Susan A. Joyce

The C-terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo.

RNase E is an essential Escherichia coli endonuclease, which controls both 5S rRNA maturation and bulk mRNA decay. While the C‐terminal half of this 1061‐residue protein associates with polynucleotide phosphorylase (PNPase) and several other enzymes into a ‘degradosome’, only the N‐terminal half, which carries the catalytic activity, is required for growth. We characterize here a mutation (rne131 ) that yields a metabolically stable polypeptide lacking the last 477 residues of RNAse E. This mutation resembles the N‐terminal conditional mutation rne1 in stabilizing mRNAs, both in bulk and individually, but differs from it in leaving rRNA processing and cell growth unaffected. Another mutation (rne105 ) removing the last 469 residues behaves similarly. Thus, the C‐terminal half of RNase E is instrumental in degrading mRNAs, but dispensable for processing rRNA. A plausible interpretation is that the former activity requires that RNase E associates with other degradosome proteins; however, PNPase is not essential, as RNase E remains fully active towards mRNAs in rne+pnp mutants. All mRNAs are not stabilized equally by the rne131 mutation: the greater their susceptibility to RNase E, the larger the stabilization. Artificial mRNAs generated by E. coli expression systems based on T7 RNA polymerase can be genuinely unstable, and we show that the mutation can improve the yield of such systems without compromising cell growth.

An antibiotic produced by an insect-pathogenic bacterium suppresses host defenses through phenoloxidase inhibition

Photorhabdus is a virulent pathogen that kills its insect host by overcoming immune responses. The bacterium also secretes a range of antibiotics to suppress the growth of other invading microorganisms. Here we show that Photorhabdus produces a small-molecule antibiotic (E)-1,3-dihydroxy-2-(isopropyl)-5-(2-phenylethenyl)benzene (ST) that also acts as an inhibitor of phenoloxidase (PO) in the insect host Manduca sexta. The Photorhabdus gene stlA encodes an enzyme that produces cinnamic acid, a key precursor for production of ST, and a mutation in stlA results in loss of ST production and PO inhibitory activity, which are both restored by genetic complementation of the mutant and also by supplying cinnamic acid. ST is produced both in vitro and in vivo in sufficient quantities to account for PO inhibition and is the only detectable solvent-extractable inhibitor. A Photorhabdus stlA− mutant is significantly less virulent, proliferates slower within the host, and provokes the formation of significantly more melanotic nodules than wild-type bacteria. Virulence of the stlA− mutant is also rescued by supplying cinnamic acid. The proximate cause of the virulence effect, however, is the inhibition of PO, because the effect of the stlA− mutation on virulence is abolished in insects in which PO has been knocked down by RNA interference (RNAi). Thus, ST has a dual function both as a PO inhibitor to counter host immune reactions and also as an antibiotic to exclude microbial competitors from the insect cadaver.

Regulation of host weight gain and lipid metabolism by bacterial bile acid modification in the gut.

Significance It is known that the gastrointestinal microbiota influences adiposity and weight gain in the host. However the mechanisms by which gut microorganisms coordinate host physiological processes are currently unclear. We demonstrate that a single, widely distributed function of the gut microbiota, bile salt hydrolase (BSH) activity, significantly influences lipid metabolism, weight gain, and cholesterol levels in the host. In our study microbial BSH activity was shown to direct expression of host signalling pathways with known roles in lipid metabolism, circadian rhythm, and epithelial cell function. The work defines the significant impact of in situ bile hydrolysis on host metabolism and indicates how this finding may be exploited as a potential intervention strategy for the control of obesity and metabolic syndrome. Alterations in the gastrointestinal microbiota have been implicated in obesity in mice and humans, but the key microbial functions influencing host energy metabolism and adiposity remain to be determined. Despite an increased understanding of the genetic content of the gastrointestinal microbiome, functional analyses of common microbial gene sets are required. We established a controlled expression system for the parallel functional analysis of microbial alleles in the murine gut. Using this approach we show that bacterial bile salt hydrolase (BSH) mediates a microbe–host dialogue that functionally regulates host lipid metabolism and plays a profound role in cholesterol metabolism and weight gain in the host. Expression of cloned BSH enzymes in the gastrointestinal tract of gnotobiotic or conventionally raised mice significantly altered plasma bile acid signatures and regulated transcription of key genes involved in lipid metabolism (Pparγ, Angptl4), cholesterol metabolism (Abcg5/8), gastrointestinal homeostasis (RegIIIγ), and circadian rhythm (Dbp, Per1/2) in the liver or small intestine. High-level expression of BSH in conventionally raised mice resulted in a significant reduction in host weight gain, plasma cholesterol, and liver triglycerides, demonstrating the overall impact of elevated BSH activity on host physiology. In addition, BSH activity in vivo varied according to BSH allele group, indicating that subtle differences in activity can have significant effects on the host. In summary, we demonstrate that bacterial BSH activity significantly impacts the systemic metabolic processes and adiposity in the host and represents a key mechanistic target for the control of obesity and hypercholesterolemia.

