bacterial repetitive extragenic palindromes (REP) Dotaz Zobrazit nápovědu
BACKGROUND: Bacterial repetitive extragenic palindromes (REPs) compose a distinct group of genomic repeats. They usually occur in high abundance (>100 copies/genome) and are often arranged in composite repetitive structures - bacterial interspersed mosaic elements (BIMEs). In BIMEs, regularly spaced REPs are present in alternating orientations. BIMEs and REPs have been shown to serve as binding sites for several proteins and suggested to play role in chromosome organization and transcription termination. Their origins are, at present, unknown. RESULTS: In this report, we describe a novel class of putative transposases related to IS200/IS605 transposase family and we demonstrate that they are obligately associated with bacterial REPs. Open reading frames coding for these REP-associated tyrosine transposases (RAYTs) are always flanked by two REPs in inverted orientation and thus constitute a unit reminiscent of typical transposable elements. Besides conserved residues involved in catalysis of DNA cleavage, RAYTs carry characteristic structural motifs that are absent in typical IS200/IS605 transposases. DNA sequences flanking rayt genes are in one third of examined cases arranged in modular BIMEs. RAYTs and their flanking REPs apparently coevolve with each other. The rayt genes themselves are subject to rapid evolution, substantially exceeding the substitution rate of neighboring genes. Strong correlation was found between the presence of a particular rayt in a genome and the abundance of its cognate REPs. CONCLUSIONS: In light of our findings, we propose that RAYTs are responsible for establishment of REPs and BIMEs in bacterial genomes, as well as for their exceptional dynamics and species-specifity. Conversely, we suggest that BIMEs are in fact a special type of nonautonomous transposable elements, mobilizable by RAYTs.
- MeSH
- Bacteria genetika chemie MeSH
- fylogeneze MeSH
- genom bakteriální MeSH
- konzervovaná sekvence MeSH
- molekulární evoluce MeSH
- molekulární sekvence - údaje MeSH
- obrácené repetice MeSH
- otevřené čtecí rámce MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- transposasy analýza genetika chemická syntéza metabolismus MeSH
- tyrosin metabolismus MeSH
- Publikační typ
- práce podpořená grantem MeSH
Repetitive extragenic palindrome (REP)-associated tyrosine transposase enzymes (RAYTs) bind REP DNA domains and catalyze their cleavage. Genomic sequence analyses identify potential noncoding REP sequences associated with RAYT-encoding genes. To probe the conformational space of potential RAYT DNA binding domains, we report here spectroscopic and calorimetric measurements that detect and partially characterize the solution conformational heterogeneity of REP oligonucleotides from six bacterial species. Our data reveal most of these REP oligonucleotides adopt multiple conformations, suggesting that RAYTs confront a landscape of potential DNA substrates in dynamic equilibrium that could be selected, enriched, and/or induced via differential binding. Thus, the transposase-bound DNA motif may not be the predominant conformation of the isolated REP domain. Intriguingly, for several REPs, the circular dichroism spectra suggest guanine tetraplexes as potential alternative or additional RAYT recognition elements, an observation consistent with these REP domains being highly nonrandom, with tetraplex-favoring 5'-G and 3'-C-rich segments. In fact, the conformational heterogeneity of REP domains detected and reported here, including the formation of noncanonical DNA secondary structures, may reflect a general feature required for recognition by RAYT transposases. Based on our biophysical data, we propose guanine tetraplexes as an additional DNA recognition element for binding by RAYT transposase enzymes.
