Comparative genome analysis provides insights into the evolution and adaptation of Pseudomonas syringae pv. aesculi on Aesculus hippocastanum.

A recently emerging bleeding canker disease, caused by Pseudomonas syringae pathovar aesculi (Pae), is threatening European horse chestnut in northwest Europe. Very little is known about the origin and biology of this new disease. We used the nucleotide sequences of seven commonly used marker genes to investigate the phylogeny of three strains isolated recently from bleeding stem cankers on European horse chestnut in Britain (E-Pae). On the basis of these sequences alone, the E-Pae strains were identical to the Pae type-strain (I-Pae), isolated from leaf spots on Indian horse chestnut in India in 1969. The phylogenetic analyses also showed that Pae belongs to a distinct clade of P. syringae pathovars adapted to woody hosts. We generated genome-wide Illumina sequence data from the three E-Pae strains and one strain of I-Pae. Comparative genomic analyses revealed pathovar-specific genomic regions in Pae potentially implicated in virulence on a tree host, including genes for the catabolism of plant-derived aromatic compounds and enterobactin synthesis. Several gene clusters displayed intra-pathovar variation, including those encoding type IV secretion, a novel fatty acid biosynthesis pathway and a sucrose uptake pathway. Rates of single nucleotide polymorphisms in the four Pae genomes indicate that the three E-Pae strains diverged from each other much more recently than they diverged from I-Pae. The very low genetic diversity among the three geographically distinct E-Pae strains suggests that they originate from a single, recent introduction into Britain, thus highlighting the serious environmental risks posed by the spread of an exotic plant pathogenic bacterium to a new geographic location. The genomic regions in Pae that are absent from other P. syringae pathovars that infect herbaceous hosts may represent candidate genetic adaptations to infection of the woody parts of the tree.

Molecular systematics of the Cactaceae

Bayesian, maximum-likelihood, and maximum-parsimony phylogenies, constructed using nucleotide sequences from the plastid gene region trnK-matK, are employed to investigate relationships within the Cactaceae. These phylogenies sample 666 plants representing 532 of the 1438 species recognized in the family. All four subfamilies, all nine tribes, and 69% of currently recognized genera of Cactaceae are sampled. We found strong support for three of the four currently recognized subfamilies, although relationships between subfamilies were not well defined. Major clades recovered within the largest subfamilies, Opuntioideae and Cactoideae, are reviewed; only three of the nine currently accepted tribes delimited within these subfamilies, the Cacteae, Rhipsalideae, and Opuntieae, are monophyletic, although the Opuntieae were recovered in only the Bayesian and maximum-likelihood analyses, not in the maximum-parsimony analysis, and more data are needed to reveal the status of the Cylindropuntieae, which may yet be monophyletic. Of the 42 genera with more than one exemplar in our study, only 17 were monophyletic; 14 of these genera were from subfamily Cactoideae and three from subfamily Opuntioideae. We present a synopsis of the status of the currently recognized genera

Partial identification of spirochaetes from two dairy cows with digital dermatitis by polymerase chain reaction analysis of the 16S ribosomal RNA gene

Specimens taken postmortem from typical lesions of digital dermatitis in two dairy cows were tested by the polymerase chain reaction (PCR) for the presence of a spirochaetal 16S rRNA gene. Seven different assays detected the gene in the samples from both cows. Two of the PCR products were sequenced and a comparison of the nucleotide sequences revealed that the spirochaete belonged to the genus Treponema and was closely related to Treponema denticola. A PCR specific for the detection of the digital dermatitis-associated treponeme was developed.

Comparative genomics of Salmonella enterica serovars Derby and Mbandaka, two prevalent serovars associated with different livestock species in the UK

