Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7

DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5′-TGGTC-3′) than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain.     

A direct proofreader–clamp interaction stabilizes the Pol III replicase in the polymerization mode

Processive DNA synthesis by the αεθ core of the Escherichia coli Pol III replicase requires it to be bound to the β₂ clamp via a site in the α polymerase subunit. How the ε proofreading exonuclease subunit influences DNA synthesis by α was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non‐proofreading activity of ε. Combination of mutagenesis with biophysical studies and single‐molecule leading‐strand replication assays traced this activity to a novel β‐binding site in ε that, in conjunction with the site in α, maintains a closed state of the αεθ–β₂ replicase in the polymerization mode of DNA synthesis. The ε–β interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein–protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states.     

Microbial sensitivity to temperature and sulfate deposition modulates greenhouse gas emissions from peat soils

Peatlands are among the largest natural sources of atmospheric methane (CH₄) worldwide. Microbial processes play a key role in regulating CH₄ emissions from peatland ecosystems, yet the complex interplay between soil substrates and microbial communities in controlling CH₄ emissions as a function of global change remains unclear. Herein, we performed an integrated analysis of multi-omics data sets to provide a comprehensive understanding of the molecular processes driving changes in greenhouse gas (GHG) emissions in peatland ecosystems with increasing temperature and sulfate deposition in a laboratory incubation study. We sought to first investigate how increasing temperatures (4, 21, and 35°C) impact soil microbiome–metabolome interactions; then explore the competition between methanogens and sulfate-reducing bacteria (SRBs) with increasing sulfate concentrations at the optimum temperature for methanogenesis. Our results revealed that peat soil organic matter degradation, mediated by biotic and potentially abiotic processes, is the main driver of the increase in CO₂ production with temperature. In contrast, the decrease in CH₄ production at 35°C was linked to the absence of syntrophic communities and the potential inhibitory effect of phenols on methanogens. Elevated temperatures further induced the microbial communities to develop high growth yield and stress tolerator trait-based strategies leading to a shift in their composition and function. On the other hand, SRBs were able to outcompete methanogens in the presence of non-limiting sulfate concentrations at 21°C, thereby reducing CH₄ emissions. At higher sulfate concentrations, however, the prevalence of communities capable of producing sufficient low-molecular-weight carbon substrates for the coexistence of SRBs and methanogens was translated into elevated CH₄ emissions. The use of omics in this study enhanced our understanding of the structure and interactions among microbes with the abiotic components of the system that can be useful for mitigating GHG emissions from peatland ecosystems in the face of global change.     

Chemostat optimization of biomass production of a mixed bacterial culture utilizing methanol

Summary A continuous culture technique was used to optimize the medium composition and growth conditions of a mixed bacterial culture utilizing methanol. The improved medium resulted in satisfactory growth, high-yield coefficients and gave a product containing reduced polysaccharide concentrations. Optimal growth and biomass yields occurred at pH 6.8 a temperature of 37° C and dissolved oxygen at >20% saturation. The maximum growth rate was 0.58 h^–1 and maximum biomass yield 0.48 g g^–1. The protein content of the product ranged between 81%–83%, and nucleic acid content between 10%–12%, increasing with growth rate. The amino acid profile of the mixed culture product met and, in some cases, exceeded the UN Food and Agricultural Organization standard, indicating a good source of feed protein.Offprint requests to: A. S. Abu-Ruwaida     

Biological yields through agricultural extension activities and services: A case study from Al-Baha region – Kingdom of Saudi Arabia

PurposeThis research determines the extent to which farmers benefit from agricultural extension activities and services through realizing higher biological crop yields in the Al-Baha region of the Kingdom of Saudi Arabia. Additionally, the nature of the relationship between the personal, economic and social characteristics of farmers and the degree to which they benefit from agricultural extension activities and services are studied.MethodologyA simple random sample representing the community of 315 farmers was drawn. Data were collected through a personal interview with a pre-tested questionnaire to satisfy the objectives of the study. Data are subjected to analyses and are interpreted in terms of percentages, arithmetic averages, and standard deviations in addition to the simple Pearson correlation coefficients.ResultsThe results show that most of the respondents depend on personal experience, friends, family and neighbors, merchants and sellers of agricultural production supplies as sources of agricultural information for enhanced crop yields. Multiple regression analysis reveals that both the educational status and the agricultural information sources are important factors that increase farmers benefit from extension activities. Farmers receive most benefits from the extension activities and services from bringing in foreign workers, conducting field trips to farms, and identifying problems faced by farmers.Research limitations/implicationsThe outcome of this questionnaire limits generalization of the findings for other parts of the Kingdom due to geographical and natural resources variations.Practical implicationsThe personal and socio-economic characteristics of the surveyed population provide the extension service with insight into their composition – a basis to formulate workable extension programs. For the extension planners of the area, this effort provides a baseline study and may assist with targeting the appropriate farmers and catering the right solutions to the problems. Such a study will help devise meaningful extension programs and save time and resources.Originality/valueLittle previous research on the usefulness of the Extension Service has been conducted in the region.ConclusionsEffective, well-planned extension programs targeting the educated and mature farmers provided more benefits and helped them obtain higher crop yields.     

Yield depression in methanol-utilizing batch cultures

Summary Yield depression, as opposed to growth inhibition, in batch cultures of methanol-utilizing microorganisms is discussed. Under conditions where the yield coefficient varies, the effect on oxygen demand has been predicted for exponentionally growing cultures.     

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness¹ and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them². This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia³. The male-to-female ratio of mutation rate is estimated at about 4–6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males⁴. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.     

Reduction of nucleic acid content in cell biomass of methanol-utilizing mixed bacterial culture by heat treatment

Summary A heat treatment method to reduce nucleic acid content in cell biomass of a mixed methanol-utilizing bacterial culture was studied. Maximum nucleic acid reduction in the bacterial cells was achieved by using heat shock at 65°C for 5–10 min followed by 2 h incubation at 55°C and 7.2±0.2 pH. In this treatment, 81–85% nucleic acid content was removed from the cells without affecting their true protein content and essential amino acids profile.