Altered Growth and Envelope Properties of Polylysogens Containing Bacteriophage Lambda N-cI- Prophages.

The bacterial virus lambda (λ) is a temperate bacteriophage that can lysogenize host Escherichia coli (E. coli) cells. Lysogeny requires λ repressor, the cI gene product, which shuts off transcription of the phage genome. The λ N protein, in contrast, is a transcriptional antiterminator, required for expression of the terminator-distal genes, and thus, λ N mutants are growth-defective. When E. coli is infected with a λ double mutant that is defective in both N and cI (i.e., λN-cI-), at high multiplicities of 50 or more, it forms polylysogens that contain 20-30 copies of the λN-cI- genome integrated in the E. coli chromosome. Early studies revealed that the polylysogens underwent "conversion" to long filamentous cells that form tiny colonies on agar. Here, we report a large set of altered biochemical properties associated with this conversion, documenting an overall degeneration of the bacterial envelope. These properties reverted back to those of nonlysogenic E. coli as the metastable polylysogen spontaneously lost the λN-cI- genomes, suggesting that conversion is a direct result of the multiple copies of the prophage. Preliminary attempts to identify lambda genes that may be responsible for conversion ruled out several candidates, implicating a potentially novel lambda function that awaits further studies.

A chemical defence against phage infection.

The arms race between bacteria and the phages that infect them drives the continual evolution of diverse anti-phage defences. Previously described anti-phage systems have highly varied defence mechanisms1-11; however, all mechanisms rely on protein components to mediate defence. Here we report a chemical anti-phage defence system that is widespread in Streptomyces. We show that three naturally produced molecules that insert into DNA are able to block phage replication, whereas molecules that target DNA by other mechanisms do not. Because double-stranded DNA phages are the most numerous group in the biosphere and the production of secondary metabolites by bacteria is ubiquitous12, this mechanism of anti-phage defence probably has a major evolutionary role in shaping bacterial communities.

A fast and reliable method for monitoring of prophage-activating chemicals.

Bacteriophages, that is viruses that infect bacteria, either lyse bacteria directly or integrate their genome into the bacterial genome as so-called prophages, where they remain at a silent state. Both phages and bacteria are able to survive in this state. However, prophages can be reactivated with the introduction of chemicals, followed by the release of a high number of phage particles, which could infect other bacteria, thus harming ecosystems by a viral bloom. The basics for a fast, automatable analytical method for the detection of prophage-activating chemicals are developed and successfully tested here. The method exploits the differences in metabolic heat produced by Escherichia coli with (λ+) and without the lambda prophages (λ-). Since the metabolic heat primarily reflects opposing effects (i.e. the reduction of heat-producing cells by lysis and enhanced heat production to deliver the energetic costs for the synthesis of phages), a systematic analysis of the influence of the different conditions (experimentally and in silico) was performed and revealed anoxic conditions to be best suited. The main advantages of the suggested monitoring method are not only the possibility of obtaining fast results (after only few hours), but also the option for automation, the low workload (requires only few minutes) and the suitability of using commercially available instruments. The future challenge following this proof of principle is the development of thermal transducers which allow for the electronic subtraction of the λ+ from the λ- signal.

Acylamino acid chiral fungicides on toxiciepigenetics in lambda DNA methylation.

Acylamino acid chiral fungicides (AACFs) are low-toxicity pesticides and considered as non-carcinogenic chemicals to laboratory animals. Though AACFs have potential toxicological effects on mammals by non-genotoxic mechanisms, the toxicoepigenomics of AACFs has not been documented. In this article, we explored toxiciepigenetics of metalaxyl, benalaxyl and furalaxyl through epigenetics research on lambda DNA under different concentration exposure. The toxicoepigenomic difference of stereoisomers was examined also. Our results showed that AACFs would affect methyltransferase activity resulting in modulating DNA methylation levels and pattern. The LOAEL of R-metalaxyl and S-metalaxyl were 30 mM and 0.3 mM, respectively. The LOAEL of (R, S)-benalaxyl and (R, S)-furalaxyl were 0.3 Mm and 30 mM, respectively. A significant dose-response effect between (R, S)-benalaxyl and global methylation level was observed. Global methylation level was more susceptible to S-enantiomer compared to R-enantiomer, which indicated enantiomers of AACFs have the enantioselectivity in toxiciepigenetics. Moreover, the dependence of the methylation inhibition on the chiral center of metalaxyl may suggest a considerable specificity of the compound of AACFs for DNA methyltransferases. The inhibition effect between R-enantiomer and S-enantiomer of AACFs on DNA methylation levels generated in this study is important for low-toxicity pesticides toxicoepigenomics evaluation.

