Regulation of Indole Signalling during the Transition of E. coli from Exponential to Stationary Phase.

During the transition from exponential to stationary phase E. coli produces a substantial quantity of the small, aromatic signalling molecule indole. In LB medium the supernatant indole concentration reaches a maximum of 0.5-1 mM. At this concentration indole has been implicated in many processes inducing acid resistance and the modulation of virulence. It has recently been shown that cell-associated indole transiently reaches a very high concentration (approx. 60 mM) during stationary phase entry, presumably because indole is being produced more rapidly than it can leave the cell. It is proposed that this indole pulse inhibits growth and cell division, causing the culture to enter stationary phase before nutrients are completely exhausted, with benefits for survival in long-term stationary phase. This study asks how E. coli cells rapidly upregulate indole production during stationary phase entry and why the indole pulse has a duration of only 10-15 min. We find that at the start of the pulse tryptophanase synthesis is triggered by glucose depletion and that this is correlates with the up-regulation of indole synthesis. The magnitude and duration of the resulting indole pulse are dependent upon the availability of exogenous tryptophan. Indole production stops when all the available tryptophan is depleted and the cell-associated indole equilibrates with the supernatant.HG was funded by a Biotechnology and Biological Sciences Research Council Doctoral Training Grant studentship (http://www.bbsrc.ac.uk/home/home.aspx), grant number PCAG-EJNF.This is the final published version. It first appeared at http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0136691

Plasmids in the driving seat: The regulatory RNA Rcd gives plasmid ColE1 control over division and growth of its E. coli host.

Regulation by non-coding RNAs was found to be widespread among plasmids and other mobile elements of bacteria well before its ubiquity in the eukaryotic world was suspected. As an increasing number of examples was characterised, a common mechanism began to emerge. Non-coding RNAs, such as CopA and Sok from plasmid R1, or RNAI from ColE1, exerted regulation by refolding the secondary structures of their target RNAs or modifying their translation. One regulatory RNA that seemed to swim against the tide was Rcd, encoded within the multimer resolution site of ColE1. Required for high fidelity maintenance of the plasmid in recombination-proficient hosts, Rcd was found to have a protein target, elevating indole production by stimulating tryptophanase. Rcd production is up-regulated in dimer-containing cells and the consequent increase in indole is part of the response to the rapid accumulation of dimers by over-replication (known as the dimer catastrophe). It is proposed that indole simultaneously inhibits cell division and plasmid replication, stopping the catastrophe and allowing time for the resolution of dimers to monomers. The idea of a plasmid-mediated cell division checkpoint, proposed but then discarded in the 1980s, appears to be enjoying a revival.HG was funded by a BBSRC DTG studentship, grant number PCAG-EJNF.This is the final version. It was first published by Elsevier at http://www.sciencedirect.com/science/article/pii/S0147619X1400088

Teaching and Professional Fellowship Report 2003/4. Access, Acrobat and Animations: Enhancing the Blackboard Virtual Learning Environment

Dr Gaimster's contextual considerations were - the impact of the Special Educational Needs and Disabillty Act 2001 (SENDA); that Blackboard has been used mainly as a vehicle for communication and that much was text based; staff wanted to use images and multimedia to enhance their sites, but did not have the technical skills required; the potential within Blackboard for the streamlining of course administration and a reduction in the use of paper based forms. She explored the potential for integrating Adobe Acrobat 6 into the Blackboard VLE, researching the benefits for staff and students in terms of improved usability, functionality and interactivity. This was achieved through the creation of interactive forms and course documents, and templates for the inclusion of multimedia elements within teaching and learning material.Amongst Julia's recommendations are the adoption of a strategic approach to accessibility; the integration of accessibility training into Blackboard courses, CLTAD and IT training programmes; the production of guidelines and templates for the production of teaching and learning materials both on- and off-line; training for users with disabilities to ensure that they are aware of and are able to use all accessibility functions

