As a leading cause of bacterial dysentery, Shigella represents a significant threat to public health and food safety. Related, but often overlooked, enteroinvasive Escherichia coli (EIEC) can also cause dysentery. Current typing methods have limited ability to identify and differentiate between these pathogens despite the need for rapid and accurate identification of pathogens for clinical treatment and outbreak response. We present a comprehensive phylogeny of Shigella and EIEC using whole genome sequencing of 169 samples, constituting unparalleled strain diversity, and observe a lack of monophyly between Shigella and EIEC and among Shigella taxonomic groups. The evolutionary relationships in the phylogeny are supported by analyses of population structure and hierarchical clustering patterns of translated gene homolog abundance. Lastly, we identified a panel of 254 single nucleotide polymorphism (SNP) markers specific to each phylogenetic cluster for more accurate identification of Shigella and EIEC. Our findings show that Shigella and EIEC are not distinct evolutionary groups within the E. coli genus and, thus, EIEC as a group is not the ancestor to Shigella. The multiple analyses presented provide evidence for reconsidering the taxonomic placement of Shigella. The SNP markers offer more discriminatory power to molecular epidemiological typing methods involving these bacterial pathogens.

BackgroundQuantitative PCR (qPCR) is a widely used technique for gene expression analysis. A common normalization method for accurate qPCR data analysis involves stable reference genes to determine relative gene expression. Despite extensive research in the forest tree species Populus, there is not a resource for reference genes that meet the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) standards for qPCR techniques and analysis. Since Populus is a woody perennial species, studies of seasonal changes in gene expression are important towards advancing knowledge of this important developmental and physiological trait. The objective of this study was to evaluate reference gene expression stability in various tissues and growth conditions in two important Populus genotypes (P. trichocarpa “Nisqually 1” and P. tremula x P. alba 717 1-B4) following MIQE guidelines.ResultsWe evaluated gene expression stability in shoot tips, young leaves, mature leaves and bark tissues from P. trichocarpa and P. tremula. x P. alba grown under long-day (LD), short-day (SD) or SD plus low-temperatures conditions. Gene expression data were analyzed for stable reference genes among 18S rRNA, ACT2, CDC2, CYC063, TIP4-like, UBQ7, PT1 and ANT using two software packages, geNormPLUS and BestKeeper. GeNormPLUS ranked TIP4-like and PT1 among the most stable genes in most genotype/tissue combinations while BestKeeper ranked CDC2 and ACT2 among the most stable genes.ConclusionsThis is the first comprehensive evaluation of reference genes in two important Populus genotypes and the only study in Populus that meets MIQE standards. Both analysis programs identified stable reference genes in both genotypes and all tissues grown under different photoperiods. This set of reference genes was found to be suitable for either genotype considered here and may potentially be suitable for other Populus species and genotypes. These results provide a valuable resource for the Populus research community.

BackgroundNucleoside phosphorylases (NPs) have been extensively investigated in human and bacterial systems for their role in metabolic nucleotide salvaging and links to oncogenesis. In plants, NP-like proteins have not been comprehensively studied, likely because there is no evidence of a metabolic function in nucleoside salvage. However, in the forest trees genus Populus a family of NP-like proteins function as an important ecophysiological adaptation for inter- and intra-seasonal nitrogen storage and cycling.ResultsWe conducted phylogenetic analyses to determine the distribution and evolution of NP-like proteins in plants. These analyses revealed two major clusters of NP-like proteins in plants. Group I proteins were encoded by genes across a wide range of plant taxa while proteins encoded by Group II genes were dominated by species belonging to the order Malpighiales and included the Populus Bark Storage Protein (BSP) and WIN4-like proteins. Additionally, we evaluated the NP-like genes in Populus by examining the transcript abundance of the 13 NP-like genes found in the Populus genome in various tissues of plants exposed to long-day (LD) and short-day (SD) photoperiods. We found that all 13 of the Populus NP-like genes belonging to either Group I or II are expressed in various tissues in both LD and SD conditions. Tests of natural selection and expression evolution analysis of the Populus genes suggests that divergence in gene expression may have occurred recently during the evolution of Populus, which supports the adaptive maintenance models. Lastly, in silico analysis of cis-regulatory elements in the promoters of the 13 NP-like genes in Populus revealed common regulatory elements known to be involved in light regulation, stress/pathogenesis and phytohormone responses.ConclusionIn Populus, the evolution of the NP-like protein and gene family has been shaped by duplication events and natural selection. Expression data suggest that previously uncharacterized NP-like proteins may function in nutrient sensing and/or signaling. These proteins are members of Group I NP-like proteins, which are widely distributed in many plant taxa. We conclude that NP-like proteins may function in plants, although this function is undefined.

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