An inhibitory RNA aptamer against the lambda cI repressor shows transcriptional activator activity in vivo.

RNA aptamers are one of the promising components for constructing artificial genetic circuits. In this study, we developed a transcriptional activator based on an RNA aptamer against one of the most frequently applied repressor proteins, lambda phage cI. In vitro selection (Systematic Evolution of Ligands by EXponential enrichment) and following in vivo screening identified an RNA aptamer with the intended transcriptional activator activity from an RNA pool containing a 40-nucleotide long random region. Quantitative analysis showed a 35-fold elevation of reporter expression upon aptamer expression. These results suggest that the diversity of artificial transcriptional activators can be extended by employing RNA aptamers against repressor proteins to broaden the parts for constructing genetic circuits.

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).

Evidence that bacteriophage λ lysogens may induce in response to the proton motive force uncoupler CCCP.

We describe a genetic ß-galactoside reporter system using a disk diffusion assay on MacConkey Lactose agar petri plates to monitor maintenance of the bacteriophage λ prophage state and viral induction in Escherichia coli K-12. Evidence is presented that the phage λ major lytic promoters, pL and pR, are activated when cells containing the reporters are exposed to the energy poison carbonyl cyanide m-chlorophenyl hydrazine, CCCP. This uncoupler of oxidative phosphorylation inhibits ATP synthesis by collapsing the proton motive force. Expression of the λ lytic promoters in response to CCCP requires host RecA function and an autocleavable CI repressor, as does SOS induction of the λ prophage that occurs by a DNA damage-dependent pathway. λ Cro function is required for CCCP-mediated activation of the λ lytic promoters. CCCP does not induce an sfi-lacZ SOS reporter.

Genotoxicity assessments of alluvial soil irrigated with wastewater from a pesticide manufacturing industry.

In this study, organochlorine pesticides (OCP) and heavy metals were analyzed from wastewater- and groundwater- irrigated soils (control samples) by gas chromatography (GC) and atomic absorption spectrophotometry (AAS), respectively. Gas chromatographic analysis revealed the presence of high concentration of pesticides in soil irrigated with wastewater (WWS). These concentrations were far above the maximum residue permissible limits indicating that alluvial soils have high binding capacity of OCP. AAS analyses revealed higher concentration of heavy metals in WWS as compared to groundwater (GWS). Also, the DNA repair (SOS)-defective Escherichia coli K-12 mutant assay and the bacteriophage lambda system were employed to estimate the genotoxicity of soils. Therefore, soil samples were extracted by hexane, acetonitrile, methanol, chloroform, and acetone. Both bioassays revealed that hexane-extracted soils from WWS were most genotoxic. A maximum survival of 15.2% and decline of colony-forming units (CFUs) was observed in polA mutants of DNA repair-defective E. coli K-12 strains when hexane was used as solvent. However, the damage of polA (-) mutants triggered by acetonitrile, methanol, chloroform, and acetone extracts was 80.0, 69.8, 65.0, and 60.7%, respectively. These results were also confirmed by the bacteriophage λ test system as hexane extracts of WWS exhibited a maximum decline of plaque-forming units for lexA mutants of E. coli K-12 pointing to an elevated genotoxic potential. The lowest survival was observed for lexA (12%) treated with hexane extracts while the percentage of survival was 25, 49.2, 55, and 78% with acetonitrile, methanol, chloroform, and acetone, respectively, after 6 h of treatment. Thus, our results suggest that agricultural soils irrigated with wastewater from pesticide industries have a notably high genotoxic potential.

Recombineering: using drug cassettes to knock out genes in vivo.

A 'gene knockout' or 'knockout' is a mutation that inactivates a gene function. These mutations are very useful for classical genetic studies as well as for modern techniques including functional genomics. In the past, knockouts of bacterial genes were often made by transposon mutagenesis. In this case, laborious screens are required to find a knockout in the gene of interest. Knockouts of other organisms have traditionally been made by first using in vitro genetic engineering to modify genes contained on plasmids or bacterial artificial chromosomes (BACs) and later moving these modified constructs to the organism of interest by cell culture techniques. Other methods utilizing a combination of genetic engineering and in vivo homologous recombination were inefficient at best. Recombineering provides a new way to generate knockout mutations directly on the bacterial chromosome or to modify any plasmid or BAC in vivo as a prelude to making knockouts in other organisms. The constructs are designed to the base pair and are not dependent on suitable restriction sites. A drug cassette can be placed anywhere within a gene or the open reading frame of the gene can be replaced with the drug cassette. Either way, the desired construct is selected for.

