Complex array of endobionts in Petalomonas sphagnophila, a large heterotrophic euglenid protist from Sphagnum-dominated peatlands.

Petalomonas sphagnophila is a poorly studied plastid-lacking euglenid flagellate living in Sphagnum-dominated peatlands. Here we present a broad-ranging microscopic, molecular and microspectrophotometric analysis of uncultured P. sphagnophila collected from four field locations in Nova Scotia, Canada. Consistent with its morphological characteristics, 18S ribosomal DNA (rDNA) phylogenies indicate that P. sphagnophila is specifically related to Petalomonas cantuscygni, the only other Petalomonas species sequenced to date. One of the peculiar characteristics of P. sphagnophila is the presence of several green-pigmented particles approximately 5 mum in diameter in its cytoplasm, which a previously published study suggested to be cyanobacterial endosymbionts. New data presented here, however, suggest that the green intracellular body may not be a cyanobacterium but rather an uncharacterized prokaryote yet to be identified by molecular sequencing. 16S rDNA library sequencing and fluorescence in situ hybridizations show that P. sphagnophila also harbors several other endobionts, including bacteria that represent five novel genus-level groups (one firmicute and four different proteobacteria). 16S rDNA phylogenies suggest that three of these endobionts are related to obligate intracellular bacteria such as Rickettsiales and Coxiella, while the others are related to the Daphnia pathogen Spirobacillus cienkowskii or belong to the Thermoactinomycetaceae. TEM, 16S rDNA library sequencing and a battery of PCR experiments show that the presence of the five P. sphagnophila endobionts varies markedly among the four geographic collections and even among individuals collected from the same location but at different time points. Our study adds significantly to the growing evidence for complex and dynamic protist-bacterial associations in nature.

Actinorhodopsin genes discovered in diverse freshwater habitats and among cultivated freshwater Actinobacteria.

Microbial rhodopsins are membrane proteins that utilize a retinal chromophore to harvest sunlight for energetic and photosensory functions. Recently, a group of novel rhodopsin sequences named 'actinorhodopsins' (ActRs) was hypothesized to exist among uncultured planktonic Actinobacteria. ActRs were discovered by mining metagenomic data obtained during the Venter Institute's Global Ocean Sampling expedition, from a hypersaline lagoon, two estuaries and a freshwater lake. On the basis of these findings, and many studies that show Actinobacteria are common inhabitants of lakes, we predicted that ActR genes would likely be present in other freshwater habitats and among the genomes of cultivated Actinobacteria. Using degenerate polymerase chain reaction primers, we discovered an ActR gene present in an actinobacterial isolate of the family Microbacteriaceae. Isolate MWH-Uga1 was cultivated prior to this study from a freshwater pond in Uganda and belongs to a group of Actinobacteria previously identified in freshwater ecosystems. ActR genes were also discovered present in numerous mixed cultures containing freshwater Actinobacteria and among environmental DNA samples obtained from three freshwater sources; a small woodland pond and the Laurentian Great Lakes Superior and Erie. An analysis of small subunit ribosomal RNA genes from metagenomic DNA samples harboring ActR genes suggests that organisms belonging to the acI lineage, an uncultured group of Actinobacteria commonly present in fresh waters, may utilize rhodopsins. The co-occurrence of an acI organism with a specific ActR variant in a mixed culture supports our hypothesis.

The gut microbiota of toll-like receptor 2-deficient mice exhibits lineage-specific modifications.

Mutations in toll-like receptors that mediate bacterial recognition by the mammalian innate immune system have the potential to substantially alter the composition of an individual's microbiota. Here we tested this hypothesis by comparing the intestinal microbiota of toll-like receptor 2-deficient mice, both young and middle aged, with that of wild-type mice of the same genetic background, housed together under specific pathogen-free conditions. Bacterial DNA was extracted from mouse caecal tissue samples, amplified using universal bacterial 16S ribosomal RNA gene primers, and cloned into a plasmid vector. Insert-containing colonies were picked for high-throughput sequencing, and sequence data were analysed yielding species-level phylogenetic data. Clone libraries were compared by phylogenetic composition analysis using UniFrac. While pairwise differences in phylogenetic population structure between mutant and wild-type mice were not statistically significant, anosim analysis did demonstrate a significant difference between toll-like receptor 2-deficient mice and their wild-type counterparts. The difference observed was probably due to a high level of colonization of the toll-like receptor 2-deficient mice by two distinct Helicobacter phylotypes that were totally absent from wild-type mice. Principal coordinate analysis clustering indicated that age is a weaker determinate than genotype and maternal heritage in the mouse caecal microbiota. The findings suggest that although mutations in toll-like receptors may cause increased susceptibility to specific opportunistic bacteria, they do not dramatically alter the phylogenetic structure of microbiota.

Searching for species in haloarchaea.

Prokaryotic (bacterial and archaeal) species definitions and the biological concepts that underpin them entail clustering (cohesion) among individuals, in terms of genome content and gene sequence similarity. Homologous recombination can maintain gene sequence similarity within, while permitting divergence between, clusters and is thus the basis for recent efforts to apply the Biological Species Concept in prokaryote systematics and ecology. In this study, we examine isolates of the haloarchaeal genus Halorubrum from two adjacent ponds of different salinities at a Spanish saltern and a natural saline lake in Algeria by using multilocus sequence analysis. We show that, although clusters can be defined by concatenation of multiple marker sequences, barriers to exchange between them are leaky. We suggest that no nonarbitrary way to circumscribe "species" is likely to emerge for this group, or by extension, to apply generally across prokaryotes. Arbitrary criteria might have limited practical use, but still must be agreed upon by the community.

Differences between tissue-associated intestinal microfloras of patients with Crohn's disease and ulcerative colitis.

A leading hypothesis for the role of bacteria in inflammatory bowel diseases is that an imbalance in normal gut flora is a prerequisite for inflammation. Testing this hypothesis requires comparisons between the microbiota compositions of ulcerative colitis and Crohn's disease patients and those of healthy individuals. In this study, we obtained biopsy samples from patients with Crohn's disease and ulcerative colitis and from healthy controls. Bacterial DNA was extracted from the tissue samples, amplified using universal bacterial 16S rRNA gene primers, and cloned into a plasmid vector. Insert-containing colonies were picked for high-throughput sequencing, and sequence data were analyzed, yielding species-level phylogenetic data. The clone libraries yielded 3,305 sequenced clones, representing 151 operational taxonomical units. There was no significant difference between floras from inflamed and healthy tissues from within the same individual. Proteobacteria were significantly (P = 0.0007) increased in Crohn's disease patients, as were Bacteroidetes (P < 0.0001), while Clostridia were decreased in that group (P < 0.0001) in comparison with the healthy and ulcerative colitis groups, which displayed no significant differences. Thus, the bacterial flora composition of Crohn's patients appears to be significantly altered from that of healthy controls, unlike that of ulcerative colitis patients. Imbalance in flora in Crohn's disease is probably not sufficient to cause inflammation, since microbiotas from inflamed and noninflamed tissues were of similar compositions within the same individual.