Background Despite the frequent isolation of Salmonella enterica sub. enterica serovars Derby and Mbandaka from livestock in the UK and USA little is known about the biological processes maintaining their prevalence. Statistics for Salmonella isolations from livestock production in the UK show that S. Derby is most commonly associated with pigs and turkeys and S. Mbandaka with cattle and chickens. Here we compare the first sequenced genomes of S. Derby and S. Mbandaka as a basis for further analysis of the potential host adaptations that contribute to their distinct host species distributions. Results Comparative functional genomics using the RAST annotation system showed that predominantly mechanisms that relate to metabolite utilisation, in vivo and ex vivo persistence and pathogenesis distinguish S. Derby from S. Mbandaka. Alignment of the genome nucleotide sequences of S. Derby D1 and D2 and S. Mbandaka M1 and M2 with Salmonella pathogenicity islands (SPI) identified unique complements of genes associated with host adaptation. We also describe a new genomic island with a putative role in pathogenesis, SPI-23. SPI-23 is present in several S. enterica serovars, including S. Agona, S. Dublin and S. Gallinarum, it is absent in its entirety from S. Mbandaka. Conclusions We discovered a new 37 Kb genomic island, SPI-23, in the chromosome sequence of S. Derby, encoding 42 ORFS, ten of which are putative TTSS effector proteins. We infer from full-genome synonymous SNP analysis that these two serovars diverged, between 182kya and 625kya coinciding with the divergence of domestic pigs. The differences between the genomes of these serovars suggest they have been exposed to different stresses including, phage, transposons and prolonged externalisation. The two serovars possess distinct complements of metabolic genes; many of which cluster into pathways for catabolism of carbon sources.

Analysis of 16S rDNA sequences from pathogenic Leptospira serovars and use of single nucleotide polymorphisms for rapid speciation by D-HPLC

Leptospira have a worldwide distribution and include important zoonotic pathogens yet diagnosis and differentiation still tend to rely on traditional bacteriological and serological approaches. In this study a 1.3 kb fragment of the rrs gene (16S rDNA) was sequenced from a panel of 22 control strains, representing serovars within the pathogenic species Leptospira interrogans, Leptospira borgpetersenii, and Leptospira kirschneri, to identify single nucleotide polymorphisms (SNPs). These were identified in the 5' variable region of the 16S sequence and a 181 bp PCR fragment encompassing this region was used for speciation by Denaturing High Performance Liquid Chromatography (D-HPLC). This method was applied to eleven additional species, representing pathogenic, non-pathogenic and intermediate species and was demonstrated to rapidly differentiate all but 2 of the non-pathogenic Leptospira species. The method was applied successfully to infected tissues from field samples proving its value for diagnosing leptospiral infections found in animals in the UK. Crown Copyright (C) 2010 Published by Elsevier Ltd. All rights reserved.

Modified rare earth semiconductor oxide as a new nucleotide probe

Recent rapid developments in biological analysis, medical diagnosis, pharmaceutical industry, and environmental control fuel the urgent need for recognition of particular DNA sequences from samples. Currently, DNA detection techniques use radiochemical, enzymatic, fluorescent, or electrochemiluminescent methods; however, these techniques require costly labeled DNA and highly skilled and cumbersome procedure, which prohibit any in-situ monitoring. Here, we report that hybridization of surface-immobilized single-stranded oligonucleotide on praseodymium oxide (evaluated as a biosensor surface for the first time) with complimentary strands in solution provokes a significant shift of electrical impedance curve. This shift is attributed to a change in electrical characteristics through modification of surface charge of the underlying modified praseodymium oxide upon hybridization with the complementary oligonucelotide strand. On the other hand, using a noncomplementary single strand in solution does not create an equivalent change in the impedance value. This result clearly suggests that a new and simple electrochemical technique based on the change in electrical properties of the modified praseodymium oxide semiconductor surface upon recognition and transduction of a biological event without using labeled species is revealed.

Patterns of nucleotide substitution in angiosperm cpDNA trnL (UAA)-trnF (GAA) regions

Patterns of substitution in chloroplast encoded trnL_F regions were compared between species of Actaea (Ranunculales), Digitalis (Scrophulariales), Drosera (Caryophyllales), Panicoideae (Poales), the small chromosome species clade of Pelargonium (Geraniales), each representing a different order of flowering plants, and Huperzia (Lycopodiales). In total, the study included 265 taxa, each with > 900-bp sequences, totaling 0.24 Mb. Both pairwise and phylogeny-based comparisons were used to assess nucleotide substitution patterns. In all six groups, we found that transition/transversion ratios, as estimated by maximum likelihood on most-parsimonious trees, ranged between 0.8 and 1.0 for ingroups. These values occurred both at low sequence divergences, where substitutional saturation, i.e., multiple substitutions having occurred at the same (homologous) nucleotide position, was not expected, and at higher levels of divergence. This suggests that the angiosperm trnL-F regions evolve in a pattern different from that generally observed for nuclear and animal mtDNA (transitional/transversion ratio > or = 2). Transition/transversion ratios in the intron and the spacer region differed in all alignments compared, yet base compositions between the regions were highly similar in all six groups. A>-C transversions were significantly less frequent than the other four substitution types. This correlates with results from studies on fidelity mechanisms in DNA replication that predict A<->T and G<->C transversions to be least likely to occur. It therefore strengthens confidence in the link between mutation bias at the polymerase level and the actual fixation of substitutions as recorded on evolutionary trees, and concomitantly, in the neutrality of nucleotide substitutions as phylogenetic markers.

