Phylogenomic Analyses of Members of the Widespread Marine Heterotrophic Genus Pseudovibrio Suggest Distinct Evolutionary Trajectories and a Novel Genus, Polycladidibacter gen. nov.

Bacteria belonging to the Pseudovibrio genus are widespread, metabolically versatile, and able to thrive as both free-living and host-associated organisms. Although more than 50 genomes are available, a comprehensive comparative genomics study to resolve taxonomic inconsistencies is currently missing. We analyzed all available genomes and used 552 core genes to perform a robust phylogenomic reconstruction. This in-depth analysis revealed the divergence of two monophyletic basal lineages of strains isolated from polyclad flatworm hosts, namely, Pseudovibrio hongkongensis and Pseudovibrio stylochi These strains have reduced genomes and lack sulfur-related metabolisms and major biosynthetic gene clusters, and their environmental distribution appears to be tightly associated with invertebrate hosts. We showed experimentally that the divergent strains are unable to utilize various sulfur compounds that, in contrast, can be utilized by the type strain Pseudovibrio denitrificans Our analyses suggest that the lineage leading to these two strains has been subject to relaxed purifying selection resulting in great gene loss. Overall genome relatedness indices (OGRI) indicate substantial differences between the divergent strains and the rest of the genus. While 16S rRNA gene analyses do not support the establishment of a different genus for the divergent strains, their substantial genomic, phylogenomic, and physiological differences strongly suggest a divergent evolutionary trajectory and the need for their reclassification. Therefore, we propose the novel genus Polycladidibacter gen. nov.IMPORTANCE The genus Pseudovibrio is commonly associated with marine invertebrates, which are essential for ocean health and marine nutrient cycling. Traditionally, the phylogeny of the genus has been based on 16S rRNA gene analysis. The use of the 16S rRNA gene or any other single marker gene for robust phylogenetic placement has recently been questioned. We used a large set of marker genes from all available Pseudovibrio genomes for in-depth phylogenomic analyses. We identified divergent monophyletic basal lineages within the Pseudovibrio genus, including two strains isolated from polyclad flatworms. These strains showed reduced sulfur metabolism and biosynthesis capacities. The phylogenomic analyses revealed distinct evolutionary trajectories and ecological adaptations that differentiate the divergent strains from the other Pseudovibrio members and suggest that they fall into a novel genus. Our data show the importance of widening the use of phylogenomics for better understanding bacterial physiology, phylogeny, and evolution.

Preliminary evaluation of a combined microPET-MR system.

There are many motivations for adding simultaneously acquired MR images to PET scanning. The most straight forward are, superior registration of MR and PET images, the addition of morphological detail when there is non-rigid motion and for pre-clinical studies simultaneous imaging could lead to a significant reduction in the time that animals are required to be anesthetised. In addition simultaneous MR has the potential to provide accurate motion correction for PET image reconstruction. For functional imaging simultaneous acquisition is required to assess the subject in the same physiological state, such as acute stroke studies. The elimination of the additional radiation associated with combining CT with PET, by providing anatomic detail with MR, would be a crucial advantage for cancer screening. Combining the two instruments necessitates some engineering tradeoffs, especially associated with the use of the highly developed photomultiplier tube (PMT) used for light amplification, because of its incompatibility with strong magnetic fields. Our approach is to provide a split in the magnet and gradients to locate the magnetic sensitive components, the PMTs, in regions of low magnetic field, leaving only the essential PET components, the scintillator blocks, in the strong magnetic field region. The crystals are coupled to the PMTs by extending the optical fibres. A further advantage accrues by moving the PET electronics out of the region seen by the MR radio-frequency (RF) and gradient coils as electromagnetic interference effects between the PET and MR systems, which could cause artefacts in either modality, are eliminated. Here we describe a preliminary evaluation of the system, which is essentially a microPET Focus-120 located in a 1T split magnet, and compare its performance to previous microPET instruments.

Development of a combined microPET-MR system.

