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Encapsulated ferritins belong to the universally distributed ferritin superfamily, whose members function as iron detoxification and storage systems. Encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store that is capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question because of the differences in organization of the ferroxidase catalytic site and neighboring secondary metal-binding sites. We have previously identified a putative metal-binding site on the inner surface of the Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase center. Here we present a comprehensive structural and functional study to investigate the functional relevance of this putative iron-entry site by means of enzymatic assays, MS, and X-ray crystallography. We show that catalysis occurs in the ferroxidase center and suggest a dual role for the secondary site, which both serves to attract metal ions to the ferroxidase center and acts as a flow-restricting valve to limit the activity of the ferroxidase center. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, although enhancing the ability of the encapsulated ferritin to undergo catalysis, does not influence the function of the secondary site. Our study demonstrates a novel molecular mechanism by which substrate flux to the ferroxidase center is controlled, potentially to ensure that iron oxidation is productively coupled to mineralization.

Leupeptin is a bacterial small molecule that is used worldwide as a protease inhibitor. However, its biosynthesis and genetic distribution remain unknown. We identified a family of leupeptins in gammaproteobacterial pathogens, including Photorhabdus, Xenorhabdus, and Klebsiella species, amongst others. Through genetic, metabolomic, and heterologous expression analyses, we established their construction by discretely expressed ligases and accessory enzymes. In Photorhabdus species, a hypothetical protein required for colonizing nematode hosts was established as a new class of proteases. This enzyme cleaved the tripeptide aldehyde protease inhibitors, leading to the formation of "pro-pyrazinones" featuring a hetero-tricyclic architecture. In Klebsiella oxytoca, the pathway was enriched in clinical isolates associated with respiratory tract infections. Thus, the bacterial production and proteolytic degradation of leupeptins can be associated with animal colonization phenotypes.

Different model systems have, over the years, contributed to our current understanding of the molecular mechanisms underpinning the various types of interaction between bacteria and their animal hosts. The genus Photorhabdus comprises Gram-negative insect pathogenic bacteria that are normally found as symbionts that colonize the gut of the infective juvenile stage of soil-dwelling nematodes from the family Heterorhabditis. The nematodes infect susceptible insects and release the bacteria into the insect haemolymph where the bacteria grow, resulting in the death of the insect. At this stage the nematodes feed on the bacterial biomass and, following several rounds of reproduction, the nematodes develop into infective juveniles that leave the insect cadaver in search of new hosts. Therefore Photorhabdus has three distinct and obligate roles to play during this life-cycle: (1) Photorhabdus must kill the insect host; (2) Photorhabdus must be capable of supporting nematode growth and development; and (3) Photorhabdus must be able to colonize the gut of the next generation of infective juveniles before they leave the insect cadaver. In this review I will discuss how genetic analysis has identified key genes involved in mediating, and regulating, the interaction between Photorhabdus and each of its invertebrate hosts. These studies have resulted in the characterization of several new families of toxins and a novel inter-kingdom signalling molecule and have also uncovered an important role for phase variation in the regulation of these different roles.

Photosynthetic efficiencies in plants are restricted by the CO2-fixing enzyme Rubisco but could be enhanced by introducing a CO2-concentrating mechanism (CCM) from green algae, such as Chlamydomonas reinhardtii (hereafter Chlamydomonas). A key feature of the algal CCM is aggregation of Rubisco in the pyrenoid, a liquid-like organelle in the chloroplast. Here we have used a yeast two-hybrid system and higher plants to investigate the protein-protein interaction between Rubisco and essential pyrenoid component 1 (EPYC1), a linker protein required for Rubisco aggregation. We showed that EPYC1 interacts with the small subunit of Rubisco (SSU) from Chlamydomonas and that EPYC1 has at least five SSU interaction sites. Interaction is crucially dependent on the two surface-exposed α-helices of the Chlamydomonas SSU. EPYC1 could be localized to the chloroplast in higher plants and was not detrimental to growth when expressed stably in Arabidopsis with or without a Chlamydomonas SSU. Although EPYC1 interacted with Rubisco in planta, EPYC1 was a target for proteolytic degradation. Plants expressing EPYC1 did not show obvious evidence of Rubisco aggregation. Nevertheless, hybrid Arabidopsis Rubisco containing the Chlamydomonas SSU could phase separate into liquid droplets with purified EPYC1 in vitro, providing the first evidence of pyrenoid-like aggregation for Rubisco derived from a higher plant.

Members of the Gram-negative bacterial genus Photorhabdus are all highly insect pathogenic and exist in an obligate symbiosis with the entomopathogenic nematode worm Heterorhabditis. All members of the genus produce the small-molecule 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS) as part of their secondary metabolism. IPS is a multi-potent compound that has antimicrobial, antifungal, immunomodulatory and anti-cancer activities and also plays an important role in symbiosis with the nematode. In this study we have examined the response of Photorhabdus itself to exogenous ectopic addition of IPS at physiologically relevant concentrations. We observed that the bacteria had a measureable phenotypic response, which included a decrease in bioluminescence and pigment production. This was reflected in changes in its transcriptomic response, in which we reveal a reduction in transcript levels of genes relating to many fundamental cellular processes, such as translation and oxidative phosphorylation. Our observations suggest that IPS plays an important role in the biology of Photorhabdus bacteria, fulfilling roles in quorum sensing, antibiotic-competition advantage and maintenance of the symbiotic developmental cycle.

Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using electron cryo-microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using HDX-MS, we observed dynamic behavior of the major five-fold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.TeaserCryo-EM and HDX-MS analysis of an encapsulin nanocompartment shows that the pores at the five-fold icosahedral vertex of the shell are flexible.

Coprolites (fossilized faeces) can provide valuable insights into species’ diet and related habits. In archaeozoological contexts, they are a potential source of information on human-animal interactions as well as human and animal subsistence. However, despite a broad discussion on coprolites in archaeology, such finds are rarely subject to detailed examination by researchers, perhaps due to the destructive nature of traditional analytical methods. Here, we have examined coprolitic remains from the Neolithic (third millennium BCE) settlement at Skara Brae, Orkney, using a range of modern methods: X-ray computed tomography, scanning electron microscopy, lipid and protein analysis (shotgun proteomics of the coprolite matrix as well as collagen peptide mass fingerprinting of isolated bone fragments). This combined approach minimised destructiveness of sampling, leaving sufficient material for subsequent study, while providing more information than traditional morphological examination alone. Based on gross visual examination, coprolites were predominantly attributed to domestic dogs (Canis familiaris), with morphologically identified bone inclusions derived from domestic sheep (Ovis aries) and common voles (Microtus arvalis). Partial dissection of a coprolite provided bone samples containing protein markers akin to those of domestic sheep. Considering the predominance of vertebral and distal limb bone fragments, Skara Brae dogs were probably consuming human butchery or meal refuse, either routinely fed to them or scavenged. The presumably opportunistic consumption of rodents may also have played a role in pest control.

