R-loop-dependent replication and genomic instability in bacteria

DNA replication, the faithful copying of genetic material, must be tightly regulated to produce daughter cells with intact copies of the chromosome(s). This regulated replication is initiated by binding of specific proteins at replication origins, such as DnaA to oriC in bacteria. However, unregulated replication can sometimes be initiated at other sites, which can threaten genomic stability. One of the first systems of unregulated replication to be described is the one activated in Escherichia coli mutants lacking RNase HI (rnhA). In fact, rnhA mutants can replicate their chromosomes in a DnaA- and oriC-independent process. Because this replication occurs in cells lacking RNase HI, it is proposed that RNA from R-loops is used as a DNA polymerase primer. Replication from R-loops has recently attracted increased attention due to the advent of DNA:RNA hybrid immunoprecipitation coupled with high-throughput DNA sequencing that revealed the high prevalence of R-loop formation in many organisms, and the demonstration that R-loops can severely threaten genomic stability. Although R-loops have been linked to genomic instability mostly via replication stress, evidence of their toxic effects via unregulated replication has also been presented. Replication from R-loops may also beneficially trigger stress-induced mutagenesis (SIM) that assists bacterial adaptation to stress. Here, we describe the cis- and trans-acting elements involved in R-loop-dependent replication in bacteria, with an emphasis on new data obtained with type 1A topoisomerase mutants and new available technologies. Furthermore, we discuss about the mechanism(s) by which R-loops can reshape the genome with both negative and positive outcomes.     

Fabrication of agar-gelatin hybrid scaffolds using a novel entrapment method for in vitro tissue engineering applications

Scaffolds of agar and gelatin were developed using a novel entrapment method where agar and gelatin molecules mutually entrapped one another forming stable cell adhesive matrices. Glutaraldehyde was used as a crosslinking agent for gelatin. Three types of hybrid matrices were prepared using agar and gelatin in different proportions in the weight ratio of 1:1, 2:1, and 3:1. Surface characterization of dry scaffolds was carried out by scanning electron microscope. Swelling studies were carried out in phosphate buffer saline (PBS) at physiological pH 7.4. The integral stability of the scaffolds was evaluated by estimating the released disintegrated gelatin from them in PBS at pH 7.4. The attachment kinetics of the cells was evaluated by culturing mouse fibroblast cell line NIH 3T3 on films. The cytocompatibility of these matrices was determined by studying growth kinetics of NIH 3T3 cells on them and morphology of cells was observed through optical photographs taken at various days of culture. It was found that the matrices containing agar and gelatin in 2:1 weight ratio exhibited best growth kinetics. The results obtained from these studies have suggested that the above-described method is a cheap and easy way to fabricate agar-gelatin hybrid scaffolds to grow cells which can be used in various in vitro tissue engineering applications like screening of drugs.     

Alterations to plasma membrane lipid contents affect the biophysical properties of erythrocytes from individuals with hypertension

Genetic and environmental factors may contribute to high blood pressure, which is termed essential hypertension. Hypertension is a major independent risk factor for cardiovascular disease, stroke and renal failure; thus, elucidation of the etiopathology of hypertension merits further research. We recently reported that the platelets and neutrophils of patients with hypertension exhibit altered biophysical characteristics. In the present study, we assessed whether the major structural elements of erythrocyte plasma membranes are altered in individuals with hypertension. We compared the phospholipid (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingosine) and cholesterol contents of erythrocytes from individuals with hypertension (HTN) and healthy individuals (HI) using LC/MS-MS. HTN erythrocytes contained higher phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine contents and a lower cholesterol content than HI erythrocytes. Furthermore, atomic force microscopy revealed important morphological changes in HTN erythrocytes, which reflected the increased membrane fragility and fluidity and higher levels of oxidative stress observed in HTN erythrocytes using spectrophotofluorometry, flow cytometry and spectrometry. This study reveals that alterations to the lipid contents of erythrocyte plasma membranes occur in hypertension, and these alterations in lipid composition result in morphological and physiological abnormalities that modify the dynamic properties of erythrocytes and contribute to the pathophysiology of hypertension.     

Contrasting patterns of genetic structure and phylogeography in the marine agarophytes Gelidiophycus divaricatus and G. freshwateri (Gelidiales,Rhodophyta) from East Asia

The evolutionary and population demographic history of marine red algae in East Asia is poorly understood. Here, we reconstructed the phylogeographies of two upper intertidal species endemic to East Asia, Gelidiophycus divaricatus and G. freshwateri. Phylogenetic and phylogeographic inferences of 393 mitochondrial cox1, 128 plastid rbcL, and 342 nuclear ITS2 sequences were complemented with ecological niche models. Gelidiophycus divaricatus, a southern species adapted to warm water, is characterized by a high genetic diversity and a strong geographical population structure, characteristic of stable population sizes and sudden reduction to recent expansion. In contrast, G. freshwateri, a northern species adapted to cold temperate conditions, is genetically relatively homogeneous with a shallow population structure resulting from steady population growth and recent equilibrium. The overlap zone of the two species roughly matches summer and winter isotherms, indicating that surface seawater temperature is a key feature influencing species range. Unidirectional genetic introgression was detected at two sites on Jeju Island where G. divaricatus was rare while G. freshwateri was common, suggesting the occurrence of asymmetric natural hybrids, a rarely reported event for rhodophytes. Our results illustrate that Quaternary climate oscillations have left strong imprints on the current day genetic structure and highlight the importance of seawater temperature and sea level change in driving speciation in upper intertidal seaweed species.     

