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

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

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

MG1655 and MG1655 TnaA-GFP grown in minimal medium with 0.2 (panels A+B), 0.1(C+D) or 0.05% (E+F) glucose produce TnaA and indole when 0.5 mM tryptophan is added.

<p>MG1655 and MG1655 TnaA- GFP cells were grown in minimal medium with 0.2, 0.1 or 0.05% glucose, with and without 0.5 mM tryptophan. Samples were removed hourly for 8 hours. The fluorescence intensity (excitation 480, emission 510 nm) (panels A, C, E) was measured and the supernatant was assayed for external indole using Kovacs assay (panel B,D F)). Data shown are the mean values ± standard deviation for three independent repeats.</p

List of <i>E</i>. <i>coli</i> strains used in this work.

<p>List of <i>E</i>. <i>coli</i> strains used in this work.</p

The addition of tryptophan to a stationary phase BW25113 culture leads to further indole synthesis.

<p>Stationary phase (24 hours) BW25113 cells were incubated with no additional tryptophan or 2 mM tryptophan added every subsequent 24 hours (arrows indicate times of addition). Samples were removed then centrifuged to remove cells. The supernatant was assayed for external indole using Kovacs assay. Data shown are the mean values ± standard deviation for three independent repeats.</p

The effect of tryptophan supplementation on the indole pulse during stationary phase entry.

<p>A culture of growing BW25113 in LB medium with 0 or 0.5 mM tryptophan added was sampled regularly. The supernatant was assayed for external indole using Kovacs assay (panel A) and the cell pellet was assayed for indole using Kovacs assay (panel B). Data shown are the mean values ± standard deviation for three independent repeats.</p

Local and Universal Action: The Paradoxes of Indole Signalling in Bacteria.

Indole is a signalling molecule produced by many bacterial species and involved in intraspecies, interspecies, and interkingdom signalling. Despite the increasing volume of research published in this area, many aspects of indole signalling remain enigmatic. There is disagreement over the mechanism of indole import and export and no clearly defined target through which its effects are exerted. Progress is hindered further by the confused and sometimes contradictory body of indole research literature. We explore the reasons behind this lack of consistency and speculate whether the discovery of a new, pulse mode of indole signalling, together with a move away from the idea of a conventional protein target, might help to overcome these problems and enable the field to move forward.The Leverhulme Trust, UK (RPG-2015-184