Enterotoxigenic Escherichia coli (ETEC) and Shigella ssp. infections are associated with high rates of mortality, especially in infants in developing countries. Due to increasing levels of global antibiotic resistance exhibited by many pathogenic organisms, alternative strategies to combat such infections are urgently required. In this study, we evaluated the stability of five coliphages (four Myoviridae and one Siphoviridae phage) over a range of pH conditions and in simulated gastric conditions. The Myoviridae phages were stable across the range of pH 2 to 7, while the Siphoviridae phage, JK16, exhibited higher sensitivity to low pH. A composite mixture of these five phages was tested in vivo in a Galleria mellonella model. The obtained data clearly shows potential in treating E. coli infections prophylactically.

Four new callipeltin A derivatives (N-Q) have been isolated from the Solomon Island marine sponge Asteropus sp. Their structures were established by spectroscopic techniques followed by acid hydrolysis and derivatisation of the free amino acids, and subsequent LCMS analysis of the derivatives. The compounds were evaluated for their activity against cancer cell lines A2058 (melanoma), HT-29 (colorectal adenocarcinoma) and MCF-7 (breast adenocarcinoma) and non-malignant MRC-5 fibroblast cells. While the acyclic callipeltins P and Q were inactive the cyclic callipeltins N and O showed significant cytotoxicity against all exposed cell lines with IC50 values as low as 0.16 mu M confirming the role of cyclic configuration in biological activity. (C) 2016 Elsevier Ltd. All rights reserved.

Isotope labels are frequently used tools to track metabolites through complex biochemical pathways and to discern the mechanisms of enzyme-catalysed reactions. Isotopically-labelled L-serine is often used to monitor the activity of the first enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT) as well as labelling downstream cellular metabolites. Intrigued by the effect that isotope labels may be having on SPT catalysis, we characterised the impact of different L-serine isotopologues on the catalytic activity of recombinant SPT isozymes from humans and the bacterium Sphingomonas paucimobilis. Our data show that S. paucimobilis SPT activity displays a clear isotope effect with [2,3,3-D] L-serine, whereas the human SPT isoform does not. This suggests that whilst both human and S. paucimobilis SPT catalyse the same chemical reaction, there may well be underlying subtle differences in their catalytic mechanisms. Our results suggest that it is that the activating small subunits of human SPT that play a key role in these mechanistic variations. This study also highlight that it is important to consider the type and location of isotope labels on a substrate when they are to be used in in vitro and in vivo studies.

Acylated amino acids function as important components of the cellular membrane in some bacteria. Biosynthesis is initiated by the N-acylation of the amino acid, and this is followed by subsequent O-acylation of the acylated molecule, resulting in the production of the mature diacylated amino acid lipid. In this study, we use both genetics and liquid chromatography-mass spectrometry (LC-MS) to characterize the biosynthesis and function of a diacylated glycine lipid (GL) species produced in Bacteroides thetaiotaomicron. We, and others, have previously reported the identification of a gene, named glsB in this study, that encodes an N-acyltransferase activity responsible for the production of a monoacylated glycine called N-acyl-3-hydroxy-palmitoyl glycine (or commendamide). In all of the Bacteroidales genomes sequenced so far, the glsB gene is located immediately downstream from a gene, named glsA, that is also predicted to encode a protein with acyltransferase activity. We use LC-MS to show that the coexpression of glsB and glsA results in the production of GL in Escherichia coli. We constructed a deletion mutant of the glsB gene in B. thetaiotaomicron, and we confirm that glsB is required for the production of GL in B. thetaiotaomicron. Moreover, we show that glsB is important for the ability of B. thetaiotaomicron to adapt to stress and colonize the mammalian gut. Therefore, this report describes the genetic requirements for the biosynthesis of GL, a diacylated amino acid species that contributes to fitness in the human gut bacterium B. thetaiotaomicron. IMPORTANCE The gut microbiome has an important role in both health and disease of the host. The mammalian gut microbiome is often dominated by bacteria from the Bacteroidales, an order that includes Bacteroides and Prevotella. In this study, we have identified an acylated amino acid, called glycine lipid, produced by Bacteroides thetaiotaomicron, a beneficial bacterium originally isolated from the human gut. In addition to identifying the genes required for the production of glycine lipids, we show that glycine lipids have an important role during the adaptation of B. thetaiotaomicron to a number of environmental stresses, including exposure to either bile or air. We also show that glycine lipids are important for the normal colonization of the murine gut by B. thetaiotaomicron. This work identifies glycine lipids as an important fitness determinant in B. thetaiotaomicron and therefore increases our understanding of the molecular mechanisms underpinning colonization of the mammalian gut by beneficial bacteria.

Background A hallmark feature of Parkinson's disease (PD) is the build-up of α-synuclein protein aggregates throughout the brain; however α-synuclein is also expressed in enteric neurons. Gastrointestinal (GI) symptoms and pathology are frequently reported in PD, including constipation, increased intestinal permeability, glial pathology, and alterations to gut microbiota composition. α-synuclein can propagate through neuronal systems but the site of origin of α-synuclein pathology, whether it be the gut or the brain, is still unknown. Physical exercise is associated with alleviating symptoms of PD and with altering the composition of the gut microbiota. Methods This study investigated the effects of bilateral nigral injection of adeno-associated virus (AAV)-α-synuclein on enteric neurons, glia and neurochemistry, the gut microbiome, and bile acid metabolism in rats, some of whom were exposed to voluntary exercise. Key results Nigral overexpression of α-synuclein resulted in significant neuronal loss in the ileal submucosal plexus with no change in enteric glia. In contrast, the myenteric plexus showed a significant increase in glial expression, while neuronal numbers were maintained. Concomitant alterations were observed in the gut microbiome and related bile acid metabolism. Voluntary running protected against neuronal loss, increased enteric glial expression, and modified gut microbiome composition in the brain-injected AAV-α-synuclein PD model. Conclusions and inferences These results show that developing nigral α-synuclein pathology in this PD model exerts significant alterations on the enteric nervous system (ENS) and gut microbiome that are receptive to modification by exercise. This highlights brain to gut communication as an important mechanism in PD pathology.

