Nucleotide second messengers in bacterial decision making

Since the initial discovery of bacterial nucleotide second messengers (NSMs), we have made huge progress towards understanding these complex signalling networks. Many NSM networks contain dozens of metabolic enzymes and binding targets, whose activity is tightly controlled at every regulatory level. They function as global regulators and in specific signalling circuits, controlling multiple aspects of bacterial behaviour and development. Despite these advances there is much still to discover, with current research focussing on the molecular mechanisms of signalling circuits, the role of the environment in controlling NSM pathways and attempts to understand signalling at the whole cell/community level. Here we examine recent developments in the NSM signalling field and discuss their implications for understanding this important driver of microbial behaviour

The biochemical and biophysical characterisation of protein antibiotics targeting Pectobacterium spp. and Streptococcus agalactiae

Food security is the idea by which a population has enough food to sustain itself without famine. A large number of factors can influence the stability of food production, including diseases caused by microorganisms. Pectobacterium spp. is one of the leading causes of soft rot disease, resulting in crop losses both pre- and post-harvest. Bacteriocins are potent narrow spectrum protein antibiotics which target closely related bacteria to the producing strain. Ferredoxin-containing bacteriocins produced by and targeted towards Pectobacterium species have both a different domain organisation and uptake mechanism to all known Gram-negative bacteriocins. This work has shown that pectocins are able to pass through the outer membrane of Pectobacterium spp. by parasitising the ferredoxin uptake receptor, FusA. This uptake requires the pectocins to be flexible in order to pass through the lumen of the barrel and enter the periplasm. This work has shown an interaction between FusB and pectocin M1, suggesting a novel mechanism of uptake. Streptococcus agalactiae is the causative agent of disease in a wide range of hosts, ranging from human neonates to farmed Tilapia. S. agalactiae infection has a detrimental effect on the dairy industry each year as it is the leading cause of mastitis in cattle. As well as this, the prevalence of S. agalactiae in farmed fish has resulted in large numbers of infected fish and subsequently the infection of consumers. Bacteriocins produced by Gram-positive bacteria are often small modified peptides which target the peptidoglycan layer or cytoplasmic membrane of the target cell. However, a small number of protein bacteriocins produced by Gram-positive bacteria have been discovered, with the best characterised of these being lysostaphin. It has been shown that bacteriocins similar to lysostaphin are also produced by other Gram-positive bacteria, such as zoocin A produced by Streptococcus zooepidemicus. Prior to this work a novel protein bacteriocin produced by and targeted towards S. agalactiae, named agalacticin A, was discovered. It was predicted that this bacteriocin was similar in structure and function to zoocin A. This work has gone some way to structurally characterising agalacticin A, showing a two-domain structure joined by a flexible linker region allowing for the two domains to move independently. As well as this it has shown the importance of the histidine residues at the predicted active site confirming the similarities between agalacticin A and zoocin A. Together this work has gone some way to showing the potential of agalacticin A as a novel therapeutic. Altogether this work has characterised three novel bacteriocins active against pathogenic bacteria to gain a better understanding of their structure, mechanism of action and uptake

Brain cell reservoirs of latent virus in presymptomatic HIV-infected individuals

We detected HIV-1 DNA in pure populations of perivascular macrophages, parenchymal microglia, and astrocytes, isolated using laser microdissection from brain tissue of five untreated individuals who died in the presymptomatic stage of infection from non-HIV causes. HIV-1 DNA was detected in the three cell populations, most consistently in perivascular macrophages, without evidence of productive infection. The percentage of PCR reactions detecting HIV-1 DNA in perivascular macrophages correlated inversely with peripheral blood CD4 counts. These findings demonstrate that brain cell reservoirs of latent HIV-1 exist before pathological HIV encephalitis and suggest that perivascular macrophage trafficking of latent virus into the brain increases with immunosuppression

CCL2 nitration is a negative regulator of chemokine-mediated inflammation.

