Evidence for iron-mediated anaerobic methane oxidation in a crude oil-contaminated aquifer.

In a methanogenic crude oil contaminated aquifer near Bemidji, Minnesota, the decrease in dissolved CH(4) concentrations along the groundwater flow path, along with the positive shift in δ(13) C(CH) (4) and negative shift in δ(13) C(DIC) , is indicative of microbially mediated CH(4) oxidation. Calculations of electron acceptor transport across the water table, through diffusion, recharge, and the entrapment and release of gas bubbles, suggest that these processes can account for at most 15% of the observed total reduced carbon oxidation, including CH(4) . In the anaerobic plume, the characteristic Fe(III)-reducing genus Geobacter was the most abundant of the microbial groups tested, and depletion of labile sediment iron is observed over time, confirming that reduced carbon oxidation coupled to iron reduction is an important process. Electron mass balance calculations suggest that organic carbon sources in the aquifer, BTEX and non-volatile dissolved organic carbon, are insufficient to account for the loss in sediment Fe(III), implying that CH(4) oxidation may also be related to Fe(III) reduction. The results support a hypothesis of Fe(III)-mediated CH(4) oxidation in the contaminated aquifer.

Novel bacterial and archaeal lineages from an in situ growth chamber deployed at a Mid-Atlantic Ridge hydrothermal vent.

The phylogenetic diversity was determined for a microbial community obtained from an in situ growth chamber placed on a deep-sea hydrothermal vent on the Mid-Atlantic Ridge (23 degrees 22' N, 44 degrees 57' W). The chamber was deployed for 5 days, and the temperature within the chamber gradually decreased from 70 to 20 degrees C. Upon retrieval of the chamber, the DNA was extracted and the small-subunit rRNA genes (16S rDNA) were amplified by PCR using primers specific for the Archaea or Bacteria domain and cloned. Unique rDNA sequences were identified by restriction fragment length polymorphisms, and 38 different archaeal and bacterial phylotypes were identified from the 85 clones screened. The majority of the archaeal sequences were affiliated with the Thermococcales (71%) and Archaeoglobales (22%) orders. A sequence belonging to the Thermoplasmales confirms that thermoacidophiles may have escaped enrichment culturing attempts of deep-sea hydrothermal vent samples. Additional sequences that represented deeply rooted lineages in the low-temperature eurarchaeal (marine group II) and crenarchaeal clades were obtained. The majority of the bacterial sequences obtained were restricted to the Aquificales (18%), the epsilon subclass of the Proteobacteria (epsilon-Proteobacteria) (40%), and the genus Desulfurobacterium (25%). Most of the clones (28%) were confined to a monophyletic clade within the epsilon-Proteobacteria with no known close relatives. The prevalence of clones related to thermophilic microbes that use hydrogen as an electron donor and sulfur compounds (S(0), SO(4), thiosulfate) indicates the importance of hydrogen oxidation and sulfur metabolism at deep-sea hydrothermal vents. The presence of sequences that are related to sequences from hyperthermophiles, moderate thermophiles, and mesophiles suggests that the diversity obtained from this analysis may reflect the microbial succession that occurred in response to the shift in temperature and possible associated changes in the chemistry of the hydrothermal fluid.

Geomicrobiology of subglacial ice above Lake Vostok, Antarctica.

Data from ice 3590 meters below Vostok Station indicate that the ice was accreted from liquid water associated with Lake Vostok. Microbes were observed at concentrations ranging from 2.8 x 10(3) to 3.6 x 10(4) cells per milliliter; no biological incorporation of selected organic substrates or bicarbonate was detected. Bacterial 16S ribosomal DNA genes revealed low diversity in the gene population. The phylotypes were closely related to extant members of the alpha- and beta-Proteobacteria and the Actinomycetes. Extrapolation of the data from accretion ice to Lake Vostok implies that Lake Vostok may support a microbial population, despite more than 10(6) years of isolation from the atmosphere.

Amplification, cloning, and sequencing of a nifH segment from aquatic microorganisms and natural communities.

