Visualizing the Replisome, Chromosome Breaks, and Replication Restart in Bacillus subtilis.

Research over the last two decades has revealed that bacterial genomes are highly organized and that bacteria have sophisticated mechanisms in place to ensure their correct replication and segregation into progeny cells. Here we discuss techniques that can be used with live bacterial cells to analyze DNA replisome dynamics, double-strand chromosome breaks, and restart of repaired replication forks.

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.

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 arises from microbial transformation of nitrogen-based fertilizers during denitrification, making microbial N2O emissions a key target for greenhouse gas reduction strategies. The genetic, physiological, and environmental regulation of microbially mediated N2O flux is poorly understood and therefore represents a critical knowledge gap in the development of successful mitigation approaches. We have previously mapped the transcriptional landscape of the model soil-denitrifying bacterium Paracoccus denitrificans Here, we show that a single bacterial small RNA (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 sequencing (RNA-seq) analysis revealed 53 genes that are controlled by sRNA-29, one of which is a previously uncharacterized GntR-type transcriptional regulator. Overexpression of this regulator phenocopies sRNA-29 overexpression and allows us to propose a model whereby sRNA-29 enhances levels of the regulator to repress denitrification under 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 be closed to support future biological and chemical N2O mitigation strategies.IMPORTANCE N2O is an important greenhouse gas and a major cause of ozone depletion. Denitrifying bacteria play vital roles in the production and consumption of N2O in many environments. Complete denitrification consists of the conversion of a soluble N-oxyanion, nitrate (NO3-), to an inert gaseous N-oxide, dinitrogen (N2). Incomplete denitrification can occur if conditions are prohibitive, for example, under conditions of low soil copper concentrations, leading to emission of N2O rather than N2 Although enzymatically well characterized, the genetic drivers that regulate denitrification in response to environmental and physiological cues are not fully understood. This study identified a new regulatory sRNA-based control mechanism for denitrification in the model denitrifying bacterium P. denitrificans Overexpression of this sRNA slows the rate of denitrification. This report highlights that there are gaps in understanding the regulation of this important pathway which need to be filled if strategies for N2O mitigation can be rationally and carefully developed.

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 minireview describes 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 in regulation of denitrification is discussed. We 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.

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.

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.

The indole pulse: a new perspective on indole signalling in Escherichia coli.

Indole has diverse signalling roles, including modulation of biofilm formation, virulence and stress responses. Changes are induced by indole concentrations of 0.5-1.0 mM, similar to those found in the supernatant of Escherichia coli stationary phase culture. Here we describe an alternative mode of indole signalling that promotes the survival of E. coli cells during long-term stationary phase. A mutant that has lost the ability to produce indole demonstrates reduced survival under these conditions. Significantly, the addition of 1 mM indole to the culture supernatant is insufficient to restore long-term survival to the mutant. We provide evidence that the pertinent signal in this case is not 1 mM indole in the culture supernatant but a transient pulse of intra-cellular indole at the transition from exponential growth to stationary phase. During this pulse the cell-associated indole reaches a maximum of approximately 60 mM. We argue that this is sufficient to inhibit growth and division by an ionophore-based mechanism and causes the cells to enter stationary phase before resources are exhausted. The unused resources are used to repair and maintain cells during the extended period of starvation.