High level indole signalling in Escherichia coli

Indole is a small signalling molecule, produced by many species of bacteria, including Escherichia coli. It is made by the enzyme tryptophanase, which converts tryptophan into indole, pyruvate and ammonia. Indole has diverse roles in E. coli, including regulation of biofilm formation, acid resistance and pathogenicity. In these cases, E. coli responds to a low, persistent level of indole (0.5-1 mM), similar to the concentration found in an E. coli culture supernatant in stationary phase (typically 0.3-0.8 mM). Recently, it has been shown that much higher concentrations of indole (3-5 mM) inhibit cell division by acting as an ionophore to dissipate the membrane potential. However the biological relevance of such high concentrations, and therefore these aspects of indole signalling, has been questioned. This work has investigated the role of indole signalling during entry into stationary phase, when indole production is quickly upregulated. The viability of non indole producing mutants was compared to wild-type indole producing cells. In the short term indole producers suffered a growth disadvantage, but in the long term they were significantly more viable than their indole non-producing counterparts. The addition of 1 mM indole to the indole non-producing culture failed to complement the phenotype. A hypothesis was developed that a high rate of indole production during stationary phase entry leads to a transient, high concentration of indole inside the cell. This regulates cell growth and division via the ionophore mechanism. The validity of this indole pulse signalling hypothesis was tested by measuring cellassociated indole. For a brief time during stationary phase entry cell-associated concentrations reached 60 mM. Cell-associated indole represents an average of indole in the cytoplasm and the cell membrane. It was shown that indole has an approximately 100-fold greater affinity for the cell membrane. 60 mM cell associated indole is equivalent to approximately 4 mM in the culture supernatant, suggesting that the indole ‘pulse’ is sufficient to inhibit growth and cell division on entry into stationary phase. The indole pulse was dependent on the stationary phase sigma factor, SigmaS, which increases tryptophanase expression on entry into stationary phase. This increased tryptophanase expression occurs immediately prior to increased indole production. The end of the pulse seems to correlate with the exhaustion of tryptophan in the growth medium

Transcriptional and Environmental Control of Bacterial Denitrification and N2O Emissions

In oxygen-limited environments, denitrifying bacteria can switch from oxygen-dependent respiration to nitrate (NO3−) respiration in which the NO3− is sequentially reduced via nitrite (NO2−), nitric oxide (NO) and nitrous oxide (N2O) to dinitrogen (N2). However, atmospheric N2O continues to rise, a significant proportion of which is microbial in origin. This implies that the enzyme responsible for N2O reduction, nitrous oxide reductase (NosZ), does not always carry out the final step of denitrification either efficiently, or in synchrony with the rest of the pathway. Despite a solid understanding of the biochemistry underpinning denitrification, there is a relatively poor understanding of how environmental signals and respective transcriptional regulators control expression of the denitrification apparatus. This mini-review will describe the current picture for transcriptional regulation of denitrification in the model bacterium, Paracoccus denitrificans, highlighting differences in other denitrifying bacteria where appropriate, as well as gaps in our understanding. Alongside this, the emerging role of small regulatory RNAs (sRNAs) in regulation of denitrification will be discussed. We will conclude by speculating how this information, aside from providing a better understanding of the denitrification process, can be translated into development of novel greenhouse gas mitigation strategies

The Geoff Egan Memorial Lecture 2011. Artefacts, art and artifice: reconsidering iconographic sources for archaeological objects in early modern Europe

A first systematic analysis of historic domestic material culture depicted in contemporaneous Western painting and prints, c.1400-1800. Drawing on an extensive data set, the paper proposes to methodologies and hermeneutics for historical analysis and archaeological correspondence

Genome-Wide Discovery of Putative sRNAs in Paracoccus denitrificans Expressed under Nitrous Oxide Emitting Conditions

