Phylogenetic Relatedness Among Plasmids Harbored by Campylobacter jejuni and Campylobacter coli Isolated From Retail Meats.

Campylobacter jejuni and Campylobacter coli are two of the major causes of foodborne illness. In this study, 29 plasmids isolated from 20 retail meat isolates of Campylobacter jejuni and Campylobacter coli were fully-sequenced individually or as a part of a whole genome sequencing approach. The fully-sequenced plasmids ranged in size from 3 to 119 kb. Molecular characterization of the sequenced plasmids was based on pangenomic analysis and types of genes present on these plasmids and similar ones from GenBank. The plasmids were categorized into four different groups. These groups include type-1 that consisted mainly of pTet plasmids with the tetO gene, type-2 plasmids commonly found in C. coli strains, type-3 which has pVir plasmids, and type-4 that consisted mainly of smaller plasmids. The type-2 plasmids were unique, common among C. coli strains, and carried several conjugative transfer genes. The type-2 plasmids were most similar to a plasmid from Helicobacter pullorum. Maximum parsimony analysis and NeighborNet analysis were used to assess the phylogenetic relatedness among the 29 plasmid sequences presented in this study in addition to the other 104 plasmid sequences of Campylobacter species available in GenBank to date. Results from MP analysis revealed multiple lineages among Campylobacter plasmids which was supported by NeighborNet analysis. Clustering of plasmids did not conform to species-specific clades which suggested an intra-species dissemination of plasmids among Campylobacter species. To our knowledge, this is the first extensive phylogenetic analysis of Campylobacter plasmids sequenced to date.

The internal transcribed spacer 2 of Jenufa (Chlorophyta, Chlorophyceae) is extraordinarily long: A hypothesis.

Aided by a host of bioinformatics tools, primary and secondary structural analyses of the internal transcribed spacer 2 (ITS2) from the eukaryotic ribosomal RNA repeat have a long and enviable record of service to diversity studies of fungi, plants and protists. Automation of annotation, secondary structure estimation and sequence alignment have become routine for the vast majority of ITS2 sequences. Challenges to the bioinformatics pipeline for ITS2 analysis generally arise in cases where the sequence length lies well outside the norm. These sequences generally defy protocols for annotation and secondary structure prediction. The long ITS2 sequences (ca. 600 nucleotides) from the green alga, Jenufa, offered an opportunity to explore this problem. Custom BLAST parameters revealed the presence of 4-helix structures (200-250 nucleotides) embedded in the 5' portion of several long ITS2 sequences of Jenufa. Of special note is the ITS2 sequence of J. lobulosa where a 4-helix structure was obtained for both the embedded ITS2 and for the complete sequence. Phylogenetic analysis of these typically-sized sequences resolved Golenkinia longispicula as the sister to Jenufa. Our observations indicate that other long ITS2 sequences should be examined for evidence of expansion or duplication. In addition, if the embedded ITS2 sequences are functional, then ribogenesis is almost certainly more diverse than is already apparent from studies of humans and yeast.

Phylogenetic analysis of Dunaliella (Chlorophyta) emphasizing new benthic and supralittoral isolates from Great Salt Lake.

Dunaliella, a commercially important chlorophyte, is globally distributed in saline habitats. Morphological species have not been definitively reconciled with phylogenetic analyses. Considerable genetic diversity continues to be discovered in new isolates, especially from soil and benthic habitats. Twenty-nine new isolates from Great Salt Lake, Utah, many from benthic or supralittoral habitats, were phylogenetically analyzed using ITS1+5.8S+ITS2 in comparison to a broad sampling of available sequences. A few new isolates align in one branch of a bifurcated monophyletic Dunaliella salina clade and several cluster within monophyletic D. viridis. Several others align with relatively few unnamed strains from other locations, comprising a diverse clade that may represent two or more new species. The overall Dunaliella clade is relatively robust, but the nearest outgroups are ambiguously placed with extremely long branches. About half of the isolates, all from benthic or supralittoral habitats, have been persistently sarcinoid in liquid media since isolation. This trait is spread across the Dunaliella phylogeny. The morphology of two sarcinoid strains was documented with light microscopy, revealing an extensive glycocalyx. Clumping behavior of unicellular and sarcinoid strains was unaffected by presence or absence of Mg2+ or Ca2+ , addition of lectin-inhibiting monosaccharides, or water-soluble factors from morphologically opposite strains. Results from this investigation have significantly expanded our current understanding of Dunaliella diversity, but it seems likely that much remains to be discovered with additional sampling.

