Induction Of Lambda Prophage Research Articles

Release mechanisms and molecular interactions of Pseudomonas aeruginosa extracellular DNA.

Pseudomonas aeruginosa infection is a significant threat for clinicians. Increasing incidents of resistant biofilm infection result in high mortality rates worldwide. There is a considerable current interest in the field of extracellular DNA (eDNA)-mediated P. aeruginosa biofilm formation. eDNA acts as a glue to make biofilm more stable. This review focuses on the diverse mechanisms and factors, which enhance the eDNA release into the extracellular milieu. Furthermore, eDNA-mediated molecular interactions within the biofilm are emphasized. In addition, drug resistance mechanisms due to the versatility of eDNA are discussed. Spatial physiological diversity is expected due to different metabolic activity of bacterial subpopulation present in P. aeruginosa biofilm layers. In P. aeruginosa, eDNA release is accomplished by cell lysis and OMVs (outer membrane vesicles). eDNA release is a spontaneous and multifactorial process, which may be accomplished by PQS, pyocyanin, and lambda prophage induction. Hydrogen peroxide and pyocin trigger cell death, which may facilitate eDNA release. Lung mucosa of cystic fibrosis patients is enriched with eDNA, which acidifies biofilm and develops P. aeruginosa resistance to aminoglycosides. Further studies on spatial and molecular characterization of bacterial subpopulation in biofilm will shed light on eDNA-biofilm interaction more precisely.Key Points• Extracellular DNA (eDNA) is a key component of Pseudomonas aeruginosa biofilm.• P. aeruginosa eDNA acts as a glue to make biofilm more stronger.• Bacterial cell death or lysis may be the potential way to release P. aeruginosa eDNA into extracellular milieu.• P. aeruginosa eDNA contributes to develop resistance to antimicrobials.

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.

Calorimetric real time monitoring of lambda prophage induction

Calorimetry can be used for a noninvasive monitoring of metabolic and energy changes in bacterial cultures in real time. This potentially includes changes occurring during phage infection cycles. To demonstrate this, the growth of Escherichia coli K124 F − carrying the lambda prophage was monitored in a reaction calorimeter, while inducing the shift from lysogeny to lysis using methotrexate (MTX) as the inducer. Different phases of the phage infection were distinguishable calorimetrically. The calorimetric signals corresponded well to changes in growth stoichiometry and kinetics that were measured non-calorimetrically. The comparison of the calorimetric results with other measurements of activity revealed that the calorimetric method was superior since the calorimetric data mirrored the progress of infection in more detail. Contradictions between the calorimetric data and the appearance of infectious phages could result from inactivation of phage by MTX.

Lambda-prophage induction modeled as a cooperative failure mode of lytic repression.

We analyze a system-level model for lytic repression of lambda phage in E. coli using reliability theory, showing that the repressor circuit comprises four redundant components whose failure mode is prophage induction. Our model reflects the specific biochemical mechanisms involved in regulation, including long-range cooperative binding, and its detailed predictions for prophage induction in E. coli under ultraviolet radiation are in good agreement with experimental data.

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 λ 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.

Role of the lytic repressor in prophage induction of phage lambda as analyzed by a module-replacement approach.

Using a module exchange approach, we have tested a long-standing model for the role of Cro repressor in lambda prophage induction. This epigenetic switch from lysogeny to the lytic state occurs on activation of the host SOS system, which leads to specific cleavage of CI repressor. It has been proposed that Cro repressor, which operates during lytic growth and which we shall term the lytic repressor, is crucial to prophage induction. In this view, Cro binds to the O(R)3 operator, thereby repressing the cI gene and making the switch irreversible. Here we tested this model by replacing lambda Cro with a dimeric form of Lac repressor and adding several lac operators. This approach allowed us to regulate the function of the lytic repressor at will and to prevent it from repressing cI, because lac repressor could not repress P(RM) in our constructs. Repression of cI by the lytic repressor was not required for prophage induction to occur. However, our evidence suggests that this binding can make induction more efficient, particularly at intermediate levels of DNA damage that otherwise cause induction of only a fraction of the population. These results indicate that this strategy of module exchange will have broad applications for analysis of gene regulatory circuits.

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.

