Identification of basepairs within Tn5 termini that are critical sfor H-NS binding to the transpososome and regulation of Tn5 transposition

<p>Abstract</p> <p>Background</p> <p>The H-NS protein is a global regulator of gene expression in bacteria and can also bind transposition complexes (transpososomes). In Tn5 transposition H-NS promotes transpososome assembly <it>in vitro </it>and disruption of the <it>hns </it>gene causes a modest decrease in Tn5 transposition (three- to five-fold). This is consistent with H-NS acting as a positive regulator of Tn5 transposition. Molecular determinants for H-NS binding to the Tn5 transpososome have not been determined, nor has the strength of the interaction been established. There is also uncertainty as to whether H-NS regulates Tn5 transposition <it>in vivo </it>through an interaction with the transposition machinery as disruption of the <it>hns </it>gene has pleiotropic effects on <it>Escherichia coli</it>, the organism used in this study.</p> <p>Results</p> <p>In the current work we have further examined determinants for H-NS binding to the Tn5 transpososome through both mutational studies on Tn5 termini (or 'transposon ends') and protein-protein cross-linking analysis. We identify mutations in two different segments of the transposon ends that abrogate H-NS binding and characterize the affinity of H-NS for wild type transposon ends in the context of the transpososome. We also show that H-NS forms cross-links with the Tn5 transposase protein specifically in the transpososome, an observation consistent with the two proteins occupying overlapping binding sites in the transposon ends. Finally, we make use of the end mutations to test the idea that H-NS exerts its impact on Tn5 transposition <it>in vivo </it>by binding directly to the transpososome. Consistent with this possibility, we show that two different end mutations reduce the sensitivity of the Tn5 system to H-NS regulation.</p> <p>Conclusions</p> <p>H-NS typically regulates cellular functions through its potent transcriptional repressor function. Work presented here provides support for an alternative mechanism of H-NS-based regulation, and adds to our understanding of how bacterial transposition can be regulated.</p

The global bacterial regulator H-NS promotes transpososome formation and transposition in the Tn5 system

The histone-like nucleoid structuring protein (H-NS) is an important regulator of stress response and virulence genes in gram-negative bacteria. In addition to binding regulatory regions of genes in a structure-specific manner, H-NS also binds in a structure-specific manner to sites in the Tn10 transpososome, allowing it to act as a positive regulator of Tn10 transposition. This is the only example to date of H-NS regulating a transposition system by interacting directly with the transposition machinery. In general, transposition complexes tend to include segments of deformed DNA and given the capacity of H-NS to bind such structures, and the results from the Tn10 system, we asked if H-NS might regulate another transposition system (Tn5) by directly binding the transposition machinery. We show in the current work that H-NS does bind Tn5 transposition complexes and use hydroxyl radical footprinting to characterize the H-NS interaction with the Tn5 transpososome. We also show that H-NS can promote Tn5 transpososome formation in vitro, which correlates with the Tn5 system showing a dependence on H-NS for transposition in vivo. Taken together the results suggest that H-NS might play an important role in the regulation of many different bacterial transposition systems and thereby contribute directly to lateral gene transfer

Application of microarray technology in pulmonary diseases

Microarrays are a powerful tool that have multiple applications both in clinical and cell biology arenas of common lung diseases. To exemplify how this tool can be useful, in this review, we will provide an overview of the application of microarray technology in research relevant to common lung diseases and present some of the future perspectives

Restricting diet for perceived health benefit: A mixed‐methods exploration of peripartum food taboos in rural Cambodia

Abstract Food taboos encompass food restrictions practiced by a group that go beyond individual preferences. During pregnancy and lactation, food taboos may contribute to inadequate nutrition and poor maternal and infant health. Restriction of specific fish, meat, fruits and vegetables is common among peripartum women in many Southeast Asian countries, but data from Cambodia are lacking. In this mixed‐methods study, 335 Cambodian mothers were asked open‐ended questions regarding dietary behaviours during pregnancy and up to 24 weeks postpartum. Descriptive statistics and content analysis were used to characterize food taboos and multiple logistic regression analyses were conducted to identify predictors of this practice. Participants were 18–44 years of age, all of Khmer ethnicity and 31% were primiparous. Sixty‐six per cent of women followed food taboos during the first 2 weeks postpartum, whereas ~20% of women restricted foods during other peripartum periods. Pregnancy taboos were often beneficial, including avoidance of sugar‐sweetened beverages, coffee and alcohol. Conversely, postpartum avoidances typically included nutrient‐dense foods such as fish, raw vegetables and chicken. Food taboos were generally followed to support maternal and child health. No significant predictors of food taboos during pregnancy were identified. Postpartum, each additional live birth a woman had reduced her odds of following food taboos by 24% (odds ratio [95% confidence interval]: 0.76 [0.61–0.95]). Specific food taboo practices and rationales varied greatly between women, suggesting that food taboos are shaped less by a strict belief system within the Khmer culture and more by individual or household understandings of food and health during pregnancy and postpartum