Seaweeds are of huge interest in the food, pharmaceutical and agricultural industries due to their high nutritional content and the prevalence of useful bioactive compounds. Current extraction methods of macroalgal-derived metabolites are however problematic due to the complexity of the algal cell wall which hinders extraction efficiencies. The use of advanced extraction methods such as enzyme-assisted extraction (EAE), which involve the application of commercial algal cell wall degrading enzymes to hydrolyze the cell wall carbohydrate network, are becoming more popular as they allow the development of more efficient and eco-friendly processes. Ascophyllum nodosum samples were collected from the Irish coast and incubated in artificial seawater for six weeks at three different temperatures (18 °C, 25°C and 30 °C) to induce decay. Microbial communities associated with the intact and decaying macroalga were examined using Illumina Miseq sequencing and culture-dependent approaches, including the novel iChip device. The bacterial populations associated with the seaweed were observed to change markedly upon decay with a substantial decrease in the relative abundances of certain phyla including Planctomycetes and Verrucomicrobia observed during the decay period. Over 800 bacterial isolates cultured from the macroalga were screened for the production of algal cell wall polysaccharidases and a range of species from the phylum Bacteroidetes together with a number of Vibrio species which displayed multiple hydrolytic enzyme activities were identified. Extracts from these enzyme-active bacterial isolates were then used in EAE of phenolics from Fucus vesiculous and were shown to be equally efficient as commercial enzymes in their extraction efficiencies.

Photorhabdus luminescensis an entomopathogenic bacterium found in symbiosis with the nematodeHeterorhabditis. Dam DNA methylation is involved in the pathogenicity of many bacteria, includingP. luminescens,whereas studies about the role of bacterial DNA methylation during symbiosis are scarce. The aim of this study was to determine the role of Dam DNA methylation inP. luminescenssymbiosis withH. bacteriophora. We constructed a strain overexpressingdamby inserting an additional copy of thedamgene under the control of a constitutive promoter in the chromosome ofP. luminescensand then achieved association between this recombinant strain and nematodes. Thedamoverexpressing strain was able to feed the nematodein vitroandin vivosimilarly as a control strain, and to re-associate with Infective Juvenile (IJ) stages in the insect. No difference in the amount of emerging IJs from the cadaver was observed between the two strains. Compared to the nematode in symbiosis with the control strain, a significant increase in LT50was observed during insect infestation with the nematode associated with thedamoverexpressing strain. These results suggest that theP. luminescensDam plays a role in the pathogenicity of the nemato-bacterial complex.

Members of the Gram-negative bacterial genusPhotorhabdusare all highly insect pathogenic and exist in an obligate symbiosis with the entomopathogenic nematode wormHeterorhabditis. All members of the genus produce the small molecule 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS) as part of their secondary metabolism. IPS is a multi-potent compound that has antimicrobial, antifungal and anticancer activities and also plays an important role in symbiosis with the nematode. In this study we have examined the response ofPhotorhabdusitself to exogenous ectopic addition of IPS at physiologically relevant concentrations. We observed that the bacteria had a measureable phenotypic response, which included a decrease in bioluminescence and pigment production. This was reflected in changes in its transcriptomic response, in which we reveal a reduction in transcript levels of genes relating to many fundamental cellular processes, such as translation and oxidative phosphorylation. Our observations suggest that IPS plays an important role in the biology ofPhotorhabdusbacteria, fulfilling roles in quorum sensing, antibiotic-competition advantage and maintenance of the symbiotic developmental cycle.

Iron is an essential element for many biological processes; however, due to its high reactivity iron can also be very toxic, producing reactive oxygen species through Fenton chemistry. Ferritins protect the cell from oxidative stress by catalytically converting Fe(II) into less toxic Fe(III) and storing the resulting iron minerals within their core. Encapsulated ferritins (EncFtn) are a sub-family of ferritin-like proteins, which are widely distributed in all bacterial and archaeal phyla. We recently characterised theRhodospirillum rubrumEncFtn, showing that although enzymatically active, due to its open structure it requires the association with an encapsulin nanocage in order to act as an iron store. Given the wide distribution of the EncFtn family in organisms with diverse environmental niches, a question arises as to whether the structure and catalytic activity is conserved across the family. Here we structurally characterise two EncFtn members from the halophileHaliangium ochraceumand the thermophilePyrococcus furiosus, which show the same distinct annular decamer topology observed inR. rubrumEncFtn, with the ferroxidase centre (FOC) formed between one of the dimer interfaces. Solution and native mass spectrometr analyses show that the stability of the protein quaternary structure differs between EncFtn proteins from different species. The catalytic role of the EncFtn proteins was confirmed by biochemical assays, and we show that Zn(II) ions inhibit the ferroxidase activity of the EncFtn proteins to varying degrees. Our results represent a further step in the characterisation of the recently discovered EncFtn ferritin-like sub-family, indicating a common structural organisation and catalytic activity, despite diverse host environments.

Lignin and lignin components of woody biomass have been identified as an attractive alternative to fossil fuels. However, the complex composition of this plant polymer is one of the drawbacks that limits its exploitation. Biocatalysis of lignin to produce platform chemicals has been receiving great attention as it presents a sustainable approach for lignin valorisation. Aligned with this area of research, in the present study we have applied ultra-high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to identify the preferred lignin substrates of a ligninolytic enzyme, a laccase produced by the terrestrial fungus Trametes versicolor. A commercial lignin was incubated with the laccase and acetosyringone (a laccase mediator) for up to 168 h and direct infusion electrospray FT-ICR MS enabled the identification of thousands of molecular species present in the complex lignin sample at different incubation time points. Significant changes in the chemical composition of lignin were detected upon laccase treatment, which resulted in a decrease in the molecular mass distribution of assigned species, consistent with laccase lytic activity. This reduction was predominantly in species classified as lignin-like (based on elemental ratios) and polymeric in nature (>400 Da). Of particular note was a fall in the number of species assigned containing sulfur. Changes in the chemical composition/structure of the lignin polymer were supported by FT-IR spectroscopy. We propose the use of FT-ICR MS as a rapid and efficient technique to support the biotechnological valorisation of lignin as well as the development and optimization of laccase-mediator systems for treating complex mixtures.

Four antimicrobials, namely; Articoat DLP 02 (AR), Artemix Consa 152/NL (AX), Auranta FV (AFV) and sodium octanoate (SO) were examined for their effectiveness, both before and after heat treatments, against bacterial strains Bacillus cereus, Pseudomonas fluorescens, Escherichia coli, Staphylococcus aureus and the microflora obtained from commercial beef steaks. Minimum inhibitory concentrations (MIC) using AR, AX, AFV and SO against these microbes were then obtained using the 96-well plate method. SO was the most effective against all bacterial strains, demonstrating the lowest MIC compared to the other antimicrobials. These antimicrobials were then successively incorporated into beef-derived gelatine films and these films were subsequently tested for structural, mechanical and barrier properties. Significantly (p < 0.05) enhanced water vapour barrier properties were determined only for antimicrobial films containing AX or SO when compared to control films. On the basis of FTIR spectra, significant changes in the structure of SO-containing films were determined when compared with control gelatin films. It was shown that active antimicrobial agents could potentially serve as commercial antimicrobial coatings for application onto conventional plastic-based food packaging.