Induction of Shiga Toxin-Converting Prophage inEscherichia coliby High Hydrostatic Pressure

Since high hydrostatic pressure is becoming increasingly important in modern food preservation, its potential effects on microorganisms need to be thoroughly investigated. In this context, mild pressures (<200 MPa) have recently been shown to induce an SOS response in Escherichia coli MG1655. Due to this response, we observed a RecA- and LexA-dependent induction of lambda prophage upon treating E. coli lysogens with sublethal pressures. In this report, we extend this observation to lambdoid Shiga toxin (Stx)-converting bacteriophages in MG1655, which constitute an important virulence trait in Stx-producing E. coli strains (STEC). The window of pressures capable of inducing Stx phages correlated well with the window of bacterial survival. When pressure treatments were conducted in whole milk, which is known to promote bacterial survival, Stx phage induction could be observed at up to 250 MPa in E. coli MG1655 and at up to 300 MPa in a pressure-resistant mutant of this strain. In addition, we found that the intrinsic pressure resistance of two types of Stx phages was very different, with one type surviving relatively well treatments of up to 400 MPa for 15 min at 20 degrees C. Interestingly, and in contrast to UV irradiation or mitomycin C treatment, pressure was not able to induce Stx prophage or an SOS response in several natural Stx-producing STEC isolates.

On the role of Cro in lambda prophage induction.

The lysogenic state of bacteriophage lambda is exceptionally stable yet the prophage is readily induced in response to DNA damage. This delicate epigenetic switch is believed to be regulated by two proteins; the lysogenic maintenance promoting protein CI and the early lytic protein Cro. First, we confirm, in the native configuration, the previous observation that the DNA loop mediated by oligomerization of CI bound to two distinct operator regions (O(L) and O(R)), increases repression of the early lytic promoters and is important for stable maintenance of lysogeny. Second, we show that the presence of the cro gene might be unimportant for the lysogenic to lytic switch during induction of the lambda prophage. We revisit the idea that Cro's primary role in induction is instead to mediate weak repression of the early lytic promoters.

An SOS response induced by high pressure in Escherichia coli.

Although pressure is an important environmental parameter in microbial niches such as the deep sea and is furthermore used in food preservation to inactivate microorganisms, the fundamental understanding of its effects on bacteria remains fragmentary. Our group recently initiated differential fluorescence induction screening to search for pressure-induced Escherichia coli promoters and has already reported induction of the heat shock regulon. Here the screening was continued, and we report for the first time that pressure induces a bona fide SOS response in E. coli, characterized by the RecA and LexA-dependent expression of uvrA, recA, and sulA. Moreover, it was shown that pressure is capable of triggering lambda prophage induction in E. coli lysogens. The remnant lambdoid e14 element, however, could not be induced by pressure, as opposed to UV irradiation, indicating subtle differences between the pressure-induced and the classical SOS response. Furthermore, the pressure-induced SOS response seems not to be initiated by DNA damage, since DeltarecA and lexA1 (Ind-) mutants, which are intrinsically hypersensitive to DNA damage, were not sensitized or were only very slightly sensitized for pressure-mediated killing and since pressure treatment was not found to be mutagenic. In light of these findings, the current knowledge of pressure-mediated effects on bacteria is discussed.

Inductive Effect of Hypoxanthine–Xanthine Oxidase System on Lambda Prophage

Hypoxanthine-xanthine oxidase (HX-XO) system is a classical system that can generate superoxide anions. The inductive effect of the HX-XO system for lambda prophage has been investigated in this study. The results showed that the system can induce lambda prophage from lysogenic state to lytic growth. The inductive effect was directly proportional to the concentration of HX and XO and inversely related to the time of preliminary incubation of HX with XO. The cell density of the lysogenic bacteria also greatly affected the inductive effect. The maximal PFU number of 2.9 x 10(4) PFU/ml was recorded at 0.86 mM HX, 1.6 x 10(-2) U/ml XO, and a cell density of 10(8) cells/ml. The inductive effect of the HX-XO system was inhibited in the suspensions by glutathione, superoxide dismutase, and catalase. The results provide evidence that free radicals are the primary factors in the induction of lambda prophage in lysogenic bacteria.

