Molecular Characterization of a Multidrug Resistance IncF Plasmid from the Globally Disseminated Escherichia coli ST131 Clone

Escherichia coli sequence type 131 (E. coli ST131) is a recently emerged and globally disseminated multidrug resistant clone associated with urinary tract and bloodstream infections. Plasmids represent a major vehicle for the carriage of antibiotic resistance genes in E. coli ST131. In this study, we determined the complete sequence and performed a comprehensive annotation of pEC958, an IncF plasmid from the E. coli ST131 reference strain EC958. Plasmid pEC958 is 135.6 kb in size, harbours two replicons (RepFIA and RepFII) and contains 12 antibiotic resistance genes (including the bla _CTX-M-15 gene). We also carried out hyper-saturated transposon mutagenesis and multiplexed transposon directed insertion-site sequencing (TraDIS) to investigate the biology of pEC958. TraDIS data showed that while only the RepFII replicon was required for pEC958 replication, the RepFIA replicon contains genes essential for its partitioning. Thus, our data provides direct evidence that the RepFIA and RepFII replicons in pEC958 cooperate to ensure their stable inheritance. The gene encoding the antitoxin component (ccdA) of the post-segregational killing system CcdAB was also protected from mutagenesis, demonstrating this system is active. Sequence comparison with a global collection of ST131 strains suggest that IncF represents the most common type of plasmid in this clone, and underscores the need to understand its evolution and contribution to the spread of antibiotic resistance genes in E. coli ST131.     

The spatial distribution of badgers, setts and latrines: the risk for intra-specific and badger-livestock disease transmission

The spatial distribution of wildlife hosts and the associated environmental distribution of their excretory products are important factors associated with the risk of disease transmission between wildlife and livestock. At a landscape scale, heterogeneous distribution of a wildlife host will create regional hot spots for disease risk, while at the farm level, distributional patterns of wildlife excretory products as well as habitat use are of primary importance to the assessment of disease risk to livestock. In the UK, badgers have been implicated in the transmission of bovine tuberculosis to cattle. In this study, we focus on the spatial and social organization and habitat use of badgers as well as the distributions of their excretions at latrine and sett sites to assess intra- and inter-species (badger–cattle) disease risk. Across the study site, badger latrines and setts were found in prominent clusters, at distances of up to 250 and 200 m respectively. This was partly due to small-scale clustering of latrines around sett sites, so that disease risk may be higher within the vicinity of setts. The clustered distribution suggests that sites of high risk for TB transmission may be localised within farms. Exclusion of cattle from the few sett and latrine sites within their grazing pasture is therefore likely to provide an effective way of reducing the risk of disease transmission. We also found evidence of social sub-division within badger social groups based on differences in the use of main and outlier setts. This may contribute to localised clusters of infection within the badger population, resulting in heterogeneous patterns of environmental disease risk to the wider host community. A greater understanding of variation in host behaviour and its implications for patterns of disease will allow the development of more targeted and effective management strategies for wildlife disease in group-living hosts.     

Gene Designer: a synthetic biology tool for constructing artificial DNA segments

Background  

Direct synthesis of genes is rapidly becoming the most efficient way to make functional genetic constructs and enables applications such as codon optimization, RNAi resistant genes and protein engineering. Here we introduce a software tool that drastically facilitates the design of synthetic genes.     

Temperature acclimation alters cardiac performance in the lobster Homarus americanus

The American lobster is a poikilotherm that inhabits a marine environment where temperature varies over a 25°C range and depends on the winds, the tides and the seasons. To determine how cardiac performance depends on the water temperature to which the lobsters are acclimated we measured lobster heart rates in vivo. The upper limit for cardiac function in lobsters acclimated to 20°C is approximately 29°C, 5°C warmer than that measured in lobsters acclimated to 4°C. Warm acclimation also slows the lobster heart rate within the temperature range from 4 to 12°C. Both effects are apparent after relatively short periods of warm acclimation (3–14 days). However, warm acclimation impairs cardiac function at cold temperatures: following several hours exposure to frigid (<5°C) temperatures heart rates become slow and arrhythmic in warm acclimated, but not cold acclimated, lobsters. Thus, acclimation temperature determines the thermal limits for cardiac function at both extremes of the 25°C temperature range lobsters inhabit in the wild. These observations suggest that regulation of cardiac thermal tolerance by the prevailing environmental temperature protects against the possibility of cardiac failure due to thermal stress.     

Regulation of galactokinase gene expression in Tetrahymena thermophila. I. Intracellular catecholamine control of galactokinase expression

The addition of glucose to the medium of Tetrahymena thermophila results in a 7-fold repression of galactokinase (EC 2.7.1.6; ATP:D-galactose-1-phosphotransferase). The presence of millimolar amounts of the catecholamines dopa, dopamine, norepinephrine, and epinephrine or the hormone glucagon also results in the repression of galactokinase in the absence of glucose. The addition of millimolar amounts of adrenergic agonists (isoproterenol, tyramine, 2-amino-6,7-dihydroxytetrahydronaphthalene) results in significant repression of galactokinase in the absence of glucose; concentrations of 2-amino-6,7-dihydroxytetrahydronaphthalene less than or equal to 10(-4) M result in a derepression of galactokinase specific activity. Addition of adrenergic antagonists (propranolol, dichloroisoproterenol) have no effect on galactokinase activity at concentrations less than 10(-4) M but do arrest cell growth at greater concentrations. The addition of the cAMP analogs caffeine or theophylline in millimolar amounts results in repression of galactokinase activity; however, cell growth is greatly slowed or completely arrested at these concentrations. Analysis of the repression response of several mutants demonstrates that mutants deficient in catecholamine biosynthesis are altered in their regulation of galactokinase. Measurements of intracellular cAMP levels for 0-24 h following the addition of several of the above compounds to exponentially growing cells did not demonstrate any change over this period. Measurement of intracellular cAMP levels for 24 h following the addition of glucose or galactose to exponentially growing wild-type and mutant cell strains did not demonstrate any difference in cAMP concentrations over this period although a wide range of galactokinase activity was exhibited. Starvation of wild-type cells prior to the addition of glucose in minimal medium without added carbohydrate resulted in a significant increase in cAMP following the addition of glucose. This increase is demonstrated to be dependent upon the ability of the cells to resume division after the arrest of growth and is not correlated with galactokinase regulation. These results support the conclusion that cAMP is not involved in the repression of galactokinase gene expression initiated by glucose or hormone-like effectors and demonstrate the participation of an adrenergic control system in galactokinase regulation which is subordinate to the regulation by glucose. A possible model is discussed.     

Collagen cross-linking: distribution of hydroxypyridinium cross-links among invertebrate phyla and tissues

1. Using a specific HPLC assay, a wide variety of marine invertebrate connective tissues was screened for the 3-hydroxypyridinium amino acids that are prominent intermolecular cross-linking residues in the collagens of many vertebrate connective tissues. 2. One or both of the two structural forms that exist, hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP), was found in organisms from the following phyla: coelenterata, Annelida, Echinodermata, Mollusca and Arthropoda. 3. Neither amino acid was found in tissues from representative species of Porifera and Chordata. 4. Of special note was an unusually high ratio of LP to HP in Limulus polyphemus gill cartilage.