Jaroslav Nunvar

Identification and characterization of repetitive extragenic palindromes (REP)-associated tyrosine transposases: implications for REP evolution and dynamics in bacterial genomes

BackgroundBacterial 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.ResultsIn 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.ConclusionsIn 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.

Understanding the Pathogenicity of Burkholderia contaminans, an Emerging Pathogen in Cystic Fibrosis.

Several bacterial species from the Burkholderia cepacia complex (Bcc) are feared opportunistic pathogens that lead to debilitating lung infections with a high risk of developing fatal septicemia in cystic fibrosis (CF) patients. However, the pathogenic potential of other Bcc species is yet unknown. To elucidate clinical relevance of Burkholderia contaminans, a species frequently isolated from CF respiratory samples in Ibero-American countries, we aimed to identify its key virulence factors possibly linked with an unfavorable clinical outcome. We performed a genome-wide comparative analysis of two isolates of B. contaminans ST872 from sputum and blood culture of a female CF patient in Argentina. RNA-seq data showed significant changes in expression for quorum sensing-regulated virulence factors and motility and chemotaxis. Furthermore, we detected expression changes in a recently described low-oxygen-activated (lxa) locus which encodes stress-related proteins, and for two clusters responsible for the biosynthesis of antifungal and hemolytic compounds pyrrolnitrin and occidiofungin. Based on phenotypic assays that confirmed changes in motility and in proteolytic, hemolytic and antifungal activities, we were able to distinguish two phenotypes of B. contaminans that coexisted in the host and entered her bloodstream. Whole genome sequencing revealed that the sputum and bloodstream isolates (each representing a distinct phenotype) differed by over 1,400 mutations as a result of a mismatch repair-deficient hypermutable state of the sputum isolate. The inferred lack of purifying selection against nonsynonymous mutations and the high rate of pseudogenization in the derived isolate indicated limited evolutionary pressure during evolution in the nutrient-rich, stable CF sputum environment. The present study is the first to examine the genomic and transcriptomic differences between longitudinal isolates of B. contaminans. Detected activity of a number of putative virulence factors implies a genuine pathogenic nature of this novel Bcc species.

Monitoring RAYT activity by surface plasmon resonance biosensor

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.

Evolution of REP diversity: a comparative study

BackgroundRepetitive 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.ResultsBased 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.ConclusionsThe 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.

Conformational Diversity of Single-Stranded DNA from Bacterial Repetitive Extragenic Palindromes: Implications for the DNA Recognition Elements of Transposases

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.

Agrobacterium bohemicum sp. nov. isolated from poppy seed wastes in central Bohemia

Two non-pathogenic strains R89-1 and R90T isolated from poppy seed (Papaver somniferum L.) wastes were phenotypically and genotypically characterized. Multilocus sequence analysis (MLSA) was conducted with six genes (atpD, glnA, gyrB, recA, rpoB, 16S rRNA). The strains represented a new species which clustered with Agrobacterium rubi NBRC 13261T and Agrobacterium skierniewicense Ch11T type strains. MLSA was further accompanied by whole-genome phylogeny, in silico DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses for both strains. ANI and dDDH values were deep below the species delineation threshold. Phenotypic features of the novel strains unequivocally allowed their differentiation from all other Agrobacterium species. Unlike other agrobacteria, the strains were salt sensitive and were able to biotransform morphine alkaloids. The dominant cellular fatty acids are 18:1 w7c, 16:0 and 12:0 aldehyde/16:1 iso I/14:0 3OH summed in feature 2 and the major respiratory quinine is Q-10 (87%). The DNA G+C content is 56mol%. Microbial community analysis indicated probable association with P. somniferum plant material. Altogether, these characteristics showed that strains R90T and R89-1 represent a new species of the genus Agrobacterium which we propose to name Agrobacterium bohemicum. The type strain of A. bohemicum is R90T (=CCM 8736T=DSM 104667T).