Radioprotective effect of sodium diethyldithiocarbamate (DDC) and S-2-aminoethyl-isothioronicadenosin-5-triphosphate (adeturon) in γ-irradiated Escherichia coli cells

The effect of sodium diethyldithiocarbamate (DDC) and S-2-aminoethyl-isothiouronicadenosin-5-triphosphate (adeturon) in the induction of Escherichia coli SOS response promoted by γ-irradiation was studied by measuring the induction of sulA gene and the induction of lambda prophage. Furthermore, as a way of measure the exonuclease activity in γ-irradiated cells in the presence or absence of both compounds, the DNA degradation was determined. Adeturon did not affected DNA degradation, but inhibited the induction of the SOS functions studied. On the contrary, DDC inhibited DNA degradation as well as the induction of the sulA gene, but enhanced lambda induction in E. coli lysogenic strains. These results indicate that both compounds diminish the DNA damage produced by γ-irradiation and also suggest that the mechanisms of radioprotection must be different. Thus, radioprotection mediated by DDC should involve free hydroxyl radical scavenging and a minor activity of exonuclease. The enhancement of phage induction in E. coli cells that DDC produces could be attributed to its quelant effect and this would not be not probably directly related to radioprotection. Adeturon, as thiols, may serve also as scavenging agent of free hydroxyl radicals, diminishing indirectly the DNA damage level. In addition, adeturon must interact with DNA in the same form that other aminothiol compounds do it. This interaction, mediated by amino groups of adeturon, may serve to concentrate these compounds near of the DNA damage site, increasing the potential for the thiol portion of the molecule to donate hydrogen, decreasing the damage level on DNA molecule. However, adeturon do not modify the exonuclease activity. Some topic about the possible clinical application of both compounds are discussed.

The Tum Protein of Coliphage 186 Is an Antirepressor

The tum gene of coliphage 186, encoded on a LexA controlled operon, is essential for UV induction of a 186 prophage. Primer extension analysis is used to confirm that Tum is the sole phage function required for prophage induction and that it acts against the maintenance repressor, CI, to relieve repression of the lytic promoters, pR and pB, and thereby bring about lytic development. In vitro experiments with purified proteins demonstrate that Tum prevents CI binding to its operator sites. Tum does not compete with CI for binding sites on DNA, and unlike RecA mediated induction of lambda prophage, the action of Tum on CI is reversible. Mechanisms by which Tum may act against CI are discussed.

Inhibition of prophage induction by synthetic pentapeptide.

Six peptide chains different in length and number of sequences were synthesized by the continuous-flow solid-phase method. Their effects on prophage induction were tested. It was found that pentapeptide 1 H-Val-Val-Asn-Asp-Leu-OH, at a concentration of 25 micrograms/mL inhibited the spontaneous induction of prophage Lambda in E. coli strain W 3110(lambda) and phi RLZLS in R. leguminosarum (lysogenic local isolate) by 99.5% and 65.7%, respectively. Moreover, this concentration was also able to inhibit the replication of the developed phages in the indicator strains. It was suggested that this peptide may block the attachment site of the induced phages or inhibit Rec A Protease.

INHIBITION OF PROPHAGE INDUCTION BY SYNTHETIC PENTAPEPTIDE

Six peptide chains different in length and sequences were synthesized by continuous flow solid phase method. Their effects on prophage induction were tested. It was found out that a pentapeptide at concentration of 25 µg/ml inhibited the spontaneous induction of prophage Lambda in E. coli strain W 3110 () and (0)RL7 s in R. Leguminosarum (lysogenic local isolate) by (99.5%) and (98.5%), respectively. This concentration was also able to inhibit the replication of the developed phages in the indicator strains. it is suggested that the peptide may block the receptor sites of the induced phages or inhibit Rec A Protease.

Genotoxicity of 2,4,5‐trichlorophenoxyacetic acid biodegradation products in the Salmonella reversion and lambda prophage‐induction bioassays

AbstractPseudomonas cepacia strain AC1100 has been isolated and reported to utilize 2,4,5‐trichlorophenoxyacetic acid (2,4,5 T) as sole carbon and energy source Metabolites from the 2,4,5‐T degradation pathway were tested for their mutagenicity in the Salmonella reversion bioassay and genotoxicity in the prophage‐induction bioassay The parental compound (2,4,5‐T) and three reported metabolites (2,4,5 trichlorophenol, 2,5‐dichlorohydroquinone, and 2‐chlorosuccinate) were negative with and without metabolic activation in Salmonella typhimurium strains TA98, TA100, TA102, and TA104 Conversely, 2,4,5‐T and 2,4,5 trichlorophenol were positive in the prophage‐induction bioassay with added S9 2,4,5‐Trichlorophenol was approximately 100 fold more genotoxic than 2,4,5‐T 2,5‐Dichlorohydroquinone was a weak direct‐acting genotoxicant in this assay In order to determine if strain AC1100 eliminated genotoxicity, 2,4,5 T medium was inoculated and the genotoxicity and the amount of 2,4,5‐T remaining were examined over the growth period As cell numbers increased, the percentage of 2,4,5‐T remaining in the medium decreased This decrease coincided with a decrease in genotoxicity in the prophage‐induction bioassay No mutagenic response was observed in the Salmonella reversion assay

