Affinities of Terminal Inverted Repeats to DNA Binding Domain of Transposase Affect the Transposition Activity of Bamboo Ppmar2 Mariner-Like Element

Mariner-like elements (MLE) are a super-family of DNA transposons widespread in animal and plant genomes. Based on their transposition characteristics, such as random insertions and high-frequency heterogeneous transpositions, several MLEs have been developed to be used as tools in gene tagging and gene therapy. Two active MLEs, Ppmar1 and Ppmar2, have previously been identified in moso bamboo (Phyllostachys edulis). Both of these have a preferential insertion affinity to AT-rich region and their insertion sites are close to random in the host genome. In Ppmar2 element, we studied the affinities of terminal inverted repeats (TIRs) to DNA binding domain (DBD) and their influence on the transposition activity. We could identify two putative boxes in the TIRs which play a significant role in defining the TIR’s affinities to the DBD. Seven mutated TIRs were constructed, differing in affinities based on similarities with those of other plant MLEs. Gel mobility shift assays showed that the TIR mutants with mutation sites G669A-C671A had significantly higher affinities than the mutants with mutation sites C657T-A660T. The high-affinity TIRs indicated that their transposition frequency was 1.5–2.0 times higher than that of the wild type TIRs in yeast transposition assays. The MLE mutants with low-affinity TIRs had relatively lower transposition frequency from that of wild types. We conclude that TIR affinity to DBD significantly affects the transposition activity of Ppmar2. The mutant MLEs highly active TIRs constructed in this study can be used as a tool for bamboo genetic studies.

Influence of IS256 on Genome Variability and Formation of Small-Colony Variants in Staphylococcus aureus

ABSTRACT Staphylococcus aureus has acquired resistance to nearly all antibiotics used in clinical practice. Whereas some resistance mechanisms are conferred by uptake of resistance genes, others evolve by mutation. In this study, IS256 has been shown to play a role, e.g., in S. aureus strains displaying intermediate resistance to vancomycin (VISA). To characterize the IS256 insertion sites in the genomes of two closely related sequence type 247 (ST247) VISA strains, all insertions were mapped in both VISA and a susceptible control strain. The results showed that the three ST247 strains contained the highest number so far of IS256 insertions for all sequenced S. aureus strains. Furthermore, in contrast to the case with the other IS elements in these genomes, the IS256 insertion sites were not identical in the closely related strains, indicating a high transposition frequency of IS256. When IS256 was introduced into a laboratory strain which was then cultured in the presence of antibiotics, it was possible to isolate small-colony variants (SCVs) that possessed IS256 insertions in guaA and hemY that displayed increased resistance to vancomycin and aminoglycosides, respectively. For these clones, a very rapid reversion to the wild type that resembled the fast reversion of clinical SCVs was observed. The reversion was caused by excision of IS256 in a small number of fast-growing clones that quickly outcompeted the SCVs in broth cultures. In conclusion, the presence of IS256 confers a strong genomic plasticity that is useful for adaptation to antibiotic stress.

Transposition of Tn125encoding the NDM-1 carbapenemase inAcinetobacter baumannii

TheblaNDM-1gene encodes a carbapenemase that confers resistance to almost all β-lactams, including latest resort carbapenems. It is increasingly reported worldwide in nosocomial and community-acquired Gram-negative bacteria.Acinetobacter baumanniiis an important opportunistic pathogen considered as an intermediate reservoir for theblaNDM-1gene. In this species, theblaNDM-1gene is located within the Tn125composite transposon. The mechanism driving the mobility of Tn125has not yet been elucidated. Here we experimentally demonstrated transposition of Tn125inA. baumannii. Systematic 3-bp duplication of the target site, being signature of transposition, was evidenced. The target site consensus for Tn125transposition was found to be GC-enriched at the duplicated 3 bp and AT-rich in the vicinity. Transposition frequency was not influenced by temperature changes or by exposure to sub-inhibitory concentrations of various antibiotics. This work is the first direct evidence of the functionality of a composite transposon inA. baumannii. It provides a mechanistic clue for the dissemination of theblaNDM-1gene inAcinetobacterspp. and subsequently among Enterobacteriaceae.

Antibiotic-Induced Autoactivation of IS256in Staphylococcus aureus

ABSTRACTThe 3′ end ofrsbU, encoding the positive regulator of the stress factor sigma B, was identified as a hot spot for spontaneous IS256insertion inStaphylococcus aureusSA137/93G. Interestingly, subinhibitory concentrations of chloramphenicol in combination with heat stress, as well as linezolid and spectinomycin at physiological temperatures, selected for suchrsbU::IS256insertion mutants. In consequence of the inactivation ofrsbU, the IS256transposition frequency was increased 4-fold inS. aureusHG001.

