Comparison of membrane organization in mitochondria from yeast and rat liver by nuclear magnetic resonance spectroscopy

Summary Proton magnetic resonance (PMR) and carbon-13 magnetic resonance (CMR) spectra of intact, unsonicated yeast and rat liver motochondria show differences which may be correlated with the composition of the membranes. High resolution PMR and CMR signals in intact yeast mitochondria have been assigned to regions of fluid lipid-lipid interaction on the basis of spectra of extracted lipid and protein, and the temperature dependence of NMR signals from the intact membrane. PMR spectra suggest that about 20% of total yeast phospholipid is in regions where both intramolecular fatty acid chain mobility and lateral diffusion of entire phospholipid molecules are possible. No such regions appear to exist in rat liver mitochondria. For both yeast and rat liver mitochondria, comparison of PMR and CMR spectra suggests that about 50% of phospholipid appears to be in regions where intramolecular fatty acid chain motion is considerable, but lateral diffusion is restricted. The remaining phospholipid appears to have little inter- or intramolecular mobility. Since NMR observation of lipid extracts from membranes indicates that phospholipid-sterol interactions do not account for the spectra of intact mitochondria, these effects are interpreted in terms of extensive lipid-protein interactions.     

Complementation of human adenovirus type 5 ts mutants by human adenovirus type 12.

Temperature-sensitive mutants of type 5 adenovirus belonging to eight complementation groups were complemented in mixed infection by type 12 adenovirus, whereas mutants of 7 other groups were not enhanced. In some crosses, phenotypic mixing took place. No evidence of recombination between type 5 ts mutants and type 12 was found.     

Microbial diversity of intestinal contents and mucus in rainbow trout (Oncorhynchus mykiss)

AIMS: The aim of this study was to understand the microbial community of intestinal contents and mucosal layer in the intestine of rainbow trout by means of culture-dependent conventional and independent molecular techniques. METHODS AND RESULTS: Forty-one culturable microbial phylotypes, and 39 sequences from 16S rRNA and two from 18S rRNA genes, were retrieved. Aeromonadaceae, Enterobacteriaceae and Pseudomonadaceae representatives were the dominant cultured bacteria. Genomic DNA isolated from intestinal contents and mucus was used to generate 104 random clones, which were grouped into 32 phylotypes at 99% minimum similarity, most of which were affiliated with Proteobacteria (>70% of the total). However, unlike library C (intestinal contents), the phyla Bacteroidetes and Fusobacteria were not found in intestinal mucus (library M), indicating that the microbiota in the gut mucus was different from that of the intestinal contents. Twelve sequences were retrieved from denaturing gradient gel electrophoresis analysis, and dominant bands were mostly related to Clostridium. CONCLUSIONS: Many novel sequences that have not been previously recognized as part of the intestinal flora of rainbow trout were retrieved. SIGNIFICANCE AND IMPACT OF THE STUDY: The fish gut harbours a larger bacterial diversity than previously recognized, and the diversity of gut mucus is different from that of intestinal contents.     

Periplasmic localization of nicotinate phosphoribosyltransferase in Escherichia coli.

Nicotinate phosphoribosyltransferase (NAPRTase) in Escherichia coli mediates the formation of nicotinate mononucleotide, a direct precursor of nicotinamide adenine dinucleotide (NAD), from nicotinate and 5-phosphoribosyl-1-pyrophosphate. Specifically, NAPRTase contributes to NAD synthesis by utilizing intracellular nicotinate formed from NAD degradation products, which are recycled by NAD cycle enzymes and exogenous nicotinate when it is available. In previous studies, it has been tacitly assumed that almost all NAD cycle enzymes are localized in the cytoplasm of E. coli. The results of this investigation provide evidence that NAPRTase is a periplasmic (extracytoplasmic) enzyme. The osmotic shock of exponential-phase cells of E. coli K-12 and ML 308-225 resulted in the release of 63 to 72% and 42 to 48%, respectively, of the NAPRTase into the shock medium. In addition, when exponential cells of strains K-12 and ML 308-225 were converted into spheroplasts, 75 to 84% and 54 to 68%, respectively, of the enzyme was released into the spheroplast medium. Since previous estimates of the effective levels of NAPRTase present in putative repressed and derepressed E. coli cells appeared to be very low, a more convenient and accurate alternative method for the evaluation of NAPRTase in whole cells was developed. The results show that NAPRTase is subject only to a modest degree of enzyme repression. In addition, no evidence was found for the presence of a protein or low-molecular-weight inhibitor of the enzyme in repressed cells.     