A pseudo-atomic model for the capsid shell of bacteriophage lambda using chemical cross-linking/mass spectrometry and molecular modeling.

Bacteriophage lambda is one of the most exhaustively studied of the double-stranded DNA viruses. Its assembly pathway is highly conserved among the herpesviruses and many of the bacteriophages, making it an excellent model system. Despite extensive genetic and biophysical characterization of many of the lambda proteins and the assembly pathways in which they are implicated, there is a relative dearth of structural information on many of the most critical proteins involved in lambda assembly and maturation, including that of the lambda major capsid protein. Toward this end, we have utilized a combination of chemical cross-linking/mass spectrometry and computational modeling to construct a pseudo-atomic model of the lambda major capsid protein as a monomer, as well as in the context of the assembled procapsid shell. The approach described here is generalizable and can be used to provide structural models for any biological complex of interest. The procapsid structural model is in good agreement with published biochemical data indicating that procapsid expansion exposes hydrophobic surface area and that this serves to nucleate assembly of capsid decoration protein, gpD. The model further implicates additional molecular interactions that may be critical to the assembly of the capsid shell and for the stabilization of the structure by the gpD decoration protein.

Evaluation of mutagenicity of wastewater in the vicinity of pesticide industry.

Pesticide industrial wastewater samples were taken from the Chinhat industrial area nearby Lucknow city, India. GC-MS analysis revealed the presence of pesticides lindane, α-endosulfan, ß-endosulfan, chlorpyriphos, monocrotophos, dimethoate and malathion. A pesticide mixture and wastewater extracts were studied to determine the mutagenicity by Ames Salmonella test, survival of DNA repair defective E. coli K-12 mutants and bacteriophage λ systems. Wastewater samples were concentrated with XAD-resins as an adsorbent and liquid-liquid extraction procedure. The XAD concentrated sample exhibited maximum mutagenic activity in comparison to liquid-liquid extracted sample. TA98 strain was the most responsive strain for both test samples with (+S9) and without (-S9) metabolic activation, while other strains exhibited weak response. A significant decline of DNA repair defective E. coli K-12 mutants, bacteriophage λ was observed with test samples in the survival. The intracellular damage was highest when treated with XAD concentrated sample as compared to liquid-liquid extract after 6h treatment.

[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-.

Effect of preservatives on Shiga toxigenic phages and Shiga toxin of Escherichia coli O157:H7.

Toxin synthesis by Shiga toxin-producing Escherichia coli (STEC) appears to be coregulated through the induction of the integrated bacteriophages that encode the toxin genes. These phages might be the principal means for the dissemination and release of Shiga toxins. We evaluated the effect of three common food preservatives, potassium sorbate, sodium benzoate, and sodium propionate, on the propagation of the phages and Shiga toxins. We tested each preservative at four concentrations, 1, 1.25, 2.5, and 5 mg/ml, both on free phages and on lysogenic phages in bacteria. We also evaluated the expression of a lambdoid phage, which was exposed to increasing concentrations of preservatives, by measuring ß-galactosidase activity from SPC105, a transductant strain. Furthermore, we tested the effect of the preservatives on cytotoxigenic activity of Shiga toxin on Vero cells. We detected an increase of the inhibitory effect of the phage lytic activity, both in lysogenic and free phages, as the preservative concentration increased. However, the inhibition was higher on the lysogenic phages release than on free phages. Sodium benzoate and potassium sorbate were about equal at inhibiting phages; they were more effective than sodium propionate. A significant decrease of lacZ expression, encoded in a lambda phage, was observed. We also found a reduction in Shiga toxin titer caused by exposure of E. coli O157:H7 to 5 mg/ml sodium benzoate or potassium sorbate. These results imply that these three preservatives, used to inhibit microbial spoilage of foods, also act to inhibit lytic activity and dispersion of a phage carrying the gene encoding powerful Shiga cytotoxins. Also notable was the inactivation of Shiga toxin activity, although this effect was detected using concentrations of preservatives greater than those allowed by the Argentine Food Code.

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.

