The Entomopathogenic Bacterial Endosymbionts Xenorhabdus and Photorhabdus: Convergent Lifestyles from Divergent Genomes

Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)

High iron supply inhibits the synthesis of the genotoxin colibactin by pathogenic Escherichia coli through a non-canonical Fur/RyhB-mediated pathway

The genotoxin colibactin is a secondary metabolite produced by a variety of pathogenic Enterobacteria, and is associated with colon cancer development and acute systemic infections. The colibactin biosynthesis requires the enzymatic activity of the phosphopantetheinyl transferase ClbA. We recently evidenced that two master regulators of bacterial iron homeostasis, i.e. the ferric uptake regulator (Fur) and the small regulatory non-coding RNA RyhB, were involved in the regulation of the clbA transcription and of the colibactin production. In this study, we investigated the impact of high iron supply on clbA transcription and colibactin production in wild type, Delta ryhB, Delta fur and Delta ryhB Delta fur strains. This revealed that high iron resulted in decreased synthesis of the genotoxin colibactin through both pathways dependent and independent of Fur/RyhB. This work highlights the complex regulatory mechanism that controls an important bacterial virulence and carcinogenesis factor by regulators of bacterial iron homeostasis

A proposed model for the regulation of <i>stlA</i> expression in <i>P. luminescens</i> TTO1.

<p>The expression of <i>stlA</i> is increased in response to nutrient limitation and we have identified 3 nutrient-associated regulators that all affect <i>stlA</i> expression. TyrR is required for <i>stlA</i> expression whilst σ^S and Lrp are needed for normal levels of <i>stlA</i> expression. Lrp also affects the levels of BCFA in the cell, presumably through a regulatory affect on <i>bkd</i> expression. The BarA/UvrY two-component pathway contributes to the post-transcriptional regulation of <i>stlA</i> expression, as described in the text. ST: 3’-5’-dihydroxy-4-isopropylstilbene; Phe: phenylalanine; CA: cinnamic acid; Leu: leucine; Bkd: branched-chain α-ketoacid dehydrogenase; BCFA: branched-chain fatty acids; IV-CoA: isovaleryl-CoA.</p

Both <i>lrp</i> and <i>rpoS</i> are required for normal <i>stlA</i> expression.

<p>A) The WT and mutant strains containing the <i>stlA-gfp</i> fusion were cultured in LB broth and, at the indicated times, samples were taken for OD₅₉₅ and fluorescence readings. The results shown are the mean of at least 3 experiments and the error bars represent the standard deviation. B) The WT and mutant strains were aliquoted onto LB agar and cultured for 3 days at 30°C. Colonies were then overlaid with soft agar and the ST-mediated inhibition of the growth of <i>M. luteus</i> was observed after a further 2-3 days incubation at 30°C. The difference in appearance of the zones of inhibition between the upper and lower panels is due to different lighting conditions used to take the PHOTOs. C) Complementation of <i>stlA-gfp</i> expression in the Δ<i>rpoS</i> mutant. WT or Δ<i>rpoS</i> mutant cells were transformed with vector alone (pBR322) or with a copy of the <i>rpoS</i> gene under the control of its own promoter, pBMM4292.2 (pBR-rpoS). Cultures were grown in LB broth (supplemented with Amp where appropriate) and samples were taken for fluorescence and OD₅₉₅ after 48h incubation at 30°C. Values shown are average of 3 independent experiments and the error bars represent standard deviation. </p

Nutrient limitation controls the activity of the <i>stlA</i> promoter.

<p>Cells carrying the <i>stlA-gfp</i> fusion were grown overnight and inoculated into the wells of a microtitre plate containing either 1x or 0.1x modified M9 broth, as indicated. The cells were cultured at 30°C and OD₆₀₀ and fluorescence were measured at 15 min intervals for 72h. A) Growth curves of TTO1 containing the <i>stlA-gfp</i> fusion in 1x and 0.1x modified M9 broth. B) Promoter activity. The fluorescence associated with the <i>stlA-gfp</i> fusion in 1x and 0.1x modified M9 broth has been corrected for background by subtracting the fluorescence of the (-)-<i>gfp</i> control at each time point and grown in the same medium. The data was then transformed as described in Materials and Methods using the mean value of data from at least 3 independent biological replicates. In order to reduce noise (particularly at early time points) the activity of the <i>stlA</i> promoter during growth in 1x (blue trend-line) and 0.1x (red trend-line) is represented by a trend-line calculated using the average of a sliding window across 6 data points.</p

The activity of the <i>stlA</i> promoter during growth of <i>Photorhabdus</i> in LB broth.

<p>Cells carrying the <i>stlA-gfp</i> fusion were grown overnight and inoculated into the wells of a microtitre plate. The cells were cultured at 30°C and OD₆₀₀ and fluorescence were measured at 15 min intervals for 43h. Promoter activity was calculated as described in Materials and Methods from at least 3 independent biological replicates. Promoter activity is represented by the filled diamond symbols whilst OD₆₀₀ is shown as the filled grey triangles. Also shown are the 4 distinct growth phases (labeled I-IV) discussed in the text. </p

Cinnamic acid can positively regulate <i>stlA</i> expression.

<p>Wild-type (WT) or <i>stlA</i>::Tn10 mutant cells carrying the <i>stlA-gfp</i> fusion were grown for 72h at 30°C in LB broth in the presence or absence of 0.5mM cinnamic acid (as indicated). OD₅₉₅ and fluorescence readings were taken and the results shown are the mean of at least 3 experiments and the error bars represent the standard deviation. Statistical significance was tested using the Student T-test and differences were deemed significant if <i>P</i><0.05 (*). <i>NS</i>: not significant.</p

Engineering the stambomycin modular polyketide synthase yields 37-membered mini-stambomycins

The modular organization of the type I polyketide synthases (PKSs) would seem propitious for rational engineering of desirable analogous. However, despite decades of efforts, such experiments remain largely inefficient. Here, we combine multiple, state-of-the-art approaches to reprogram the stambomycin PKS by deleting seven internal modules. One system produces the target 37-membered mini-stambomycin metabolites − a reduction in chain length of 14 carbons relative to the 51-membered parental compounds − but also substantial quantities of shunt metabolites. Our data also support an unprecedented off-loading mechanism of such stalled intermediates involving the C-terminal thioesterase domain of the PKS. The mini-stambomycin yields are reduced relative to wild type, likely reflecting the poor tolerance of the modules downstream of the modified interfaces to the non-native substrates. Overall, we identify factors contributing to the productivity of engineered whole assembly lines, but our findings also highlight the need for further research to increase production titers.ISSN:2041-172