Solution spectroscopy of dipicolinic acid interaction with nucleic acids: Role in spore heat resistance

The effect of dipicolinic acid (DPA) or its calcium chelate (CaDPA) on the spectral characteristics of nucleic acids was examined. Dipicolinic acid was found to displace ethidium gromide from DNA; this indicates that it may bind by intercalation. On interaction with DNA, the ultraviolet absorption spectrum revealed downfield shifts and caused progressive diminution in both DNA and dipicolinate chromophores. The strength and type of interaction may be ion-specific but not discriminatory to any type of base pairing. Spectral analysis also indicated that both dipicolinate and calcium dipicolinate bound to different RNA species, although the mechanism of binding was not elucidated. We conclude that the interaction of dipicolinate/ calcium dipicolinate with nucleic acids is a mechanism whereby water can be removed from spore polynucleotides, increasing their stability to denaturation.     

Familial multiple-system tauopathy with presenile dementia is localized to chromosome 17.

An autosomal dominant presenile dementia affecting 39 individuals in a seven-generation, 383-member pedigree has been studied at Indiana University. In the affected members of this family, clinical symptoms occurred early in life, with an average age at onset of 48.8 years. The presenting clinical features include disequilibrium, neck stiffness, dysphagia, and memory loss. As the disease progresses, further cognitive decline, superior-gaze palsy, and dystaxia also are observed. The average duration from onset of symptoms to death is approximately 10 years. Neuropathologic studies of nine affected individuals showed neuronal loss in several areas of the CNS, as well as argentophilic tau-immunopositive inclusions in neurons and in oligodendroglia. A limited genomic screen by use of DNA samples from 28 family members localized the gene for this disorder to a 3-cM region on chromosome 17, between the markers THRA1 and D17S791. The gene for tau also was analyzed, through samples from the family.     

Molecular Ecological Analysis of Methanogens and Methanotrophs in Blanket Bog Peat

Abstract Methane production and methane oxidation potential were measured in a 30 cm peat core from the Moorhouse Nature Reserve, UK. The distribution of known groups of methanogens and methane oxidizing bacteria throughout this peat core was assessed. Using 16S rRNA gene retrieval and functional gene probing with genes encoding key proteins in methane oxidation and methanogenesis, several major groups of microorganisms were detected. Methane production and oxidation was detected in all depths of the peat core. PCR amplification and oligonucleotide probing experiments using DNA isolated from all sections of the peat core detected methanotrophs from the groups Methylosinus and Methylococcus and methanogens from the groups Methanosarcinaceae, Methanococcaceae, and Methanobacteriaceae. 16S rDNA sequences amplified with the Methylosinus-specific primer were shown to have a high degree of identity with 16S rDNA sequences previously detected in acidic environments. However, no methanogen sequences were detected by the probes available in this study in the sections of the peat core (above 7 cm) where the majority of methanogenesis occurred, either because of low methanogen numbers or because of the presence of novel methanogen sequences. Received: 9 March 1999; Accepted: 21 June 1999     

The particulate methane monooxygenase gene pmoA and its use as a functional gene probe for methanotrophs

The particulate methane monooxygenase gene pmoA, encoding the 27 kDa polypeptide of the membrane-bound particulate methane monooxygenase, was amplified by PCR from DNA isolated from a blanket peat bog and from enrichment cultures established, from the same environment, using methane as sole carbon and energy source. The resulting 525 bp PCR products were cloned and a representative number of clones were sequenced. Phylogenetic analysis of the derived amino acid sequences of the pmoA clones retrieved directly from environmental DNA samples revealed that they form a distinct cluster within representative PmoA sequences from type II methanotrophs and may originate from a novel group of acidophilic methanotrophs. The study also demonstrated the utility of the pmoA gene as a phylogenetic marker for identifying methanotroph-specific DNA sequences in the environment.     

