Reconstitution of a functional IS608 single-strand transpososome: role of non-canonical base pairing

Single-stranded (ss) transposition, a recently identified mechanism adopted by members of the widespread IS200/IS605 family of insertion sequences (IS), is catalysed by the transposase, TnpA. The transposase of IS608, recognizes subterminal imperfect palindromes (IP) at both IS ends and cleaves at sites located at some distance. The cleavage sites, C, are not recognized directly by the protein but by short sequences 5′ to the foot of each IP, guide (G) sequences, using a network of canonical (‘Watson–Crick’) base interactions. In addition a set of non-canonical base interactions similar to those found in RNA structures are also involved. We have reconstituted a biologically relevant complex, the transpososome, including both left and right ends and TnpA, which catalyses excision of a ss DNA circle intermediate. We provide a detailed picture of the way in which the IS608 transpososome is assembled and demonstrate that both C and G sequences are essential for forming a robust transpososome detectable by EMSA. We also address several questions central to the organization and function of the ss transpososome and demonstrate the essential role of non-canonical base interactions in the IS608 ends for its stability by using point mutations which destroy individual non-canonical base interactions

The influence of the accessory genome on bacterial pathogen evolution

Bacterial pathogens exhibit significant variation in their genomic content of virulence factors. This reflects the abundance of strategies pathogens evolved to infect host organisms by suppressing host immunity. Molecular arms-races have been a strong driving force for the evolution of pathogenicity, with pathogens often encoding overlapping or redundant functions, such as type III protein secretion effectors and hosts encoding ever more sophisticated immune systems. The pathogens’ frequent exposure to other microbes, either in their host or in the environment, provides opportunities for the acquisition or interchange of mobile genetic elements. These DNA elements accessorise the core genome and can play major roles in shaping genome structure and altering the complement of virulence factors. Here, we review the different mobile genetic elements focusing on the more recent discoveries and highlighting their role in shaping bacterial pathogen evolution

Enteropathogen Resource Integration Center (ERIC): bioinformatics support for research on biodefense-relevant enterobacteria

ERIC, the Enteropathogen Resource Integration Center (www.ericbrc.org), is a new web portal serving as a rich source of information about enterobacteria on the NIAID established list of Select Agents related to biodefense—diarrheagenic Escherichia coli, Shigella spp., Salmonella spp., Yersinia enterocolitica and Yersinia pestis. More than 30 genomes have been completely sequenced, many more exist in draft form and additional projects are underway. These organisms are increasingly the focus of studies using high-throughput experimental technologies and computational approaches. This wealth of data provides unprecedented opportunities for understanding the workings of basic biological systems and discovery of novel targets for development of vaccines, diagnostics and therapeutics. ERIC brings information together from disparate sources and supports data comparison across different organisms, analysis of varying data types and visualization of analyses in human and computer-readable formats

Azospirillum Genomes Reveal Transition of Bacteria from Aquatic to Terrestrial Environments

Fossil records indicate that life appeared in marine environments ∼3.5 billion years ago (Gyr) and transitioned to terrestrial ecosystems nearly 2.5 Gyr. Sequence analysis suggests that “hydrobacteria” and “terrabacteria” might have diverged as early as 3 Gyr. Bacteria of the genus Azospirillum are associated with roots of terrestrial plants; however, virtually all their close relatives are aquatic. We obtained genome sequences of two Azospirillum species and analyzed their gene origins. While most Azospirillum house-keeping genes have orthologs in its close aquatic relatives, this lineage has obtained nearly half of its genome from terrestrial organisms. The majority of genes encoding functions critical for association with plants are among horizontally transferred genes. Our results show that transition of some aquatic bacteria to terrestrial habitats occurred much later than the suggested initial divergence of hydro- and terrabacterial clades. The birth of the genus Azospirillum approximately coincided with the emergence of vascular plants on land

CGAT: a comparative genome analysis tool for visualizing alignments in the analysis of complex evolutionary changes between closely related genomes

