Easy and Effective Method for Extracting and Purifying Wolbachia Genomic DNA.

A number of methods for extracting the DNA of maternally inherited obligate intracellular bacteria Wolbachia from an insect host and its subsequent purification have been described in previous scholarship. As Wolbachia is present in the hosts' organisms in rather low quantities, these techniques used to be quite labor-intensive. For this paper, we analyzed them in detail, searched for a possibility to simplify and accelerate the protocol, and proposed an easy and effective method for isolating Wolbachia DNA from Drosophila melanogaster with a purity sufficient for genomic sequencing. Our method involves the centrifugation of homogenized flies or just their ovaries, as the most Wolbachia-enriched tissue, followed by the filtration of homogenate and extraction of DNA using a modified version of the Livak buffer protocol. The proportion of Wolbachia DNA in the total DNA was quantified based on the results of sequencing with the use of the Illumina MiSeq platform and a pipeline of bioinformatic analysis. For the two analyzed D. melanogaster lines infected with two different Wolbachia strains, the proportion was at least 68 and 94%, respectively.

New Wolbachia pipientis Genotype Increasing Heat Stress Resistance of Drosophila melanogaster Host Is Characterized by a Large Chromosomal Inversion.

The maternally transmitted endocellular bacteria Wolbachia is a well-known symbiont of insects, demonstrating both negative and positive effects on host fitness. The previously found Wolbachia strain wMelPlus is characterized by a positive effect on the stress-resistance of its host Drosophila melanogaster, under heat stress conditions. This investigation is dedicated to studying the genomic underpinnings of such an effect. We sequenced two closely related Wolbachia strains, wMelPlus and wMelCS112, assembled their complete genomes, and performed comparative genomic analysis engaging available Wolbachia genomes from the wMel and wMelCS groups. Despite the two strains under study sharing very close gene-composition, we discovered a large (>1/6 of total genome) chromosomal inversion in wMelPlus, spanning through the region that includes the area of the inversion earlier found in the wMel group of Wolbachia genotypes. A number of genes in unique inversion blocks of wMelPlus were identified that might be involved in the induction of a stress-resistant phenotype in the host. We hypothesize that such an inversion could rearrange established genetic regulatory-networks, causing the observed effects of such a complex fly phenotype as a modulation of heat stress resistance. Based on our findings, we propose that wMelPlus be distinguished as a separate genotype of the wMelCS group, named wMelCS3.

Bioinformatic Assessment of Factors Affecting the Correlation between Protein Abundance and Elongation Efficiency in Prokaryotes.

Protein abundance is crucial for the majority of genetically regulated cell functions to act properly in prokaryotic organisms. Therefore, developing bioinformatic methods for assessing the efficiency of different stages of gene expression is of great importance for predicting the actual protein abundance. One of these steps is the evaluation of translation elongation efficiency based on mRNA sequence features, such as codon usage bias and mRNA secondary structure properties. In this study, we have evaluated correlation coefficients between experimentally measured protein abundance and predicted elongation efficiency characteristics for 26 prokaryotes, including non-model organisms, belonging to diverse taxonomic groups The algorithm for assessing elongation efficiency takes into account not only codon bias, but also number and energy of secondary structures in mRNA if those demonstrate an impact on predicted elongation efficiency of the ribosomal protein genes. The results show that, for a number of organisms, secondary structures are a better predictor of protein abundance than codon usage bias. The bioinformatic analysis has revealed several factors associated with the value of the correlation coefficient. The first factor is the elongation efficiency optimization type-the organisms whose genomes are optimized for codon usage only have significantly higher correlation coefficients. The second factor is taxonomical identity-bacteria that belong to the class Bacilli tend to have higher correlation coefficients among the analyzed set. The third is growth rate, which is shown to be higher for the organisms with higher correlation coefficients between protein abundance and predicted translation elongation efficiency. The obtained results can be useful for further improvement of methods for protein abundance prediction.