Genomic and Gene-Expression Comparisons among Phage-Resistant Type-IV Pilus Mutants of Pseudomonas syringae pathovar phaseolicola

Pseudomonas syringae pv. phaseolicola (Pph) is a significant bacterial pathogen of agricultural crops, and phage Φ6 and other members of the dsRNA virus family Cystoviridae undergo lytic (virulent) infection of Pph, using the type IV pilus as the initial site of cellular attachment. Despite the popularity of Pph/phage Φ6 as a model system in evolutionary biology, Pph resistance to phage Φ6 remains poorly characterized. To investigate differences between phage Φ6 resistant Pph strains, we examined genomic and gene expression variation among three bacterial genotypes that differ in the number of type IV pili expressed per cell: ordinary (wild-type), non-piliated, and super-piliated. Genome sequencing of non-piliated and super-piliated Pph identified few mutations that separate these genotypes from wild type Pph–and none present in genes known to be directly involved in type IV pilus expression. Expression analysis revealed that 81.1% of gene ontology (GO) terms up-regulated in the non-piliated strain were down-regulated in the super-piliated strain. This differential expression is particularly prevalent in genes associated with respiration—specifically genes in the tricarboxylic acid cycle (TCA) cycle, aerobic respiration, and acetyl-CoA metabolism. The expression patterns of the TCA pathway appear to be generally up and down-regulated, in non-piliated and super-piliated Pph respectively. As pilus retraction is mediated by an ATP motor, loss of retraction ability might lead to a lower energy draw on the bacterial cell, leading to a different energy balance than wild type. The lower metabolic rate of the super-piliated strain is potentially a result of its loss of ability to retract.     

Interactions of the NPXY microdomains of the low density lipoprotein receptor‐related protein 1

The low density lipoprotein receptor‐related protein 1 (LRP1) mediates internalization of a large number of proteins and protein–lipid complexes and is widely implicated in Alzheimer's disease. The cytoplasmic domain of LRP1 (LRP1‐CT) can be phosphorylated by activated protein‐tyrosine kinases at two NPXY motifs in LRP1‐CT; Tyr 4507 is readily phosphorylated and must be phosphorylated before phosphorylation of Tyr 4473 occurs. Pull‐down experiments from brain lysate revealed numerous proteins binding to LRP1‐CT, but the results were highly variable. To separate which proteins bind to each NPXY motif and their phosphorylation dependence, each NPXY motif microdomain was prepared in both phosphorylated and non‐phosphorylated forms and used to probe rodent brain extracts for binding proteins. Proteins that bound specifically to the microdomains were identified by LC‐MS/MS, and confirmed by Western blot. Recombinant proteins were then tested for binding to each NPXY motif. The NPXY₄₅₀₇ (membrane distal) was found to interact with a large number of proteins, many of which only bound the tyrosine‐phosphorylated form. This microdomain also bound a significant number of other proteins in the unphosphorylated state. Many of the interactions were later confirmed to be direct with recombinant proteins. The NPXY₄₄₇₃ (membrane proximal) bound many fewer proteins and only to the phosphorylated form.     

Evolutionary divergence of β–expansin structure and function in grasses parallels emergence of distinctive primary cell wall traits

Expansins are wall‐loosening proteins that promote the extension of primary cell walls without the hydrolysis of major structural components. Previously, proteins from the EXPA (α–expansin) family were found to loosen eudicot cell walls but to be less effective on grass cell walls, whereas the reverse pattern was found for EXPB (β–expansin) proteins obtained from grass pollen. To understand the evolutionary and structural bases for the selectivity of EXPB action, we assessed the extension (creep) response of cell walls from diverse monocot families to EXPA and EXPB treatments. Cell walls from Cyperaceae and Juncaceae (families closely related to grasses) displayed a typical grass response (‘β–response’). Walls from more distant monocots, including some species that share with grasses high levels of arabinoxylan, responded preferentially to α–expansins (‘α–response’), behaving in this regard like eudicots. An expansin with selective activity for grass cell walls was detected in Cyperaceae pollen, coinciding with the expression of genes from the divergent EXPB–I branch that includes grass pollen β–expansins. The evolutionary origin of this branch was located within Poales on the basis of phylogenetic analyses and its association with the ‘sigma’ whole‐genome duplication. Accelerated evolution in this branch has remodeled the protein surface in contact with the substrate, potentially for binding highly substituted arabinoxylan. We propose that the evolution of the divergent EXPB–I group made a fundamental change in the target and mechanism of wall loosening in the grass lineage possible, involving a new structural role for xylans and the expansins that target them.     

A novel mode of chromosomal evolution peculiar to filamentous Ascomycete fungi

Background  

Gene loss, inversions, translocations, and other chromosomal rearrangements vary among species, resulting in different rates of structural genome evolution. Major chromosomal rearrangements are rare in most eukaryotes, giving large regions with the same genes in the same order and orientation across species. These regions of macrosynteny have been very useful for locating homologous genes in different species and to guide the assembly of genome sequences. Previous analyses in the fungi have indicated that macrosynteny is rare; instead, comparisons across species show no synteny or only microsyntenic regions encompassing usually five or fewer genes. To test the hypothesis that chromosomal evolution is different in the fungi compared to other eukaryotes, synteny was compared between species of the major fungal taxa.     

The molecular basis for the broad substrate specificity of human sulfotransferase 1A1

Cytosolic sulfotransferases (SULTs) are mammalian enzymes that detoxify a wide variety of chemicals through the addition of a sulfate group. Despite extensive research, the molecular basis for the broad specificity of SULTs is still not understood. Here, structural, protein engineering and kinetic approaches were employed to obtain deep understanding of the molecular basis for the broad specificity, catalytic activity and substrate inhibition of SULT1A1. We have determined five new structures of SULT1A1 in complex with different acceptors, and utilized a directed evolution approach to generate SULT1A1 mutants with enhanced thermostability and increased catalytic activity. We found that active site plasticity enables binding of different acceptors and identified dramatic structural changes in the SULT1A1 active site leading to the binding of a second acceptor molecule in a conserved yet non-productive manner. Our combined approach highlights the dominant role of SULT1A1 structural flexibility in controlling the specificity and activity of this enzyme.     

Use of low-coverage, large-insert, short-read data for rapid and accurate generation of enhanced-quality draft Pseudomonas genome sequences

Next-generation genomic technology has both greatly accelerated the pace of genome research as well as increased our reliance on draft genome sequences. While groups such as the Genomics Standards Consortium have made strong efforts to promote genome standards there is a still a general lack of uniformity among published draft genomes, leading to challenges for downstream comparative analyses. This lack of uniformity is a particular problem when using standard draft genomes that frequently have large numbers of low-quality sequencing tracts. Here we present a proposal for an "enhanced-quality draft" genome that identifies at least 95% of the coding sequences, thereby effectively providing a full accounting of the genic component of the genome. Enhanced-quality draft genomes are easily attainable through a combination of small- and large-insert next-generation, paired-end sequencing. We illustrate the generation of an enhanced-quality draft genome by re-sequencing the plant pathogenic bacterium Pseudomonas syringae pv. phaseolicola 1448A (Pph 1448A), which has a published, closed genome sequence of 5.93 Mbp. We use a combination of Illumina paired-end and mate-pair sequencing, and surprisingly find that de novo assemblies with 100x paired-end coverage and mate-pair sequencing with as low as low as 2-5x coverage are substantially better than assemblies based on higher coverage. The rapid and low-cost generation of large numbers of enhanced-quality draft genome sequences will be of particular value for microbial diagnostics and biosecurity, which rely on precise discrimination of potentially dangerous clones from closely related benign strains.