The enteric gamma-proteobacterium Photorhabdus luminescens kills a wide range of insects, whilst also maintaining a mutualistic relationship with soil nematodes from the family Heterorhabditis. Pathogenicity is associated with bacterial exponential growth, whilst mutualism is associated with post-exponential (stationary) phase. During post-exponential growth, P. luminescens also elaborates an extensive secondary metabolism, including production of bioluminescence, antibiotics and pigment. However, the regulatory network that controls the expression of this secondary metabolism is not well understood. The stringent response is a well-described global regulatory system in bacteria and mediated by the alarmone (p)ppGpp. In this study, we disrupted the genes relA and spoT, encoding the two predicted (p)ppGpp synthases of P. luminescens TTO1, and we showed that (p)ppGpp is required for secondary metabolism. Moreover, we found the (p)ppGpp is not required for pathogenicity of P. luminescens, but is required for bacterial survival within the insect cadaver. Finally, we showed that (p)ppGpp is required for P. luminescens to support normal nematode growth and development. Therefore, the regulatory network that controls the transition from pathogenicity to mutualism in P. luminescens requires (p)ppGpp. This is the first report outlining a role for (p)ppGpp in controlling the outcome of an interaction between a bacteria and its host.

Antimalarial drugs have thus far been chiefly derived from two sources-natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.

Allergenic mites are responsible for inducing hypersensitive reactions in genetically predisposed people worldwide. Mites in dust from 30 Irish homes with pets (dogs, n = 23; cats, n = 7) were compared with those in 30 homes without pets. House dust mites constituted 78% of all mites recorded, with Dermatophagoides pteronyssinus (Acariformes: Pyroglyphidae) representing 57-72% of mites in furniture and mattresses in both home types compared with only 22% of mites in pet beds. Although storage mites accounted for just 13% of all mites recorded, they represented 46% of mites recorded in pet beds. Median levels of the dust mite allergen Der p 1 (µg/g) in dust samples from mattresses in homes without pets were significantly greater than in mattresses from homes with pets, reflecting the greater densities of D. pteronyssinus found in the former home category. Mite species richness was greater in homes with pets (17 species) than in homes without pets (13 species). This suggests that although the presence of pets can result in a wider variety of epidemiologically important mite species within households, increased competition among mite species may result in a more balanced mite fauna in the home, inhibiting the dominance of any one species and hence lowering allergen-associated risks.

American ginseng (Panax quinquefolius) roots have long been harvested for use in herbal medicine. Overharvesting has threatened long-term viability of wild American ginseng populations. Research has been ongoing to determine factors affecting the variation of ginsenosides in roots. Given the conservation concerns regarding wild American ginseng, we began experimenting with a partial-root harvest method in 2014 for extracting tissue for ginsenoside analysis without killing individual plants or causing long-term declines in wild populations. We took partial-root harvest samples from 57 plants in four wild populations throughout western North Carolina and monitored morphological attributes of these and 56 paired, unharvested plants of similar size for four years after harvest. Partial-root samples were taken from an additional 162 plants from 16 new populations in 2015 and 2016. Morphological attributes of these plants were monitored annually or biannually. In the paired plant study, annual reemergence did not differ between harvested and unharvested plants in any year after harvest. Leaf area was significantly lower in harvested plants than unharvested controls in the first year postharvest, but these differences did not persist after the first year. In the unpaired study, preharvest-postharvest comparisons were more variable, likely due to different harvest years and interannual variation in weather. Our results demonstrate that partial-root harvest could be an effective way for ginsenoside researchers to reduce their impact on wild and cultivated American ginseng populations and it may represent a non-destructive harvest protocol for root tissue phytochemical analysis.

Acylated amino acids function as important components of the cellular membrane in some bacteria. Biosynthesis is initiated by the N-acylation of the amino acid and this is followed by subsequent O-acylation of the acylated molecule resulting in the production of the mature diacylated amino acid lipid. In this study we use both genetics and liquid chromatography-mass spectrometry (LC-MS) to characterize the biosynthesis and function of novel diacylated glycine lipid (GL) species inBacteroides thetaiotaomicron. We, and others, have previously reported the identification of a gene, namedglsBin this study, that encodes a N-acyltransferase activity responsible for the production of a monoacylated glycine called N-acyl-3-hydroxy-palmitoyl glycine (or commendamide). In all of theBacteroidalesgenomes so far sequenced theglsBgene is located immediately downstream from a gene, namedglsA, also predicted to encode a protein with acyltransferase activity. We use LC-MS to show that co-expression ofglsBandglsAresults in the production of GL inEscherichia coli. We constructed a deletion mutant of theglsBgene inB. thetaiotaomicronand we confirm thatglsBis required for the production of GL inB. thetaiotaomicron. Moreover, we show thatglsBis important for the ability ofB. thetaiotaomicronto adapt to stress and colonize the mammalian gut. Therefore, this report is the first to describe the genetic requirements for the biosynthesis of GL, a novel diacylated amino acids species that contributes to fitness in the human gut bacterium,B. thetaiotaomicron.

We studied the habitat characteristics of Spiraea virginiana Britton (Virginia spiraea), a federally threatened riparian shrub, along eight rivers in western North Carolina. Comparisons between plots with and without S. virginiana revealed that S. virginiana plots were on steep, south-facing slopes and had a higher percentage of large substrate, lower herbaceous and vine cover, higher non–S. virginiana shrub density, lower tree influence, and higher visible sky than control plots. When relating these habitat attributes to the presence/absence of S. virginiana using conditional logistic regression, only substrate size and non–S. virginiana shrub density had significant effects on S. virginiana presence. Principal components analysis (PCA) of all plots (S. virginiana and control) found S. virginiana plots separating from controls that had higher vine and herb cover and lower non–S. virginiana shrub density and slope. Spiraea virginiana plots at the Cheoah River separated from other sites by having...

[b]Introduction and objective[/b]. House dust mites produce allergens which can cause or aggravate diseases such as asthma, eczema and rhinitis. The objectives of this study are to quantify typical house dust mite and Der p 1 allergen levels in child car seats, and to determine external variables that may influence mite populations in cars. [b]Materials and Methods[/b]. Dust samples were collected from the child car seats and driver seats of 106 cars using a portable vacuum sampling pump over a two minute sampling period. Mites were counted and identified and results were expressed as mites per gram (mites/g) of dust, while Der p 1 content of samples were measured by enzyme-linked immunosorbent assay (ELISA). Questionnaires were completed by participants to identify environmental and behavioural effects on mite populations. Results were analysed using General Linear Model (GLM) procedures. [b]Results[/b]. Twelve species of mites, of which nine are known to produce harmful allergens, were recorded from 212 dust samples. Over 80% of drivers’ seats and over 77% of child car seats harboured dust mites with a significant correlation (p = 0.001) between the mites/g of dust and Der p 1 content recovered from each seat. A mean of 53 mites/g of dust per seat was recovered, with a mean Der p 1 level of 1.1µg/g. Over 12% of driver seats and 15% of child car seats contained house dust mite levels sufficient to be risk factors for sensitisation and allergic reactions. [b]Conclusions[/b]. Child car seats and driver seats are habitats to a range of mite species which can be present in sufficient concentrations to cause or aggravate allergen related illnesses in individuals who are genetically predisposed.