An egg‐laying device to estimate the induction of sterility in Ceratitis capitata (Diptera: Tephritidae) sterile insect technique programmes

Area‐wide environmentally friendly pest control methods such as the sterile insect technique (SIT) are being developed and improved to contribute in managing agricultural, environmental and public health problems. A key aspect to evaluate performance of sterile males is to directly measure sterility induction in the field. Sterility induction has been estimated for tephritid fruit flies by recovering egg from host fruit in the field, the method is, however, impractical, and past efforts to develop artificial egg‐laying devices have not prospered. Here, we evaluated response of wild gravid Ceratitis capitata (Medfly) females to long‐distance fruit‐based chemical attractants, visual and tactile stimuli to develop an artificial egg‐laying device. The device combining the most attractive features was further tested under two deployment schemes. Finally, devices and deployment tactics were used to compare fertility levels between feral Medfly females under conventional management and under SIT. Agar spheres wrapped in plastic film, baited with pressed peach juice and visually enhanced with yellow discs received more egg than other combinations of attractive features. Such devices also received more eggs when deployed on fruitless trees and when placed on the orchard perimeter. The egg hatch in an orchard under conventional management was estimated at 86%, whilst egg hatch in an area under SIT was reduced to 31%. The egg‐laying devices are therefore useful to measure sterility induction and can be further improved by refining long‐distance attraction and deployment schemes.     

Detection and recovery of sublethally-injured enterotoxigenic Staphylococcus aureus

AIMS: To determine whether sublethally-injured (acid- or heat-shocked) Staphylococcus aureus cells are recoverable using selective agar overlays. METHODS AND RESULTS: Brain Heart Infusion (BHI) Agar overlaid with either Baird-Parker Agar (BPA) or Gram-Positive Agar (GPA) was compared in the ability to resuscitate heat- and acid-shocked enterotoxigenic Staph. aureus. BHI/BPA overlays allowed for greater recovery of both heat- and acid-shocked cells than BHI/GPA, although the former was not selective and allowed growth of bacteria other than Staph. aureus. No significant difference existed in percent recovery of heat- and acid-shocked cells between the two overlay approaches. Significant differences were noted in counts on BHI/GPA plates and straight selective GPA/GPA plates, however. Viability of heat- and acid-shocked Staph. aureus was also examined using fluorescence microscopy, the relative counts of which correlated well to the calculated percent recovery on selective agar overlays. CONCLUSIONS: This work has shown that an improved agar overlay technique increases the sensitivity of the standard plate count while enumerating sublethally-injured enterotoxigenic Staph. aureus compared with direct plating onto selective media. SIGNIFICANCE AND IMPACT OF THE STUDY: These data emphasize the need to develop practical and cost-effective methods that reliably detect and enumerate sublethally-injured pathogens such as Staph. aureus.     

The HI0073/HI0074 protein pair from Haemophilus influenzae is a member of a new nucleotidyltransferase family: structure,sequence analyses,and solution studies

The crystal structure of HI0074 from Haemophilus influenzae, a protein of unknown function, has been determined at a resolution of 2.4 A. The molecules form an up-down, four-helix bundle, and associate into homodimers. The fold is most closely related to the substrate-binding domain of KNTase, yet the amino acid sequences of the two proteins exhibit no significant homology. Sequence analyses of completely and incompletely sequenced genomes reveal that the two adjacent genes, HI0074 and HI0073, and their close relatives comprise a new family of nucleotidyltransferases, with 15 members at the time of writing. The analyses also indicate that this is one of eight families of a large nucleotidyltransferase superfamily, whose members were identified based on the proximity of the nucleotide- and substrate-binding domains on the respective genomes. Both HI0073 and HI0074 were annotated "hypothetical" in the original genome sequencing publication. HI0073 was cloned, expressed, and purified, and was shown to form a complex with HI0074 by polyacrylamide gel electrophoresis under nondenaturing conditions, analytic size exclusion chromatography, and dynamic light scattering. Double- and single-stranded DNA binding assays showed no evidence of DNA binding to HI0074 or to HI0073/HI0074 complex despite the suggestive shape of the putative binding cleft formed by the HI0074 dimer.     

Cell wall polysaccharides from Gelidium species: physico-chemical studies using MRI techniques

Magnetic resonance imaging (MRI) has already been successively used to investigate polysaccharide matrices. In particular, MRI at microscopic resolution (MR microscopy) is now one of the most powerful techniques for studying the physical properties of natural hydrogels. To contribute to a better understanding of the correlation between chemical and physical properties of agar gels, we report here the measurement of the water magnetic parameters for agar gels extracted from different species of Gelidium: T1 and T2 relaxation times, magnetisation transfer (Ms /M0) and diffusion (D) were measured to evaluate their use for studying the gel characteristics. MR microscopic images were acquired at 7.05 Tesla using various pulse sequences. The results obtained confirmed the possibility to use quantitative MRI for the characterisation of physical parameters correlated with the type of agar chemical structure. In particular, T2 data obtained for gels at different concentrations indicate that this magnetic parameter is very sensitive to the agar concentration and hence particularly useful for the gel strength determination. This revised version was published online in June 2006 with corrections to the Cover Date.