Novel high-resolution characterization of ancient DNA reveals C > U-type base modification events as the sole cause of post mortem miscoding lesions

Ancient DNA (aDNA) research has long depended on the power of PCR to amplify trace amounts of surviving genetic material from preserved specimens. While PCR permits specific loci to be targeted and amplified, in many ways it can be intrinsically unsuited to damaged and degraded aDNA templates. PCR amplification of aDNA can produce highly-skewed distributions with significant contributions from miscoding lesion damage and non-authentic sequence artefacts. As traditional PCR-based approaches have been unable to fully resolve the molecular nature of aDNA damage over many years, we have developed a novel single primer extension (SPEX)-based approach to generate more accurate sequence information. SPEX targets selected template strands at defined loci and can generate a quantifiable redundancy of coverage; providing new insights into the molecular nature of aDNA damage and fragmentation. SPEX sequence data reveals inherent limitations in both traditional and metagenomic PCR-based approaches to aDNA, which can make current damage analyses and correct genotyping of ancient specimens problematic. In contrast to previous aDNA studies, SPEX provides strong quantitative evidence that C > U-type base modifications are the sole cause of authentic endogenous damage-derived miscoding lesions. This new approach could allow ancient specimens to be genotyped with unprecedented accuracy.     

Identification, characterization, and spatial localization of two flagellin species in Helicobacter pylori flagella.

Flagellar filaments were isolated from Helicobacter pylori by shearing, and flagellar proteins were further purified by a variety of techniques, including CsCl density gradient ultracentrifugation, pH 2.0 acid disassociation-neutral pH reassociation, and differential ultracentrifugation followed by molecular sieving with a Sephacryl S-500 column or Mono Q anion-exchange column, and purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to an Immobilon membrane. Two flagellin species of pI 5.2 and with apparent subunit molecular weights (Mrs) of 57,000 and 56,000 were obtained. N-terminal amino acid analysis showed that the two H. pylori flagellin species were related to each other and shared sequence similarity with the N-terminal amino acid sequence of Campylobacter coli, Bacillus, Salmonella, and Caulobacter flagellins. Analysis of the amino acid composition of the predominant 56,000-Mr flagellin species isolated from two strains showed that it was comparable to the flagellins of other species. The minor 57,000-Mr flagellin species contained a higher content of proline. Immunoelectron microscopic studies with polyclonal monospecific H. pylori antiflagellin antiserum and monoclonal antibody (MAb) 72c showed that the two different-Mr flagellin species were located in different regions of the assembled flagellar filament. The minor 57,000-Mr species was located proximal to the hook, and the major 56,000-Mr flagellin composed the remainder of the filament. Western immunoblot analysis with polyclonal rabbit antisera raised against H. pylori or Campylobacter jejuni flagellins and MAb 72c showed that the 56,000-Mr flagellin carried sequences antigenetically cross-reactive with the 57,000-Mr H. pylori flagellin and the flagellins of Campylobacter species. This antigenic cross-reactivity did not extend to the flagellins of other gram-negative bacteria. The 56,000-Mr flagellin also carried H. pylori-specific sequences recognized by two additional MAbs. The epitopes for these MAbs were not surface exposed on the assembled inner flagellar filament of H. pylori but were readily detected by immunodot blot assay of sodium dodecyl sulfate-lysed cells of H. pylori, suggesting that this serological test could be a useful addition to those currently employed in the rapid identification of this important pathogen.     

Specificity switching of the P1 plasmid centromere-like site.

The P1 plasmid partition site acts like a centromere, promoting accurate segregation of copies to daughter cells. A 34 bp segment is essential for partition and binds the plasmid ParB protein. Additional sequences act as specificity elements that direct the choice of copies for partition. They include a second ParB binding site and a site for the host integration host factor protein. Sites lacking one or more of these additional elements are switched to a different specificity. Defined mutants were scored for partition specificity and protein binding. The results suggest that the wild-type site is folded in a specific DNA-protein complex. Disruption of the complex leads to an open configuration which, while still active in partition, has altered recognition specificity.     

Identification of Vibrio harveyi using PCR amplification of the toxR gene

AIMS: The aim of this study was to develop an effective method for the identification of Vibrio harveyi based on using the toxR gene as a taxonomic marker. METHODS AND RESULTS: Primers for the toxR gene were designed for specificity to V. harveyi, and incorporated in a polymerase chain reaction (PCR). The results of the PCR, which took <5 h from DNA extraction to amplification, revealed positive amplification of the toxR gene fragment in 20 V. harveyi isolates including type strains, whereas DNA from 23 other Vibrionaceae type strains and 13 Vibrio parahaemolyticus strains were negative. The detection limit of the PCR was 4.0 x 10(3) cells ml(-1). In addition, the technique enabled the recognition of V. harveyi from diseased fish. CONCLUSIONS: The PCR was specific and sensitive, enabling the identification of V. harveyi within 5 h. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR allowed the rapid and sensitive detection of V. harveyi.