Draft Nuclear Genome Sequence of the Halophilic and Beta-Carotene-Accumulating Green Alga Dunaliella salina Strain CCAP19/18.

The halotolerant alga Dunaliella salina is a model for stress tolerance and is used commercially for production of beta-carotene (=pro-vitamin A). The presented draft genome of the genuine strain CCAP19/18 will allow investigations into metabolic processes involved in regulation of stress responses, including carotenogenesis and adaptations to life in high-salinity environments.

Assessing intragenomic variation of the internal transcribed spacer two: Adapting the Illumina metagenomics protocol.

Primary and secondary structural data from the internal transcribed spacer two (ITS2) have been used extensively for diversity studies of many different eukaryotic organisms, including the green algae. Ease of amplification is due, at least in part, to the fact that ITS2 is part of the tandemly-repeated rRNA array. The potential confounding influence of intragenomic variability has yet to be addressed except in a few organisms. Moreover, few of the assessments of intragenomic variation have taken advantage of the deep sequencing capacity of sequence-by-synthesis protocols. We present results from our adaptation of the 16S Metagenomics Sequencing Library Preparation/Illumina protocol for deep sequencing of the ITS2 genes in selected isolates of the green algal genus, Haematococcus. Deep sequencing yielded from just under 20,000 to more than 500,000 merged reads, outpacing results from recent pyrosequencing efforts. Furthermore, a conservative evaluation of these data revealed a range of three to six ITS2 sequence haplotypes (defined as unique sets of nucleotide polymorphisms) across the taxon sampling. The frequency of the dominant haplotype ranged from 0.35 to 0.98. In all but two cases, the haplotype with the greatest frequency corresponded to a sequence obtained by the Sanger method using PCR templates. Our data also show that results from the sequencing-by-synthesis approach are reproducible. In addition to advancing our understanding of ribosomal RNA variation, the results of this investigation will allow us to begin testing hypotheses regarding the maintenance of homogeneity across multi-copy genes.

Molecular analysis confirms the long-distance transport of Juniperus ashei pollen.

Although considered rare, airborne pollen can be deposited far from its place of origin under a confluence of favorable conditions. Temporally anomalous records of Cupressacean pollen collected from January air samples in London, Ontario, Canada have been cited as a new case of long-distance transport. Data on pollination season implicated Juniperus ashei (mountain cedar), with populations in central Texas and south central Oklahoma, as the nearest source of the Cupressacean pollen in the Canadian air samples. This finding is of special significance given the allergenicity of mountain cedar pollen. While microscopy is used extensively to identify particles in the air spora, pollen from all members of the Cupressaceae, including Juniperus, are morphologically indistinguishable. Consequently, we implemented a molecular approach to characterize Juniperus pollen using PCR in order to test the long-distance transport hypothesis. Our PCR results using species-specific primers confirmed that the anomalous Cupressacean pollen collected in Canada was from J. ashei. Forward trajectory analysis from source areas in Texas and the Arbuckle Mountains in Oklahoma and backward trajectory analysis from the destination area near London, Ontario were completed using models implemented in HYSPLIT4 (Hybrid Single-Particle Lagrangian Integrated Trajectory). Results from these trajectory analyses strongly supported the conclusion that the J. ashei pollen detected in Canada had its origins in Texas or Oklahoma. The results from the molecular findings are significant as they provide a new method to confirm the long-distance transport of pollen that bears allergenic importance.