Single‐cell genomics based on Raman sorting reveals novel carotenoid‐containing bacteria in the Red Sea

Cell sorting coupled with single‐cell genomics is a powerful tool to circumvent cultivation of microorganisms and reveal microbial ‘dark matter’. Single‐cell Raman spectra (SCRSs) are label‐free biochemical ‘fingerprints’ of individual cells, which can link the sorted cells to their phenotypic information and ecological functions. We employed a novel Raman‐activated cell ejection (RACE) approach to sort single bacterial cells from a water sample in the Red Sea based on SCRS. Carotenoids are highly diverse pigments and play an important role in phototrophic bacteria, giving strong and distinctive Raman spectra. Here, we showed that individual carotenoid‐containing cells from a Red Sea sample were isolated based on the characteristic SCRS. RACE‐based single‐cell genomics revealed putative novel functional genes related to carotenoid and isoprenoid biosynthesis, as well as previously unknown phototrophic microorganisms including an unculturable Cyanobacteria spp. The potential of Raman sorting coupled to single‐cell genomics has been demonstrated.     

Revealing the uncultivated majority: combining DNA stable-isotope probing, multiple displacement amplification and metagenomic analyses of uncultivated Methylocystis in acidic peatlands

Peatlands represent an enormous carbon reservoir and have a potential impact on the global climate because of the active methanogenesis and methanotrophy in these soils. Uncultivated methanotrophs from seven European peatlands were studied using a combination of molecular methods. Screening for methanotroph diversity using a particulate methane monooxygenase-based diagnostic gene array revealed that Methylocystis-related species were dominant in six of the seven peatlands studied. The abundance and methane oxidation activity of Methylocystis spp. were further confirmed by DNA stable-isotope probing analysis of a sample taken from the Moor House peatland (England). After ultracentrifugation, (13)C-labelled DNA, containing genomic DNA of these Methylocystis spp., was separated from (12)C DNA and subjected to multiple displacement amplification (MDA) to generate sufficient DNA for the preparation of a fosmid metagenomic library. Potential bias of MDA was detected by fingerprint analysis of 16S rRNA using denaturing gradient gel electrophoresis for low-template amplification (0.01 ng template). Sufficient template (1-5 ng) was used in MDA to circumvent this bias and chimeric artefacts were minimized by using an enzymatic treatment of MDA-generated DNA with S1 nuclease and DNA polymerase I. Screening of the metagenomic library revealed one fosmid containing methanol dehydrogenase and two fosmids containing 16S rRNA genes from these Methylocystis-related species as well as one fosmid containing a 16S rRNA gene related to that of Methylocella/Methylocapsa. Sequencing of the 14 kb methanol dehydrogenase-containing fosmid allowed the assembly of a gene cluster encoding polypeptides involved in bacterial methanol utilization (mxaFJGIRSAC). This combination of DNA stable-isotope probing, MDA and metagenomics provided access to genomic information of a relatively large DNA fragment of these thus far uncultivated, predominant and active methanotrophs in peatland soil.     

Diversity and activity of methanotrophs in alkaline soil from a Chinese coal mine

Culture-independent molecular biological techniques, including 16S rRNA gene and functional gene clone libraries and microarray analyses using pmoA (encoding a key subunit of particulate methane monooxygenase), were applied to investigate the methanotroph community structure in alkaline soil from a Chinese coal mine. This environment contained a high diversity of methanotrophs, including the type II methanotrophs Methylosinus / Methylocystis , type I methanotrophs related to Methylobacter / Methylosoma and Methylococcus , and a number of as yet uncultivated methanotrophs. In order to identify the metabolically active methane-oxidizing bacteria from this alkaline environment, DNA stable isotope probing (DNA-SIP) experiments using ¹³CH₄ were carried out. This showed that both type I and type II methanotrophs were active, together with methanotrophs related to Methylocella , which had previously been found only in acidic environments. Methylotrophs, including Methylopila and Hyphomicrobium , were also detected in soil DNA and after DNA-SIP experiments. DNA sequence information on the most abundant, active methanotrophs in this alkaline soil will facilitate the design of oligonucleotide probes to monitor enrichment cultures when isolating key alkaliphilic methanotrophs from such environments.