Genomic library screens for genes involved in n-butanol tolerance in Escherichia coli

Background

n-Butanol is a promising emerging biofuel, and recent metabolic engineering efforts have demonstrated the use of several microbial hosts for its production. However, most organisms have very low tolerance to n-butanol (up to 2% (v/v)), limiting the economic viability of this biofuel. The rational engineering of more robust n-butanol production hosts relies upon understanding the mechanisms involved in tolerance. However, the existing knowledge of genes involved in n-butanol tolerance is limited. The goal of this study is therefore to identify E. coli genes that are involved in n-butanol tolerance.

Methodology/Principal Findings

Using a genomic library enrichment strategy, we identified approximately 270 genes that were enriched or depleted in n-butanol challenge. The effects of these candidate genes on n-butanol tolerance were experimentally determined using overexpression or deletion libraries. Among the 55 enriched genes tested, 11 were experimentally shown to confer enhanced tolerance to n-butanol when overexpressed compared to the wild-type. Among the 84 depleted genes tested, three conferred increased n-butanol resistance when deleted. The overexpressed genes that conferred the largest increase in n-butanol tolerance were related to iron transport and metabolism, entC and feoA, which increased the n-butanol tolerance by 32.8±4.0% and 49.1±3.3%, respectively. The deleted gene that resulted in the largest increase in resistance to n-butanol was astE, which enhanced n-butanol tolerance by 48.7±6.3%.

Conclusions/Significance

We identified and experimentally verified 14 genes that decreased the inhibitory effect of n-butanol tolerance on E. coli. From the data, we were able to expand the current knowledge on the genes involved in n-butanol tolerance; the results suggest that an increased iron transport and metabolism and decreased acid resistance may enhance n-butanol tolerance. The genes and mechanisms identified in this study will be helpful in the rational engineering of more robust biofuel producers.     

SPOC: A widely distributed domain associated with cancer,apoptosis and transcription

Background  

The Split ends (Spen) family are large proteins characterised by N-terminal RNA recognition motifs (RRMs) and a conserved SPOC (Spen paralog and ortholog C-terminal) domain. The aim of this study is to characterize the family at the sequence level.     

Population structure of North Atlantic and North Pacific sei whales (<Emphasis Type="Italic">Balaenoptera borealis</Emphasis>) inferred from mitochondrial control region DNA sequences and microsatellite genotypes

Currently, three stocks of sei whales (Balaenoptera borealis) are defined in the North Atlantic; the Nova Scotian, Iceland-Denmark Strait and Eastern North Atlantic stocks, which are mainly based upon historical catch and sighting data. We analyzed mitochondrial control region DNA (mtDNA) sequences and genotypes from 7 to 11 microsatellite loci in 87 samples from three sites in the North Atlantic; Iceland, the Gulf of Maine and the Azores, and compared against the North Pacific using 489 previously published samples. No statistically significant deviations from homogeneity were detected among the North Atlantic samples at mtDNA or microsatellite loci. The genealogy estimated from the mtDNA sequences revealed a clear division of the haplotypes into a North Atlantic and a North Pacific clade, with the exception of one haplotype detected in a single sample from the Azores, which was included in the North Pacific clade. Significant genetic divergence between the North Atlantic and North Pacific Oceans was detected (mtDNA Φ_ST?=?0.72, microsatellite Weir and Cockerham’s ? = 0.20; p?<?0.001). The coalescent-based estimate of the population divergence time between the North Atlantic and North Pacific populations from the sequence variation among the mtDNA sequences was at 163,000 years ago. However, the inference was limited by an absence of samples from the Southern Hemisphere and uncertainty regarding mutation rates and generation times. The estimates of inter-oceanic migration rates were low (Nm at 0.007 into the North Pacific and at 0.248 in the opposite direction). Although estimates of genetic divergence among the current North Atlantic stocks were low and consistent with the extensive range of movement observed in satellite tagged sei whales, the high uncertainty of the genetic divergence estimates precludes rejection of multiple stocks in the North Atlantic.     

Ripening in papaya fruit is altered by ACC oxidase cosuppression

Papaya (Carica papaya) is a very important crop in many tropical countries but it is highly susceptible to parasitic diseases, physiological disorders, mechanical damage and fruit overripening. Here we report a study on ACC oxidase cosuppression and its effects on papaya fruit ripening. Papaya ACC oxidase was isolated using PCR and embriogenic cells transformed by biolistic using the CaMV 35S promoter to drive the expression of the PCR fragment in sense orientation. Fifty transgenic lines were recovered and 20 of those were grown under field conditions. Southern analysis showed incorporation of the transgene in different copy numbers in the papaya genome. Fruits were evaluated in terms of texture (firmness), colour development, respiration and ethylene production. A sharp reduction in ethylene and CO2 production was detected, whereas softening and colour development of the peel were also altered. Overall, transgenic fruits showed a delay in ripening rate. A reduction in mRNA level for ACC oxidase in transgenic fruit was clearly detectable by northern blot. More studies are necessary before this technology can be used to extend the shelf life of papaya fruit.     