Castor canadensis (Beaver) is a selective forager that can modify the species composition and structure of plant communities. However, no studies have examined the use of woody plants by Beaver in temperate forests that contain a dominant nonnative plant. We investigated foraging of woody plants by Beaver in a riparian shrub community that is dominated by both native and nonnative species, including the federally threatened shrub Spiraea virginiana (Virginia Spiraea). We established 48 random, 25-m transects along a 12-km reach of the Cheoah River in the Appalachian Mountains of North Carolina. We sampled woody plants every 5 m using a modified point-centered quarter method to estimate relative abundance and to quantify browsing by Beaver. We used a mixed linear model to determine Beaver forage selection on the 9 most abundant plant species and Virginia Spiraea. We recorded 984 plants of 58 woody species (55 native, 3 nonnative). Beaver browsed 24% of the woody species sampled and 8% of all stems...

The contribution of the gut microbiota to the metabolism of cholesterol is not well understood. In this study, we identify 21 fosmid clones from a human gut microbiome metagenomic library that, when expressed in Escherichia coli, produce halos on LB agar supplemented with 0.01% (w/v) cholesterol (LBC agar). Analysis of 14 of these clones revealed that they all share a fragment of DNA with homology to the genome of Bacteroides vulgatus. The gene responsible for halo production on LBC agar, named choA, was identified as an N-acyltransferase known to produce an acylated glycine molecule called commendamide. In this study we show that commendamide is capable of producing a halo on LBC agar suggesting that this molecule is solubilizing the cholesterol micelles in LBC agar. We also show that commendamide is responsible for the previously described hemolytic activity associated with the choA orthologue in Bacteroides fragilis. A functional analysis of ChoA identified 2 amino acids that are important for commendamide biosynthesis and we present phylogenetic and functional data showing that orthologues of choA are found only in the order Bacteroidales. Therefore, the production of commendamide may be an adaptation to the environments colonized by the Bacteroidales, including the mammalian gut.

Seaweeds are of significant interest in the food, pharmaceutical, and agricultural industries as they contain several commercially relevant bioactive compounds. Current extraction methods for macroalgal-derived metabolites are, however, problematic due to the complexity of the algal cell wall which hinders extraction efficiencies. The use of advanced extraction methods, such as enzyme-assisted extraction (EAE), which involve the application of commercial algal cell wall degrading enzymes to hydrolyze the cell wall carbohydrate network, are becoming more popular. Ascophyllum nodosum samples were collected from the Irish coast and incubated in artificial seawater for six weeks at three different temperatures (18 &deg, C, 25 &deg, C, and 30 &deg, C) to induce decay. Microbial communities associated with the intact and decaying macroalga were examined using Illumina sequencing and culture-dependent approaches, including the novel ichip device. The bacterial populations associated with the seaweed were observed to change markedly upon decay. Over 800 bacterial isolates cultured from the macroalga were screened for the production of algal cell wall polysaccharidases and a range of species which displayed multiple hydrolytic enzyme activities were identified. Extracts from these enzyme-active bacterial isolates were then used in EAE of phenolics from Fucus vesiculosus and were shown to be more efficient than commercial enzyme preparations in their extraction efficiencies.

The etiology of Crohn‘s disease (CD) is complex and involves both host susceptibility factors (i.e., the presence of particular genetic alleles) and environmental factors, including bacteria. In this regard, adherent–invasive Escherichia coli (AIEC), have recently emerged as an exciting potential etiological agent of CD. AIEC are distinguished from commensal strains of E. coli through their ability to adhere to and invade epithelial cells and replicate in macrophages. Recent molecular analyses have identified genes required for both invasion of epithelial cells and replication in the macrophage. However, these genetic studies, in combination with recent genome sequencing projects, have revealed that the pathogenesis of this group of bacteria cannot be explained by the presence of AIEC-specific genes. In this article, we review the role of AIEC as a pathobiont in the pathology of CD. We also describe the emerging link between AIEC and autophagy, and we propose a model for AIEC pathogenesis.

Spiraea virginiana (Virginia Spiraea) is a federally threatened shrub endemic to the southern Blue Ridge and Appalachian Plateau physiographic provinces. Observations along the Cheoah River, where the largest population of S. virginiana occurs in North Carolina, indicate that Castor canadensis (Beaver) feed on S. virginiana. However, the effects of Beaver foraging on this imperiled shrub are unknown. To address this issue, we randomly located 50 belt transects (25 m × 2.5 m) along the center of the scour zone of the Cheoah River and assessed all basal stems (

American ginseng, Panax quinquefolius L., is an herbaceous perennial species that is destructively harvested for its bioactive compounds called ginsenosides. The demand for this herb fosters illegal poaching and over-harvesting that reduces genetic variability and population viability. Five wild populations in western North Carolina were studied to better understand the production of ginsenosides in leaf and root tissues. Total ginsenoside concentration was significantly higher in leaves than roots, though total yield was higher in roots due to greater root biomass. However, some ginsensosides (Rb2, Rd and Re) had higher or more consistent yields in leaves than roots, so might be developed into a sustainable source of these medicinally-active compounds. Additionally, we identified regional root chemotypes that differed in the production of the ginsenosides Rg1 and Re and could be developed into regional cultivars depending on the desired panel of ginsenosides.

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, plays a critical role in the orchestration of immune responses. S1P levels within the mammalian host are tightly regulated, in part through the activity of S1P lyase (S1PL) which catalyses its irreversible degradation. Herein, we describe the identification and characterization of secreted S1PL orthologues encoded by the facultative intracellular bacteria Burkholderia pseudomallei and Burkholderia thailandensis. These bacterial orthologues exhibited S1PL enzymatic activity, functionally complemented an S1PL-deficient yeast strain and conferred resistance to the antimicrobial sphingolipid D-erythro-sphingosine. We report that secretion of these bacterial S1PLs is pH-dependent, and is observed during intracellular infection. S1PL-deficient mutants displayed impaired intracellular replication in murine macrophages (associated with an inability to evade the maturing phagosome) and were significantly attenuated in murine and larval infection models. Furthermore, treatment of Burkholderia-infected macrophages with either S1P or a selective agonist of S1P receptor 1 enhanced bacterial colocalisation with LAMP-1 and reduced their intracellular survival. In summary, our studies confirm bacterial-encoded S1PL as a critical virulence determinant of B. pseudomallei and B. thailandensis, further highlighting the pivotal role of S1P in host-pathogen interactions. In addition, our data suggest that S1P pathway modulators have potential for the treatment of intracellular infection.