Meta-Analyses and Orthodontic Evidence-Based Clinical Practice in the 21st Century

Introduction:Aim of this systematic review was to assess the orthodontic related issues which currently provide the best evidence as documented by meta-analyses, by critically evaluating and discussing the methodology used in these studies.Material and Methods:Several electronic databases were searched and handsearching was also performed in order to identify the corresponding meta-analyses investigating orthodontic related subjects. In total, 197 studies were retrieved initially. After applying specific inclusion and exclusion criteria, 27 articles were identified as meta-analyses treating orthodontic-related subjects.Results:Many of these 27 papers presented sufficient quality and followed appropriate meta-analytic approaches to quantitatively synthesize data and presented adequately supported evidence. However, the methodology used in some of them presented weaknesses, limitations or deficiencies. Consequently, the topics in orthodontics which currently provide the best evidence, include some issues related to Class II or Class III treatment, treatment of transverse problems, external apical root resorption, dental anomalies, such as congenital missing teeth and tooth transposition, frequency of severe occlusal problems, nickel hypersensitivity, obstructive sleep apnea syndrome, and computer-assisted learning in orthodontic education.Conclusions:Only a few orthodontic related issues have been so far investigated by means of MAs. In addition, for some of these issues investigated in the corresponding MAs no definite conclusions could be drawn, due to significant methodological deficiencies of these studies. According to this investigation, it can be concluded that at the begin of the 21stcentury there is evidence for only a few orthodontic related issues as documented by meta-analyses, and more well-conducted high quality research studies are needed to produce strong evidence in order to support evidence-based clinical practice in orthodontics.

Mapping barley Ds insertions using wheat deletion lines reveals high insertion frequencies in gene-rich regions with high to moderate recombination rates

Gene distribution is highly uneven in the large genomes of barley and wheat; however, location, order, and gene density of gene-containing regions are very similar between the two genomes. Flanking sequences from 35 unique, single-copy, barley Ds insertion events were physically mapped using wheat nullisomic-tetrasomic, ditelosomic, and deletion lines. Of the 35 sequences, 23 (66%) detected 34 loci mapping on all 7 homoeologous wheat groups. Seven sequences were not mapped owing to lack of polymorphism and the remaining 5 (14%) were barley-specific. All 34 loci physically mapped to the previously identified gene-rich regions (GRRs) of wheat, making the contained genes candidates for targeted mutagenesis by remobilization. Transpositions occurred preferentially into GRRs with higher recombination rates. The GRRs containing 17 of the 23 Ds insertions accounted for 60%–89% of the respective arm’s recombination. The remaining 6 (17%) insertions mapped to GRRs with <15% of the arm’s recombination. Overall, kb/cM estimates for the Ds-containing GRRs were twofold higher than those for regions without insertions. These results suggest that all genes may be targeted by transposon-based gene cloning, although the transposition frequency for genes present in recombination-poor regions is significantly less than that present in highly recombinogenic regions.

σB Regulates IS256-Mediated Staphylococcus aureus Biofilm Phenotypic Variation

ABSTRACT Biofilm formation in Staphylococcus aureus is subject to phase variation, and biofilm-negative derivatives emerge sporadically from a biofilm-positive bacterial population. To date, the only known mechanism for generating biofilm phenotypic variation in staphylococci is the reversible insertion/excision of IS256 in biofilm-essential genes. In this study, we present evidence suggesting that the absence of the σB transcription factor dramatically increases the rate of switching to the biofilm-negative phenotype in the clinical isolate S. aureus 15981, under both steady-state and flow conditions. The phenotypic switching correlates with a dramatic increase in the number of IS256 copies in the chromosomes of biofilm-negative variants, as well as with an augmented IS256 insertion frequency into the icaC and the sarA genes. IS256-mediated biofilm switching is reversible, and biofilm-positive variants could emerge from biofilm-negative σB mutants. Analysis of the chromosomal insertion frequency using a recombinant IS256 element tagged with an erythromycin marker showed an almost three-times-higher transposition frequency in a ΔσB strain. However, regulation of IS256 activity by σB appears to be indirect, since transposase transcription is not affected in the absence of σB and IS256 activity is inhibited to wild-type levels in a ΔσB strain under NaCl stress. Overall, our results identify a new role for σB as a negative regulator of insertion sequence transposition and support the idea that deregulation of IS256 activity abrogates biofilm formation capacity in S. aureus.

IS1 transposition is enhanced by translation errors and by bacterial growth at extreme glucose levels.

Transposition of insertion sequences (IS) is an enzyme-mediated process that only occurs in a minority of cells within a bacterial culture. Transposition is thus a rare event, but transposition frequency may vary depending on experimental conditions. For instance in a rich broth, IS elements are known to transpose during stationary phase but not during exponential growth. Using a reporter system which involves the activation of the cryptic bgl operon in Escherichia coli, we show that the frequency of IS1 transposition is a function of glucose concentration in the growth medium, it is increased by streptomycin amounts that are below minimum inhibitory concentration (sub-MIC) and is inhibited in an rpsL150 strain with high translation accuracy. Since starved cells are known to enhance ribosome frameshifting, our data suggests that growth conditions applied in this study could affect IS1 transposition by increasing translation infidelity.