The C-terminus of the phage λ Orf recombinase is involved in DNA binding

Phage λ Orf substitutes for the activities of the Escherichia coli RecFOR proteins in vivo and is therefore implicated as a recombination mediator, encouraging the assembly of bacterial RecA onto single-stranded DNA (ssDNA) coated with SSB. Orf exists as a dimer in solution, associates with E. coli SSB and binds preferentially to ssDNA. To help identify interacting domains we analysed Orf and SSB proteins carrying mutations or truncations in the C-terminal region. A cluster of acidic residues at the carboxy-terminus of SSB is known to attract multiple protein partners to assist in DNA replication and repair. In this case an alternative domain must be utilized since Orf association with SSB was unaffected by an SSB113 point mutant (P176S) or removal of the last ten residues (ΔC10). Structurally the Orf C-terminus consists of a helix with a flexible tail that protrudes from each side of the dimer and could serve as a binding site for either SSB or DNA. Eliminating the six residue flexible tail (ΔC6) or the entire helix (ΔC19) had no significant impact on the Orf-SSB interaction. However, the OrfΔC6 protein exhibited reduced DNA binding, a feature shared by single amino acid substitutions within (W141F) or adjacent (R140A) to this region. The OrfΔC19 mutant bound poorly to DNA and secondary structure analysis in solution revealed that this truncation induces protein misfolding and aggregation. The results show that the carboxy-terminus of Orf is involved in nucleic acid recognition and also plays an unexpected role in maintaining structural integrity.     

ClpP/ClpX-mediated degradation of the bacteriophage λ O protein and regulation of λ phage and λ plasmid replication

The O protein is a replication initiator that binds to the orilambda region and promotes assembly of the bacteriophage lambda replication complex. This protein, although protected from proteases by other elements of the replication complex, in a free form is rapidly degraded in the host, Escherichia coli, by the ClpP/ClpX protease. Nevertheless, the physiological role of this rapid degradation remains unclear. Here we demonstrate that the copy number of plasmids derived from bacteriophage lambda is significantly higher in wild-type cells growing in rich media than in slowly growing bacteria. However, lambda plasmid copy number in bacteria devoid of the ClpP/ClpX protease was not dependent on the bacterial growth rate and in all minimal media tested was comparable to that observed in wildtype cells growing in a rich medium. Contrary to lambda plasmid replication, the efficiency of lytic growth of bacteriophage lambda was found to be dependent on the host growth rate in both wild-type bacteria and clpP and clpX mutants. The activities of two major lambda promoters operating during the lytic development, p(R) and p(L), were found to be slightly dependent on the host growth rate. However, when p(R) activity was significantly decreased in the dnaA mutant, production of phage progeny was completely abolished at low growth rates. These results indicate that the O protein (whose level in E. coli cells depends on the activity of ClpP/ClpX protease) is a major limiting factor in the regulation of lambda plasmid replication at low bacterial growth rates. However, this protein seems to be only one of the limiting factors in the bacteriophage lambda lytic development under poor growth conditions of host cells. Therefore, it seems that the role of the rapid ClpP/ClpX-mediated proteolysis of the O protein is to decrease the efficiency of early DNA replication of the phage in slowly growing host cells.     

Adsorption of phage λ to Salmonella typhimurium lamB + requires the presence of lipopolysaccharide in the outer membrane

Summary Two S. typhimurium strains TA1534 (rfa ⁺) and TA1538 (rfaE) were transformed with the lamB expression plasmid pAMH70. Transposition events with placMu55 hybrid phage were successful only with TA1534/pAMH70 strain. Using SDS-PAGE, the LamB protein was present in the total cell proteins but not in the outer membrane proteins of the TA1538/pAMH70 strain. The LamB protein must linked to the LPS of the outer membrane to allow adsorption of phage in S. typhimurium.     

