against mutations. The landscape approach computed from dCME is general and can be applied to study broad issues in systems biology.

Overexpression of the LexA-regulated tisAB RNA in E. coli inhibits SOS functions; implications for regulation of the SOS response.

The DNA damage induced SOS response in Escherichia coli is initiated by cleavage of the LexA repressor through activation of RecA. Here we demonstrate that overexpression of the SOS-inducible tisAB gene inhibits several SOS functions in vivo. Wild-type E. coli overexpressing tisAB showed the same UV sensitivity as a lexA mutant carrying a noncleavable version of the LexA protein unable to induce the SOS response. Immunoblotting confirmed that tisAB overexpression leads to higher levels of LexA repressor and northern experiments demonstrated delayed and reduced induction of recA mRNA. In addition, induction of prophage lambda and UV-induced filamentation was inhibited by tisAB overexpression. The tisAB gene contains antisense sequences to the SOS-inducible dinD gene (16 nt) and the uxaA gene (20 nt), the latter encoding a dehydratase essential for galacturonate catabolism. Cleavage of uxaA mRNA at the antisense sequence was dependent on tisAB RNA expression. We showed that overexpression of tisAB is less able to confer UV sensitivity to the uxaA dinD double mutant as compared to wild-type, indicating that the dinD and uxaA transcripts modulate the anti-SOS response of tisAB. These data shed new light on the complexity of SOS regulation in which the uxaA gene could link sugar metabolism to the SOS response via antisense regulation of the tisAB gene.

Host responses influence on the induction of lambda prophage.

Inactivation of bacteriophage lambda CI repressor leads almost exclusively to lytic development. Prophage induction can be initiated either by DNA damage or by heat treatment of a temperature-sensitive repressor. These two treatments also cause a concurrent activation of either the host SOS or heat-shock stress responses respectively. We studied the effects of these two methods of induction on the lytic pathway by monitoring the activation of different lambda promoters, and found that the lambda genetic network co-ordinates information from the host stress response networks. Our results show that the function of the CII transcriptional activator, which facilitates the lysogenic developmental pathway, is not observed following either method of induction. Mutations in the cro gene restore the CII function irrespective of the induction method. Deletion of the heat-shock protease gene ftsH can also restore CII function following heat induction but not following SOS induction. Our findings highlight the importance of the elimination of CII function during induction as a way to ensure an efficient lytic outcome. We also show that, despite the common inhibitory effect on CII function, there are significant differences in the heat- and SOS-induced pathways leading to the lytic cascade.

Global analysis of host response to induction of a latent bacteriophage.

BACKGROUND: The transition from viral latency to lytic growth involves complex interactions among host and viral factors, and the extent to which host physiology is buffered from the virus during induction of lysis is not known. A reasonable hypothesis is that the virus should be evolutionarily selected to ensure host health throughout induction to minimize its chance of reproductive failure. To address this question, we collected transcriptional profiles of Escherichia coli and bacteriophage lambda throughout lysogenic induction by UV light. RESULTS: We observed a temporally coordinated program of phage gene expression, with distinct early, middle and late transcriptional classes. Our study confirmed known host-phage interactions of induction of the heat shock regulon, escape replication, and suppression of genes involved in cell division and initiation of replication. We identified 728 E. coli genes responsive to prophage induction, which included pleiotropic stress response pathways, the Arc and Cpx regulons, and global regulators crp and lrp. Several hundred genes involved in central metabolism, energy metabolism, translation and transport were down-regulated late in induction. Though statistically significant, most of the changes in these genes were mild, with only 140 genes showing greater than two-fold change. CONCLUSION: Overall, we observe that prophage induction has a surprisingly low impact on host physiology. This study provides the first global dynamic picture of how host processes respond to lambda phage induction.

A novel method for evaluating free radical scavenging abilities of antioxidants using ultraviolet induction of bacteriophage lambda.

A novel biological method used to evaluate free radical scavenging abilities of antioxidants using ultraviolet (UV) induction of bacteriophage lambda in lysogenic Escherichia coli kappa12 (lambda+) has been developed. This method is based on the induction of bacteriophage lambda from lysogenic cycle to lytic cycle by ultraviolet irradiation, and formation of free radicals during the course of induction. In the experiments, 10(8)cells/ml and 30s (39J/m2) were determined as the cell density of the lysogenic bacterium and UV irradiation time, respectively. The reliability of this method was demonstrated by electron paramagnetic resonance (EPR) spectroscopy and spin trapping with DMPO. This method had good reproducibility with intra-day variations (RSD, %) of less than 4% and inter-day variations (RSD, %) of less than 5%, respectively. By this method, the free radical scavenging abilities (ID50) of well-known antioxidants such as glutathione, superoxide dismutase (SOD), catalase (CAT) and carotenoids were determined quantitatively. The results were consistent with the ones obtained by conventional methods for evaluating free radical scavenging abilities. This developed method is reliable and uses common instruments and inexpensive, stable reagents, thus it could be utilized as a routine laboratory quantitative assay to screen a large number of substances that have potential to scavenge the free radical.