Influence of the Escherichia coli oxyR gene function on lambda prophage maintenance.

In Escherichia coli hosts, hydrogen peroxide is one of the factors that may cause induction of lambda prophage. Here, we demonstrate that H2O2-mediated lambda prophage induction is significantly enhanced in the oxyR mutant host. The mRNA levels for cI gene expression were increased in a lambda 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 DeltaoxyR 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 lambda prophage induction by hydrogen peroxide in the oxyR mutant. Therefore, OxyR seems to be a factor stimulating lambda 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.

Acyl-homoserine lactones can induce virus production in lysogenic bacteria: an alternative paradigm for prophage induction.

Prophage typically are induced to a lytic cycle under stressful environmental conditions or when the host's survival is threatened. However, stress-independent, spontaneous induction also occurs in nature and may be cell density dependent, but the in vivo signal(s) that can trigger induction is unknown. In the present study, we report that acyl-homoserine lactones (AHL), the essential signaling molecules of quorum sensing in many gram-negative bacteria, can trigger phage production in soil and groundwater bacteria. This phenomenon also was operative in a lambda lysogen of Escherichia coli. In model coculture systems, we monitored the real-time AHL production from Pseudomonas aeruginosa PAO1 using an AHL bioluminescent sensor and demonstrated that lambda-prophage induction in E. coli was correlated with AHL production. As a working model in E. coli, we show that the induction responses of lambda with AHL remained unaffected when recA was deleted, suggesting that this mechanism does not involve an SOS response. In the same lambda lysogen we also demonstrated that sdiA, the AHL receptor, and rcsA, a positive transcriptional regulator of exopolysaccharide synthesis, are involved in the AHL-mediated induction process. These findings relate viral reproduction to chemical signals associated with high host cell abundance, suggesting an alternative paradigm for prophage induction.

Genotoxicity of agricultural soils in the vicinity of industrial area.

Soil samples from agricultural fields (cultivated) in the vicinity of industrial area of Ghaziabad City (India) were collected. In this city, wastewater coming from both industrial and domestic sources and without any treatment is used to irrigate the food crops. This practice has been polluting the soil and pollutants might reach the food chain. Gas chromatographic analysis show the presence of certain organochlorine (DDE, DDT, dieldrin, aldrin and endosulfan) and organophosphorus (dimethoate, malathion, methylparathion and chlorpyrifos) pesticides in soil samples. Samples were extracted using different solvents, i.e. methanol, chloroform, acetonitrile, hexane and acetone (all were HPLC-grade, SRL, India), and the extracts were assayed for genotoxic potential using Ames Salmonella/microsome test, DNA repair defective mutants and bacteriophage lambda systems. TA98 and TA100 were found to be the most sensitive strains to all the soil extracts tested. Methanol extracts exhibited a maximum mutagenicity with TA98 strain {540 (-S9) and 638 (+S9) revertants/g of soil} and 938 (-S9) and 1008 (+S9) revertants/g of soil with TA100 strain. The damage in the DNA repair defective mutants was found maximum with methanolic extract followed by acetonitrile, chloroform, hexane and acetone at the dose level of 40 microl/ml culture after 6h of treatment. The survival was 25, 30, 32, 33 and 35% in polA strain after 6h of treatment when tested with wastewater irrigated soil extracts of methanol, acetonitrile, chloroform, hexane and acetone, respectively. A significant decrease in the plaque forming units of bacteriophage lambda was also observed when treated with 40 microl of test samples. Present results showed that methanolic extracts of soil were more toxic than other soil extracts. The soil is accumulating a large number of pollutants due to wastewater irrigation and this practice of accumulation has an impact on soil health.

Genotoxicity of wastewaters used for irrigation of food crops.