Molecular approaches for the analysis of airborne pollen: A case study of Juniperus pollen.

BACKGROUND: Pollen monitoring is a common and vital tool in the field of allergy, creating awareness in pollen sensitive individuals. Traditionally, pollen monitoring has been based on conventional microscopic counting techniques that are labor intensive and limited in the identification to the genus or family level. Molecular techniques provide an alternative approach that is less labor intensive and enable identification of any species by its genetic fingerprint. OBJECTIVE: To use quantitative polymerase chain reaction (qPCR) to evaluate pollen concentrations in air samples. METHODS: Juniperus pollen was selected as our model because of the importance of this pollen in the southcentral United States. We analyzed 105 air samples collected with a Burkard spore trap from 2013 to 2015 using species-specific primers and probes. To evaluate the feasibility of a molecular approach, we used duplicate air samples that allowed us to compare results from classical identification based on light microscopy with our qPCR results. RESULTS: Pollen concentrations from the qPCR data were significantly correlated with concentrations determined through light microscopy (R = 0.902, P < .001). We also confirmed an overlap in the pollination seasons between Juniperus ashei and Juniperus pinchotii and between J ashei and Juniperus virginiana. CONCLUSION: We found that this method correctly identified different Juniperus species present in mixed air samples in the southcentral United States, an accomplishment that cannot be achieved using microscopic identification. We conclude that the qPCR method is more accurate and sensitive than current pollen monitoring techniques and, therefore, has the potential to be used in various pollen monitoring stations.

Phylogeny of Oedogoniales, Chaetophorales and Chaetopeltidales (Chlorophyceae): inferences from sequence-structure analysis of ITS2.

BACKGROUND AND AIMS: The green algal class Chlorophyceae comprises five orders (Chlamydomonadales, Sphaeropleales, Chaetophorales, Chaetopeltidales and Oedogoniales). Attempts to resolve the relationships among these groups have met with limited success. Studies of single genes (18S rRNA, 26S rRNA, rbcL or atpB) have largely failed to unambiguously resolve the relative positions of Oedogoniales, Chaetophorales and Chaetopeltidales (the OCC taxa). In contrast, recent genomics analyses of plastid data from OCC exemplars provided a robust phylogenetic analysis that supports a monophyletic OCC alliance. METHODS: An ITS2 data set was assembled to independently test the OCC hypothesis and to evaluate the performance of these data in assessing green algal phylogeny at the ordinal or class level. Sequence-structure analysis designed for use with ITS2 data was employed for phylogenetic reconstruction. KEY RESULTS: Results of this study yielded trees that were, in general, topologically congruent with the results from the genomic analyses, including support for the monophyly of the OCC alliance. CONCLUSIONS: Not all nodes from the ITS2 analyses exhibited robust support, but our investigation demonstrates that sequence-structure analyses of ITS2 provide a taxon-rich means of testing phylogenetic hypotheses at high taxonomic levels. Thus, the ITS2 data, in the context of sequence-structure analysis, provide an economical supplement or alternative to the single-marker approaches used in green algal phylogeny.

Internal transcribed spacer 2 (nu ITS2 rRNA) sequence-structure phylogenetics: towards an automated reconstruction of the green algal tree of life.

