Halophilic archaea in the human intestinal mucosa.

The human gastrointestinal tract microbiota, despite its key roles in health and disease, remains a diverse, variable and poorly understood entity. Current surveys reveal a multitude of undefined bacterial taxa and a low diversity of methanogenic archaea. In an analysis of the microbiota in colonic mucosal biopsies from patients with inflammatory bowel disease we found 16S rDNA sequences representing a phylogenetically rich diversity of halophilic archaea from the Halobacteriaceae (haloarchaea), including novel phylotypes. As the human colon is not considered a salty environment and haloarchaea are described as extreme halophiles, we evaluated and further discarded the possibility that these sequences originated from pre-colonoscopy saline lavage solutions. Furthermore, aerobic enrichment cultures prepared from a patient biopsy at low salinity (2.5% NaCl) yielded haloarchaeal sequence types. Microscopic observation after fluorescence in situ hybridization provided evidence of the presence of viable archaeal cells in these cultures. These results prove the survival of haloarchaea in the digestive system and suggest that they may be members of the mucosal microbiota, even if present in low numbers in comparison with methanogenic archaea. Investigation of a potential physiological basis of this association may lead to new insights into gastrointestinal health and disease.

In silico comparison of pKLC102-like genomic islands of Pseudomonas aeruginosa.

The genomic island pKLC102 first detected in Pseudomonas aeruginosa clone C strains can cross species barriers and exhibits the highest mobilization rate of a genomic island known to date. Homologous genomic islands of 81-108 kb in size were identified in the completely sequenced P. aeruginosa strains PA7, PA14, 2192, C3719 and PACS2, but not in strains PAO1 and LES. All pKLC102-like genomic islands are integrated in chromosomal tRNA(Lys) genes and share a syntenic set of more than 70 homologous ORFs, part of which are related to DNA replication or mobility genes. The conserved backbone has predilection sites for the uptake of island-specific gene cassettes. A major difference between the islands is the organization of the origin of replication oriV.

Gordonibacter pamelaeae gen. nov., sp. nov., a new member of the Coriobacteriaceae isolated from a patient with Crohn's disease, and reclassification of Eggerthella hongkongensis Lau et al. 2006 as Paraeggerthella hongkongensis gen. nov., comb. nov.

A strictly anaerobic, Gram-positive, short-rod/coccobacillus-shaped bacterial strain, designated 7-10-1-b(T), was isolated from the colon of a patient suffering from acute Crohn's disease. The isolate formed small, pale-white, semi-translucent colonies on solid cultivation media. The strain was catalase-positive and metabolized only a small number of carbon sources. Whole-cell fatty acids consisted predominantly of saturated fatty acids (89 %), of which 15 : 0 anteiso was the major component. The polar lipids phosphatidylglycerol and diphosphatidylglycerol as well as four glycolipids were identified. 16S rRNA gene sequence analysis revealed that the isolate represents a distinct lineage within the family Coriobacteriaceae and has 94.6 % identity to the type strain of [Eggerthella] hongkongensis, the phylogenetically closest bacterial species. On the basis of the analyses performed, the new genus and species Gordonibacter pamelaeae gen. nov., sp. nov. is described, with strain 7-10-1-b(T) (=DSM 19378(T) =CCUG 55131(T)) as the type and only strain of Gordonibacter pamelaeae. Also, based on the chemotaxonomic data obtained for all type strains of the neighbouring genus Eggerthella, we propose that Eggerthella hongkongensis Lau et al. 2006 be transferred to a new genus as Paraeggerthella hongkongensis gen. nov., comb. nov.; the type strain of Paraeggerthella hongkongensis is HKU10(T) (=DSM 16106(T) =CCUG 49250(T)).

Transcript profiling of the Pseudomonas aeruginosa genomic islands PAGI-2 and pKLC102.