Inhibitory effects of anti-SARS traditional Chinese medicines on the UV irradiation of lambda-lysogen.

By using lambda-lysogen as a model, the inhibitory effects of anti-severe acute respiratory syndrome (SARS) traditional Chinese medicines (TCMs) prescription I on the UV irradiation were investigated in this present study. It was found that the prescription I possessed obvious inhibitory effects on the UV induction of lambda-lysogen, the inhibitory rate reaching 83.87%. Among five medicinal herbs prescribed in that formula, Herba Patriniae, Radix Astragali and Radix Glycyrrhizae played important roles. When these three herbs were eliminated from the recipe separately, the inhibitory effects were prominently decreased. If only one of these five medicinal herbs was added into the medium of lambda-lysogen, the inhibitory rates ranged from 27.0% approximately 45.0%. By electron spin resonance (ESR) detection, we found that the prescription I, Herba Patriniae and other main herbs in that recipe, could quench effectively the free radicals generated in the process of lambda-lysogenic cells by UV. These results provide a novel idea for further studying the pharmacology of TCM and exploring the mechanism of SARS virus infection.

Effect of glutathione on UV induction of prophage lambda.

The effect of glutathione (GSH) on the ultraviolet (UV) induction of lambda prophage was investigated in lysogenic Escherichia coli. The data showed that extracellular GSH could inhibit the UV induction of lambda prophage. The inhibitory rates were concentration dependent, and the maximal rate obtained was 94% with 3.0 M GSH. The effect was also measured in three different lambda lysogens: a wild-type strain (wt), an isogenic GSH-deficient strain, and an isogenic strain producing increased amounts of GSH. The result showed that when subjected to UV irradiation (254 nm, 60 J m(-2)), GSH-deficient strain was approximately fivefold more sensitive to be lysed than wt, whereas the strain with higher intracellular GSH levels was only 28% susceptible to be lysed. With electron spin resonance and spin trapping techniques, we observed that free radical signals occurred in the suspensions of UV irradiated lysogenic cells and the intensity of signals was influenced by GSH levels. These results indicate that GSH can significantly inhibit the UV induction of lambda prophage, and that this effect is correlated to its capacity to scavenge free radicals generated after UV irradiation.

Error-prone DNA repair and translesion synthesis: focus on the replication fork.

Dean Rupp and Paul Howard-Flanders showed that, following exposure to ultraviolet light, bacteria deficient in nucleotide excision repair synthesised DNA with minimal delay and in pieces roughly the size of the distances between pyrimidine dimmers. The discontinuities or gaps between these pieces were subsequently sealed. This led directly to the hypothesis of translesion synthesis.

A study on the bacterial photo-toxicity of phenothiazinium based photosensitisers.

"Comet assay" showed light activated (3.15 Jcm-2 over 30 min) phenothiazinium based photosensitisers (PhBPs) to induce photo-damage of Staphylococcus aureus DNA, as indicated by DNA "tails" between 80 and 120 microm. In general, PhBPs exhibited significant singlet oxygen yields (Phi(DeltaPhBP)>0.7), suggesting the use of type II mechanisms of photo-oxidation. However, the photodynamic action of PhBPs on DNA showed generally insignificant production of 7,8-dihydro-8-oxo-2'-deoxyguanosine, normally a major product of type II DNA photo-oxidation. These combined results show DNA to be a major site of action of PhBPs and suggest that this action may involve type II attack on a nucleoside(s) other than guanosine.

An action spectrum of the riboflavin-photosensitized inactivation of Lambda phage.

The Action Spectrum of riboflavin (RB) sensitized inactivation of lambda phage was determined between 266 and 575 nm. Below 304 nm, RB depresses the phage reduction by screening phage from radiation that it would otherwise absorb directly. Between 308 and 525 nm, RB sensitizes the inactivation of phage. Enhanced phage reduction is observed at 320 and 500 nm because of binding of RB to the phage and the shifting of the absorption curve of the phage-bound flavin relative to free flavin in phosphate-buffered saline. Enhanced inactivation at 320 and 500 nm and depressed phage inactivation between 360 and 410 nm is also influenced by the inner filter effect.

