Generation of genetic diversity is a prerequisite for bacterial evolution and adaptation. Short-term diversification and selection within populations is, however, largely uncharacterised, as existing studies typically focus on fixed substitutions. Here, we use whole-genome deep-sequencing to capture the spectrum of mutations arising during biofilm development for two Pseudomonas aeruginosa strains. This approach identified single nucleotide variants with frequencies from 0.5% to 98.0% and showed that the clinical strain 18A exhibits greater genetic diversification than the type strain PA01, despite its lower per base mutation rate. Mutations were found to be strain specific: the mucoid strain 18A experienced mutations in alginate production genes and a c-di-GMP regulator gene; while PA01 acquired mutations in PilT and PilY1, possibly in response to a rapid expansion of a lytic Pf4 bacteriophage, which may use type IV pili for infection. The Pf4 population diversified with an evolutionary rate of 2.43 × 10 −3 substitutions per site per day, which is comparable to single-stranded RNA viruses. Extensive within-strain parallel evolution, often involving identical nucleotides, was also observed indicating that mutation supply is not limiting, which was contrasted by an almost complete lack of noncoding and synonymous mutations. Taken together, these results suggest that the majority of the P. aeruginosa genome is constrained by negative selection, with strong positive selection acting on an accessory subset of genes that facilitate adaptation to the biofilm lifecycle. Long-term bacterial evolution is known to proceed via few, nonsynonymous, positively selected mutations, and here we show that similar dynamics govern shortterm, within-population bacterial diversification.dispersal | prophage | haplotypes D iversifying selection within bacterial populations underpins a range of ecological and clinical phenomena, for example niche adaptation (1-3) and antibiotic resistance (4). During diversifying selection, a single population explores multiple fitness peaks, resulting in subpopulations with different adaptive mutations. This kind of within-population genetic diversity can provide the raw material on which long-term evolution acts. However, diversifying selection of bacterial populations is still poorly understood, because previous experimental evolution and epidemiological studies typically have focused on fixed substitutions at the end point of evolution (5), ignoring short-term or withinpopulation effects.Laboratory-grown Pseudomonas aeruginosa biofilms provide an ideal model for investigating within-population diversification. The bacterium P. aeruginosa is a widespread, Gram-negative generalist and can have either a planktonic, motile lifestyle or exist as a biofilm (i.e., a surface-attached cells embedded within an extracellular polymeric matrix). P. aeruginosa has been the focus of extensive research, because of both its status as a model organism and its ability to form opportunistic, chronic, often lethal biof...

Aims The microbiome-derived metabolite trimethylamine-N-oxide (TMAO) has attracted major interest and controversy both as a diagnostic biomarker and therapeutic target in atherothrombosis. Methods and results Plasma TMAO increased in mice on ‘unhealthy’ high-choline diets and notably also on ‘healthy’ high-fibre diets. Interestingly, TMAO was found to be generated by direct oxidation in the gut in addition to oxidation by hepatic flavin-monooxygenases. Unexpectedly, two well-accepted mouse models of atherosclerosis, ApoE−/− and Ldlr−/− mice, which reflect the development of stable atherosclerosis, showed no association of TMAO with the extent of atherosclerosis. This finding was validated in the Framingham Heart Study showing no correlation between plasma TMAO and coronary artery calcium score or carotid intima-media thickness (IMT), as measures of atherosclerosis in human subjects. However, in the tandem-stenosis mouse model, which reflects plaque instability as typically seen in patients, TMAO levels correlated with several characteristics of plaque instability, such as markers of inflammation, platelet activation, and intraplaque haemorrhage. Conclusions Dietary-induced changes in the microbiome, of both ‘healthy’ and ‘unhealthy’ diets, can cause an increase in the plasma level of TMAO. The gut itself is a site of significant oxidative production of TMAO. Most importantly, our findings reconcile contradictory data on TMAO. There was no direct association of plasma TMAO and the extent of atherosclerosis, both in mice and humans. However, using a mouse model of plaque instability we demonstrated an association of TMAO plasma levels with atherosclerotic plaque instability. The latter confirms TMAO as being a marker of cardiovascular risk.

In hypersaline environments, haloarchaea (halophilic members of the Archaea) are the dominant organisms, and the viruses that infect them, haloarchaeoviruses are at least ten times more abundant. Since their discovery in 1974, described haloarchaeoviruses include head-tailed, pleomorphic, spherical and spindle-shaped morphologies, representing Myoviridae, Siphoviridae, Podoviridae, Pleolipoviridae, Sphaerolipoviridae and Fuselloviridae families. This review overviews current knowledge of haloarchaeoviruses, providing information about classification, morphotypes, macromolecules, life cycles, genetic manipulation and gene regulation, and host-virus responses. In so doing, the review incorporates knowledge from laboratory studies of isolated viruses, field-based studies of environmental samples, and both genomic and metagenomic analyses of haloarchaeoviruses. What emerges is that some haloarchaeoviruses possess unique morphological and life cycle properties, while others share features with other viruses (e.g., bacteriophages). Their interactions with hosts influence community structure and evolution of populations that exist in hypersaline environments as diverse as seawater evaporation ponds, to hot desert or Antarctic lakes. The discoveries of their wide-ranging and important roles in the ecology and evolution of hypersaline communities serves as a strong motivator for future investigations of both laboratory-model and environmental systems.

Androgen receptor splice variant V7 (AR-V7) was recently identified as a valuable predictive biomarker in metastatic castrate-resistant prostate cancer. Here, we report a new, sensitive and accurate screen for AR-V7 mRNA expression directly from circulating tumor cells (CTCs): We combined EpCAM-based immunomagnetic CTC isolation using the IsoFlux microfluidic platform with droplet digital polymerase chain reaction (ddPCR) to analyze total AR and AR-V7 expression from prostate cancer patients CTCs. We demonstrate that AR-V7 is reliably detectable in enriched CTC samples with as little as five CTCs, even considering tumor heterogeneity, and confirm detection of AR-V7 in CTC samples from advanced prostate cancer (PCa) patients with AR-V7 detection limited to castrate resistant disease status in our sample set. Sensitive molecular analyses of circulating tumor cells (CTCs) or circulating tumor nucleic acids present exciting strategies to detect biomarkers, such as AR-V7 from non-invasive blood samples, so-called blood biopsies.