By use of the polymerase chain reaction and degenerate oligonucleotide primers for highly conserved regions of nifH, a segment of nifH DNA was amplified from several aquatic microorganisms, including an N2-fixing bacterium closely associated with the marine filamentous cyanobacterium Trichodesmium sp., a heterotrophic isolate from the root/rhizome of the seagrass Ruppia maritima, and the heterocystous freshwater cyanobacterium Anabaena oscillarioides. nifH segments were amplified directly from DNA extracted from the rhizosphere of roots of the seagrass Halodule wrightii. The nifH fragments were then cloned and sequenced. The DNA and deduced amino acid sequences were compared with known sequences, revealing distinct differences between taxonomic groups. This technique was shown to be useful for (i) the detection of N2-fixing microorganisms and (ii) rapidly obtaining the DNA sequence of the nifH gene, which provides information about general taxonomic groups of N2-fixing microorganisms.

The Malmö polymorphism of coagulation factor IX, an immunologic polymorphism due to dimorphism of residue 148 that is in linkage disequilibrium with two other F.IX polymorphisms.

A mouse monoclonal antibody (MAB 9.9) to coagulation factor IX (F.IX) detects a polymorphism in the plasma of normal people. Its epitope has been narrowed down to less than 6 amino acids in the activation peptide of the X-linked F.IX protein. The activation peptide contains a dimorphism--Thr:Ala--at position 148 of the protein. Using synthetic oligonucleotides, we have demonstrated that (1) the F.IX which reacts with 9.9 has Thr at position 148 and (2) that which does not has Ala. Positive reactors (148thr) are designated Malmö A, and negative reactors (148ala) are designated Malmö B. The plasma levels of AA women are indistinguishable from those of A men, and both B men and BB women are null against MAB 9.9. The plasma level of Malmö A in AB women is approximately half that of AA women, and "lyonization" is clearly operating in the heterozygotes. The dimorphism is in strong linkage disequilibrium with two other intragenic RFLPs, TaqI and XmnI. Furthermore, intragenic crossing-over--including double crossing-over--appears to have occurred between the three sites. Seven of the eight possible haplotypes have been identified, five in men and two others in women. The immunoassay that identifies approximately 50% of the AB women in the pool of Malmö A females with 95% confidence identifies men unambiguously as A or B. The assay would be very useful for population-genetic studies of the Malmö epitope if the studies were limited to men.

Population genetics of coagulant factor IX: frequencies of two DNA polymorphisms in five ethnic groups.

Two frequently used restriction-enzyme polymorphisms (RFLPs) of coagulant F.IX, TaqI and XmnI, have been examined in five ethnic groups: white Americans, black Americans, East Indians, Chinese, and Malays. There is a distinct "cline" in the frequencies of both polymorphisms, from white Americans to Malays. The rarer type 2 alleles of both polymorphisms, in which middle recognition sites are present--and which in our sample reach their highest frequencies in white Americans--are marginally higher in four groups of Europeans previously reported by others. The frequencies of the rarer alleles are significantly higher in Europeans than in black Americans and East Indians, and these alleles are essentially absent in Chinese and Malays. The frequency of heterozygosity diminishes in the same order, being zero in Malays for both polymorphisms. The polymorphisms are in strong linkage disequilibrium, and in all groups the type 1 allele for TaqI is disproportionately accompanied by the type 1 allele for XmnI. The paucity of type 2 alleles and the low rate of heterozygosity in four non-European groups suggest that the polymorphisms will be of little diagnostic value south of Gibraltar and east of Suez. This prediction is confirmed by the observed haplotype frequencies in the black American and the Oriental groups.

Leishmania donovani: immunization against infection as a function of parasite growth phase.

C57BL/6 mice were immunized against Leishmania donovani infection with a subcutaneous vaccination protocol. Groups received 3 injections at 4-day intervals combining glucan and killed promastigotes harvested from either logarithmic or stationary phase cultures. Controls were immunized with glucan alone, stationary or log phase promastigotes alone, or were untreated. All groups were challenged intravenously with stationary phase promastigotes at day 45 post-immunization. Results revealed that animals immunized with the glucan-killed parasite vaccine, utilizing promastigotes derived from either log (GPL) or stationary phase cultures (GPS), demonstrated significant resistance against infection as compared to controls or untreated mice. Additionally, the reduction in hepatic amastigote proliferation in mice immunized with GPS was significantly greater than in mice immunized with GPL.