UV- and MMS-induced mutagenesis of lambdaO(am)8 phage under nonpermissive conditions for phage DNA replication.

Mutagenesis in Escherichia coli, a subject of many years of study is considered to be related to DNA replication. DNA lesions nonrepaired by the error-free nucleotide excision repair (NER), base excision repair (BER) and recombination repair (RR), stop replication at the fork. Reinitiation needs translesion synthesis (TLS) by DNA polymerase V (UmuC), which in the presence of accessory proteins, UmuD', RecA and ssDNA-binding protein (SSB), has an ability to bypass the lesion with high mutagenicity. This enables reinitiation and extension of DNA replication by DNA polymerase III (Pol III). We studied UV- and MMS-induced mutagenesis of lambdaO(am)8 phage in E. coli 594 sup+ host, unable to replicate the phage DNA, as a possible model for mutagenesis induced in nondividing cells (e.g. somatic cells). We show that in E. coli 594 sup+ cells UV- and MMS-induced mutagenesis of lambdaO(am)8 phage may occur. This mutagenic process requires both the UmuD' and C proteins, albeit a high level of UmuD' and low level of UmuC seem to be necessary and sufficient. We compared UV-induced mutagenesis of lambdaO(am)8 in nonpermissive (594 sup+) and permissive (C600 supE) conditions for phage DNA replication. It appeared that while the mutagenesis of lambdaO(am)8 in 594 sup+ requires the UmuD' and C proteins, which can not be replaced by other SOS-inducible protein(s), in C600 supE their functions may be replaced by other inducible protein(s), possibly DNA polymerase IV (DinB). Mutations induced under nonpermissive conditions for phage DNA replication are resistant to mismatch repair (MMR), while among those induced under permissive conditions, only about 40% are resistant.

Amplified UvrA protein can ameliorate the ultraviolet sensitivity of an Escherichia coli recA mutant.

When a recA strain of Escherichia coli was transformed with the multicopy plasmid pSF11 carrying the uvrA gene of E. coli, its extreme ultraviolet (UV) sensitivity was decreased. The sensitivity of the lexA1 (Ind(-)) strain to UV was also decreased by pSF11. The recA cells expressing Neurospora crassa UV damage endonuclease (UVDE), encoding UV-endonuclease, show UV resistance. On the other hand, only partial amelioration of UV sensitivity of the recA strain was observed in the presence of the plasmid pNP10 carrying the uvrB gene. Host cell reactivation of UV-irradiated lambda phage in recA cells with pSF11 was as efficient as that in wild-type cells. Using an antibody to detect cyclobutane pyrimidine dimers, we found that UV-irradiated recA cells removed dimers from their DNA more rapidly if they carried pSF11 than if they carried a vacant control plasmid. Using anti-UvrA antibody, we observed that the expression level of UvrA protein was about 20-fold higher in the recA strain with pSF11 than in the recA strain without pSF11. Our results were consistent with the idea that constitutive level of UvrA protein in the recA cells results in constitutive levels of active UvrABC nuclease which is not enough to operate full nucleotide excision repair (NER), thus leading to extreme UV sensitivity.

Inhibitory effects of gynostemma pentaphyllum on the UV induction of bacteriophage lambda in lysogenic Escherichia coli.

Effects of gynostemma pentaphyllum (GP) on the bacteriophage lambda induced by ultraviolet (UV) irradiation have been studied. The results showed that GP could inhibit the UV induction of bacteriophage lambda in lysogenic cells. The inhibitory effects were dependent on the concentration and the reaction time of GP, and were efficient at 40 to approximately 125 microg ml(-1) for 10 min. The inhibitory rate was higher than 70% when the GP concentration was 50 microg ml(-1). By electron spin resonance (ESR) and spin-trapping techniques, the signals of free radicals were detected in the suspension of the lambda lysogenic bacteria induced by ultraviolet irradiation, but after the addition of GP the signals were decreased. These results indicate that gynostemma pentaphyllum not only is a scavenger of free radicals, but also possesses the biological function of anti-irradiation, and that there is a close relation between the UV irradiation of the bacteriaphage lambda and free radicals.

Genetic evidence that the elevated levels of Escherichia coli helicase II antagonize recombinational DNA repair.