Prophage induction by DNA topoisomerase II poisons and reactive-oxygen species: Role of DNA breaks

Various compounds were evaluated for their ability to induce prophage lambda in the Eschirichia coli WP2 5(λ) microscreen assay. The inability of a DNA gyrase subunit B inhibitor (novobiocin) to induce prophage indicated that inhibition of the gyrase's ATPase was insufficient to elicit the SOS response. In contrast, poisons of DNA gyrase subunit A (nalidixic acid and oxolinic acid) were the most potent inducers of prophage among the agents examined here. This suggested that inhibition of the ligation function of subunit A, which also has a DNA nicking activity, likely resulting in DNA breaks that were available (as single-stranded DNA) to act as strong SOS-inducing signals, leading to prophage induction. Agents that both intercalated and produced reactive-oxygen species (the mammalian DNA topoisomerase II poisons, adriamycin, ellipticine, and m-AMSA) were the next most potent inducers of prophage. Agents that produced reactive-oxygen species only (hydrogen peroxide and paraquat) were less potent than adriamycin and ellipticine but more potent than m-AMSA. Agents that intercalated but did not generate reactive-oxygen species (actinomycin D) or that did neither (teniposide) were unable to induce prophage, suggesting that intercalation alone may be insufficient to induce prophage. These results illustrate the variety of mechanisms (and the relative effectiveness of these mechanisms) by which agents can induce prophage. Nonetheless, these agents may induce prophage by producing essentially the same type of DNA damage, i.e., DNA strand breaks. The potent genotoxicity of the DNA gyrase subunit A poisons illustrates the genotoxic consequences of perturbing an important DNA-protein complex such as that formed by DNA and DNA topoisomerase.

Mutagenic effects of heavy ions in bacteria

The peculiarities and mechanisms of the mutagenic action of gamma-rays and heavy ions on bacterial cells have been investigated. Direct mutations in the lac-operon of E. coli in wild type cells and repair deficient strains have been detected. Furthermore, the induction of revertants in Salmonella tester strains was measured. It was found that the mutation rate was a linear-quadratic function of dose in the case of both gamma-rays and heavy ions with LET up to 200 keV/micrometer. The relative biological effectiveness (RBE) increased with LET up to 20 keV/micrometer. Low mutation rates were observed in repair deficient mutants with a block of SOS-induction. The induction of SOS-repair by ionizing radiation has been investigated by means of the "SOS-chromotest" and lambda-prophage induction. It was shown that the intensity of the SOS-induction in E. coli increased with increasing LET up to 40-60 keV/micrometer.

Performance of 133 compounds in the lambda prophage induction endpoint of the Microscreen assay and a comparison with S. typhimurium mutagenicity and rodent carcinogenicity assays

The Microscreen assay was developed as a means of testing very small samples, as in complex mixture fractionation. It is a multi-endpoint assay which utilizes E. coli WP2s(λ). Exposure takes place to serial dilutions of the test compound in microtitre wells (250 μl) followed by sampling from wells in which growth has occurred (‘non-toxic wells’). Although a number of different endpoints can be measured, only the prophage induction endpoint (the first one developed) has been extensively tested. Results with 133 compounds are presented. These include 111 compounds which have been tested in the S. typhimurium assay and 66 compounds for which both rodent bioassay and S. typhimurium assay data exists. The concordance for the Microscreen assay and the S. typhimurium assay was 71%. For this group of compounds, the sensitivity of the Microscreen assay in detecting carcinogens was 76% compared with 58% for the S. typhimurium assay. However, the S. typhimurium assay was somewhat more specific (69%) compared with the Microscreen (56%). The overall association between carcinogenicity and Microscreen results was statistically significant ( p = 0.029), whereas for the S. typhimurium assay the association with carcinogenicity was non-significant ( p = 0.086). The Microscreen assay was able to detect halogenated compounds better than the S. typhimurium assay. The Microscreen assay should prove useful in complex mixture fractionation, or in other situations where sample size is limiting.