Oxygen isotope effects during microbial sulfate reduction: applications to sediment cell abundances.

The majority of anaerobic biogeochemical cycling occurs within marine sediments. To understand these processes, quantifying the distribution of active cells and gross metabolic activity is essential. We present an isotope model rooted in thermodynamics to draw quantitative links between cell-specific sulfate reduction rates and active sedimentary cell abundances. This model is calibrated using data from a series of continuous culture experiments with two strains of sulfate reducing bacteria (freshwater bacterium Desulfovibrio vulgaris strain Hildenborough, and marine bacterium Desulfovibrio alaskensis strain G-20) grown on lactate across a range of metabolic rates and ambient sulfate concentrations. We use a combination of experimental sulfate oxygen isotope data and nonlinear regression fitting tools to solve for unknown kinetic, step-specific oxygen isotope effects. This approach enables identification of key isotopic reactions within the metabolic pathway, and defines a new, calibrated framework for understanding oxygen isotope variability in sulfate. This approach is then combined with porewater sulfate/sulfide concentration data and diagenetic modeling to reproduce measured 18O/16O in porewater sulfate. From here, we infer cell-specific sulfate reduction rates and predict abundance of active cells of sulfate reducing bacteria, the result of which is consistent with direct biological measurements.

Sulfur mass-independent fractionation in subsurface fracture waters indicates a long-standing sulfur cycle in Precambrian rocks.

The discovery of hydrogen-rich waters preserved below the Earth's surface in Precambrian rocks worldwide expands our understanding of the habitability of the terrestrial subsurface. Many deep microbial ecosystems in these waters survive by coupling hydrogen oxidation to sulfate reduction. Hydrogen originates from water-rock reactions including serpentinization and radiolytic decomposition of water induced by decay of radioactive elements in the host rocks. The origin of dissolved sulfate, however, remains unknown. Here we report, from anoxic saline fracture waters ∼2.4 km below surface in the Canadian Shield, a sulfur mass-independent fractionation signal in dissolved sulfate. We demonstrate that this sulfate most likely originates from oxidation of sulfide minerals in the Archaean host rocks through the action of dissolved oxidants (for example, HO· and H2O2) themselves derived from radiolysis of water, thereby providing a coherent long-term mechanism capable of supplying both an essential electron donor (H2) and a complementary acceptor (sulfate) for the deep biosphere.

Occult cytomegalovirus in vivarium-housed mice may influence transplant allograft acceptance.

We have recently shown that latent murine cytomegalovirus (MCMV) can influence murine transplant allograft acceptance. During these studies we became aware that vivarium-housed control mice can acquire occult MCMV infection. The purpose of this investigation was to confirm occult MCMV transmission and determine the timing, vehicle, and possible consequences of transmission. Mice arriving from a commercial vendor were negative for MCMV both by commercial serologic testing and by our nested PCR. Mice housed in our vivarium became positive for MCMV DNA 30-60 days after arrival, but remained negative for MCMV by commercial serologic testing. To confirm MCMV we sequenced PCR products for several genes and showed >99% homology to MCMV. Further sequence analyses show that the occult MCMV is similar to a laboratory strain of MCMV, but the vehicle of transmission remains unclear. Control tissues from historical experiments with unexplained graft losses were evaluated for occult MCMV, and mice with unexplained allograft losses showed significantly higher incidence of occult MCMV than did allograft acceptors. Deliberate infection with very low titer MCMV confirmed that viral transmission can occur without measurable virus specific antibody or T-cell responses. These data suggest that vivarium-housed mice can develop occult MCMV that is missed by currently available commercial serologic testing, and that these infections may influence transplant allograft acceptance.

Analysis and mapping of a family of 3'-coterminal transcripts containing coding sequences for human cytomegalovirus open reading frames UL93 through UL99.

