Massive Diversification in Aging Colonies of Escherichia coli

The evolutionary success of bacteria depends greatly on their capacity to continually generate phenotypic diversity. Structured environments are particularly favorable for diversification because of attenuated clonal interference, which renders selective sweeps nearly impossible and enhances opportunities for adaptive radiation. We examined at the microscale level the emergence and the spatial and temporal dynamics of phenotypic diversity and their underlying causes in Escherichia coli colonies. An important dynamic heterogeneity in the growth, metabolic activity, morphology, gene expression patterns, stress response induction, and death patterns among cells within colonies was observed. Genetic analysis indicated that the phenotypic variation resulted mostly from mutations and that indole production, oxidative stress, and the RpoS-regulated general stress response played an important role in the generation of diversity. We observed the emergence and persistence of phenotypic variants within single colonies that exhibited variable fitness compared to the parental strain. Some variants showed improved capacity to produce biofilms, whereas others were able to use different nutrients or to tolerate antibiotics or oxidative stress. Taken together, our data show that bacterial colonies provide an ecological opportunity for the generation and maintenance of vast phenotypic diversity, which may increase the probability of population survival in unpredictable environments.     

The entire N-terminal half of TatC is involved in twin-arginine precursor binding

Translocation of twin-arginine precursor proteins across the cytoplasmic membrane of Escherichia coli requires the three membrane proteins TatA, TatB, and TatC. TatC and TatB were shown to be involved in precursor binding. We have analyzed in vitro a number of single alanine substitutions in tatC that were previously shown to compromise in vivo the function of the Tat translocase. All tatC mutants that were defective in precursor translocation into cytoplasmic membrane vesicles concomitantly interfered with precursor binding not only to TatC but also to TatB. Hence structural changes of TatC that affect precursor targeting simultaneously abolish engagement of the twin-arginine signal sequence with TatB and block the formation of a functional Tat translocase. Since these phenotypes were observed for tatC mutations spread over the first half of TatC, this entire part of the molecule must globally be involved in precursor binding.     

Cysteine scanning mutagenesis and topological mapping of the Escherichia coli twin-arginine translocase TatC Component

The TatC protein is an essential component of the Escherichia coli twin-arginine (Tat) protein translocation pathway. It is a polytopic membrane protein that forms a complex with TatB, together acting as the receptor for Tat substrates. In this study we have constructed 57 individual cysteine substitutions throughout the protein. Each of the substitutions resulted in a TatC protein that was competent to support Tat-dependent protein translocation. Accessibility studies with membrane-permeant and -impermeant thiol-reactive reagents demonstrated that TatC has six transmembrane helices, rather than the four suggested by a previous study (K. Gouffi, C.-L. Santini, and L.-F. Wu, FEBS Lett. 525:65-70, 2002). Disulfide cross-linking experiments with TatC proteins containing single cysteine residues showed that each transmembrane domain of TatC was able to interact with the same domain from a neighboring TatC protein. Surprisingly, only three of these cysteine variants retained the ability to cross-link at low temperatures. These results are consistent with the likelihood that most of the disulfide cross-links are between TatC proteins in separate TatBC complexes, suggesting that TatC is located on the periphery of the complex.     

Efficient optimization of crystallization conditions by manipulation of drop volume ratio and temperature

An efficient optimization method for the crystallization of biological macromolecules has been developed and tested. This builds on a successful high-throughput technique for the determination of initial crystallization conditions. The optimization method takes an initial condition identified through screening and then varies the concentration of the macromolecule, precipitant, and the growth temperature in a systematic manner. The amount of sample and number of steps is minimized and no biochemical reformulation is required. In the current application a robotic liquid handling system enables high-throughput use, but the technique can easily be adapted in a nonautomated setting. This method has been applied successfully for the rapid optimization of crystallization conditions in nine representative cases.     

PRTFDC1 is a genetic modifier of HPRT-deficiency in the mouse

Lesch-Nyhan disease (LND) is a severe X-linked neurological disorder caused by a deficiency of hypoxanthine phosphoribosyltransferase (HPRT). In contrast, HPRT-deficiency in the mouse does not result in the profound phenotypes such as self-injurious behavior observed in humans, and the genetic basis for this phenotypic disparity between HPRT-deficient humans and mice is unknown. To test the hypothesis that HPRT deficiency is modified by the presence/absence of phosphoribosyltransferase domain containing 1 (PRTFDC1), a paralog of HPRT that is a functional gene in humans but an inactivated pseudogene in mice, we created transgenic mice that express human PRTFDC1 in wild-type and HPRT-deficient backgrounds. Male mice expressing PRTFDC1 on either genetic background were viable and fertile. However, the presence of PRTFDC1 in the HPRT-deficient, but not wild-type mice, increased aggression as well as sensitivity to a specific amphetamine-induced stereotypy, both of which are reminiscent of the increased aggressive and self-injurious behavior exhibited by patients with LND. These results demonstrate that PRTFDC1 is a genetic modifier of HPRT-deficiency in the mouse and could therefore have important implications for unraveling the molecular etiology of LND.     