Calcrete profile development in quaternary alluvial sequences, southeast Spain: Implications for using calcretes as a basis for landform chronologies

A detailed study of the morphology and micro-morphology of Quaternary alluvial calcrete profiles from the Sorbas Basin shows that calcretes may be morphologically simple or complex. The 'simple' profiles reflect pedogenesis occurring after alluvial terrace formation and consist of a single pedogenic horizon near the land surface. The 'complex' profiles reflect the occurrence of multiple calcrete events during terrace sediment aggradation and further periods of pedogenesis after terrace formation. These 'complex' calcrete profiles are consequently described as composite profiles. The exact morphology of the composite profiles depends upon: (1) the number of calcrete-forming events occurring during terrace sediment aggradation; (2) the amount of sediment accretion that occurs between each period of calcrete formation; and (3) the degree of pedogenesis after terrace formation. Simple calcrete profiles are most useful in establishing landform chronologies because they represent a single phase of pedogenesis after terrace formation. Composite profiles are more problematic. Pedogenic calcretes that form within them may inherit carbonate from calcrete horizons occurring lower down in the terrace sediments. In addition erosion may lead to the exhumation of older calcretes within the terrace sediment. Calcrete 'inheritance' may make pedogenic horizons appear more mature than they actually are and produce horizons containing carbonate embracing a range of ages. Calcrete exhumation exposes calcrete horizons whose morphology and radiometric ages are wholly unrelated to terrace surface age. Composite profiles are, therefore, only suitable for chronological studies if the pedogenic horizon capping the terrace sequence can be clearly distinguished from earlier calcrete-forming events. Thus, a detailed morphological/micro-morphological study is required before any chronological study is undertaken. This is the only way to establish whether particular calcrete profiles are suitable for dating purposes. Copyright (C) 2003 John Wiley Sons, Ltd.

Producing Inequalities: Regional Sequences in Later Prehistoric Southern Spain

This paper presents regional sequences of production, consumption and Social relations ill Southern Spain from the beginning of the Neolithic to the Early Bronze Age (c. 5600-1550 BC). The regions Studied are southeast Spain, Valencia, the southern Meseta and central/western Andalucia. The details presented for each region and period vary in quality but Show how Much our knowledge of the archaeological record of southern Spain has changed during the last four decades. Among the Surprises are the rapidity of agricultural adoption. the emergence of regional centres of aggregated population in enclosed/fortified settlements of up to 400 hectares in the fourth and third millennia BC. the use of copper objects as instruments of production, rather than as items With 11 purely symbolic of 'prestige' value, large-scale copper production in western Andalucia in the third millennium BC (as opposed to the usual domestic production model), and the inference of societies based oil relations of class.

Wiggle-match dating of tree-ring sequences

Given the non-monotonic form of the radiocarbon calibration curve, the precision of single C-14 dates on the calendar timescale will always be limited. One way around this limitation is through comparison of time-series, which should exhibit the same irregular patterning as the calibration curve. This approach can be employed most directly in the case of wood samples with many years growth present (but not able to be dated by dendrochronology), where the tree-ring series of unknown date can be compared against the similarly constructed C-14 calibration curve built from known-age wood. This process of curve-fitting has come to be called "wiggle-matching." In this paper, we look at the requirements for getting good precision by this method: sequence length, sampling frequency, and measurement precision. We also look at 3 case studies: one a piece of wood which has been independently dendrochronologically dated, and two others of unknown age relating to archaeological activity at Silchester, UK (Roman) and Miletos, Anatolia (relating to the volcanic eruption at Thera).