Regulation of replication of λ phage and λ plasmid DNAs at low temperature

It was previously demonstrated that while lysogenic development of bacteriophage λ in Escherichia coli proceeds normally at low temperature (20–25° C), lytic development is blocked under these conditions owing to the increased stability of the phage CII protein. This effect was proposed to be responsible for the increased stimulation of the p _E promoter, which interferes with expression of the replication genes, leading to inhibition of phage DNA synthesis. Here we demonstrate that the burst size of phage λcIb2, which is incapable of lysogenic development, increases gradually over the temperature range from 20 to 37° C, while no phage progeny are observed at 20° C. Contrary to previous reports, it is possible to demonstrate that p _E promoter activation by CII may be more efficient at lower temperature. Using density-shift experiments, we found that phage DNA replication is completely blocked at 20° C. Phage growth was also inhibited in cells overexpressing cII, which confirms that CII is responsible for inhibition of phage DNA replication. Unexpectedly, we found that replication of plasmids derived from bacteriophage λ is neither inhibited at 20° C nor in cells overexpressing cII. We propose a model to explanation the differences in replication observed between λ phage and λ plasmid DNA at low temperature. Received: 30 December 1997 / Accepted: 25 February 1998     

Host controlled modification of phage λ in zygotic induction inEscherichia coli B

Experiments on zygotic induction of prophage inE. coli Hfr K 12 ×E. coli B F^– crosses are described. The prophage is not restricted inE. coli B zygotes. Most of the zygotes produce the host modified.B; about 10–50% of the zygotes also produce a few.K particles.     

Joining of λ bacteriophage DNA moleculesin vitro using the gene product of the λint phage

The ability of cell extracts ofEscherichia coli 1100 prepared at different times after infection with bacteriophage λint 6 cI 857 or λ 857 to attach molecules of λ³²P-DNA to λ DNA adsorbed on a nitrocellulose membrane filter was compared. It was found that only with extracts from cells infected with bacteriophage λ cI 857 with an active int gene was it possible to attach molecules of λ³²P-DNA to λ DNA. This ability was reflected best in extracts prepared from cells 10 min after infection. A similar ability was observed also with extracts prepared from cells ofEscherichia coli 1100 (λ cI 857) after heat induction of the prophage. The maximum efficiency in this case was observed with cell extracts 15 min after the temperature rise.     

Nutrition in the life cycle assessment of foods—function or impact?

Purpose

This work provides an unambiguous conceptual framework for inclusion of nutrition in Life Cycle Assessments (LCAs) of food that enables the distinction between two different roles of nutrition, namely serving as the basis of food comparisons via the functional unit and as an impact pathway that links food ingestion with human health effects.

Methods

We compare how nutritional aspects have been considered in the functional unit of published LCAs of food with the procedural requirements for ensuring comparability of the functional units. We consider the relevance of nutrient profiling models for assessing food- and diet-related health damages and benefits in the context of LCAs of food. We finally recommend a method that will enable a systematic, comparative, and holistic assessment of the marginal health effect of food products on human health.

Results and discussion

Satiety is proposed as a central attribute for comparisons of food products, while weighted measures of nutrient content are suggested to be largely misplaced as part of the functional unit. In contrast, nutritional measures have a large role to play in assessing the human health impacts of the marginal ingestion of specific food products. Such measures should enable a direct quantification of human health effect and benefits and should take advantage of robust epidemiological evidence.

Conclusions

Nutritional measures enter into both the functional unit in the form of satiety measures and into the calculation of impacts in the form of the marginal influence of the specific food item on the human health impact of the overall diet. To enhance the differentiation of health impacts at the level of individual food items, it is recommended to combine the nutrient balance indicator with the DALY Nutritional Index (DANI) in each specific dietary context.

     

Damages induced in λ phage DNA by enzyme-generated triplet acetone

Exposure of λ phage to triplet acetone, generated via the oxidation of isobutanal by peroxidase, leads to genome lesions. The majority of these lesions are detected as DNA single-strand breaks only under alkaline conditions, and so true breaks do not occur. Also, no sites sensitive to UV-endonuclease from Micrococcus leteus were found in DNA from treated phage. The participation of triplet acetone in the generation of such DNA damage is discussed.     

How can a life cycle inventory parametric model streamline life cycle assessment in the wooden pallet sector?