Ferritins are ubiquitous proteins that oxidise and store iron within a protein shell to protect cells from oxidative damage. We have characterized the structure and function of a new member of the ferritin superfamily that is sequestered within an encapsulin capsid. We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface. EncFtn adopts an open decameric structure that is topologically distinct from other ferritins. While EncFtn acts as a ferroxidase, it cannot mineralize iron. Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage. This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system, where the oxidation and mineralization of iron are distributed between two proteins. DOI: http://dx.doi.org/10.7554/eLife.18972.001, eLife digest Iron is essential for life as it is a key component of many different enzymes that participate in processes such as energy production and metabolism. However, iron can also be highly toxic to cells because it readily reacts with oxygen. This reaction can damage DNA, proteins and the membranes that surround cells. To balance the cell’s need for iron against its potential damaging effects, organisms have evolved iron storage proteins known as ferritins that form cage-like structures. The ferritins convert iron into a less reactive form that is mineralised and safely stored in the central cavity of the ferritin cage and is available for cells when they need it. Recently, a new family of ferritins known as encapsulated ferritins have been found in some microorganisms. These ferritins are found in bacterial genomes with a gene that codes for a protein cage called an encapsulin. Although the structure of the encapsulin cage is known to look like the shell of a virus, the structure that the encapsulated ferritin itself forms is not known. It is also not clear how encapsulin and the encapsulated ferritin work together to store iron. He et al. have now used the techniques of X-ray crystallography and mass spectrometry to determine the structure of the encapsulated ferritin found in some bacteria. The encapsulated ferritin forms a ring-shaped doughnut in which ten subunits of ferritin are arranged in a ring; this is totally different from the enclosed cages that other ferritins form. Biochemical studies revealed that the encapsulated ferritin is able to convert iron into a less reactive form, but it cannot store iron on its own since it does not form a cage. Thus, the encapsulated ferritin needs to be housed within the encapsulin cage to store iron. Further work is needed to investigate how iron moves into the encapsulin cage to reach the ferritin proteins. Some organisms have both standard ferritin cages and encapsulated ferritins; why this is the case also remains to be discovered. DOI: http://dx.doi.org/10.7554/eLife.18972.002

The breakdown of fucose and rhamnose released from plant cell walls by the cellulolytic soil bacterium Clostridium phytofermentans produces toxic aldehyde intermediates. To enable growth on these carbon sources, the pathway for the breakdown of fucose and rhamnose is encapsulated within a bacterial microcompartment (BMC). These proteinaceous organelles sequester the toxic aldehyde intermediates and allow the efficient action of acylating aldehyde dehydrogenase enzymes to produce an acyl-CoA that is ultimately used in substrate-level phosphorylation to produce ATP. Here we analyse the kinetics of the aldehyde dehydrogenase enzyme from the fucose/rhamnose utilisation BMC with different short-chain fatty aldehydes and show that it has activity against substrates with up to six carbon atoms, with optimal activity against propionaldehyde. We have also determined the X-ray crystal structure of this enzyme in complex with CoA and show that the adenine nucleotide of this cofactor is bound in a distinct pocket to the same group in NAD+. This work is the first report of the structure of CoA bound to an aldehyde dehydrogenase enzyme and our crystallographic model provides important insight into the differences within the active site that distinguish the acylating from non-acylating aldehyde dehydrogenase enzymes.

A general definition of secondary metabolism is that it consists of the metabolic pathways and the products of metabolism that are not absolutely required for the survival of the organism. Using this definition, it is now well established that Photorhabdus elaborate an extensive secondary metabolism during the post-exponential phase of bacterial growth. This secondary metabolism includes, but is not limited to, the production of light, a stilbene antibiotic and an anthraquinone pigment. In this chapter, the role of secondary metabolism during the life cycle of Photorhabdus will be discussed. Recent work has shown that secondary metabolism in Photorhabdus is required for the mutualistic association between the bacteria and its nematode partner, in particular bacterial secondary metabolism is required to support normal nematode growth and development. An isogenic population of Photorhabdus is phenotypically heterogenous and this facilitates functional partitioning within the population. The relationship between secondary metabolism and the various phenotypic and phase variants that exist in populations of Photorhabdus will also be discussed. Finally, this chapter will also describe the various regulatory nodes that have been identified as being part of the complex regulatory network that is used to control the temporal expression of secondary metabolism in Photorhabdus.

© 2016 Australian Society for Parasitology Plasmodium vivax is the most geographically widespread malaria parasite. Unique features of transmission biology complicate P. vivax control. Interventions targeting transmission are required for malaria eradication. In the absence of an in vitro culture, transmission studies rely on live isolates from non-human primates or endemic regions. Here, we demonstrate P. vivax gametocytes from both India and Brazil are stable during cryopreservation. Importantly, cryopreserved gametocytes from Brazil were capable of infecting three anopheline mosquito species in feedings done in the United States. These findings create new opportunities for transmission studies in diverse locales.

The bacterium, Photorhabdus luminescens, is an insect pathogen that also maintains a mutualistic interaction with nematodes from the family Heterorhabditis. Photorhabdus luminescens is carried in the gut of the infective juvenile (IJ), a nematode stage that infects soft-cuticled insect larvae in the soil. Photorhabdus luminescens is released into the insect blood to convert the insect into a biomass that is able to support nematode growth and development. Nematode reproduction continues for 2-3 generations before the nematodes enter an alternative developmental pathway leading to a new generation of IJs that are efficiently colonized by P. luminescens in a process called transmission. Transmission begins with the adherence of P. luminescens to the rectal gland cells (RGC) located in the gut of the hermaphrodite. The bacteria enter, and replicate within, the RGC resulting in vacuolization, and ultimately lysis, of the RGC. In this way P. luminescens are released throughout the body cavity of the hermaphrodite to encounter, and colonize, the developing IJs. In this study we show that the LysR-type regulator, HdfR, is required for normal transmission in P. luminescens TTO1. Transcriptome analysis revealed that HdfR regulates the expression of 124 genes, including genes involved in arginine metabolism, hydroxyphenylacetate catabolism and pigment production. Using fluorescence microscopy we show that the ΔhdfR mutant is able to attach to, and grow within, the hermaphrodite as well as wild-type bacteria but subsequent steps in transmission are delayed. Therefore, HdfR plays an important role in coordinating the interaction between P. luminescens and its nematode partner during transmission.