Chemokines promote leukocyte recruitment during inflammation. The oxidative burst is an important effector mechanism, this leads to the generation of reactive nitrogen species (RNS), including peroxynitrite (ONOO). The current study was performed to determine the potential for nitration to alter the chemical and biological properties of the prototypical CC chemokine, CCL2. Immunofluorescence was performed to assess the presence of RNS in kidney biopsies. Co-localisation was observed between RNS-modified tyrosine residues and the chemokine CCL2 in diseased kidneys. Nitration reduced the potential of CCL2 to stimulate monocyte migration in diffusion gradient chemotaxis assays (p < 0.05). This was consistent with a trend towards reduced affinity of the nitrated chemokine for its cognate receptor CCR2b. The nitrated chemokine was unable to induce transendothelial monocyte migration in vitro and failed to promote leukocyte recruitment when added to murine air pouches (p < 0.05). This could potentially be attributed to reduced glycosaminoglycan binding ability, as surface plasmon resonance spectroscopy showed that nitration reduced heparan sulphate binding by CCL2. Importantly, intravenous administration of nitrated CCL2 also inhibited the normal recruitment of leukocytes to murine air pouches filled with unmodified CCL2. Together these data suggest that nitration of CCL2 during inflammation provides a mechanism to limit and resolve acute inflammation

Interaction specificity of Arabidopsis 14-3-3 proteins with phototropin receptor kinases

Phototropin receptor kinases play an important roles in optimising plant growth in response to blue light. Much is known regarding their photochemical reactivity, yet little progress has been made to identify downstream signalling components. Here, we isolated several interacting proteins for Arabidopsis phototropin 1 (phot1) by yeast two-hybrid screening. These include members of the NPH3/RPT2 (NRL) protein family, proteins associated with vesicle trafficking, and the 14-3-3 lambda (?) isoform from Arabidopsis . 14-3-3? and phot1 were found to colocalise and interact in vivo. Moreover, 14-3-3 binding to phot1 was limited to non-epsilon 14-3-3 isoforms and was dependent on key sites of receptor autophosphorylation. No 14-3-3 binding was detected for Arabidopsis phot2, suggesting that 14-3-3 proteins represent specific mode of phot1 signalling

The UK Crop Microbiome Cryobank: a utility and model for supporting Phytobiomes research

Plant microbiomes are the microbial communities essential to the functioning of the phytobiome—the system that consist of plants, their environment, and their associated communities of organisms. A healthy, functional phytobiome is critical to crop health, improved yields and quality food. However, crop microbiomes are relatively under-researched, and this is associated with a fundamental need to underpin phytobiome research through the provision of a supporting infrastructure. The UK Crop Microbiome Cryobank (UKCMC) project is developing a unique, integrated and open-access resource to enable the development of solutions to improve soil and crop health. Six economically important crops (Barley, Fava Bean, Oats, Oil Seed Rape, Sugar Beet and Wheat) are targeted, and the methods as well as data outputs will underpin research activity both in the UK and internationally. This manuscript describes the approaches being taken, from characterisation, cryopreservation and analysis of the crop microbiome through to potential applications. We believe that the model research framework proposed is transferable to different crop and soil systems, acting not only as a mechanism to conserve biodiversity, but as a potential facilitator of sustainable agriculture systems

Structural insights into the mechanism of adaptive ribosomal modification by Pseudomonas RimK

Bacteria are equipped with a diverse set of regulatory tools that allow them to quickly adapt to their environment. The RimK system allows for Pseudomonas spp. to adapt through post-transcriptional regulation by altering the ribosomal subunit RpsF. RimK is found in a wide range of bacteria with a conserved amino acid sequence, however, the genetic context and the role of this protein is highly diverse. By solving and comparing the structures of RimK homologs from two related but functionally divergent systems, we uncovered key structural differences that likely contribute to the different activity levels of each of these homologs. Moreover, we were able to clearly resolve the active site of this protein for the first time, resolving binding of the glutamate substrate. This work advances our understanding of how subtle differences in protein sequence and structure can have profound effects on protein activity, which can in turn result in widespread mechanistic changes