Pleistocene geochronologies for fluvial sedimentary sequences: an archaeologist's perspective

Current models of Pleistocene fluvial system development and dynamics are assessed from the perspective of European Lower and Middle Palaeolithic stone tool assemblages recovered from fluvial secondary contexts. Fluvial activity is reviewed both in terms of Milankovitch-scale processes across the glacial/interglacial cycles of the Middle and Late Pleistocene, and in response to sub-Milankovitch scale, high-frequency, low-magnitude climatic oscillations. The chronological magnitude of individual phases of fluvial activity is explored in terms of radiocarbon-dated sequences from the Late Glacial and early Holocene periods. It is apparent that fluvial activity is associated with periods of climatic transition, both high and low magnitude, although system response is far more universal in the case of the high magnitude glacial/ interglacial transitions. Current geochronological tools do not permit the development of high-resolution sequences for Middle Pleistocene sediments, while localised erosion and variable system responses do not facilitate direct comparison with the ice core records. However, Late Glacial and early Holocene sequences indicate that individual fluvial activity phases are relatively brief in duration (e.g. 10(2) and 10(3) yr). From an archaeological perspective, secondary context assemblages can only be interpreted in terms of a floating geochronology, although the data also permit a reinvestigation of the problems of artefact reworking. Copyright (c) 2005 John Wiley I Sons, Ltd.

Aligning sequences to structures

Most newly sequenced proteins are likely to adopt a similar structure to one which has already been experimentally determined. For this reason, the most successful approaches to protein structure prediction have been template-based methods. Such prediction methods attempt to identify and model the folds of unknown structures by aligning the target sequences to a set of representative template structures within a fold library. In this chapter, I discuss the development of template-based approaches to fold prediction, from the traditional techniques to the recent state-of-the-art methods. I also discuss the recent development of structural annotation databases, which contain models built by aligning the sequences from entire proteomes against known structures. Finally, I run through a practical step-by-step guide for aligning target sequences to known structures and contemplate the future direction of template-based structure prediction.

Origin and evolution of 3'UTR of flaviviruses: long direct repeats as a basis for the formation of secondary structures and their significance for virus transmission

The 3' untranslated regions (3'UTRs) of flaviviruses are reviewed and analyzed in relation to short sequences conserved as direct repeats (DRs). Previously, alignments of the 3'UTRs have been constructed for three of the four recognized flavivirus groups, namely mosquito-borne, tick-borne, and nonclassified flaviviruses (MBFV, TBFV, and NCFV, respectively). This revealed (1) six long repeat sequences (LRSs) in the 3'UTR and open-reading frame (ORF) of the TBFV, (2) duplication of the 3'UTR of the NCFV by intramolecular recombination, and (3) the possibility of a common origin for all DRs within the MBFV. We have now extended this analysis and review it in the context of all previous published analyses. This has been achieved by constructing a robust alignment between all flaviviruses using the published DRs and secondary RNA structures as "anchors" to reveal additional homologies along the 3'UTR. This approach identified nucleotide regions within the MBFV, NKV (no-known vector viruses), and NCFV 3'UTRs that are homologous to different LRSs in the TBFV 3'UTR and ORF. The analysis revealed that some of the DRs and secondary RNA structures described individually within each flavivirus group share common evolutionary origins. The 3'UTR of flaviviruses, and possibly the ORF, therefore probably evolved through multiple duplication of an RNA domain, homologous to the LRS previously identified only in the TBFV. The short DRs in all virus groups appear to represent the evolutionary remnants of these domains rather than resulting from new duplications. The relevance of these flavivirus DRs to evolution, diversity, 3'UTR enhancer function, and virus transmission is reviewed.

Direct repeats in the flavivirus 3' untranslated region; a strategy for survival in the environment?

Previously, direct repeats (DRs) of 20-70 nucleotides were identified in the 3' untranslated regions (3'UTR) of flavivirus sequences. To address their functional significance, we have manually generated a pan-flavivirus 3'UTR alignment and correlated it with the corresponding predicted RNA secondary structures. This approach revealed that intra-group-conserved DRs evolved from six long repeated sequences (LRSs) which, as approximately 200-nucleotide domains were preserved only in the genomes of the slowly evolving tick-borne flaviviruses. We propose that short DRs represent the evolutionary remnants of LRSs rather than distinct molecular duplications. The relevance of DRs to virus replication enhancer function, and thus survival, is discussed.