Phylogenetic tree of tRNAs using a simple algorithm

A simple method for phylogenetic tree construction is described. In this method each node is calculated considering the distance between the elements and the difference between these elements and an average element, allowing the selection of the most probable node.Two examples of tRNA phylogenies (E. coli set and Phe family) are analyzed, giving both reliable trees. Data from these dendrograms give support to the idea of an early cloverleaf arising.     

Identification of critical batch operating parameters in fed-batch recombinant E. coli fermentations using decision tree analysis

To develop a useful fermentation process model, it is first necessary to identify which batch operating parameters are critical in determining the process outcome. To identify critical processing inputs in large databases, we have explored the use of Decision Tree Analysis with the decision metrics of Gain (i.e., Shannon Entropy changes), Gain Ratio, and a multiple hypergeometric distribution. The usefulness of this approach lies in its ability to treat "categorical" variables, which are typical of archived fermentation databases, as well as "continuous" variables. In this work, we demonstrate the use of Decision Tree Analysis for the problem of optimizing recombinant green fluorescent protein production in E. coli. A database of 85 fermentations was generated to examine the effect of 15 process input parameters on final biomass yield, maximum recombinant protein concentration, and productivity. The use of Decision Tree Analysis led to a considerable reduction in the fermentation database through the identification of the significant as well as insignificant inputs. However, different decision metrics selected different inputs and different numbers of inputs to classify the data for each output.     

Phylogenetic tree construction using sequential stochastic approximation Monte Carlo

Monte Carlo methods have received much attention recently in the literature of phylogenetic tree construction. However, they often suffer from two difficulties, the curse of dimensionality and the local-trap problem. The former one is due to that the number of possible phylogenetic trees increases at a super-exponential rate as the number of taxa increases. The latter one is due to that the phylogenetic tree has often a rugged energy landscape. In this paper, we propose a new phylogenetic tree construction method, which attempts to alleviate these two difficulties simultaneously by making use of the sequential structure of phylogenetic trees in conjunction with stochastic approximation Monte Carlo (SAMC) simulations. The use of the sequential structure of the problem provides substantial help to reduce the curse of dimensionality in simulations, and SAMC effectively prevents the system from getting trapped in local energy minima. The new method is compared with a variety of existing Bayesian and non-Bayesian methods on simulated and real datasets. Numerical results are in favor of the new method in terms of quality of the resulting phylogenetic trees.     

Elucidating acetate tolerance in E. coli using a genome-wide approach

Engineering organisms for improved performance using lignocellulose feedstocks is an important step towards a sustainable fuel and chemical industry. Cellulosic feedstocks contain carbon and energy in the form of cellulosic and hemicellulosic sugars that are not metabolized by most industrial microorganisms. Pretreatment processes that hydrolyze these polysaccharides often also result in the accumulation of growth inhibitory compounds, such as acetate and furfural among others. Here, we have applied a recently reported strategy for engineering tolerance towards the goal of increasing Escherichia coli growth in the presence of elevated acetate concentrations (Lynch et al., 2007). We performed growth selections upon an E. coli genome library developed using a moderate selection pressure to identify genomic regions implicated in acetate toxicity and tolerance. These studies identified a range of high-fitness genes that are normally involved in membrane and extracellular processes, are key regulated steps in pathways, and are involved in pathways that yield specific amino acids and nucleotides. Supplementation of the products and metabolically related metabolites of these pathways significantly increased growth rate (a 130% increase in specific growth) at inhibitory acetate concentrations. Our results suggest that acetate tolerance will not involve engineering of a single pathway; rather we observe a range of potential mechanisms for overcoming acetate based inhibition.     

Binding of E. coli DNA photolyase to a defined substrate containing a single T mean value of T dimer.

The E. coli DNA photolyase is a flavoprotein that catalyzes the photoreversal of pyrimidine dimers. The enzyme binds to DNA containing pyrimidine dimers in a light-independent step and repairs the dimer upon absorbing a photon in the 300-600 nm range. The rate and equilibrium constants for the light-independent reaction were determined before, using randomly modified substrates that contained T mean value of T, T mean value of C and C mean value of C dimers in random sequence surrounding. In this paper we have determined these constants for a defined substrate (a 43 bp oligomer containing a T mean value of T dimer) using the gel retardation assay. We find that: the equilibrium constant and the off rate obtained with this substrate by this technique are similar to those obtained with randomly modified DNA using filter binding and flash photolysis techniques. the off rate with the defined substrate is heterogeneous indicating heterogeneity in the enzyme population or in the enzyme-substrate complexes, and the enzyme has 7.5 X 10(4)-fold higher affinity for pyrimidine dimer compared to non-dimer DNA nucleotides.     

Phylogenetic mixtures on a single tree can mimic a tree of another topology

Phylogenetic mixtures model the inhomogeneous molecular evolution commonly observed in data. The performance of phylogenetic reconstruction methods where the underlying data are generated by a mixture model has stimulated considerable recent debate. Much of the controversy stems from simulations of mixture model data on a given tree topology for which reconstruction algorithms output a tree of a different topology; these findings were held up to show the shortcomings of particular tree reconstruction methods. In so doing, the underlying assumption was that mixture model data on one topology can be distinguished from data evolved on an unmixed tree of another topology given enough data and the "correct" method. Here we show that this assumption can be false. For biologists, our results imply that, for example, the combined data from two genes whose phylogenetic trees differ only in terms of branch lengths can perfectly fit a tree of a different topology.     