BACKGROUND: Repetitive extragenic palindromic elements (REPs) constitute a group of bacterial genomic repeats known for their high abundance and several roles in host cells´ physiology. We analyzed the phylogenetic distribution of particular REP classes in genomic sequences of sixty-three bacterial strains belonging to the Pseudomonas fluorescens species complex and ten strains of Stenotrophomonas sp., in order to assess intraspecific REP diversity and to gain insight into long-term REP evolution. RESULTS: Based on proximity to RAYT (REP-associated tyrosine transposase) genes, twenty-two and thirteen unique REP classes were determined in fluorescent pseudomonads and stenotrophomonads, respectively. In stenotrophomonads, REP elements were typically found in tens or a few hundred copies per genome. REPs of fluorescent pseudomonads were generally more numerous, occurring in hundreds or even over a thousand perfect copies of particular REP class per genome. REP sequences showed highly heterogeneous distribution. The abundances of REP classes roughly followed host strains´ phylogeny, differing markedly among individual clades. High abundances of particular REP classes appeared to depend on the presence of the cognate RAYT gene, and deviations from this state could be attributed to recent or ancient mutations of rayt-flanking REPs, or RAYT loss. RAYTs of both studied bacterial groups are monophyletic, and their cognate REPs show species-specific characteristics, suggesting shared evolutionary history of REPs, RAYTs and their hosts. CONCLUSIONS: The results of our large-scale analysis show that REP elements constitute intriguingly dynamic components of genomes of fluorescent pseudomonads and stenotrophomonads, and indicate that REP diversification and proliferation are ongoing processes. High numbers of REPs have apparently been retained during the entire evolutionary time since the establishment of these two bacterial lineages, probably because of their beneficial effect on host long-term fitness. REP elements in these bacteria represent a suitable platform to study the interplay between repeated elements, their mobilizers and host bacterial cells.
AIMS: The aim of this study was to develop a simple protocol for a PCR-based fingerprinting of Stenotrophomonas maltophilia (SmrepPCR) that utilizes primers complementary to repetitive extragenic palindromic elements (REPs) of this micro-organism. METHODS AND RESULTS: The relatedness of 34 isolates of environmental and clinical origin was investigated by two SmrepPCRs with two different primers, gyrB sequencing and XbaI macrorestriction followed by pulsed-field gel electrophoresis. While SmrepPCR (with primer DIR) results matched data obtained from the analysis of gyrB nucleotide sequences and identified several clonal complexes, XbaI macrorestriction showed high level of heterogeneity between isolates. The macrorestriction-based clustering of isolates did not correspond to both gyrB and DIR-SmrepPCR grouping. CONCLUSIONS: Our results show that SmrepPCR-inferred relationship of isolates is in a good agreement with sequence-based methods. The combined information from all methods used suggests that rapid evolution of S. maltophilia genomes might be predominantly due to high rate of rearrangements caused by mobile genetic elements. SIGNIFICANCE AND IMPACT OF THE STUDY: The presented method is an inexpensive and easy to perform alternative to genotype S. maltophilia isolates and to study their population genetics. SmrepPCR demonstrates the usefulness of species-specific repetitive elements in genomic analyses.
- MeSH
- DNA bakterií analýza genetika MeSH
- DNA fingerprinting MeSH
- druhová specificita MeSH
- fylogeneze MeSH
- lidé MeSH
- obrácené repetice MeSH
- polymerázová řetězová reakce metody MeSH
- pulzní gelová elektroforéza MeSH
- Stenotrophomonas maltophilia genetika izolace a purifikace MeSH
- variabilita počtu kopií segmentů DNA MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The process of DNA transposition involves the binding, cleavage, and recombination of specific DNA segments (transposable elements, TE) and is catalyzed by special enzymes encoded by the TE transposases. REP-associated tyrosine transposases (RAYTs) are a class of Y1 nucleases related to the IS200/IS605 transposases associated with a bacterial TE known as repetitive extragenic palindrome elements (REPs). Although RAYT has been subject of numerous studies, where DNA binding and cleavage by RAYT have been confirmed for Escherichia coli, the molecular mechanism of DNA insertion has not been fully understood. In this work, it is demonstrated that surface plasmon resonance (SPR) biosensor technology combined with a system of DNA hairpin probes (mimicking the natural REP sequence) and short oligonucleotides (ONs) can provide a rapid and real-time platform for monitoring and quantification of RAYT activity. We utilized RAYT from E. coli (strain MG1655) as a model system, where we evaluated its activity towards both a natural REP sequence as well as REP sequences having modifications targeting specific features of the DNA crucial for the DNA binding and cleavage. The characteristics of the RAYT-DNA interaction obtained by means of the SPR approach were compared with the results of SDS-PAGE analysis.
- MeSH
- biosenzitivní techniky přístrojové vybavení metody MeSH
- Escherichia coli enzymologie MeSH
- povrchová plasmonová rezonance přístrojové vybavení metody MeSH
- proteiny z Escherichia coli chemie genetika metabolismus MeSH
- regulace genové exprese enzymů fyziologie MeSH
- regulace genové exprese u bakterií fyziologie MeSH
- transposasy chemie genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