Human cytomegalovirus (HCMV) open reading frames (ORFs) UL93 through UL99 are contained within a region of viral genome that is well conserved in all herpesviruses. Previous reports detailing the expression of ORF UL99 (also referred to as the 28-kDa virion phosphoprotein or pp28) indicated that the pattern of transcription proximal to pp28 is extremely complex and involves a number of large overlapping transcripts, none of which have been characterized. We have used an RNA-mapping approach consisting of Northern (RNA) hybridization, RNase protection, and primer extensions to determine the coding capacity of several large-molecular-weight transcripts which overlap the 1.3- and 1.6-kb UL99-specific transcripts. Our results suggest that six differentially regulated transcripts with sizes of 2.6, 4.7, 5.6, 7.3, 9.1, and 10.5 kb, and derived from the same strand of the viral genome overlap, are 3'-coterminal with the smaller UL99-specific transcripts. On the basis of 5'-end mapping via primer extension and RNase protection, we have determined that the 2.6- to 10.5-kb messages initiate upstream of each of the potential ORFs in this region, UL98, UL97, UL96, UL95, UL94, and UL93. By using cycloheximide and ganciclovir [9-(1,3-dihydroxy-2-propoxymethyl)guanine] to block de novo viral protein synthesis and viral DNA replication, respectively, we have determined that the 2.6-, 4.7-, 5.6-, and 7.3-kb messages have characteristics of early or early-late transcripts, whereas the 9.1- and 10.5-kb messages appear to be true late transcripts. The evolutionary conservation of ORFs UL93 through UL99 and their transcriptional regulation in other herpesviruses are discussed.

Identification of positive and negative regulatory regions involved in regulating expression of the human cytomegalovirus UL94 late promoter: role of IE2-86 and cellular p53 in mediating negative regulatory function.

The human cytomegalovirus (HCMV) UL94 gene product is a herpesvirus-common virion protein that is expressed with true late kinetics. To identify the important cis- and trans-acting factors which contribute to UL94 transcriptional regulation, we have cloned, sequenced, and analyzed UL94 promoter function by transient transfection analysis. Transfection of UL94 promoter-reporter gene constructs into permissive human fibroblasts or U373(MG) cells indicated that promoter activity was detected following infection with HCMV. Point mutations within a TATA-like element located upstream of the RNA start site significantly reduced UL94 promoter activity. Deletion mutagenesis of the promoter indicated that a positive regulatory element (PRE) was likely to exist downstream of the UL94 mRNA start site, while a negative regulatory element (NRE) was present upstream of the TATA box. At late times of infection, the PRE appeared to have a dominant effect over the NRE to stimulate maximum levels of UL94 promoter activity, while at earlier times of infection, no activity associated with the PRE could be detected. The NRE, however, appeared to cause constitutive down-regulation of UL94 promoter activity. Binding sites for the cellular p53 protein located within the NRE appeared to contribute to NRE function, and NRE function could be recapitulated in cotransfection assays by concomitant expression of p53 and HCMV IE2-86 protein. Our results suggest a novel mechanism by which the cellular protein p53, which is involved in both transcriptional regulation and progression of cellular DNA synthesis, plays a central role in the regulation of a viral promoter which is not activated prior the onset of viral DNA replication.

The human cytomegalovirus UL94 open reading frame encodes a conserved herpesvirus capsid/tegument-associated virion protein that is expressed with true late kinetics.

In this report, we provide a detailed characterization of the human cytomegalovirus (HCMV) UL94 gene product. Northern (RNA) blot analysis of infected cell RNA demonstrated that UL94 message was found only at late times of infection and was not synthesized in the presence of the viral DNA replication inhibitor ganciclovir. Expression of the UL94 open reading frame in vitro and in vivo yielded a protein with the predicted molecular mass of 36 kDa. A monoclonal antibody raised to a UL94-specific peptide reacted specifically with a 36-kDa protein in HCMV-infected fibroblasts. This protein was found only at late times of infection and was also present in purified HCMV virions. Fractionation of purified virions and HCMV-infected cells revealed an association of UL94 immunoreactivity with the capsid/tegument and nuclear fractions, respectively. The evolutionary conservation of UL94 protein sequence and an analysis of potential functional regions of the protein are discussed.

Mass-independent sulfur of inclusions in diamond and sulfur recycling on early Earth.

Populations of sulfide inclusions in diamonds from the Orapa kimberlite pipe in the Kaapvaal-Zimbabwe craton, Botswana, preserve mass-independent sulfur isotope fractionations. The data indicate that material was transferred from the atmosphere to the mantle in the Archean. The data also imply that sulfur is not well mixed in the diamond source regions, allowing for reconstruction of the Archean sulfur cycle and possibly offering insight into the nature of mantle convection through time.