As evidenced by the success of PET-CT, there are many benefits from combining imaging modalities into a single scanner. The combination of PET and MR offers potential advantages over PET-CT, including improved soft tissue contrast, access to the multiplicity of contrast mechanisms available to MR, simultaneous imaging and fast MR sequences for motion correction. In addition, PET-MR is more suitable than PET-CT for cancer screening due to the elimination of the radiation dose from CT. A key issue associated with combining PET and MR is the fact that the performance of the photomultiplier tubes (PMTs) used in conventional PET detectors is degraded in the magnetic field required for MR. Two approaches have been adopted to circumvent that issue: retention of conventional, magnetic field-sensitive PMT-based PET detectors by modification of other features of the MR or PET system, or the use of new, magnetic field-insensitive devices in the PET detectors including avalanche photo-diodes (APDs) and silicon photomultipliers (SiPMs). Taking the former approach, we are assembling a modified microPET Focus 120 within a gap in a novel, 1T superconducting magnet. The PMTs are located in a low magnetic field (approximately 30mT) through a combination of magnet design and the use of fiber optic 'bundles'. Two main features of the modified PET system have been tested, namely the effect of using long fiber optic bundles in the PET detector, and the impact of magnetic field upon the performance of the position sensitive PMTs. The design of a modified microPET-MR system for small animal imaging is completed, and assembly and testing is underway.

A locally adaptive registration technique for high precision registration of 3-D MRI data.

This study demonstrates how the rigid body registration parameters for good registration of serially acquired 3-D magnetic resonance images vary systematically when the registration routine is presented with a series of cropped data sets that are systematically positioned throughout the entire volume. The results of the registration of these subcubes are compared with the results of a single registration of the complete volume for two consecutive 3-D scans of the brain of a normal volunteer, with one scan having optimized shim coil currents and the other having all second-order shim coil currents set to zero. The technique is sensitive and able to reveal subvoxel misregistrations.

Low-light imaging technology in the life sciences.

Photon imaging is an increasingly important technique for the measurement and analysis of chemiluminescence and bioluminescence. New high-performance low-light level imaging systems have recently become available for the life sciences. These systems use advances in camcera design and digital image processing and are now being used for a wide range of luminescence applications. They offer good sensitivity for photon detection and large dynamic range, and are suitable for quantitative analysis. This is achieved using a range of software techniques including image arithmetic, histogramming or summing regions of interest, feature extraction and multiple image processing for kinetics or assay screening. Improvements in image-processing hardware and software have increased the usefulness of these systems in the biosciences. Low-light imaging is a rapid and non-invasive method for the sensitive detection and analysis of luminescent assays. As such it offers a powerful and sensitive tool for investigating processes, both at the cellular level (luc and lux reporter genes, intracellular signalling) and for measurement of macro samples (immunoassays, gels and blots, tissue sections).

CCD imaging of luciferase gene expression in single mammalian cells.

Quantitative and sensitive imaging of chemiluminescence, bioluminescence and fluorescence emissions is emerging as an increasingly important technique for a range of biomedical applications (Hooper et al., 1990). A brief review of low-light-level imaging is presented, with particular reference to charge-coupled devices (CCD). Detectors for sensitive imaging are described and compared, including various CCDs and photon-counting devices. Image analysis techniques based on digital image processing, may be applied to quantify luminescent processes with these detectors. Images of luciferase gene expression in single mammalian cells have been obtained using a particular high-sensitivity intensified CCD camera. The method is illustrated using cell monolayers infected with recombinant vaccinia virus encoding the firefly luciferase, luc gene (Rodriguez et al., 1988). The CCD camera has been used to detect luciferase expression in single, recombinant infected cells amongst over one million non-infected cells. The rapid detection of luciferase-expressing viruses may be used for the selection of virus deletion mutants into which the luciferase gene has been cloned at specific sites. This is particularly useful in the case of viruses such as cytomegalovirus which have slow replication cycles. This direct imaging technique is simple and versatile. It offers a rapid, non-invasive method for the sensitive detection of luciferase activity in single, luciferase-expressing cells. One can envisage the use of luciferase as a sensitive and convenient co-selection marker gene in the analysis of both gene expression and protein function. These methods offer tremendous potential in the fields of molecular and cellular biology.