BACKGROUND: The recent accumulation of closely related genomic sequences provides a valuable resource for the elucidation of the evolutionary histories of various organisms. However, although numerous alignment calculation and visualization tools have been developed to date, the analysis of complex genomic changes, such as large insertions, deletions, inversions, translocations and duplications, still presents certain difficulties. RESULTS: We have developed a comparative genome analysis tool, named CGAT, which allows detailed comparisons of closely related bacteria-sized genomes mainly through visualizing middle-to-large-scale changes to infer underlying mechanisms. CGAT displays precomputed pairwise genome alignments on both dotplot and alignment viewers with scrolling and zooming functions, and allows users to move along the pre-identified orthologous alignments. Users can place several types of information on this alignment, such as the presence of tandem repeats or interspersed repetitive sequences and changes in G+C contents or codon usage bias, thereby facilitating the interpretation of the observed genomic changes. In addition to displaying precomputed alignments, the viewer can dynamically calculate the alignments between specified regions; this feature is especially useful for examining the alignment boundaries, as these boundaries are often obscure and can vary between programs. Besides the alignment browser functionalities, CGAT also contains an alignment data construction module, which contains various procedures that are commonly used for pre- and post-processing for large-scale alignment calculation, such as the split-and-merge protocol for calculating long alignments, chaining adjacent alignments, and ortholog identification. Indeed, CGAT provides a general framework for the calculation of genome-scale alignments using various existing programs as alignment engines, which allows users to compare the outputs of different alignment programs. Earlier versions of this program have been used successfully in our research to infer the evolutionary history of apparently complex genome changes between closely related eubacteria and archaea. CONCLUSION: CGAT is a practical tool for analyzing complex genomic changes between closely related genomes using existing alignment programs and other sequence analysis tools combined with extensive manual inspection

Complex organic matter in Titan's atmospheric aerosols from in situ pyrolysis and analysis

Aerosols in Titan's atmosphere play an important role in determining its thermal structure(1-3). They also serve as sinks for organic vapours(4) and can act as condensation nuclei for the formation of clouds(5,6), where the condensation efficiency will depend on the chemical composition of the aerosols(5,7). So far, however, no direct information has been available on the chemical composition of these particles. Here we report an in situ chemical analysis of Titan's aerosols by pyrolysis at 600 degrees C. Ammonia (NH3) and hydrogen cyanide (HCN) have been identified as the main pyrolysis products. This clearly shows that the aerosol particles include a solid organic refractory core. NH3 and HCN are gaseous chemical fingerprints of the complex organics that constitute this core, and their presence demonstrates that carbon and nitrogen are in the aerosols.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62786/1/nature04349.pd

Insight from the draft genome of Dietzia cinnamea P4 reveals mechanisms of survival in complex tropical soil habitats and biotechnology potential

The draft genome of Dietzia cinnamea strain P4 was determined using pyrosequencing. In total, 428 supercontigs were obtained and analyzed. We here describe and interpret the main features of the draft genome. The genome contained a total of 3,555,295 bp, arranged in a single replicon with an average G+C percentage of 70.9%. It revealed the presence of complete pathways for basically all central metabolic routes. Also present were complete sets of genes for the glyoxalate and reductive carboxylate cycles. Autotrophic growth was suggested to occur by the presence of genes for aerobic CO oxidation, formate/formaldehyde oxidation, the reverse tricarboxylic acid cycle and the 3-hydropropionate cycle for CO2 fixation. Secondary metabolism was evidenced by the presence of genes for the biosynthesis of terpene compounds, frenolicin, nanaomycin and avilamycin A antibiotics. Furthermore, a probable role in azinomycin B synthesis, an important product with antitumor activity, was indicated. The complete alk operon for the degradation of n-alkanes was found to be present, as were clusters of genes for biphenyl ring dihydroxylation. This study brings new insights in the genetics and physiology of D. cinnamea P4, which is useful in biotechnology and bioremediation

Comparative Genomics Study of Multi-Drug-Resistance Mechanisms in the Antibiotic-Resistant Streptococcus suis R61 Strain

BACKGROUND: Streptococcus suis infections are a serious problem for both humans and pigs worldwide. The emergence and increasing prevalence of antibiotic-resistant S. suis strains pose significant clinical and societal challenges. RESULTS: In our study, we sequenced one multi-drug-resistant S. suis strain, R61, and one S. suis strain, A7, which is fully sensitive to all tested antibiotics. Comparative genomic analysis revealed that the R61 strain is phylogenetically distinct from other S. suis strains, and the genome of R61 exhibits extreme levels of evolutionary plasticity with high levels of gene gain and loss. Our results indicate that the multi-drug-resistant strain R61 has evolved three main categories of resistance. CONCLUSIONS: Comparative genomic analysis of S. suis strains with diverse drug-resistant phenotypes provided evidence that horizontal gene transfer is an important evolutionary force in shaping the genome of multi-drug-resistant strain R61. In this study, we discovered novel and previously unexamined mutations that are strong candidates for conferring drug resistance. We believe that these mutations will provide crucial clues for designing new drugs against this pathogen. In addition, our work provides a clear demonstration that the use of drugs has driven the emergence of the multi-drug-resistant strain R61