Non-canonical Structures in Promoter Modulate Gene Expression in Escherichia coli

Herein we show how sequences that can form different non-canonical structures affect gene expression levels when inserted in the core of σ70-dependent promoter, between the −35 and −10 elements recognized by RNA polymerase, in E. coli. We note that influence on level of GFP expression varies considerably depending on introduction of non-canonical structural elements in the antisense and sense strands as well as with their propensities to form G-triplex, G-hairpin, hairpin or G-quadruplex structures. Moreover, the extent of repression of expression does not relate to the in vitro thermal stability in a simple manner. Repression is most likely caused by steric interference rather than improper distance between the −35 and −10 elements. Although properties like thermal stability and topology can be somewhat different under in vivo and in vitro conditions, our results suggest that the extent of expression suppression cannot be dependent solely on thermal stabilities of G-rich structures alone. This work is licensed under a Creative Commons Attribution 4.0 International License

CPMAS NMR platform for direct compositional analysis of mycobacterial cell-wall complexes and whole cells

Tuberculosis and non-tuberculosis mycobacterial infections are rising each year and often result in chronic incurable disease. Important antibiotics target cell-wall biosynthesis, yet some mycobacteria are alarmingly resistant or tolerant to currently available antibiotics. This resistance is often attributed to assumed differences in composition of the complex cell wall of different mycobacterial strains and species. However, due to the highly crosslinked and insoluble nature of mycobacterial cell walls, direct comparative determinations of cell-wall composition pose a challenge to analysis through conventional biochemical analyses. We introduce an approach to directly observe the chemical composition of mycobacterial cell walls using solid-state NMR spectroscopy. 13C CPMAS spectra are provided of individual components (peptidoglycan, arabinogalactan, and mycolic acids) and of in situ cell-wall complexes. We assigned the spectroscopic contributions of each component in the cell-wall spectrum. We uncovered a higher arabinogalactan-to-peptidoglycan ratio in the cell wall of M. abscessus, an organism noted for its antibiotic resistance, relative to M. smegmatis. Furthermore, differentiating influences of different types of cell-wall targeting antibiotics were observed in spectra of antibiotic-treated whole cells. This platform will be of value in evaluating cell-wall composition and antibiotic activity among different mycobacteria and in considering the most effective combination treatment regimens

Effects of Maternal Cigarette Smoking on Trace Element Levels and Steroidogenesis in the Maternal–Placental–Fetal Unit

This study evaluates the interaction of toxic elements cadmium (Cd) and lead (Pb) due to exposure from cigarette smoking, essential elements, and steroidogenesis in the maternal–placental–fetal unit. In a cohort of 155 healthy, postpartum women with vaginal term deliveries in clinical hospitals in Zagreb, Croatia, samples of maternal blood/serum and urine, placental tissue, and umbilical cord blood/serum were collected at childbirth. The biomarkers determined were concentrations of Cd, Pb, iron (Fe), zinc (Zn), copper (Cu), and selenium (Se), and steroid hormones progesterone and estradiol in maternal and umbilical cord blood and the placenta. Three study groups were designated based on self-reported data on cigarette smoking habits and confirmed by urine cotinine levels: never smokers (n = 71), former smokers (n = 48), and active smokers (n = 36). Metal(loid)s, steroid hormones, urine cotinine, and creatinine levels were analyzed by ICP–MS, ELISA, GC–MS, and spectrophotometry. Cigarette smoking during pregnancy was associated with increased Cd levels in maternal, placental, and fetal compartments, Pb in the placenta, and with decreased Fe in the placenta. In active smokers, decreased progesterone and estradiol concentrations in cord blood serum were found, while sex steroid hormones did not change in either maternal serum or placenta. This study provides further evidence regarding toxic and essential metal(loid) interactions during prenatal life, and new data on sex steroid disruption in cord serum related to cigarette smoking. The results indicate that umbilical cord sex steroid levels may be a putative early marker of developmental origins of the future burden of disease related to harmful prenatal exposure to cigarette smoke