The gut microbiota and the metabolic health of the host.

Purpose of review It is clear that the metabolic activities of the gut microbiota significantly impact upon human health and disease. Recent findings Recent analyses have correlated alterations in microbial community structure with the onset of diabetes, obesity and cardiovascular disease as well as inflammatory conditions of the intestine. This work has demonstrated the influence of diet upon the microbiota in disease states and has identified a number of microbial metabolites that orchestrate the crucial aspects of the host–microbe dialog. The microbial production of short-chain fatty acids, trimethylamine, acetaldehyde and inflammatory mediators has been shown to significantly impact upon the metabolic health of the host through pathways that influence satiety, gut permeability and immune function. In the small intestine, microbial metabolism alters the host bile acid profile affecting the interactions with dedicated bile acid receptors (including FXR and TGR5) to influence both local and systemic cellular responses. Recent findings have, therefore, identified specific microbiota profiles and metabolites as predictors of disease risk as well as determining the microbial species (such as Akkermansia muciniphila and Bilophila wadsworthia) which correlate with health and disease. Summary This work identifies the microbiota as an important target for new diagnostic and therapeutic approaches in metabolic disease.

High Resolution In Vivo Bioluminescent Imaging for the Study of Bacterial Tumour Targeting

The ability to track microbes in real time in vivo is of enormous value for preclinical investigations in infectious disease or gene therapy research. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumours following systemic administration. Bioluminescent Imaging (BLI) represents a powerful tool for use with bacteria engineered to express reporter genes such as lux. BLI is traditionally used as a 2D modality resulting in images that are limited in their ability to anatomically locate cell populations. Use of 3D diffuse optical tomography can localize the signals but still need to be combined with an anatomical imaging modality like micro-Computed Tomography (μCT) for interpretation. In this study, the non-pathogenic commensal bacteria E.coli K-12 MG1655 and Bifidobacterium breve UCC2003, or Salmonella Typhimurium SL7207 each expressing the luxABCDE operon were intravenously (IV) administered to mice bearing subcutaneous (s.c) FLuc-expressing xenograft tumours. Bacterial lux signal was detected specifically in tumours of mice post IV-administration and bioluminescence correlated with the numbers of bacteria recovered from tissue. Through whole body imaging for both lux and FLuc, bacteria and tumour cells were co-localised. 3D BLI and μCT image analysis revealed a pattern of multiple clusters of bacteria within tumours. Investigation of spatial resolution of 3D optical imaging was supported by ex vivo histological analyses. In vivo imaging of orally-administered commensal bacteria in the gastrointestinal tract (GIT) was also achieved using 3D BLI. This study demonstrates for the first time the potential to simultaneously image multiple BLI reporter genes three dimensionally in vivo using approaches that provide unique information on spatial locations.