The phylogenetically ancient genomic islands of the abundant PAGI-2/pKLC102 family are prone to horizontal gene transfer amongst proteobacteria, and account for most genomic diversity in Pseudomonas aeruginosa. The mRNA expression levels of the sequenced PAGI-2 and pKLC102 islands were determined in P. aeruginosa clone C strains C and SG17M during exponential and stationary growth in Luria broth or Vogel-Bonner mineral medium. Of the 111 ORFs of PAGI-2, only one gene was significantly expressed at a level of more than 0.0001% of total RNA. The individual mRNA transcripts of the 103 pKLC102 ORFs, however, were present in the range of 0.001% to more than 1% in the bacterial RNA population, and amounted altogether to more than 10% of cellular RNA. Homologous genes were strongly transcribed from pKLC102, but not at all from PAGI-2 under the tested conditions. Thus PAGI-2, which was stably captured by its host chromosome, was transcriptionally silent, whereas the mRNA transcripts derived from the mobile and episomally replicating pKLC102 were constitutively more abundant in the cell than the mRNA pool transcribed from the core genome.

Diversity of the abundant pKLC102/PAGI-2 family of genomic islands in Pseudomonas aeruginosa.

The known genomic islands of Pseudomonas aeruginosa clone C strains are integrated into tRNA(Lys) (pKLC102) or tRNA(Gly) (PAGI-2 and PAGI-3) genes and differ from their core genomes by distinctive tetranucleotide usage patterns. pKLC102 and the related island PAPI-1 from P. aeruginosa PA14 were spontaneously mobilized from their host chromosomes at frequencies of 10% and 0.3%, making pKLC102 the most mobile genomic island known with a copy number of 30 episomal circular pKLC102 molecules per cell. The incidence of islands of the pKLC102/PAGI-2 type was investigated in 71 unrelated P. aeruginosa strains from diverse habitats and geographic origins. pKLC102- and PAGI-2-like islands were identified in 50 and 31 strains, respectively, and 15 and 10 subtypes were differentiated by hybridization on pKLC102 and PAGI-2 macroarrays. The diversity of PAGI-2-type islands was mainly caused by one large block of strain-specific genes, whereas the diversity of pKLC102-type islands was primarily generated by subtype-specific combination of gene cassettes. Chromosomal loss of PAGI-2 could be documented in sequential P. aeruginosa isolates from individuals with cystic fibrosis. PAGI-2 was present in most tested Cupriavidus metallidurans and Cupriavidus campinensis isolates from polluted environments, demonstrating the spread of PAGI-2 across habitats and species barriers. The pKLC102/PAGI-2 family is prevalent in numerous beta- and gammaproteobacteria and is characterized by high asymmetry of the cDNA strands. This evolutionarily ancient family of genomic islands retained its oligonucleotide signature during horizontal spread within and among taxa.

Bacteria displaying interleukin-4 mutants stimulate mammalian cells and reflect the biological activities of variant soluble cytokines.

We describe a novel procedure that allows the rapid determination of cytokine activity on cells that express their cognate receptor. The four-helix bundle cytokine interleukin-4 (IL-4) was inducibly expressed as a fusion with the E. coli outer-membrane protein intimin, such that IL-4 was presented on the surfaces of the bacteria. Expression and accessibility of the cytokine on the cell exteriors were monitored by Western blotting and fluorescence microscopy, making use of two epitopes flanking the IL-4 component of the fusion protein. To demonstrate the biological activity of the immobilized cytokine, a Ba/F3-derived cell line stably transfected with both the bipartite human IL-4 receptor and an IL-4-specific luciferase reporter gene construct was employed. Bacterial cells displaying interleukin-4 elicited a specific, dose-dependent response in the reporter cells. Two variants of IL-4 with previously characterized (partial) antagonistic properties were also expressed as membrane-bound fusion proteins and were tested for their activity in the immobilized state. In comparison with bacteria displaying wild-type IL-4, E. coli clones presenting variants IL-4 Y124G and Y124D showed diminished or abolished activity, respectively, on murine reporter cells. The relative signaling potencies of the immobilized IL-4 variants thus closely mirror the agonistic properties of the corresponding soluble cytokines. This approach should be generally applicable for the mutational analysis of numerous signal mediators that trigger cellular responses through dimerization of transmembrane receptors.