Background: Proteolytic degradation of the extracellular matrix, a feature of mucosal homeostasis and tissue renewal, also contributes to the complications of intestinal inflammation. Whether this proteolytic activity is entirely host-derived, or, in part, produced by the gut microbiota, is unknown. Methods: We screened the bacterial colonies for gelatinolytic activity from fecal samples of 20 healthy controls, 23 patients with ulcerative colitis, and 18 with Crohn's disease (CD). In addition, the genes encoding metalloproteases were detected by conventional or real-time polymerase chain reaction (PCR). Results: Gelatinolytic activity was found in approximately one-quarter of samples regardless of the presence of inflammation and without any attempt to enhance the sensitivity of the culture-based screen. This was associated with a diversity of bacteria, particularly in CD, but was predominantly linked with Clostridium perfringens. Culture supernatants from C. perfringens degraded gelatin, azocoll, type I collagen, and basement membrane type IV collagen, but different isolates varied in the degree of proteolytic activity. Results were confirmed by detection of the C. perfringens colA gene (encoding collagenase) in fecal DNA, again regardless of the presence or absence of inflammation. However, the biologic significance and potential implications of microbial-derived proteolytic activity were confirmed by reduced transepithelial resistance (TER) after exposure of rat distal colon to culture supernatants of C. perfringens in Ussing chambers. Conclusions: The study shows that microbial-derived proteolytic activity has the capacity to contribute to mucosal homeostasis and may participate in the pathogenesis of inflammatory bowel disease. (Inflamm Bowel Dis 2010)

Photorhabdus luminescens is a species of Gram-negative bacteria that is pathogenic to insects while also maintaining a mutualistic association with nematodes from the family Heterorhabditis. P. luminescens elaborates an extensive secondary metabolism during the post-exponential phase of growth that includes the production of an antibiotic called 3-5-dihydroxy-4-isopropylstilbene (ST), an anthraquinone pigment (AQ) and bioluminescence. In this study we identified a mutant that was unable to produce ST, AQ and light. This mutation was found to be in the mdh gene, encoding malate dehydrogenase, a key enzyme in the tricarboxylic acid (TCA) cycle. Interestingly the mdh mutant was unaffected in virulence but was unable to support nematode growth and development in vivo or in vitro. This clearly establishes that secondary metabolism in P. luminescens is required for the mutualistic interaction with the nematode. Furthermore, the construction of mutations in key genes in other central metabolic pathways confirmed the critical role for the TCA cycle in both secondary metabolism and mutualism, but not in virulence. Therefore, we conclude that the TCA cycle is required for the transition of P. luminescens from pathogen to mutualist implicating the involvement of a metabolic switch in the regulation of lifestyle decisions in this bacterium.

Peroxiredoxins are ubiquitous proteins that catalyze the reduction of hydroperoxides, thus conferring resistance to oxidative stress. Using high-resolution mass spectrometry, we recently reclassified one such peroxiredoxin, bacterioferritin comigratory protein (BCP) of Escherichia coli, as an atypical 2-Cys peroxiredoxin that functions through the formation of an intramolecular disulfide bond between the active and resolving cysteine. An engineered E. coli BCP, which lacked the resolving cysteine, retained enzyme activity through a novel catalytic pathway. Unlike the active cysteine, the resolving cysteine of BCP peroxiredoxins is not conserved across all members of the family. To clarify the catalytic mechanism of native BCP enzymes that lack the resolving cysteine, we have investigated the BCP homologue of Burkholderia cenocepacia. We demonstrate that the B. cenocepacia BCP (BcBCP) homologue functions through a 1-Cys catalytic pathway. During catalysis, BcBCP can utilize thioredoxin as a reductant for the sulfenic acid intermediate. However, significantly higher peroxidase activity is observed utilizing glutathione as a resolving cysteine and glutaredoxin as a redox partner. Introduction of a resolving cysteine into BcBCP changes the activity from a 1-Cys pathway to an atypical 2-Cys pathway, analogous to the E. coli enzyme. In contrast to the native B. cenocepacia enzyme, thioredoxin is the preferred redox partner for this atypical 2-Cys variant. BCP-deficient B. cenocepacia exhibit a growth-phase-dependent hypersensitivity to oxidative killing. On the basis of sequence alignments, we believe that BcBCP described herein is representative of the major class of bacterial BCP peroxiredoxins. To our knowledge, this is the first detailed characterization of their catalytic activity. These studies support the subdivision of the BCP family of peroxiredoxins into two classes based on their catalytic activity.

The Rcs phosphorelay is composed of RcsC, RcsD and the response regulator RcsB, and this signalling pathway has been implicated in virulence and biofilm formation in many enteric bacteria. It was previously shown that a mutation in rcsC resulted in defective biofilm formation in Escherichia coli [Ferrieres, L. & Clarke, D. J. (2003) Mol Microbiol 50, 1665-1682]. To identify the molecular mechanisms underlying the observed biofilm defect we carried out a screen looking for suppressor mutants that restored biofilm formation in the rcsC mutant background. One of the mutants was identified to be in rprA, a gene encoding a small RNA molecule that is involved in the post-transcriptional control of the alternative sigma factor, sigma(S). The expression of rprA is regulated by the Rcs phosphorelay, and there are elevated sigma(S) levels present in the rcsC mutant due to the overexpression of rprA in this background. Using different approaches, we have established that the increase in sigma(S) levels is responsible for the biofilm defect. Therefore, the Rcs phosphorelay is involved in maintaining appropriate levels of sigma(S) during biofilm formation in E. coli.

The Rcs phosphorelay is an important signalling pathway that is conserved throughout the Enterobacteriaceae. The Rcs phosphorelay is composed of the RcsC sensor kinase, a membrane-localized HPt-containing protein RcsD and the cytoplasmic response regulator RcsB. In this study we were interested in studying the degree of functional conservation between the different enteric RcsC homologues. Therefore, we tested for the ability of RcsC homologues from pathogenic Escherichia coli, Salmonella enterica and Yersinia pestis to functionally complement an rcsC mutation in E. coli K-12. Complementation was measured as (1) an increase in cpsB-lacZ expression in response to DjlA overproduction, a well-established signal for RcsC activation and (2) the ability to restore biofilm formation. All of the homologues increased cpsB-lacZ expression in response to DjlA overproduction confirming that the different RcsC homologues are able to sense this stimulus and to transduce the signal to the downstream components of the Rcs pathway in E. coli. This suggests that the core signalling domains of RcsC are functionally conserved throughout the Enterobacteriaceae. However, we also show that RcsC from Y. pestis was unable to restore normal biofilm formation in the E. coli K-12 rcsC mutant and we argue that this is due to the increased net kinase activity observed with this homologue.