Draft Genome Sequence of Stenotrophomonas maltophilia Strain 5BA-I-2, a Soil Isolate and a Member of a Phylogenetically Basal Lineage

ABSTRACT Stenotrophomonas maltophilia is an omnipresent environmental bacterium emerging as an opportunistic human pathogen and exhibiting multidrug resistance. Here, we report the draft genome sequence of S. maltophilia strain 5BA-I-2, a soil isolate and a member of a phylogenetically basal lineage.

DNA profiling of Stenotrophomonas maltophilia by PCR targeted to its species-specific repetitive palindromic sequences

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.

The crystal structure of XdpB, the bacterial old yellow enzyme, in an FMN-free form

Old Yellow Enzymes (OYEs) are NAD(P)H dehydrogenases of not fully resolved physiological roles that are widespread among bacteria, plants, and fungi and have a great potential for biotechnological applications. We determined the apo form crystal structure of a member of the OYE class, glycerol trinitrate reductase XdpB, from Agrobacterium bohemicum R89-1 at 2.1 Å resolution. In agreement with the structures of the related bacterial OYEs, the structure revealed the TIM barrel fold with an N-terminal β-hairpin lid, but surprisingly, the structure did not contain its cofactor FMN. Its putative binding site was occupied by a pentapeptide TTSDN from the C-terminus of a symmetry related molecule. Biochemical experiments confirmed a specific concentration-dependent oligomerization and a low FMN content. The blocking of the FMN binding site can exist in vivo and regulates enzyme activity. Our bioinformatic analysis indicated that a similar self-inhibition could be expected in more OYEs which we designated as subgroup OYE C1. This subgroup is widespread among G-bacteria and can be recognized by the conserved sequence GxxDYP in proximity of the C termini. In proteobacteria, the C1 subgroup OYEs are typically coded in one operon with short-chain dehydrogenase. This operon is controlled by the tetR-like transcriptional regulator. OYEs coded in these operons are unlikely to be involved in the oxidative stress response as the other known members of the OYE family because no upregulation of XdpB was observed after exposing A. bohemicum R89-1 to oxidative stress.

What matters in chronic Burkholderia cenocepacia infection in cystic fibrosis: Insights from comparative genomics

Burkholderia cenocepacia causes severe pulmonary infections in cystic fibrosis (CF) patients. Since the bacterium is virtually untreatable by antibiotics, chronic infections persist for years and might develop into fatal septic pneumonia (cepacia syndrome, CS). To devise new strategies to combat chronic B. cenocepacia infections, it is essential to obtain comprehensive knowledge about their pathogenesis. We conducted a comparative genomic analysis of 32 Czech isolates of epidemic clone B. cenocepacia ST32 isolated from various stages of chronic infection in 8 CF patients. High numbers of large-scale deletions were found to occur during chronic infection, affecting preferentially genomic islands and nonessential replicons. Recombination between insertion sequences (IS) was inferred as the mechanism behind deletion formation; the most numerous IS group was specific for the ST32 clone and has undergone transposition burst since its divergence. Genes functionally related to transition metal metabolism were identified as hotspots for deletions and IS insertions. This functional category was also represented among genes where nonsynonymous point mutations and indels occurred parallelly among patients. Another category exhibiting parallel mutations was oxidative stress protection; mutations in catalase KatG resulted in impaired detoxification of hydrogen peroxide. Deep sequencing revealed substantial polymorphism in genes of both categories within the sputum B. cenocepacia ST32 populations, indicating extensive adaptive evolution. Neither oxidative stress response nor transition metal metabolism genes were previously reported to undergo parallel evolution during chronic CF infection. Mutations in katG and copper metabolism genes were overrepresented in patients where chronic infection developed into CS. Among professional phagocytes, macrophages use both hydrogen peroxide and copper for their bactericidal activity; our results thus tentatively point to macrophages as suspects in pathogenesis towards the fatal CS.