Effects of temperature and water availability on light energy utilization in photosynthetic processes of Deschampsia antarctica

Regional climate change in Antarctica would favor the carbon assimilation of Antarctic vascular plants, since rising temperatures are approaching their photosynthetic optimum (10–19°C). This could be detrimental for photoprotection mechanisms, mainly those associated with thermal dissipation, making plants more susceptible to eventual drought predicted by climate change models. With the purpose to study the effect of temperature and water availability on light energy utilization and putative adjustments in photoprotective mechanisms of Deschampsia antarctica Desv., plants were collected from two Antarctic provenances: King George Island and Lagotellerie Island. Plants were cultivated at 5, 10 and 16°C under well‐watered (WW) and water‐deficit (WD, at 35% of the field capacity) conditions. Chlorophyll fluorescence, pigment content and de‐epoxidation state were evaluated. Regardless of provenances, D. antarctica showed similar morphological, biochemical and functional responses to growth temperature. Higher temperature triggered an increase in photochemical activity (i.e. electron transport rate and photochemical quenching), and a decrease in thermal dissipation capacity (i.e. lower xanthophyll pool, Chl a/b and β carotene/neoxanthin ratios). Leaf mass per unit area was reduced at higher temperature, and was only affected in plants exposed to WD at 16°C and exhibiting lower electron transport rate and amount of chlorophylls. D. antarctica is adapted to frequent freezing events, which may induce a form of physiological water stress. Photoprotective responses observed under WD contribute to maintain a stable photochemical activity. Thus, it is possible that short‐term temperature increases could favor the photochemical activity of this species. However, long‐term effects will depend on the magnitude of changes and the plant's ability to adjust to new growth temperature.     

Hospital costs associated with delirium in older medical patients

Introduction

Delirium is a common and serious complication in older patients, associated with increased, potentially preventable, morbidity and mortality. The aim of this study was to evaluate the associated costs of delirium during hospitalization in a university affiliated hospital in Chile.

Materials and methods

Prospective cohort study of consecutive patients 65 years and older, admitted to a medical ward. A psychogeriatric team assessed patients during the first and every 48 h until discharge using the Confusion Assessment Method (CAM-S), length of hospital stay, pharmacy and total hospitalization costs were analyzed. Statistical analysis was performed using bivariate and multivariate analysis according to delirium diagnosis.

Results

Data from 454 patients was analyzed, 160 of them in a delirium cohort (35.2%) and 294 in a non-delirium cohort (64.8%). The delirium cohort had a longer hospital stay (DATA) and higher mortality (7.0% versus 1.7%). The median of total costs of delirium during hospital stay was 38.7% higher than the non-delirium cohort (P < .001). Total costs were significantly higher in the delirium cohort after adjustment of covariables (P = .01).

Conclusions

This study confirms that delirium is associated with significantly greater costs. Considering that effective delirium prevention is possible, the knowledge of associated costs can help health care providers to justify prevention strategies and finally give better care for older patients.     

Water management practices and climate in ancient agriculture: inferences from the stable isotope composition of archaeobotanical remains

Carbon isotope discrimination (Δ¹³C) in charred grains from archaeological sites provides reliable information about water availability of ancient crops. However, as cereals are cultivated plants, they may reflect not only climatic fluctuations, but also the effect on water status of certain agronomic practices, such as sowing in naturally wet soils or irrigation. In this work, we propose a methodological approach to combine Δ¹³C data from different plant species, in order to discriminate between climate-derived and anthropogenic effects on ancient crops. We updated previous models for estimating water inputs from Δ¹³C of cereal grains of Hordeum vulgare and Triticum aestivum/durum, and we applied them to published data from several archaeological sites, including samples from the Neolithic to the present day in northeast and southeast Spain, as well as from the Neolithic site of Tell Halula (northwest Syria). We found an important decrease in water availability from the Neolithic to the present time in the three areas of study, especially clear for the two driest areas (southeast Spain and northwest Syria). Potential differences in water management practices between wheat and barley, as well as between cereal and legume crops (Vicia faba and Lens culinaris), are also discussed on the basis of the comparison of Δ¹³C values across several archaeological sites.     

Patterns of diversifying selection in the phytotoxin-like scr74 gene family of Phytophthora infestans

Phytophthora infestans, the organism responsible for the Irish famine, causes late blight, a re-emerging disease of potato and tomato. Little is known about the molecular evolution of P. infestans genes. To identify candidate effector genes (virulence or avirulence genes) that may have co-evolved with the host, we mined expressed sequence tag (EST) data from infection stages of P. infestans for secreted and potentially polymorphic genes. This led to the identification of scr74, a gene that encodes a predicted 74-amino acid secreted cysteine-rich protein with similarity to the Phytophthora cactorum phytotoxin PcF. The expression of scr74 was upregulated approximately 60-fold 2 to 4 days after inoculation of tomato and was also significantly induced during early stages of colonization of potato. The scr74 gene was found to belong to a highly polymorphic gene family within P. infestans with 21 different sequences identified. Using the approximate and maximum likelihood (ML) methods, we found that diversifying selection likely caused the extensive polymorphism observed within the scr74 gene family. Pairwise comparisons of 17 scr74 sequences revealed elevated ratios of nonsynonymous to synonymous nucleotide-substitution rates, particularly in the mature region of the proteins. Using ML, all 21 polymorphic amino acid sites were identified to be under diversifying selection. Of these 21 amino acids, 19 are located in the mature protein region, suggesting that selection may have acted on the functional portions of the proteins. Further investigation of gene copy number and organization revealed that the scr74 gene family comprises at least three copies located in a region of no more than 300 kb of the P. infestans genome. We found evidence that recombination contributed to sequence divergence within at least one gene locus. These results led us to propose an evolutionary model that involves gene duplication and recombination, followed by functional divergence of scr74 genes. This study provides support for using diversifying selection as a criterion for identifying candidate effector genes from sequence databases.