Some phages survive irradiation much better upon multiple than upon single infection, a phenomenon known as multiplicity reactivation (MR). Long ago MR of UV-irradiated lambda red phage in E. coli cells was shown to be a manifestation of recA-dependent recombinational DNA repair. We used this experimental model to assess the influence of helicase II on the type of recombinational repair responsible for MR. Since helicase II is encoded by the SOS-inducible uvrD gene, SOS-inducing treatments such as irradiating recA(+) or heating recA441 cells were used. We found: i) that MR was abolished by the SOS-inducing treatments; ii) that in uvrD background these treatments did not affect MR; and iii) that the presence of a high-copy plasmid vector carrying the uvrD(+) allele together with its natural promoter region was sufficient to block MR. From these results we infer that helicase II is able to antagonize the type of recA-dependent recombinational repair acting on multiple copies of UV-damaged lambda DNA and that its anti-recombinogenic activity is operative at elevated levels only.

ENU induces mutations in the heart of lacZ transgenic mice.

The use of transgenic mouse models as somatic mutation assays allows determination of mutation in all tissues of the mouse, including non-dividing tissues. In this regard, these models can be used to study the possibility that mutations can be induced in mitotically quiescent organs such as the heart. Mutations are generally thought to be associated with mitotic processes of DNA replication. Mutations, however, are also postulated to occur in the absence of mitosis as the result of DNA repair. In order to determine whether or not mutations could be induced in the heart, we analyzed the mutant frequency in the hearts of F(1) (Muta Mouse X SWR) mice that had been treated acutely with 250 mg/kg ENU and sampled at days 10, 35, and 70 post-treatment. A significant increase in mutant frequency at day 70 shows that mutations can be induced in the heart. Since the heart contains small numbers of non-muscle cells, additional mechanisms that could explain these results were also considered. The effect of ENU-induced cell proliferation or a sub-population of rapidly dividing cells is ruled out by C(14)-thymidine uptake studies which showed minimal proliferation. By the same token, the influence of ex vivo mutations (i.e., DNA adducts fixed as mutations during replication in the bacteria) is ruled out by the observed time course of mutations, as well as experimental evidence showing that such mutations are not detected in the lacZ assay.

Mutagenic effects of gamma-rays and incorporated 8-3H-purines on extracellular lambda phage: influence of mutY and mutM host mutations.

The lethal and mutagenic effects on phage lambdacI857 of 60Co gamma-rays and of decay of 3H incorporated into phage DNA both as 8-3H-deoxyadenosine and 8-3H-deoxyguanosine (using 8-3H-adenine as a labelled DNA precursor) were studied on four isogenic Escherichia coli strains: AB1157 M(+)Y(+) (wild type, mutM(+) mutY(+)), AB1157 M(-)Y(+) (mutM::kan mutY(+) mutant deficient in the formamidopyrimidine-DNA glycosylase MutM), AB1157 M(+)Y(-) (mutM(+) mutY mutant deficient in the A:G mismatch DNA glycosylase MutY), and AB1157 M(-)Y(-) (mutM::kan mutY double mutant deficient in both DNA glycosylases). The main products of transmutation component of 3H decay in position 8 of purine residues are 8-oxo-7, 8-dihydroadenine (8-oxoA) and 8-oxo-7,8-dihydroguanine (8-oxoG), the latter being responsible for the most part of the mutagenic effect. The lethal effects of both gamma-rays and tritium decay virtually did not depend on the repair phenotypes of the host strains used. Therefore, the MutM and MutY glycosylases are not involved in the repair of lethal DNA damages induced by ionizing radiation or by the transmutation component of 3H decay in purine residues of phage DNA. The efficiencies of mutagenic action of 3H-purines E(m) (frequencies of c-mutations per one 3H decay in phage genome) were 2.4-, 3.8- and 55-fold higher in the M(-)Y(+), M(+)Y(-) and M(-)Y(-) mutants, respectively, in comparison to the wild-type host. The mutagenic efficiencies E(m) for gamma-rays were nearly identical in the M(+)Y(+) and M(-)Y(+) hosts, but were increased 1.8- and 8.3-fold, respectively, in the M(+)Y(-) and M(-)Y(-) mutants. These data suggest that: (1) the MutY and MutM DNA glycosylases are important for prevention of mutations caused not only by spontaneous oxidation of guanine residues, but also by ionizing radiation or by decay of 3H incorporated into purine bases of DNA; (2) the MutY and MutM enzymes functionally cooperate in elimination of mutagenic damages induced by these agents.