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.

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.