Purpose

This study discusses the use of parameterization within the life cycle inventory (LCI) in the wooden pallet sector, in order to test the effectiveness of LCI parametric models to calculate the environmental impacts of similar products. Starting from a single case study, the objectives of this paper are (1) to develop a LCI parametric model adaptable to a range of wooden pallets, (2) to test this model with a reference product (non-reversible pallet with four-way blocks) and (3) to determine numerical correlations between the environmental impacts and the most significant LCI parameters; these correlations can be used to improve the design of new wooden pallets.

Methods

The conceptual scheme for defining the model is based on ISO14040-44 standards. First of all, the product system was defined identifying the life cycle of a generic wood pallet, as well as its life cycle stages. A list of independent and dependent parameters was used to describe the LCI flows of a generic wooden pallet. The LCI parametric model was applied to calculate the environmental impacts of the reference product, with regard to a selection of impact categories at midpoint level (climate change, human toxicity, particulate matter formation, agricultural land occupation, fossil depletion). The model was then applied to further 11 wooden pallets belonging to the same category.

Results and discussion

The definition of a LCI parametric model based on 31 independent parameters and 21 dependent parameters streamlined the data collection process, as the information required for fulfilling the LCI are standard information about the features of the wooden pallet and its manufacturing process. The contribution analysis on the reference product revealed that the most contributing life cycle stages are wood and nails extraction and manufacturing (positive value of environmental impact) and end-of-life (avoided impact). This result is driven by two parameters: mass of wood and average distance for transport of wood. Based on the results of the application of the LCI parametric model to the identified products, one parameter-based regression and one multiple non-linear regression allowed to define a correlation between the life cycle impact assessment (LCIA) category indicators considered and the most influencing parameters.

Conclusions

The definition of LCI parametric model in the wooden pallet sector can effectively be used for calculating the environmental impacts of products with different designs, as well as for obtaining a preliminary estimation of the life cycle environmental impacts of new products.     

Protein composition of different stages in the life cycle of Ulva mutabilis,Føyn

Summary Soluble protein, about 15% of the total cellular protein, was extracted from different stages in the haplodiplontic life cycle of Ulva mutabilis. The electrophoretic band pattern of the protein extracts from the haploid gametophyte and the diploid sporophyte were found to be the same, except for one slow moving band present in the gametophyte, but lacking in the diploid sporophyte. This band was also missing in the extract from the haploid parthenosporophyte, but is seen in the extract from the zoospores. It was found that the synthesis of the protein in this band occurred during most of the preparation period preceding meiosis. The band is not seen in extracts from gametes, and it is inferred that this protein is broken down during the period preceding the mitotic gametangial division in the gametophyte. So far the protein making up this band behaves as should be required for a factor determining the shift in generation during the life cycle.     

The kidney form of Trypanosoma musculi: A distinct stage in the life cycle?

Trypanosoma musculi is a parasite specific to mice, which resides in the blood and lacks intracellular stages. After immune clearance of the flagellates from the general circulation, mice are resistant to reinfection. Yet, long after parasites are no longer detected in the peripheral blood, they persist in the vasa recta of the kidneys and it has been proposed that this is an immunologically privileged site for T. musculi. This relationship provides a useful model for studies of latent or chronic infections in immune hosts. Here, Fernando Monroy and Donald Dusanic consider the immune responses of mice to T. musculi and compare characteristics of the parasites from the vasa recta (kidney forms, KFs) of mice with latent infections to trypanosomes from the peripheral blood (bloodstream forms, BSFs) of animals during active infections. They consider how KFs evade immune destruction and suggest that these sequestered parasites represent a distinct stage in the life cycle.     