BACKGROUND: Chloroplast-encoded genes (matK and rbcL) have been formally proposed for use in DNA barcoding efforts targeting embryophytes. Extending such a protocol to chlorophytan green algae, though, is fraught with problems including non homology (matK) and heterogeneity that prevents the creation of a universal PCR toolkit (rbcL). Some have advocated the use of the nuclear-encoded, internal transcribed spacer two (ITS2) as an alternative to the traditional chloroplast markers. However, the ITS2 is broadly perceived to be insufficiently conserved or to be confounded by introgression or biparental inheritance patterns, precluding its broad use in phylogenetic reconstruction or as a DNA barcode. A growing body of evidence has shown that simultaneous analysis of nucleotide data with secondary structure information can overcome at least some of the limitations of ITS2. The goal of this investigation was to assess the feasibility of an automated, sequence-structure approach for analysis of IT2 data from a large sampling of phylum Chlorophyta. METHODOLOGY/PRINCIPAL FINDINGS: Sequences and secondary structures from 591 chlorophycean, 741 trebouxiophycean and 938 ulvophycean algae, all obtained from the ITS2 Database, were aligned using a sequence structure-specific scoring matrix. Phylogenetic relationships were reconstructed by Profile Neighbor-Joining coupled with a sequence structure-specific, general time reversible substitution model. Results from analyses of the ITS2 data were robust at multiple nodes and showed considerable congruence with results from published phylogenetic analyses. CONCLUSIONS/SIGNIFICANCE: Our observations on the power of automated, sequence-structure analyses of ITS2 to reconstruct phylum-level phylogenies of the green algae validate this approach to assessing diversity for large sets of chlorophytan taxa. Moreover, our results indicate that objections to the use of ITS2 for DNA barcoding should be weighed against the utility of an automated, data analysis approach with demonstrated power to reconstruct evolutionary patterns for highly divergent lineages.

Dynamic evolutionary pattern of alpha2-macroglobulin in a model organism, the zebrafish (Danio rerio).

Studies of alpha-2-macroglobulin (alpha(2)M), a universal protease inhibitor, have indicated that it plays a unique and critical role in the innate immune system of vertebrate and invertebrate animals. The distinctive mechanism of pathogen inhibition--through physical entrapment of the pathogen-derived protease--makes alpha(2)M an ideal candidate for molecular evolutionary analysis. Furthermore, recent studies revealed that the Osteichthyes are characterized by levels of alpha(2)M diversification that exceed those recorded in other animal groups. Our study of Danio rerio (zebrafish) indicated that (1) two distinct lineages of alpha(2)M and alpha(2)M-like isoforms exist and (2) at least some codons in the functional domains of alpha(2)M have been subjected to positive Darwinian selection. The findings of several hot-spots for nonsynonymous substitutions in the two functional domains such as bait region and receptor binding domain, suggest that host-immune selection have played a dominant role in these two genomic regions of alpha(2)M. The presence of two, non-monophyletic alpha(2)M lineages in zebrafish provides compelling evidence of an ancient gene duplication event. The accelerated rate of nucleotide substitution in the functional domains of alpha(2)M is consistent with similar observations of other immune system components.

Cyanobacterial diversity and halotolerance in a variable hypersaline environment.

The Great Salt Plains (GSP) in north-central Oklahoma, USA is an expansive salt flat (approximately 65 km(2)) that is part of the federally protected Salt Plains National Wildlife Refuge. The GSP serves as an ideal environment to study the microbial diversity of a terrestrial, hypersaline system that experiences wide fluctuations in freshwater influx and diel temperature. Our study assessed cyanobacterial diversity at the GSP by focusing on the taxonomic and physiological diversity of GSP isolates, and the 16S rRNA phylogenetic diversity of isolates and environmental clones from three sites (north, central, and south). Taxonomic diversity of isolates was limited to a few genera (mostly Phormidium and Geitlerinema), but physiological diversity based on halotolerance ranges was strikingly more diverse, even between strains of the same phylotype. The phylogenetic tree revealed diversity that spanned a number of cyanobacterial lineages, although diversity at each site was dominated by only a few phylotypes. Unlike other hypersaline systems, a number of environmental clones from the GSP were members of the heterocystous lineage. Although a number of cyanobacterial isolates were close matches with prevalent environmental clones, it is not certain if these clones reflect the same halotolerance ranges of their matching isolates. This caveat is based on the notable disparities we found between strains of the same phylotype and their inherent halotolerance. Our findings support the hypothesis that variable or poikilotrophic environments promote diversification, and in particular, select for variation in ecotype more than phylotype.