Pyocin S5 import into Pseudomonas aeruginosa reveals a generic mode of bacteriocin transport

Pyocin S5 (PyoS5) is a potent protein bacteriocin that eradicates the human pathogen Pseudomonas aeruginosa in animal infection models, but its import mechanism is poorly understood. Here, using crystallography, biophysical and biochemical analyses, and live-cell imaging, we define the entry process of PyoS5 and reveal links to the transport mechanisms of other bacteriocins. In addition to its C-terminal pore-forming domain, elongated PyoS5 comprises two novel tandemly repeated kinked 3-helix bundle domains that structure-based alignments identify as key import domains in other pyocins. The central domain binds the lipid-bound common polysaccharide antigen, allowing the pyocin to accumulate on the cell surface. The N-terminal domain binds the ferric pyochelin transporter FptA while its associated disordered region binds the inner membrane protein TonB1, which together drive import of the bacteriocin across the outer membrane. Finally, we identify the minimal requirements for sensitizing Escherichia coli toward PyoS5, as well as other pyocins, and suggest that a generic pathway likely underpins the import of all TonB-dependent bacteriocins across the outer membrane of Gram-negative bacteria

Evaluating the Influence of Epidemiological Parameters and Host Ecology on the Spread of Phocine Distemper Virus through Populations of Harbour Seals

Catriona Harris was supported by a grant from the UK Natural Environment Research Council. The funders had no role in study design, data collections and analysis, decision to publish, or preparation of the manuscript.Background: Outbreaks of phocine distemper virus (PDV) in Europe during 1988 and 2002 were responsible for the death of around 23,000 and 30,000 harbour seals, respectively. These epidemics, particularly the one in 2002, provided an unusual opportunity to estimate epidemic parameters for a wildlife disease. There were marked regional differences in the values of some parameters both within and between epidemics. Methodology and Principal Findings: We used an individual-based model of seal movement that allowed us to incorporate realistic representations of space, time and animal behaviour into a traditional epidemiological modelling framework. We explored the potential influence of a range of ecological (foraging trip duration, time of epidemic onset, population size) and epidemiological (length of infectious period, contact rate between infectious and susceptible individuals, case mortality) parameters on four readily-measurable epidemic characteristics (number of dead individuals, duration of epidemic, peak mortality date and prevalence) and on the probability that an epidemic would occur in a particular region. We analysed the outputs as if they were the results of a series of virtual experiments, using Generalised Linear Modelling. All six variables had a significant effect on the probability that an epidemic would be recognised as an unusual mortality event by human observers. Conclusions: Regional and temporal variation in contact rate was the most likely cause of the observed differences between the two epidemics. This variation could be a consequence of differences in the way individuals divide their time between land and sea at different times of the year.Publisher PDFPeer reviewe

Compensatory mutations reducing the fitness cost of plasmid carriage occur in plant rhizosphere communities

Plasmids drive bacterial evolutionary innovation by transferring ecologically important functions between lineages, but acquiring a plasmid often comes at a fitness cost to the host cell. Compensatory mutations, which ameliorate the cost of plasmid carriage, promote plasmid maintenance in simplified laboratory media across diverse plasmid-host associations. Whether such compensatory evolution can occur in more complex communities inhabiting natural environmental niches where evolutionary paths may be more constrained is, however, unclear. Here, we show a substantial fitness cost of carrying the large conjugative plasmid pQBR103 in Pseudomonas fluorescens SBW25 in the plant rhizosphere. This plasmid fitness cost could be ameliorated by compensatory mutations affecting the chromosomal global regulatory system gacA/gacS, which arose rapidly in plant rhizosphere communities and were exclusive to plasmid carriers. These findings expand our understanding of the importance of compensatory evolution in plasmid dynamics beyond simplified lab media. Compensatory mutations contribute to plasmid survival in bacterial populations living within complex microbial communities in their environmental niche