Calculating life? Duelling discourses in interdisciplinary systems biology

A high profile context in which physics and biology meet today is in the new field of systems biology. Systems biology is a fascinating subject for sociological investigation because the demands of interdisciplinary collaboration have brought epistemological issues and debates front and centre in discussions amongst systems biologists in conference settings, in publications, and in laboratory coffee rooms. One could argue that systems biologists are conducting their own philosophy of science. This paper explores the epistemic aspirations of the field by drawing on interviews with scientists working in systems biology, attendance at systems biology conferences and workshops, and visits to systems biology laboratories. It examines the discourses of systems biologists, looking at how they position their work in relation to previous types of biological inquiry, particularly molecular biology. For example, they raise the issue of reductionism to distinguish systems biology from molecular biology. This comparison with molecular biology leads to discussions about the goals and aspirations of systems biology, including epistemic commitments to quantification, rigor and predictability. Some systems biologists aspire to make biology more similar to physics and engineering by making living systems calculable, modelable and ultimately predictable-a research programme that is perhaps taken to its most extreme form in systems biology's sister discipline: synthetic biology. Other systems biologists, however, do not think that the standards of the physical sciences are the standards by which we should measure the achievements of systems biology, and doubt whether such standards will ever be applicable to 'dirty, unruly living systems'. This paper explores these epistemic tensions and reflects on their sociological dimensions and their consequences for future work in the life sciences.     

Side-effects of pesticides on the life cycle of the mite pathogenic fungus Neozygites floridana

The tomato red spider mite, Tetranychus evansi Baker and Pritchard, is an invasive species in Africa causing considerable damage to Solanaceous crops. The fungal pathogen Neozygites floridana Weiser and Muma from Brazil has been considered a potential candidate for introduction into Africa for the control of T. evansi. To be incorporated in the tomato production system, N. floridana has to be compatible with the pesticides used for the control of other pests and diseases. Pesticides used in tomatoes that might affect the fungus were therefore studied by the use of different methods. Two insecticides (Lambda-cyhalothrin and Methomyl), two acaricides (Propargite and Abamectin), and two fungicides (Captan and Mancozeb) were tested in two concentrations: the mean commercial rate (CR) and 50% of the mean commercial rate (CR/2). Fungus-killed mite cadavers or the substrates used for sporulation (leaf discs and coverslips) were either immersed or sprayed with the pesticides before testing their effects on sporulation, germination of primary conidia and infectivity of N. floridana. Direct immersion of cadavers, coverslips or leaf discs into pesticides affected sporulation and germination stronger than the spray tower method, although infectivity of capilliconidia was neither affected by the method of application nor the concentration of the pesticides. The fungicides Captan and Mancozeb resulted in a high reduction in sporulation and germination at both concentrations. Propargite did not inhibit sporulation but affected germination of primary conidia. Methomyl and Abamectin resulted in less effects on N. floridana.     

Chain Bias in Chi-Stimulated Heteroduplex Patches in the λ Ren Gene Is Determined by the Orientation of λ Cos

Heteroduplex patch recombinants have received information in one DNA chain but have not recombined flanking markers. Evidence regarding which chain is exchanged bears on the structure of recombination intermediates. The direction of travel along DNA of RecBCD recombinase, the central enzyme in the Escherichia coli RecBCD pathway of homologous recombination, is determined in phage lambda by the orientation of the packaging origin, cos. cos is a double-chain cut site which serves as a preferred entry site for RecBCD. Using partially denaturing gels to resolve heteroduplex molecules, we have examined patch recombinants at the lambda ren gene. We report that the transferred information in Chi-stimulated patches at ren can occur on either chain, but is biased to the chain ending 5' at the right of the lambda map (the lambda r chain) in phage carrying cos in its normal orientation. The chain bias switches in favor of the chain that ends 3' at the right (the lambda l chain) when RecBCD travel direction is reversed by inverting cos. We entertain models that accommodate these and other results pertaining to the structure of RecBCD-mediated recombinants.     

Homologous interactions of λ repressor and λ Cro with the λ operator

Lambda repressor and lambda Cro bind to the same six sites on the phage chromosome but with different relative affinities. Nucleotides at certain positions in the operator are conserved in all sites, as are amino acids at certain positions in the recognition α-helices of repressor and Cro. Here we focus on one of the conserved amino acids, a serine found at position 2 of each recognition helix. We show that, contrary to a previous model, both serines contact the same conserved position in the operator, position 4. We suggest a simplified view of how repressor and Cro recognize similar operator sites but distinguish differently among them.