Purification of histidine-tagged T4 RNA ligase from E. coli

Here we report the construction of a histidine-tagged T4 RNA ligase expression plasmid (pRHT4). The construct, when overexpressed in BL21 (DE3) cells, allows the preparation of large quantities of T4 RNA ligase in high purity using only a single purification column. The histidine affinity tag does not inhibit enzyme function, and we were able to purify 1-3 mg pure protein/g cell pellet. A simple purification procedure ensures that the enzyme is de-adenylated to levels comparable to those found for many commercial preparations. The purified protein has very low levels of RNase contamination and functioned normally in a variety of activity assays.     

A simple algorithm to infer gene duplication and speciation events on a gene tree

MOTIVATION: When analyzing protein sequences using sequence similarity searches, orthologous sequences (that diverged by speciation) are more reliable predictors of a new protein's function than paralogous sequences (that diverged by gene duplication), because duplication enables functional diversification. The utility of phylogenetic information in high-throughput genome annotation ('phylogenomics') is widely recognized, but existing approaches are either manual or indirect (e.g. not based on phylogenetic trees). Our goal is to automate phylogenomics using explicit phylogenetic inference. A necessary component is an algorithm to infer speciation and duplication events in a given gene tree. RESULTS: We give an algorithm to infer speciation and duplication events on a gene tree by comparison to a trusted species tree. This algorithm has a worst-case running time of O(n(2)) which is inferior to two previous algorithms that are approximately O(n) for a gene tree of sequences. However, our algorithm is extremely simple, and its asymptotic worst case behavior is only realized on pathological data sets. We show empirically, using 1750 gene trees constructed from the Pfam protein family database, that it appears to be a practical (and often superior) algorithm for analyzing real gene trees. AVAILABILITY: http://www.genetics.wustl.edu/eddy/forester.     

Laser crosslinking of E. coli RNA polymerase and T7 DNA.

The first photochemical crosslinking of a protein to a nucleic acid using laser excitation is reported. A single, 120 mJ, 20 ns pulse at 248 nm crosslinks about 10% of bound E. coli RNA polymerase to T7 DNA under the conditions studied. The crosslinking yield depends on mercaptoethanol concentration, and is a linear function of laser intensity. The protein subunits crosslinked to DNA are beta, beta' and sigma.     

Phylogenetic analysis using insertion sequence fingerprinting in Escherichia coli

Chromosomal DNA from 23 closely related, pathogenic strains of Escherichia coli was digested and probed for the insertion sequences IS1, IS2, IS4, IS5, and IS30. Under the assumption that elements residing in DNA restriction fragments of the same apparent length are identical by descent, parsimony analysis of these characters yielded a unique phylogenetic tree. This analysis not only distinguished among bacterial strains that were otherwise identical in their biochemical characteristics and enzyme electrophoretic mobilities, but certain aspects of the topology of the tree were consistent across several unrelated insertion elements. The distribution of IS elements was then reexamined in light of the inferred phylogenetic relationships to investigate the biological properties of the elements, such as rates of insertion and deletion, and to discover apparent recombinational events. The analysis shows that the pattern of distribution of insertion elements in the bacterial genome is sufficiently stable for epidemiological studies. Although the rate of recombination by conjugation has been postulated to be low, at least two such events appear to have taken place.      

Nucleotide sequences of the gal E gene and the gal T gene of E. coli.

The nucleotide sequences of the gal E gene coding for UDP-galactose-4-epimerase and the gal T gene coding for galactose-1-P uridyltransferase of Escherichia coli have been determined. UDP-galactose-4-epimerase and galactose-1-P uridyltransferase are predicted to consist of 338 and 347 residues, respectively, NH2-terminal methionines included.     

Evaluation of a simple,rapid and field-adapted diagnostic assay for enterotoxigenic E. coli and Shigella

Understanding the global burden of enterotoxigenic E. coli (ETEC) and Shigella diarrhea as well as estimating the cost effectiveness of vaccines to control these two significant pathogens have been hindered by the lack of a diagnostic test that is rapid, simple, sensitive, and can be applied to the endemic countries. We previously developed a simple and rapid assay, Rapid Loop mediated isothermal amplification based Diagnostic Test (RLDT) for the detection of ETEC and Shigella spp. (Shigella). In this study, the RLDT assay was evaluated in comparison with quantitative PCR (qPCR), culture and conventional PCR for the detection of ETEC and Shigella. This validation was performed using previously collected stool samples from endemic countries, from the travelers to the endemic countries, as well as samples from a controlled human infection model study of ETEC. The performance of RLDT from dried stool spots was also validated. RLDT resulted in excellent sensitivity and specificity compared to qPCR (99% and 99.2% respectively) ranging from 92.3 to 100% for the individual toxin genes of ETEC and 100% for Shigella. Culture was less sensitive compared to RLDT. No significant differences were noted in the performance of RLDT using samples from various sources or stool samples from moderate to severe diarrhea or asymptomatic infections. RLDT performed equally well in detection of ETEC and Shigella from the dried stool samples on filter papers. This study established that RLDT is sufficiently sensitive and specific to be used as a simple and rapid diagnostic assay to detect ETEC and Shigella in endemic countries to determine disease burden of these pathogens in the national and subnational levels. This information will be important to guide public health and policy makers to prioritize resources for accelerating the development and introduction of effective preventative and/or treatment interventions against these enteric infections.