Continuous enrichment culturing of thermophiles under sulfate and nitrate-reducing conditions and at deep-sea hydrostatic pressures.

A continuous culture bioreactor was developed to enrich for nitrate and sulfate reducing thermophiles under in situ deep-sea pressures. The ultimate objective of this experimental design was to be able to study microbial activities at chemical and physical conditions relevant to seafloor hydrothermal vents. Sulfide, sulfate and oxide minerals from sampled seafloor vent-chimney structures [East Pacific Rise (9 degrees 46'N)] served as source mineral and microbial inoculum for enrichment culturing using nitrate and sulfate-enriched media at 70 and 90 degrees C and 250 bars. Changes in microbial diversity during the continuous reaction flow were monitored using denaturing gradient gel electrophoresis (DGGE) of PCR amplified 16S rRNA gene fragments. Time series changes in fluid chemistry were also monitored throughout the experiment to assess the feedback between mineral-fluid reaction and metabolic processes. Data indicate a shift from the dominance of epsilon Proteobacteria in the initial inoculum to the several Aquificales-like phylotypes in nitrate-reducing enrichment media and Thermodesulfobacteriales in the sulfate-reducing enrichment media. Methanogens were detected in the original sulfide sample and grew in selected sulfate-enriched experiments. Microbial interactions with anhydrite and pyrrhotite in the chimney material resulted in measurable changes in fluid chemistry despite a fluid residence time only 75 min in the reactor. Changes in temperature rather than source material resulted in greater differences in microbial enrichments and mediated geochemical reactions.

Diversity of 16S rRNA gene, ITS region and aclB gene of the Aquificales.

The Aquificales are prevalent members of the microbial communities inhabiting many marine and terrestrial hydrothermal systems. Numerous new strains were obtained from deep-sea and terrestrial hydrothermal systems. In order to resolve the phylogenetic relationships within this group, three different phylogenetic datasets were used, namely the 16S rRNA gene, the intergenic transcribed spacer region between the 16S rRNA and 23S rRNA genes (ITS) and the gene coding for the ATP citrate lyase (aclB), a key enzyme in the reductive TCA cycle. The data were analyzed using neighbor-joining, parsimony and maximum likelihood. The resulting phylogenies appeared to be consistent between the three markers. The three genes confirmed the presence of isolates that merit further characterization and descriptions as new species and perhaps even new genera. The detailed phylogenetic interrelationships of these isolates are described here.

Characterization, chromosomal localization, and the complete 30-kb DNA sequence of the human Jagged2 (JAG2) gene.

The genomic sequence of the human Jagged2 (JAG2) gene, which encodes a ligand for the Notch receptors, was determined. The 30-kb DNA sequence spanning the JAG2 gene contains 26 exons and a putative promoter region. Several potential binding sites for transcription factors, including NF-kappab, E47, E12, E2F, Ets-1, MyoD, and OCT-1, were found in the human JAG2 promoter region. The JAG2 gene was also mapped to the chromosomal region 14q32 using fluorescence in situ hybridization.

Cloning, characterization, and the complete 56.8-kilobase DNA sequence of the human NOTCH4 gene.

The first complete mammalian genomic sequence reported thus far in the Notch gene family, including a putative promoter region and 30 exons of the human NOTCH4 gene spanning 56.8 kb of DNA, were sequenced. The NOTCH4 locus contains a TATA-less promoter with two putative transcription initiation sites (Inr), three RBP-Jkappa sites, and two GATA recognition sites. Two cDNA isoforms, NOTCH4(L) and NOTCH4(S),were identified. Whereas the NOTCH4(S) isoform contains the entire coding sequence, the NOTCH4(L) isoform has two unspliced intronic sequences between exons 11 and 12 and exons 20 and 21 and a misspliced exon 6. Consistent with these results, two alternatively spliced isoforms of transcripts of approximately 9.3 and 6.7 kb were detected by Northern blot analysis. The predicted amino acid sequence of the NOTCH4 protein based on the NOTCH4(S) cDNA sequence contains 2003 amino acids and includes the predominant motifs of the Notch family: 29 epidermal growth factor (EGF)-like repeats, 3 Notch/lin-12 repeats, a transmembrane region, 6 cdc10/Ankyrin repeats, and a PEST domain.

Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1.

Alagille syndrome is an autosomal dominant disorder characterized by abnormal development of liver, heart, skeleton, eye, face and, less frequently, kidney. Analyses of many patients with cytogenetic deletions or rearrangements have mapped the gene to chromosome 20p12, although deletions are found in a relatively small proportion of patients (< 7%). We have mapped the human Jagged1 gene (JAG1), encoding a ligand for the developmentally important Notch transmembrane receptor, to the Alagille syndrome critical region within 20p12. The Notch intercellular signalling pathway has been shown to mediate cell fate decisions during development in invertebrates and vertebrates. We demonstrate four distinct coding mutations in JAG1 from four Alagille syndrome families, providing evidence that it is the causal gene for Alagille syndrome. All four mutations lie within conserved regions of the gene and cause translational frameshifts, resulting in gross alterations of the protein product Patients with cytogenetically detectable deletions including JAG1 have Alagille syndrome, supporting the hypothesis that haploinsufficiency for this gene is one of the mechanisms causing the Alagille syndrome phenotype.

Structure and evolution of ribonuclease P RNA in Gram-positive bacteria.

The sequences and structures of RNase P RNAs of some Gram-positive bacteria, e.g. Bacillus subtilis, are very different than those of other bacteria. In order to expand our understanding of the structure and evolution of RNase P RNA in Gram-positive bacteria, gene sequences encoding RNase P RNAs from 10 additional species from this evolutionary group have been determined, doubling the number of sequences available for comparative analysis. The enlarged data set allows refinement of the secondary structure model of these unusual RNase P RNAs and the identification of potential tertiary interactions between P10.1 and L12, and between L5.1 and L15.1. The newly-obtained sequences suggest that RNase P RNA underwent an abrupt, dramatic restructuring in the ancestry of the low-G+C Gram-positive bacteria after the divergence of the branches leading to the 'Clostridia and relatives' and the remaining low-G+C Gram-positive species. The unusual structures of the RNase P RNAs of Mycoplasma hyopneumoniae and M.floccularre are apparently derived from RNAs with Bacillus-like structure rather than from intermediate, partially restructured ancestral RNAs. The structure of the RNase P RNA from the photosynthetic Heliobacillus mobilis supports the relationship of this specie with Bacillus and Staphylococcus rather than the 'Clostridia and relatives' as suggested by the sequences of their small-subunit ribosomal RNAs.

Mycoplasma fermentans simplifies our view of the catalytic core of ribonuclease P RNA.

The catalytic RNA moiety of (eu)bacterial RNase P is responsible for cleavage of the 5' leader sequence from precursor tRNAs. We report the sequence, the catalytic properties, and a phylogenetic-comparative structural analysis of the RNase P RNA from Mycoplasma fermentans, at 276 nt the smallest known RNase P RNA. This RNA is noteworthy in that it lacks a stem-loop structure (helix P12) that was thought previously to be universally present in bacterial RNase P RNAs. This finding suggests that helix P12 is not required for catalytic activity in vivo. In order to test this possibility in vitro, the kinetic properties of M. fermentans RNase P RNA and a mutant Escherichia coli RNase P RNA that was engineered to lack helix P12 were determined. These RNase P RNAs are catalytically active with efficiencies (Kcat/Km) comparable to that of native E. coli RNase P RNA. These results show that helix P12 is dispensable in vivo in some organisms, and therefore is unlikely to be essential for the mechanism of RNase P action. The notion that all phylogenetically volatile structures in RNase P RNA are dispensable for the catalytic mechanism was tested. A synthetic RNA representing the phylogenetic minimum RNase P RNA was constructed by deleting all evolutionarily variable structures from the M. fermentans RNA. This simplified RNA (Micro P RNA) was catalytically active in vitro with approximately 600-fold decrease in catalytic efficiency relative to the native RNA.