Antiangiogenic peptides and proteins: from experimental tools to clinical drugs

The formation of a 'tumor-associated vasculature', a process referred to as tumor angiogenesis, is a stromal reaction essential for tumor progression. Inhibition of tumor angiogenesis suppresses tumor growth in many experimental models, thereby indicating that tumor-associated vasculature may be a relevant target to inhibit tumor progression. Among the antiangiogenic molecules reported to date many are peptides and proteins. They include cytokines, chemokines, antibodies to vascular growth factors and growth factor receptors, soluble receptors, fragments derived from extracellular matrix proteins and small synthetic peptides. The polypeptide tumor necrosis factor (TNF, Beromun) was the first drug registered for the regional treatment of human cancer, whose mechanisms of action involved selective disruption of the tumor vasculature. More recently, bevacizumab (Avastin), an antibody against vascular endothelial growth factor (VEGF)-A, was approved as the first systemic antiangiogenic drug that had a significant impact on the survival of patients with advanced colorectal cancer, in combination with chemotherapy. Several additional peptides and antibodies with antiangiogenic activity are currently tested in clinical trials for their therapeutic efficacy. Thus, peptides, polypeptides and antibodies are emerging as leading molecules among the plethora of compounds with antiangiogenic activity. In this article, we will review some of these molecules and discuss their mechanism of action and their potential therapeutic use as anticancer agents in humans.     

The harmful alga Aureococcus anophagefferens utilizes 19′-butanoyloxyfucoxanthin as well as xanthophyll cycle carotenoids in acclimating to higher light intensities

Aureococcus anophagefferens is a picoplanktonic microalga that is very well adapted to growth at low nutrient and low light levels, causing devastating blooms (“brown tides”) in estuarine waters. To study the factors involved in long-term acclimation to different light intensities, cells were acclimated for a number of generations to growth under low light (20 μmol photons m^? 2 s^? 1), medium light (60 or 90 μmol photons m^? 2 s^? 1) and high light (200 μmol photons m^? 2 s^? 1), and were analyzed for their contents of xanthophyll cycle carotenoids (the D pool), fucoxanthin and its derivatives (the F pool), Chls c₂ and c₃, and fucoxanthin Chl a/c polypeptides (FCPs). Higher growth light intensities resulted in increased steady state levels of both diadinoxanthin and diatoxanthin. However, it also resulted in the conversion of a significant fraction of fucoxanthin to 19′-butanoyloxyfucoxanthin without a change in the total F pool. The increase in 19′-butanoyloxyfucoxanthin was paralleled by a decrease in the effective antenna size, determined from the slope of the change in F₀ as a function of increasing light intensity. Transfer of acclimated cultures to a higher light intensity showed that the conversion of fucoxanthin to its derivative was a relatively slow process (time-frame of hours). We suggest the replacement of fucoxanthin with the bulkier 19′-butanoyloxyfucoxanthin results in a decrease in the light-harvesting efficiency of the FCP antenna and is part of the long-term acclimative response to growth at higher light intensities.     

Sex-Dependent Alterations in Social Behaviour and Cortical Synaptic Activity Coincide at Different Ages in a Model of Alzheimer’s Disease

Besides memory deficits, Alzheimer’s disease (AD) patients suffer from neuropsychiatric symptoms, including alterations in social interactions, which are subject of a growing number of investigations in transgenic models of AD. Yet the biological mechanisms underlying these behavioural alterations are poorly understood. Here, a social interaction paradigm was used to assess social dysfunction in the triple-transgenic mouse model of AD (3xTg-AD). We observed that transgenic mice displayed dimorphic behavioural abnormalities at different ages. Social disinhibition was observed in 18 months old 3xTg-AD males compared to age and sex-matched control mice. In 3xTg-AD females, social disinhibition was present at 12 months followed by reduced social interactions at 18 months. These dimorphic behavioural alterations were not associated with alterations in AD neuropathological markers such as Aβ or tau levels in the frontal cortex. However, patch-clamp recordings revealed that enhanced social interactions coincided temporally with an increase in both excitatory and inhibitory basal synaptic inputs to layer 2–3 pyramidal neurons in the prefrontal cortex. These findings uncover a novel pattern of occurrence of psychiatric-like symptoms between sexes in an AD model. Our results also reveal that functional alterations in synapse activity appear as a potentially significant substrate underlying behavioural correlates of AD.