In most towns of India, wastewater coming from both industrial and domestic sources and without any treatment is used to irrigate the agricultural crops. This practice has been polluting the soil, and pollutants could possibly reach the food chain. For the above reasons, the wastewaters of Ghaziabad City (India), which is used for irrigation, were sampled (at two different sites) and monitored for the presence of genotoxic agents from January 2005 to June 2007. Gas chromatographic analysis showed the presence of certain OC (DDE, DDT, Dieldrin, Aldrin, and Endosulfan) and OP (Dimethoate, Malathion, Methlyparathion, and Chlorpyrifos) pesticides in both the sampling sites. Wastewater samples were concentrated using XAD resins (XAD-4 and XAD-8) and liquid-liquid extraction procedures, and the extracts were assayed for genotoxic potential by Ames Salmonella/microsome test, DNA repair defective mutants, and bacteriophage lambda systems. The test samples exhibited significant mutagenicity with TA98, TA97a, and TA100 strains with the probable role of contaminating pesticides in the wastewater. However, XAD-concentrated samples were more mutagenic in both sites as compared to liquid-liquid-extracted samples. The damage in the DNA repair defective mutants in the presence of XAD-concentrated water samples were also found to be higher to that of liquid-liquid-extracted water samples at the dose level of 20 muL/mL culture. All the mutants invariably exhibited significant decline in their colony-forming units as compared to their isogenic wild-type counterparts. The survival was decreased by 81.7 and 75.5% in polA(-) strain in site I, and 76.0 and 73.5% in site II in polA(-) under the same experimental conditions after 6 h of treatment with XAD-concentrated and liquid-liquid-extracted samples, respectively. A significant decrease in the survival of bacteriophage lambda was also observed when treated with the test samples.

Studies on modulation of DNA integrity in Fenton's system by phytochemicals.

Chronic exposure to oxidative stress especially to highly reactive hydroxyl radicals (HO*) could damage biomolecules, particularly DNA, that in turn would accelerate onset of degenerative diseases. In the present study a few standard phytochemicals (vitamin C, gallic acid, catechin, apigenin, naringenin and naringin) and plant extracts (Hippophae rhamnoides kernel (HRK), Syzygium cumini kernel (SCK) and Punica granatum pericarp (PGP)) were evaluated for their potential to protect/damage DNA in Fenton's system using in vitro models. The results indicated a significant DNA protective effect for naringin and PGP whereas other phytochemicals/extracts showed DNA damaging effect similar to or more than that of control value. The phytochemicals/extracts were also evaluated for their antioxidant and iron chelation properties. In general, the phytochemicals/extracts with high antioxidant activity but without iron chelation capacity failed to protect DNA in Fenton's system, suggesting that iron chelation was an essential requirement for the phytochemicals studied here to retard HO* generation by Fenton's reaction. This was demonstrated by the high iron chelation capacity of naringin and PGP (83.67% and 68.67% respectively) and their DNA protective effect. Commonly consumed phytochemicals such as vitamin C and gallic acid with their high reducing power and at higher physiological concentration, could regenerate free iron for Fenton's reaction leading to DNA damage as shown here.

Packaging of a unit-length viral genome: the role of nucleotides and the gpD decoration protein in stable nucleocapsid assembly in bacteriophage lambda.

The developmental pathways for a variety of eukaryotic and prokaryotic double-stranded DNA viruses include packaging of viral DNA into a preformed procapsid structure, catalyzed by terminase enzymes and fueled by ATP hydrolysis. In most instances, a capsid expansion process accompanies DNA packaging, which significantly increases the volume of the capsid to accommodate the full-length viral genome. "Decoration" proteins add to the surface of the expanded capsid lattice, and the terminase motors tightly package DNA, generating up to approximately 20 atm of internal capsid pressure. Herein we describe biochemical studies on genome packaging using bacteriophage lambda as a model system. Kinetic analysis suggests that the packaging motor possesses at least four ATPase catalytic sites that act cooperatively to effect DNA translocation, and that the motor is highly processive. While not required for DNA translocation into the capsid, the phage lambda capsid decoration protein gpD is essential for the packaging of the penultimate 8-10 kb (15-20%) of the viral genome; virtually no DNA is packaged in the absence of gpD when large DNA substrates are used, most likely due to a loss of capsid structural integrity. Finally, we show that ATP hydrolysis is required to retain the genome in a packaged state subsequent to condensation within the capsid. Presumably, the packaging motor continues to "idle" at the genome end and to maintain a positive pressure towards the packaged state. Surprisingly, ADP, guanosine triphosphate, and the nonhydrolyzable ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) similarly stabilize the packaged viral genome despite the fact that they fail to support genome packaging. In contrast, the poorly hydrolyzed ATP analog ATP-gammaS only partially stabilizes the nucleocapsid, and a DNA is released in "quantized" steps. We interpret the ensemble of data to indicate that (i) the viral procapsid possesses a degree of plasticity that is required to accommodate the packaging of large DNA substrates; (ii) the gpD decoration protein is required to stabilize the fully expanded capsid; and (iii) nucleotides regulate high-affinity DNA binding interactions that are required to maintain DNA in the packaged state.