Nitrous oxide (N2O) is a stable, ozone depleting greenhouse gas. Emissions of N2O into the atmosphere continue to rise, primarily due to the use of nitrogen-containing fertilizers by soil denitrifying microbes. It is clear more effective mitigation strategies are required to reduce emissions. One way to help develop future mitigation strategies is to address the currently poor understanding of transcriptional regulation of the enzymes used to produce and consume N2O. With this ultimate aim in mind we performed RNA-seq on a model soil denitrifier, Paracoccus denitrificans, cultured anaerobically under high N2O and low N2O emitting conditions, and aerobically under zero N2O emitting conditions to identify small RNAs (sRNAs) with potential regulatory functions transcribed under these conditions. sRNAs are short (∼40–500 nucleotides) non-coding RNAs that regulate a wide range of activities in many bacteria. Hundred and sixty seven sRNAs were identified throughout the P. denitrificans genome which are either present in intergenic regions or located antisense to ORFs. Furthermore, many of these sRNAs are differentially expressed under high N2O and low N2O emitting conditions respectively, suggesting they may play a role in production or reduction of N2O. Expression of 16 of these sRNAs have been confirmed by RT-PCR. Ninety percent of the sRNAs are predicted to form secondary structures. Predicted targets include transporters and a number of transcriptional regulators. A number of sRNAs were conserved in other members of the α-proteobacteria. Better understanding of the sRNA factors which contribute to expression of the machinery required to reduce N2O will, in turn, help to inform strategies for mitigation of N2O emissions

Spirit, mind and body: the archaeology of monastic healing

Archaeology and material culture are used in this chapter to consider how monastic experience responded to illness, ageing and disability. The approach taken is influenced by the material study of religion, which interrogates how bodies and things engage to construct the sensory experience of religion, and by practice-based approaches in archaeology, which examine the active role of space and material culture in shaping religious agency and embodiment. The archaeology of monastic healing focuses on the full spectrum of healing technologies, from managing the body in order to prevent illness, through to the treatment of the sick and preparation of the corpse for burial

A central small RNA regulatory circuit controlling bacterial denitrification and N2O emissions

Global atmospheric loading of the climate active gas nitrous oxide (N2O) continues to increase. A significant proportion of anthropogenic N2O emissions arise from microbial transformation of nitrogen-based fertilisers during denitrification, making microbial N2O emissions a key target for greenhouse gas reduction strategies. The genetic, physiological and environmental regulation of microbial mediated N2O flux is poorly  understood and is therefore a critical knowledge gap in development of successful  mitigation approaches. We have previously mapped the transcriptional landscape of 1 the model soil denitrifying bacterium, Paracoccus denitrificans. Here, we show that a 32 single bacterial sRNA can control the denitrification rate of P. denitrificans by stalling denitrification at nitrite reduction to limit production of downstream pathway intermediates and N2O emissions. Overexpression of sRNA-29 downregulates nitrite reductase and limits NO and N2O production by cells. RNA-seq analysis revealed 53 genes are controlled by sRNA-29, one of which is a previously uncharacterised GntR- type transcriptional regulator. Overexpression of this regulator phenocopies sRNA-29 overexpression and allows us propose a model whereby sRNA-29 enhances levels of  the regulator to repress denitrification in appropriate conditions. Our identification of a new regulatory pathway controlling the core denitrification pathway in bacteria highlights the current chasm in knowledge regarding genetic regulation of this pivotal biogeochemical process, which needs to close to support future biological and chemical N2O mitigation strategies

A multi-spectroscopic study for the characterization and definition of production techniques of German ceramic sherds

The aim of this archaeometric study is to recover information regarding technological processes and raw materials used for the production of ceramic sherds coming from five central and Eastern German sites, between Lower Saxony and Saxony states. The ceramic fragments have been investigated by a multi-spectroscopic approach: Fourier transform infrared spectroscopy (FT-IR), micro-Raman spectroscopy and X-ray Fluorescence (XRF) were employed to characterize both ceramic bodies and glazes. Moreover the innovative application of Laser Induced Fluorescence (LIF) on ceramic findings has been proposed and evaluated. Chemical and mineralogical composition, as well as microstructure, of ceramic mixture and glaze are correlated to native material composition and firing temperature, which have become a fundamental features in archaeometric research and play a key role in understanding the provenance of the pottery and its production techniques.The multi-spectroscopic approach applied in this work has enabled the ability to characterize the ceramic sherds and to investigate through non-destructive techniques both ceramic glaze and matrix giving information regarding the raw materials and pigments/colourants used, and regarding firing temperature and technology. The present study carried on using complementary methods suggests different raw material sources and temperature kilns. These data are in agreement with the location of ceramic sites and with data in literature. Furthermore, the interesting results suggest that non-destructive techniques, such as LIF and Raman spectroscopy, are promising methods for ceramic and glaze characterization. (C) 2015 Elsevier B.V. All rights reserved