A hexA homologue from Photorhabdus regulates pathogenicity, symbiosis and phenotypic variation

Photorhabdus is a genus of entomopathogenic Gram‐negative bacteria that belong to the family Enterobactericeae. Remarkably, at the same time as being pathogenic to insect larvae, Photorhabdus also have a mutualistic relationship with entomophagous nematodes of the family Heterorhabditiae. Photorhabdus can be isolated in two phenotypically distinct forms, termed the primary and secondary variant. Both variants grow equally well and are equally virulent when injected into insect larvae. However, only the primary variant can colonize the intestinal tract of the IJ stage of the nematode and support nematode growth and development. The primary variant expresses several phenotypes that are absent from the secondary variant, including the production of extracellular enzymes, pigments, antibiotics and light. In this study, we use Photorhabdus temperata strain K122 to show that these primary‐specific products are symbiosis factors, i.e. factors that are required for nematode growth and development. We also show that, in P. temperata K122, the production of these symbiosis factors is repressed in the secondary variant by the protein encoded by a gene with homology to hexA from Erwinia . Moreover, the derepression of the symbiosis factors in the secondary variant results in a significant attenuation of virulence to larvae of the greater wax moth, Galleria mellonella . This suggests that, during a normal infection, pathogenicity and symbiosis must be temporally separated and that HexA is involved in the regulation of this pathogen–symbiont transition.

A Type II Polyketide Synthase is Responsible for Anthraquinone Biosynthesis in Photorhabdus luminescens

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.

Nematode symbiont for Photorhabdus asymbiotica

Photorhabdus asymbiotica is an emerging bacterial pathogen that causes locally invasive soft tissue and disseminated bacteremic infections in the United States and Australia. Although the source of infection was previously unknown, we report that the bacterium is found in a symbiotic association with an insect-pathogenic soil nematode of the genus Heterorhabditis.

Molecular pathogenesis of Listeria monocytogenes in the alternative model host Galleria mellonella.

Larvae of Galleria mellonella, the greater wax moth, provide an alternative infection model for many human pathogens as they are amenable to use at elevated incubation temperatures (37 °C). This study and a parallel study by Mukherjee et al. [Mukherjee, K., Altincicek, B., Hain, T., Domann, E., Vilcinskas, A. & Chakraborty, T. (2010). Appl Environ Microbiol 76, 310-317] establish this insect host as an appropriate model to investigate the pathogenesis of Listeria species. In this study we show that inoculation with Listeria monocytogenes initiates a dynamic infection in G. mellonella and that production of the cytolysin listeriolysin O (LLO) is necessary for toxicity and bacterial growth. Production of LLO by the non-pathogenic species Lactococcus lactis is sufficient to induce mortality in the insect model. We employed real-time bioluminescence imaging to examine the dynamics of listerial growth and virulence gene expression in the G. mellonella model. Analysis of lux promoter fusions demonstrated significant induction of virulence gene expression upon introduction of the pathogen into insects at both 30 and 37 °C. The host response to listerial infection was examined which demonstrated that haemocyte destruction accompanies L. monocytogenes pathogenesis and is preceded by activation of the phenoloxidase system. Furthermore, we demonstrate that Listeria innocua is pathogenic to G. mellonella through a persistence mechanism that implicates an alternative mechanism for pathogenicity in this model.

The exbD gene of Photorhabdus temperata is required for full virulence in insects and symbiosis with the nematode Heterorhabditis

Photorhabdus are bacteria found colonizing the gut of a specialized stage of the nematode Heterorhabditis, called the infective juvenile (IJ). The IJ is a free‐living stage of the nematode that seeks out and infects insect larvae. Once inside the insect the IJ release Photorhabdus into the haemolymph where the bacteria rapidly proliferate, killing the insect within 48–72 h. The nematodes grow and reproduce in the insect cadaver by feeding on the Photorhabdus biomass. In this study we use Photorhabdus temperata K122 to show that genes involved in iron acquisition play a key role during the course of the tripartite bacteria–nematode–insect interaction. We show that a strain carrying a mutation in a gene with homology to exbD, encoding a component of the TonB complex, is unable to grow well in conditions where iron is not freely available. In addition, this mutant, BMM417, requires a longer time to kill the insect larvae than the wild‐type bacteria and this defect in pathogenicity is complemented by the co‐injection of iron. Moreover, the increase in LT50 observed with BMM417 is correlated with a significantly slower in vivo growth rate suggesting that iron is limiting in the insect. We also show that BMM417 is unable to support the growth and development of the Heterorhabditis nematode. Addition of exogenous iron to the growth media restores nematode growth and development on BMM417, suggesting that aspects of iron metaboism in Photorhabdus are important during the symbiosis with the nematode.