Twelve Caribbean species of Vachellia (formerlyAcacia subgenus Acacia) are formally monographed with taxonomic, habitat, geographic distribution descriptions, and lists of representative specimens examined. The species treated here are all part of the informal Vachellia acuifera group, which is characterized by generally enlarged stipular spines borne in clusters on the stems, 1–3 pairs of pinnae per leaf, and 20–50 stamens. Ordination analyses of vegetative characters show that these species form discrete groups in two- and three-dimensional plots. Vachellia baessleria Clarke, Seigler, & Ebinger, endemic to Guantánamo and Las Tunas, Cuba, is newly described. Vachellia acuifera is wide ranging, occurring from the Bahamas to Cuba. The other species are highly restricted in their distribution and ecological setting. Four of the species are only known from Hispaniola (V. barahonensis, V. caurina, V. cucuyo, and V. oviedoensis) and six only from Cuba (V. baessleria, V. belairioides, V. bucheri, V. daemon, V. roigii, and V. zapatensis). Another species, V. anagadensis, is only found on the island of Anegada in the British Virgin Islands.

Biotin protein ligase (BPL; EC 6.3.4.15) catalyses the formation of biotinyl-5'-AMP from biotin and ATP, and the succeeding biotinylation of the biotin carboxyl carrier protein. We describe the crystal structures, at 2.4 A resolution, of the class I BPL from the hyperthermophilic bacteria Aquifex aeolicus (AaBPL) in its ligand-free form and in complex with biotin and ATP. The solvent-exposed β- and γ-phosphates of ATP are located in the inter-subunit cavity formed by the N- and C-terminal domains. The Arg40 residue from the conserved GXGRXG motif is shown to interact with the carboxyl group of biotin and to stabilise the alpha- and β-phosphates of the nucleotide. The structure of the mutant AaBPL R40G in both the ligand-free and biotin-bound forms reveals that the mutated loop has collapsed, thus hindering ATP binding. Isothermal titration calorimetry indicated that the presence of biotin is not required for ATP binding to wild-type AaBPL in the absence of Mg(2+), and the binding of biotin and ATP has been determined to occur via a random but cooperative process. The affinity for biotin is relatively unaffected by the R40G mutation. In contrast, the thermodynamic data indicate that binding of ATP to AaBPL R40G is very weak in the absence or in the presence of biotin. The AaBPL R40G mutant remains catalytically active but shows poor substrate specificity; mass spectrometry and Western blot studies revealed that the mutant biotinylates both the target A. aeolicus BCCPΔ67 fragment and BSA, and is subject to self-biotinylation.

Photorhabdus is a member of the family Enterobacteriaceae that lives in a mutualistic association with a Heterorhabditis nematode worm. The nematode worm burrows into insect prey and regurgitates Photorhabdus, which goes on to kill the insect. The nematode feeds off the growing bacteria until the insect tissues are exhausted, whereupon they reassociate and leave the cadaver in search of new prey. This highly efficient partnership has been used for many years as a biological crop protection agent. The dual nature of Photorhabdus as a pathogen and mutualist makes it a superb model for understanding these apparently exclusive activities. Furthermore, recently identified clinical isolates of Photorhabdus are helping us to understand how human pathogens can emerge from the enormous reservoir of invertebrate pathogens in the environment. As Photorhabdus has never been found outside a host animal, its niche represents an entirely biotic landscape. In this review we discuss what molecular adaptations allow this bacterium to complete this fascinating and complex life cycle.

Photorhabdus are entomopathogenic members of the family Enterobacteriaceae. In addition to killing insects Photorhabdus also have a mutualistic association with nematodes from the family Heterorhabditidiae. Therefore, the bacteria have a complex life cycle that involves temporally separated pathogenic and mutualistic associations with two different invertebrate hosts. This tripartite Photorhabdus-insect-nematode association provides researchers with a unique opportunity to characterize the prokaryotic contribution to two different symbioses, i.e. pathogenicity and mutualism while also studying the role of the host in determining the outcome of association with the bacteria. In this review I will outline the life cycle of Photorhabdus and describe recent important advances in our understanding of the symbiology of Photorhabdus. Finally, the contribution made by this model to our understanding of the nature of symbiotic associations will be discussed.

Type II polyketide synthases are involved in the biosynthesis of numerous clinically relevant secondary metabolites with potent antibiotic or anticancer activity. Until recently the only known producers of type II PKSs were members of the Gram-positive actimomycetes, well-known producers of secondary metabolites in general. Here we present the second example of a type II PKS from Gram-negative bacteria. We have identified the biosynthesis gene cluster responsible for the production of anthraquinones (AQs) from the entomopathogenic bacterium Photorhabdus luminescens. This is the first example of AQ production in Gram-negative bacteria, and their heptaketide origin was confirmed by feeding experiments. Deletion of a cyclase/aromatase involved in AQ biosynthesis resulted in accumulation of mutactin and dehydromutactin, which have been described as shunt products of typical octaketide compounds from streptomycetes, and a pathway for AQ formation from octaketide intermediates is discussed.

Photorhabdus and Xenorhabdus bacteria colonize the intestines of the infective soil-dwelling stage of entomophagous nematodes, Heterorhabditis and Steinernema, respectively. These nematodes infect susceptible insect larvae and release the bacteria into the insect blood. The bacteria kill the insect larvae and convert the cadaver into a food source suitable for nematode growth and development. After several rounds of reproduction the nematodes are recolonized by the bacteria before emerging from the insect cadaver into the soil to search for a new host. Photorhabdus and Xenorhabdus bacteria therefore engage in both pathogenic and mutualistic interactions with different invertebrate hosts as obligate components of their life cycle. In this review we aim to describe current knowledge of the molecular mechanisms utilized by Photorhabdus and Xenorhabdus to control their host-dependent interactions. Recent work has established that there is a trade-off between pathogenicity and mutualism in both these species of bacteria suggesting that the transition between these interactions must be under regulatory control. Despite the superficial similarity between the life cycles of these bacteria, it is now apparent that the molecular components of the regulatory networks controlling pathogenicity and mutualism in Photorhabdus and Xenorhabdus are very different.

A workshop on viral vectors for malaria vaccine development, organized by the PATH Malaria Vaccine Initiative, was held in Bethesda, MD on October 20, 2005. Recent advancements in viral-vectored malaria vaccine development and emerging vector technologies were presented and discussed. Classic viral vectors such as poxvirus, adenovirus and alphavirus vectors have been successfully used to deliver malaria antigens. Some of the vaccine candidates have demonstrated their potential in inducing malaria-specific immunity in animal models and human trials. In addition, emerging viral-vector technologies, such as measles virus (MV), vesicular stomatitis virus (VSV) and yellow fever (YF) virus, may also be useful for malaria vaccine development. Studies in animal models suggest that each viral vector is unique in its ability to induce humoral and/or cellular immune responses. Those studies have also revealed that optimization of Plasmodium genes for mammalian expression is an important aspect of vaccine design. Codon-optimization, surface-trafficking, de-glycosylation and removal of toxic domains can lead to improved immunogenicity. Understanding the vector's ability to induce an immune response and the expression of malaria antigens in mammalian cells will be critical in designing the next generation of viral-vectored malaria vaccines.