Description: Over recent years, several PCR primers have been described to amplify genes encoding the structural subunits of ammonia monooxygenase (AMO) from ammonia-oxidizing bacteria (AOB). Most of them target amoA, while amoB and amoC have been neglected so far. This study compared the nucleotide sequence of 33 primers that have been used to amplify different regions of the amoCAB operon with alignments of all available sequences in public databases. The advantages and disadvantages of these primers are discussed based on the original description and the spectrum of matching sequences obtained. Additionally, new primers to amplify the almost complete amoCAB operon of AOB belonging to Betaproteobacteria (betaproteobacterial AOB), a primer pair for DGGE analysis of amoA and specific primers for gammaproteobacterial AOB, are also described. The specificity of these new primers was also evaluated using the databases of the sequences created during this study.

Description: The oxidation of ammonia plays a significant role in the transformation of fixed nitrogen in the global nitrogen cycle. Autotrophic ammonia oxidation is known in three groups of microorganisms. Aerobic ammonia-oxidizing bacteria and archaea convert ammonia into nitrite during nitrification. Anaerobic ammonia-oxidizing bacteria (anammox) oxidize ammonia using nitrite as electron acceptor and producing atmospheric dinitrogen. The isolation and cultivation of all three groups in the laboratory are quite problematic due to their slow growth rates, poor growth yields, unpredictable lag phases, and sensitivity to certain organic compounds. Culture-independent approaches have contributed importantly to our understanding of the diversity and distribution of these microorganisms in the environment. In this review, we present an overview of approaches that have been used for the molecular study of ammonia oxidizers and discuss their application in different environments.

Description: The genes and intergenic regions of the amoCAB operon were analyzed to establish their potential as molecular markers for analyzing ammonia-oxidizing betaproteobacterial (beta-AOB) communities. Initially, sequence similarity for related taxa, evolutionary rates from linear regressions, and the presence of conserved and variable regions were analyzed for all available sequences of the complete amoCAB operon. The gene amoB showed the highest sequence variability of the three amo genes, suggesting that it might be a better molecular marker than the most frequently used amoA to resolve closely related AOB species. To test the suitability of using the amoCAB genes for community studies, a strategy involving nested PCR was employed. Primers to amplify the whole amoCAB operon and each individual gene were tested. The specificity of the products generated was analyzed by denaturing gradient gel electrophoresis, cloning, and sequencing. The fragments obtained showed different grades of sequence identity to amoCAB sequences in the GenBank database. The nested PCR approach provides a possibility to increase the sensitivity of detection of amo genes in samples with low abundance of AOB. It also allows the amplification of the almost complete amoA gene, with about 300 bp more sequence information than the previous approaches. The coupled study of all three amo genes and the intergenic spacer regions that are under different selection pressure might allow a more detailed analysis of the evolutionary processes, which are responsible for the differentiation of AOB communities in different habitats.

Description: The mammalian mitochondrial genomes differ from the nuclear genomes by maternal inheritance, absence of recombination, and higher mutation rate. All these differences decrease the effective population size of mitochondrial genome and make it more susceptible to accumulation of slightly deleterious mutations. It was hypothesized that mitochondrial genes, especially in species with low effective population size, irreversibly degrade leading to decrease of organismal fitness and even to extinction of species through the mutational meltdown. To interrogate this hypothesis, we compared the purifying selections acting on the representative set of mitochondrial (potentially degrading) and nuclear (potentially not degrading) protein-coding genes in species with different effective population size. For 21 mammalian species, we calculated the ratios of accumulation of slightly deleterious mutations approximated by Kn/Ks separately for mitochondrial and nuclear genomes. The 75% of variation in Kn/Ks is explained by two independent variables: type of a genome (mitochondrial or nuclear) and effective population size of species approximated by generation time. First, we observed that purifying selection is more effective in mitochondria than in the nucleus that implies strong evolutionary constraints of mitochondrial genome. Mitochondrial de novo nonsynonymous mutations have at least 5-fold more harmful effect when compared with nuclear. Second, Kn/Ks of mitochondrial and nuclear genomes is positively correlated with generation time of species, indicating relaxation of purifying selection with decrease of species-specific effective population size. Most importantly, the linear regression lines of mitochondrial and nuclear Kn/Ks's from generation times of species are parallel, indicating congruent relaxation of purifying selection in both genomes. Thus, our results reveal that the distribution of selection coefficients of de novo nonsynonymous mitochondrial mutations has a similar shape with the distribution of de novo nonsynonymous nuclear mutations, but its mean is five times smaller. The harmful effect of mitochondrial de novo nonsynonymous mutations triggers highly effective purifying selection, which maintains the fitness of the mammalian mitochondrial genome.

Description: Bacterial endospores are highly specialized cellular forms that allow endospore-forming Firmicutes (EFF) to tolerate harsh environmental conditions. EFF are considered ubiquitous in natural environments, in particular, those subjected to stress conditions. In addition to natural habitats, EFF are often the cause of contamination problems in anthropogenic environments, such as industrial production plants or hospitals. It is therefore desirable to assess their prevalence in environmental and industrial fields. To this end, a high-sensitivity detection method is still needed. The aim of this study was to develop and evaluate an approach based on quantitative PCR (qPCR). For this, the suitability of functional genes specific for and common to all EFF were evaluated. Seven genes were considered, but only spo0A was retained to identify conserved regions for qPCR primer design. An approach based on multivariate analysis was developed for primer design. Two primer sets were obtained and evaluated with 16 pure cultures, including representatives of the genera Bacillus , Paenibacillus , Brevibacillus , Geobacillus , Alicyclobacillus , Sulfobacillus , Clostridium , and Desulfotomaculum , as well as with environmental samples. The primer sets developed gave a reliable quantification when tested on laboratory strains, with the exception of Sulfobacillus and Desulfotomaculum . A test using sediment samples with a diverse EFF community also gave a reliable quantification compared to 16S rRNA gene pyrosequencing. A detection limit of about 10 4 cells (or spores) per gram of initial material was calculated, indicating this method has a promising potential for the detection of EFF over a wide range of applications.

Description: By identifying genomic sequence regions conserved among several species, comparative genomics offers opportunities to discover putatively functional elements without any prior knowledge of what these functions might be. Comparative analyses across mammals estimated 4–5% of the human genome to be functionally constrained, a much larger fraction than the 1–2% occupied by annotated protein-coding or RNA genes. Such functionally constrained yet unannotated regions have been referred to as conserved non-coding sequences (CNCs) or ultra-conserved elements (UCEs), which remain largely uncharacterized but probably form a highly heterogeneous group of elements including enhancers, promoters, motifs, and others. To facilitate the study of such CNCs/UCEs, we present our resource of Conserved Elements from Genomic Alignments (CEGA), accessible from http://cega.ezlab.org . Harnessing the power of multiple species comparisons to detect genomic elements under purifying selection, CEGA provides a comprehensive set of CNCs identified at different radiations along the vertebrate lineage. Evolutionary constraint is identified using threshold-free phylogenetic modeling of unbiased and sensitive global alignments of genomic synteny blocks identified using protein orthology. We identified CNCs independently for five vertebrate clades, each referring to a different last common ancestor and therefore to an overlapping but varying set of CNCs with 24 488 in vertebrates, 241 575 in amniotes, 709 743 in Eutheria, 642 701 in Boreoeutheria and 612 364 in Euarchontoglires, spanning from 6 Mbp in vertebrates to 119 Mbp in Euarchontoglires. The dynamic CEGA web interface displays alignments, genomic locations, as well as biologically relevant data to help prioritize and select CNCs of interest for further functional investigations.